Livret complet.pdf

4th Annual Meeting November 4-6, 2015 Toulouse, France

Conseil Régional Midi-Pyrénées

22, Bd du Maréchal Juin

GDR 3545

G Protein-coupled Receptors

From physiology to drugs

(RCPG-Physio-Med)

Thanks to Sponsors and Partners of the GDR3545 in 2015

Thanks to the Sponsors of the 4th meeting of the GDR3545

4th Annual meeting of the GDR3545 « RCPG-Physio-Med » 1

SCIENTIFIC PROGRAM


Wednesday november 4

12:30 Registration

14:00 Welcome

SESSION 1 - GPCR REGULATION AND SIGNALING (Chairperson L Moulédous)

14:15 Ulla PETAJA-REPO, University of Oulu (Finland)

GPCR folding, maturation and trafficking

15:00 Andrew TOBIN, University of Leicester (UK)

Cellular and physiological impact of GPCR phosphorylation

15:45 Young Scientist selected by the British Pharmacological Society

Joaquin Botta, University of East Anglia, Norwich (UK)

Cognitive impairment induced by delta9-tetrahydrocannabinol occurs through

heteromers between cannabinoid CB1 and serotonin 5-HT2A receptors

16:00 Coffee break

16:30 Franck VANDERMOERE, IGF Université de Montpellier (France)

Phosphoproteomics of 5-HT2A/mGlu2 receptors: toward new insights into the

mechanism of action of hallucinogens and antipsychotics

17:00 Technological Lecture: Sébastien IGONET, Calixar

Stabilization of native & functional membrane proteins: CALIXAR approach

17:15 Young Scientists selected from posters

Lauriane Onfroy, I2MC Université de Toulouse (France)

Relationship between heterotrimeric G proteins and beta-adrenergic ligand efficiency

Wissem Deraredj Nadim, CBM Orleans (France)

The GTPase-activating Protein-related Domain of neurofibromin regulates MC1R-

mediated pigmentation in human melanocytes

Flavie Landomiel, INRA Tours (France)

-arrestins directly interact with PKA, promoting its translocation to the nucleus and

form a transcriptionally active complex with CREB upon stimulation of Gs-coupled

GPCRs

Nathalie Langonné, INRA Tours (France)

β-arrestin-dependent transcriptome & proteome: a comparative study

18:30-21:00 Poster Session 1 (odd numbers)

19:00-21:00 Buffet


Thursday november 5

SESSION 2 - NANOSCALE ORGANIZATION AND DYNAMICS OF GPCR SIGNALING

(Chairperson L Salomé)

9:00 Jörg STANDFUSS, Paul Scherrer Institute (Switzerland)

Molecular mechanisms of phosphorylation-dependent arrestin activation

9:45 Jean-Louis BANERES, IBMM Université de Montpellier (France)

Organization of a signaling complex between ghrelin receptor oligomers and G

proteins

10:30 Coffee break

11:00 Julie PERROY, IGF Université de Montpellier (France)

Improving single-cell BRET imaging using NanoLuc- Application to ERK activity sensing


Romain Gerbier, Institut Cochin Paris (France)

In vivo characterization of MTR/5-HT2c heteromers in mouse brain

Chady Nasrallah, IGF Montpellier (France)

Expression and purification of the human metabotropic Glutamate Receptor 5

12:00 Buffet

SESSION 3 - GPCR TARGETING IN PATHOPHYSIOLOGY

Special Session sponsored by Astrazeneca

GPCR targeting in atherothrombosis and bleeding (Chairperson J-M Sénard)

13:30 Stuart MUNDELL, University of Bristol (UK)

Mind the GAPP: Linking patient bleeding with G protein-coupled receptor function

14:15 Kenneth A. JACOBSON, NIH Bethesda (USA)

Structure and pharmacochemistry of purinergic receptors

15:00 Thibault LHERMUSIER/ Pierre SIE, CHU Toulouse (France)

Benefits and risks of new antiplatelet agents

15:30 Coffee break

Session 3 (Chairperson D Fourmy)

16 :00 Bernard LAGANE, Institut Pasteur Paris (France)

Mechanisms of HIV-1 escape/resistance to inhibition by CCR5 ligands

16:45 Jean-Claude REUBI, University of Berne (Switzerland)

Regulatory peptide receptors as targets for tumor diagnosis and therapy


17:30 Guenhael SANZ, INRA Jouy en Josas (France)

Stimulation of olfactory receptors expressed in cancer cells promotes their

invasiveness and proliferation, leading to increased metastasis emergence in vivo


Zuzana Hajasova, Université Paris Descartes (France)

The mGlu7 receptor: a new target in the therapy of cocaine and opiates addiction?

Pierre-Michaël Coly, Université de Rouen (France)

Chemotactic G-protein coupled receptors control cell migration by suppressing

autophagosome biogenesis

Messal Nassima, CRI Université Paris Diderot (France)

Anti-inflammatory properties of the neuropeptide orexins in ulcerative colitis: a new

promising therapeutical molecule

18:45 General Assembly of GDR members.

Hybrigenics Yeast two-hybrid screen project selected in 2014: Report

19:30-22:00 Poster Session 2 (even numbers)

20:00-22:00 Buffet

Friday november 6

SESSION 4 - DRUG-DISCOVERY AND GENETIC APPROACHES TO ADDRESS GPCR FUNCTION

(Chairperson A Milon)

9:00 Timo Müller, Helmholtz Zentrum München and Technische Universität München

(Germany)

A rationally designed monomeric peptide triagonist corrects obesity and diabetes in

rodents

9:45 Gregg SIEGAL, ZoBio Leidein (The Netherlands) Stabilized GPCRs Enable Biophysics and Structure Based Drug Discovery

10:30 Coffe break

11:00 Frédéric SIMONIN, ESBS Université de Strasbourg (France)

Genetic models for the study of RFamide receptors


Arturo Roca-Rivada, Institut Cochin Paris (France)

Regulation of energy metabolism by Endospanin 1, a negative regulator of the leptin

receptor


Inna Slynko, LIT Université de Strasbourg (France)

Searching for ligands targeting a GPCR homodimeric interface

12:00 Ralf JOCKERS, Institut Cochin Université Descartes Paris (France)

Therapeutic potential of orphan GPCR

12:45-13:30 Awards and Conclusion

09:00

12:00

12:30

13:30

14:15

16:00

18:30

19:00

19:30 Poster Session 2 (even

20:00 Buffet

21:00

22:00

Wednesday november 4 Thursday november 5

SESSION 2 - NANOSCALE ORGANIZATION AND

DYNAMICS OF GPCR SIGNALING

Poster Session 1 (odd

numbers)

SESSION 1 - GPCR REGULATION AND

SIGNALING

Buffet

Registration

Friday november 6

SESSION 4 - DRUG-DISCOVERY AND GENETIC

APPROACHES TO ADDRESS GPCR FUNCTION

SESSION 3 - GPCR TARGETING IN

PATHOPHYSIOLOGY

Awards and Conclusions

General Assembly

SESSION 3 - Special session GPCR targeting in

atherothrombosis and bleeding

Buffet


WEDNESDAY 4th Session 1 - GPCR regulation and signaling

Lecture 01

GPCR folding, maturation and trafficking

Ulla Petäjä-Repo

Medical Research Center Oulu and Cancer and Translational Medicine Research Unit University of Oulu FI-90014 Oulu, Finland

G protein-coupled receptors (GPCRs) are synthesized at the endoplasmic reticulum (ER) membrane, and their folding status is scrutinized by the ER quality control machinery, involving a number of molecular chaperones and folding factors. Only correctly folded receptors are transported through the secretory pathway to the plasma membrane, whereas misfolded or incompletely folded receptors are targeted to ER-associated degradation in cytosolic proteasomes. The cell surface receptors are eventually internalized and degraded in lysosomes either constitutively or in an activation-dependent manner. The overall level of functionally active GPCRs is thus governed by the maturation efficiency of newly-synthesized receptors and the internalization/lysosomal degradation of cell surface receptors. In this presentation I will focus on the delta-opioid receptor biogenesis, especially on its two processing events that occur in different sub-compartments of the secretory pathway: N- and O-glycosylation. N-glycosylation is initiated co-translationally in the ER and it has an important role in the quality control of nascent receptors, whereas the post-translational N-acetylgalatosamine-type O-glycosylation occurs in the Golgi complex, affecting fully folded receptors.





Lecture 02

Cellular and physiological impact of GPCR phosphorylation

Andrew TOBIN, University of Leicester (UK)





Lecture 03

Phosphoproteomics of 5-HT2A/mGlu2 receptors: toward new insights into the mechanism of action of hallucinogens and antipsychotics

Samy Murat (1-3), Samah Karaki (1-3), Carine Becamel (1-3), Clotilde Mannoury la Cour (4), Mark J. Millan (4) , Philippe Rondard (1-3), Laurent Prezeau (1-3), Joel Bockaert (1-3), Philippe Marin (1-3*), Franck Vandermoere (1-3*)

CNRS, UMR-5203, Institut de Genomique Fonctionnelle, Montpellier; (2) INSERM, U1191, Montpellier; (3) Universite Montpellier, F-34094 Montpellier, France ; (4) Institut de Recherches Servier, Croissy-sur-Seine, France. (*) These authors equally contributed to the study

The serotonin 5-HT2A receptor is the primary target of psychedelic hallucinogens such as LSD, mescaline and psilocybin (agonists), which reproduce some of the core symptoms of schizophrenia and of second-generation antipsychotics such as clozapine, olanzapine and risperidone (antagonists or inverse agonists). Recent findings demonstrate that 5-HT2A receptors form heteromers with metabotropic glutamate mGlu2 receptors, another target of last-generation antipsychotics (agonists or positive allosteric modulators). The association of both receptors has profound consequences on their pharmacology and signal transduction properties as well as on the behavioural effects of drugs that bind to either 5-HT2A receptors or mGlu2 receptors. For instance, 5-HT2A receptor/mGlu2 heteromer formation is essential for the expression of psychotropic-like effects of hallucinogens and imbalanced activity and coupling properties of 5-HT2A and mGlu2 receptors within the heterocomplex might be one of the molecular substrates for a susceptibility to schizophrenia. To get further insight into the mechanism of action of drugs acting at 5-HT2A/mGlu2 heteromers, we explored their impact upon the phosphorylation pattern of each receptor by high-resolution mass spectrometry. We showed in HEK-293 cells and in mice prefrontal cortex that hallucinogenic 5-HT2A receptor agonists (LSD, DOI) but not non-hallucinogenic 5-HT2A receptor agonists promote 5-HT2A receptor phosphorylation at Ser280 located in the i3 loop. Site mutagenesis indicated that Ser280 phosphorylation is responsible for the lower capacity of hallucinogens to promote receptor desensitization and internalization, compared with non-hallucinogenic agonists. Conversely, several phosphorylated residues were identified in the C-terminal domain of mGlu2 receptors co-expressed with 5-HT2A receptors in HEK-293 cells. LY379268, an orthosteric agonist of mGlu2, slightly increases the phosphorylation of a particular cluster when mGlu2 was expressed alone, strongly increases this phosphorylation when 5-HT2A receptor was co-expressed, a potentiation prevented by the co-application of the synthetic hallucinogen DOI. Site mutagenesis indicated that phosphorylation of this cluster is responsible for the lower negative coupling of mGlu2 to Gio / Adenylate cyclase pathway. Collectively, these findings reveal both ligand-biased and heteromerization-biased subtle modifications of 5-HT2A/mGlu2 phosphorylation barcodes that might underlie the behavioural effects of drugs acting at each subunit of 5-HT2A/mGlu2 heteromers.





Short communication 01

P01 Cognitive impairment induced by delta9-tetrahydrocannabinol occurs through heteromers between cannabinoid CB1 and serotonin 5-HT2A receptors

Viñals X1,2*, Moreno E3,4*, Lanfumey L5, Cordomi A6, Pastor A2, de La Torre R2, Gasperini P 3,4,7, Botta J7, Howell LA7 , Pardo L6, Lluís C3,4, Canela EI3,4, McCormick PJ3,4,7#, Maldonado R1#, Robledo P1,2#

1Neuropharmacology Laboratory, University Pompeu Fabra, Barcelona, Spain. 2Human Pharmacology and Clinical Neurosciences Research Group, IMIM-Hospital del Mar Medical Research Institute, Barcelona, Spain 3Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas. 4Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Spain. 5CPN, INSERM UMR S894, Université Paris Descartes, UMR S894, Paris, France. 6 Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 7 School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ. *These authors contributed equally to this work #These authors equally supervised this work




P20 Relationship between heterotrimeric G proteins and beta-adrenergic ligand efficiency

ONFROY Lauriane, SEGUELAS Marie-Hélène, HEYMES Christophe, SENARD Jean-Michel, GALES Céline

UMR 1048, Institut des Maladies Métaboliques et cardiovasculaires, INSERM, Université Toulouse III Paul Sabatier, Toulouse, France




P29 The GTPase-activating Protein-related Domain of neurofibromin regulates MC1R-mediated pigmentation in human melanocytes

W Deraredj Nadim, S Hassanaly, C Kieda, H Bénédetti, C Grillon and S Morisset-Lopez

Centre de Biophysique Moléculaire, CNRS UPR4301 affiliated to the University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France




P35 -arrestins directly interact with PKA, promoting its translocation to the nucleus and form a transcriptionally active complex with CREB upon stimulation of Gs-coupled GPCRs.

Landomiel Flavie, Tranchant Thibaud, Bourquard Thomas, Langonné Nathalie, Gauthier Christophe, Boulo Thomas, Bigot Yves, Akli-Ayoub Mohammed, Poupon Anne, Crépieux Pascale et Reiter Eric

INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France CNRS, UMR6175, F-37380 Nouzilly, France ; Université François Rabelais, F-37041 Tours, France.




P31 β-arrestin-dependent transcriptome & proteome: a comparative study.

LANGONNÉ-GALLAY N(1), VANDERMOERE F(2), BOURQUARD T(1), CASSIER E(2), GAUTHIER C(1), BOULO T(1), POUPON A(1), CREPIEUX P(1), MARIN P(2), REITER E(1).

(1)BIOS group, UMR0085, INRA, F37380, Nouzilly, France. (2)Neuroproteomics and Signaling of Neurological and Psychiatric Disorders, UMR CNRS 5203, INSERM U1191, Université de Montpellier, F34094, Montpellier, France.



Technological Lecture

P04 Native and stabilized membrane proteins for drug discovery: CALIXAR approach

Emmanuel DEJEAN (1), Elodie MANDON (1), Sébastien IGONET (1), Julien DAUVERGNE (1), Vincent CORVEST (1), Thomas IWEMA (1), Morgane AGEZ (1), Daniel LEAMAN (2), Michael ZWICK (2) & Anass JAWHARI (1)

1) CALIXAR, 60 avenue Rockefeller, 69008 Lyon, France ; www.calixar.com. 2) The Scripps Research Institute, La Jolla, California

Membrane proteins represents up to 70% of therapeutic targets that are involved in different key cellular processes and signaling events. Surprisingly, less than 1% of protein structures in the Protein Data Bank correspond to membrane proteins. Those targets are very often instable which represents a bottleneck for their production in homogenous and stable state suitable for structural studies, antibody development and HTS ligand screening. CALIXAR has developed a native approach consisting on native isolation of therapeutic targets from the expression, solubilization/stabilisation, and purification to functional as well as structural characterization. Instead of modifying the protein (by truncations and mutations) to adapt to the environment, we modify the chemical environment to adapt to the protein, using innovative proprietary detergents/ surfactants compounds and combination of compounds to help solubilize and stabilize in the same time. The starting mater ial can be either endogenous (Organs, Primary cells, bacteria, and viruses), or recombinant (E.coli, Yeast, insect cells, CHO and HEK cells). Here we will describe examples of highly druggable targets that were produced in their most native state without any single mutation, truncation or fusion and that could exhibit striking thermo-stability increase and homogeneity improvement, while keeping their functional features. Indeed, more than 30°C thermostability improvement was obtained for the human transporter BCRP while keeping its native sequence and modifying only the chemical environment. We show striking stability improvement of 7TM proteins: bacteriorhodopsin and wild type and non-truncated adenosine receptor for which crystals could be obtained and antibodies were derived. Conditions of functional stabilization of the trimer of HIV envelop protein were also found which open doors to future structural investigations and conformational antibody development of native HIV envelop. Taken together this data show that despite the diversity of membrane protein classes and the variety of the biological starting materials, CALIXAR approach is successfully applied in customize manner to produce high quality proteins for pharmaceutical, biotech and academic partners as well as in the context of internal researches (CALIXAR’s pipeline) on CNS disorders, cancer and infectious diseases. We are convinced that this work will pave a way to more secure discovery programs and therefore to better drugs in the near future.


THURSDAY 5th Session 2- Nanoscale organization and dynamics of GPCR signaling

Lecture 04

Molecular mechanisms of phosphorylation-dependent arrestin activation

Jörg Standfuss, Paul Scherrer Institute (Switzerland)





Lecture 05

Organization of a signaling complex between ghrelin receptor oligomers and G proteins

Jean-Louis Banères

Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS – Université de Montpellier – ENSCM, Faculté de Pharmacie, 15 avenue Charles Flahault, 34093 Montpellier cedex 05. [email protected]

There is increasing evidence that G protein-coupled receptors (GPCRs) can arrange as homo- and hetero-oligomers. This is the case for the ghrelin receptor that has been shown to interact with the dopamine D2 receptor. By doing so, it modulates the response triggered by dopaminergic ligands. In this context, we used isolated recombinant proteins reconstituted in a model membrane-like environment to delineate the arrangement of such a hetero-oligomeric complex and how it interacts with the G protein partner of each receptor.





Lecture 06

Improving single-cell BRET imaging using NanoLuc – Application to ERK activity sensing

Elise Goyet 123, Vincent Ollendorff 4 and Julie Perroy 123

1 CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France 2 INSERM, U1191, Montpellier, F-34094, France 3 Universités de Montpellier 1 & 2, UMR-5203, Montpellier, F-34094, France 4 INRA, UMR866 Dynamique Musculaire et Mébabolisme,, Université Montpellier, 34060 Montpellier, France Better understanding of physiological processes implies to elucidate the spatio-temporal patterns of signaling pathways activation as well as dynamic reorganization of protein-protein interactions in living cells. Bioluminescent Resonance Energy Transfer is a powerful technology to monitor dynamic intracellular events in live cells. Despite the excellent signal/ background ratio provided by BRET, the use of BRET in microscopy has been restricted largely due to the low level of light emission intrinsic to the bioluminescent luciferase reaction. In the present work, we addressed this issue using Nanoluc, an extremely bright luciferase recently described. We characterized the benefits of NanoLuc in single-cell BRET imaging by using BRET positive fusion constructs. The time resolution, the stability overtime, the dynamic range as well as the sensitivity of the BRET signal were substantially improved within the NanoLuc-Venus fusion. In line with these benefits, we optimized the BRET-based sensor of ERK activity using NanoLuc as energy donor, so called YEN. The higher sensitivity of YEN together with an improved spatio-temporal resolution enabled the detection of subtle, transient and localized variations of ERK activity in dendritic spines. The YEN sensor should thus help to unravel the complex role of ERK signaling in many physiological processes including neuronal plasticity. Extending the use of NanoLuc in BRET imaging would thus provide better description of spatio-temporal dynamics of protein-protein interactions as well as activation patterns of specific signaling pathways.






P49 In vivo characterization of MTR/5-HT2c heteromers in mouse brain

Romain Gerbier, Florence Gbahou, Delphine Ndiaye-Lobry, Philippe Delagrange, Ralf Jockers

Inserm, U1016, Institut Cochin, Paris, France; (2) Institut de Recherches Servier, Suresnes, France




P47 Expression and purification of the human metabotropic Glutamate Receptor 5

Chady Nasrallah, Karine Rottier, Gaetan Bellot & Guillaume Lebon

Institut de Genomique Fonctionnelle, UMR 5203 CNRS – U 1191 INSERM – Univ. Montpellier, 141 rue de la Cardonille, Montpellier, France.


THURSDAY 5th Session 3- GPCR targeting in pathophysiology

Special Session GPCR targeting in atherothrombosis and bleeding

Lecture 07

Mind the GAPP: Linking patient bleeding with G protein-coupled receptor function

Stuart Mundell, University of Bristol (UK). [email protected]

Platelet-expressed GPCRs are critical regulators of platelet function with pharmacological blockade of

these receptors forming a powerful therapeutic tool in the treatment and prevention of arterial

thrombosis. Antithrombotic drug therapy is however associated with high inter-patient variability in

therapeutic response and adverse bleeding effects. In order to optimise the use of existing anti-

platelet drugs and to develop new therapies, more detailed knowledge is required to understand the

molecular mechanisms that regulate GPCR and therefore platelet function. The study of patients

with bleeding problems is a powerful approach in determining the function and regulation of

important proteins, including G protein-coupled receptors (GPCRs) in human platelets. As part of an

integrated functional genomic strategy (the Genotyping and Phenotyping of Platelets (GAPP)

consortium), we have identified a number of mutations in GPCR genes that could contribute to

bleeding tendency in patients (Reviewed in 1) including a series of mutations in the platelet P2Y12

receptor (P2Y12R). Through this approach we have shown that disruption of the PDZ ligand found at

the extreme C-terminus of the P2Y12R, results in loss of surface P2Y12R and dysregulation of

receptor function in human platelets (2,3). Mass spectrometry analysis has revealed a number of

platelet expressed proteins that bind to the P2Y12R in a PDZ-dependent manner. Subsequent studies

have examined the ability of these binding partners to regulate P2Y12R function in both cell lines and

platelets showing they regulate receptor activity likely by regulating surface receptor expression and

agonist-dependent traffic.

This work is supported by the British Heart Foundation. SJM is a Senior BHF Research Fellow

References

1) Nisar et al., (2015) Rare platelet GPCR variants: what can we learn? Br J Pharmacol. 172, 3242-53.

2) Nisar et al., (2011) An intact PDZ-motif is essential for correct P2Y12 purinoceptor traffic in human platelets. Blood 118,

5641-51.

3) Cunningham et al, (2013) Differential endosomal sorting of a novel P2Y12 purinoreceptor mutant Traffic 14, 585-98.







Lecture 08

Structure and Pharmacochemistry of Purinergic Receptors

Kenneth A. Jacobson

NIDDK, National Institutes of Health, Bethesda, MD, 20892, USA, [email protected]

We establish structure activity relationships at G protein-coupled receptors (GPCRs) that respond to extracellular nucleosides and nucleotides, e.g. adenosine receptors (ARs) and P2Y receptors (P2YRs), respectively, to design pharmacological probes and potential therapeutic agents. Recent, detailed structural information derived from the X-ray crystallographic structures within these families enables the rational design of novel ligands, guides the modification of known agonists and antagonists, and helps predict polypharmacology [1]. We have introduced high affinity probes for pharmacological characterization of P2YRs and for drug discovery. Conformational constraint of normally flexible ribose with bicyclic analogues, i.e. either of two orientations of the methanocarba ([3.1.0]bicyclohexane) ring system, increases the ligand selectivity at P2Y1R (requires North-conformation), and other P2YR subtypes require a South-conformation. We recently reported high-resolution X-ray structures for the P2Y1 (Gq-coupled) and P2Y12 (Gi-coupled) receptors for ADP, which are both anti-thrombotic targets that represent distinct GPCR subfamilies [2]. Nucleotides bind to P2Y1R and P2Y12R at different sites. Nucleotide and nonnucleotide antagonists bind to P2Y1R at two entirely unexpected and distinct sites, i.e. orthosteric and allosteric [3]. Allosteric antagonist, urea BPTU binds to a novel site on the exterior of the 7TM bundle in contact with the phospholipid bilayer. Comparison of agonist- and antagonist-bound P2Y12Rs indicates unprecedented structural plasticity in the outer transmembrane (TM) regions and extracellular loops. Antagonist-bound P2Y12R conformation is open and lacks a normally conserved disulfide bond. We performed extensive molecular modeling studies based on these structures and mutagenesis, to predict the binding modes of major classes of P2Y12R ligands previously reported [4]. Various nucleotide derivatives docked readily to the agonist-bound P2Y12R, but uncharged nucleotide-like antagonist ticagrelor required a hybrid receptor resembling the agonist-bound P2Y12R except for the top portion of TM6. Thus, 3D knowledge of P2Y receptor structure and function facilitates the understanding of recognition of known ligands and the identification of novel ligands. References [1] K. A. Jacobson, S. Costanzi, S. Paoletta, Trends Pharmacol. Sci. 2014, 35, 658-663. [2] K. A. Jacobson, S. Paoletta, V. Katritch, B. Wu, Z.G. Gao, Q. Zhao, R.C. Stevens, E. Kiselev, Mol. Pharmacol. 2015, 88, 220-230. [3] D. Zhang, Z.G. Gao, K. Zhang, E. Kiselev, S. Crane, J. Wang, S. Paoletta, C. Yi, L. Ma, W. Zhang, G. W. Han, H. Liu, V. Cherezov, V. Katritch, H. Jiang, R. C. Stevens, K. A. Jacobson, Q. Zhao, B. Wu, Nature 2015, 520, 317-321. [4] S. Paoletta, D. Sabbadin, I. von Kügelgen, S. Hinz, V. Katritch, K. Hoffmann, A. Abdelrahman, J. Straßburger, Y. Baqi, Q. Zhao, R.C. Stevens, S. Moro, C.E. Müller, K.A. Jacobson. J. Computer-Aided Mol. Des. 2015, 29, 737-756.







Lecture 09

Benefits and risks of new antiplatelet agents

Pierre Sié / Thibault Lhermusier, CHU Toulouse (France)






Lecture 10

The conformational plasticity of CC chemokine receptor 5 (CCR5) and its consequences in the development and inhibition of HIV infection

Bernard Lagane1, 2

1 Viral Pathogenesis Unit, Institut Pasteur and 2 INSERM Unit 1108, Paris, France.

CCR5 is a seven-transmembrane domain, G protein-coupled receptor (GPCR) for chemokines playing a role in

innate immunity. It is also involved in the development of HIV infection. CCR5 acts as a CD4-associated

coreceptor required for entry of R5-tropic strains of HIV-1 into CD4+ T-lymphocytes and macrophages. During

viral entry, CCR5 interacts with the CD4-bound form of the HIV-1 envelope glycoprotein gp120, thereby leading

to fusion between the viral and cell membranes and release of the viral genome into the host cell cytoplasm.

R5-tropic viruses predominate after transmission and in the asymptomatic stages of infection. As infection

progresses, viruses using the chemokine receptor CXCR4 (X4- or R5X4-tropic viruses) as a coreceptor may also

emerge, which often correlates with a poor clinical outcome and development of AIDS.

Over the last few years, it has become evident that CCR5, similarly to other GPCRs, may adopt different

conformations at the cell surface, although the significance of these observations for the receptor functions

remains largely unknown. Studies showed that structurally unrelated CCR5 ligands may stabilize different

receptor conformational ensembles. While doing so, some CCR5 native chemokines and chemokine analogs

can exhibit different signaling outcomes. In the same line, non-peptidic, low molecular weight CCR5 ligands

including the marketed drug maraviroc inhibit R5 HIV-1 entry through stabilization of a G-protein uncoupled

form of CCR5, which HIV-1 gp120 cannot bind to. Finally, different anti-CCR5 monoclonal antibodies (mAbs)

recognize distinct subsets of the CCR5 conformations at the cell surface. Interestingly, the anti-HIV activity of

mAbs does not necessarily correlate with the amount of the CCR5 molecules they recognize, suggesting that

HIV itself also uses only a subset of cell surface CCR5 proteins.

In this talk, I will present recent data from our laboratory showing how CCR5 conformational heterogeneity

influences development of HIV infection and antiviral strategies aimed at blocking the coreceptor. In vitro,

chemokine binding to CCR5 inhibits viral entry into cells both by sterically preventing gp120 from binding and

by inducing the coreceptor endocytosis, suggesting that chemokines may act as a natural barrier against HIV in

vivo. Our data however led us to challenge this hypothesis, HIV taking advantage of the CCR5 conformational

heterogeneity to escape inhibition by chemokines. More precisely, CCR5 conformations of low-affinity for

chemokines are (i) exploited by R5 HIV-1 for entry into cells and (ii) involved in CCR5 endocytosis on which the

antiviral activity of R5-CHKs is partially dependent. Interestingly, this fraction of CCR5 receptors, which we

propose to name “spare receptors” as it probably does not take part in the chemokine-mediated functional

responses, remains recognized by chemokine analogs displaying improved anti-HIV-1 activity. Finally, more

recent data revealed that envelope glycoproteins isolated from distinct primary HIV-1 isolates also

recognize/stabilize distinct subsets of the CCR5 conformations, suggesting that a link could exist between the

phenotypic properties of viruses (efficiency of entry, cell tropism, infectivity, triggering of intracellular signaling

useful for steps of the viral cycle, and sensitivity to inhibitors) and the nature and/or number of receptor

conformations to which they bind. Elucidating these hypotheses could thus highlight a new aspect in the field

of HIV infection, i.e. the link between CCR5 conformational plasticity, gp120 functional selectivity and

physiopathology of HIV infection and provide important clues for developing future CCR5 entry inhibitors.





Lecture 11

Regulatory Peptide Receptors as Targets for Tumor Diagnosis and Therapy

Jean Claude Reubi

Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, PO Box 62, Murtenstrasse 31, CH-3010 Berne, Switzerland. E-mail: [email protected] Recent examples of clinical applications of overexpressed peptide receptors in cancer are somatostatin receptor imaging and targeted radiotherapy. In vivo receptor targeting is now emerging for other overexpressed peptide receptors, such as GLP-1, neurotensin or GRP receptors, suggesting a general principle for oncology. The role of in vitro receptor detecting methodologies that are truly predictive for cancer target definition and its subsequent in vivo applications will be emphasized. Further, mechanisms of actions will be discussed and clinical examples of peptide receptor targeting in vivo in cancer will be given.

mailto:[email protected]





Lecture 12

Stimulation of olfactory receptors expressed in cancer cells promotes their invasiveness and proliferation, leading to increased metastasis emergence in vivo

Sanz Guenhaël1, Leray Isabelle2, Grébert Denise1, Antoine Sharmilee1, Acquistapace Adrien1, Boukadiri Abdelhak3, Mir Lluis M.2

1INRA UR1197 Unite de Neurobiologie de l’olfaction Jouy-en-Josas France, 2CNRS Gustave Roussy UMR8203 Laboratoire de Vectorologie et thérapeutiques anti-cancéreuses Villejuif France, 3INRA UMR 1313 Génétique Animale et Biologie Intégrative Jouy-en-Josas France

Olfactory receptors (ORs) are G protein-coupled receptors mainly expressed in the olfactory epithelium where they allow the detection and discrimination of a myriad of odorant molecules. Moreover, ORs are expressed in other tissues such as in some cancer cells, with additional functions. For instance, the OR51E2 olfactory receptor, also named prostate specific G protein-coupled receptor (PSGR), is overexpressed in prostate cancer cells and is considered a tumor marker of prostate cancer. We demonstrated its involvement in tumor progression: we showed that the PSGR is able to promote LNCaP prostate cancer cell invasiveness in vitro and in vivo upon stimulation with its odorant agonist β-ionone (G. Sanz et al, 9(1): e85110. 2014). We report now that a relatively short exposure to β-ionone (two weeks) is already able to promote metastasis emergence while a longer treatment exacerbates this committed effect. We also report that α-ionone, usually considered as an OR51E2 antagonist, promotes LNCaP tumor growth in vivo. Moreover, the combination of α-ionone with β-ionone displays a higher effect on tumor growth than each molecule alone. In vitro assessment of LNCaP cell growth upon exposure to α-ionone further demonstrates that α-ionone is not an OR51E2 antagonist as previously reported and supports the in vivo results. Altogether, our results show that α-ionone and β-ionone induce different cellular effects, each leading to increased tumor aggressiveness.






P27 The mGlu7 receptor: a new target in the therapy of cocaine and opiates addiction?

Zuzana Hajasova (1), Corinne Canestrelli (1), Delphine Rigault (2), Isabelle Brabet (3), Cyril Goudet (3), Francine Acher (2), Florence Noble (1), Nicolas Marie (1)

(1) CNRS ERL 3649 « Neuroplasticité et thérapies des addictions » / Inserm UMR-S 1124 / Université Paris Descartes (2) CNRS UMR 8601 « Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques » / Université Paris Descartes (3) CNRS UMR 5203 « Institut de Génomique Fonctionnelle » / Inserm U 1191 / Université de Montpellier




P46 Chemotactic G-protein coupled receptors control cell migration by suppressing autophagosome biogenesis

Coly Pierre-Michaël, Perzo Nicolas, Le Joncour Vadim, Lecointre Céline, Shouft Marie-Thérèse, Tonon Marie-Christine, Wurtz Olivier, Gandolfo Pierrick, Castel Hélène, and Morin Fabrice

Inserm U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation, DC2N, Astrocyte and Vascular Niche, Biomedical Research Institute (IRIB), University of Rouen, 76821, France.




P12 Anti-inflammatory properties of the neuropeptide orexins in ulcerative colitis: a

new promising therapeutical molecule

Messal N1., Fernandez N2., Bergere M1., Pedruzzi E2., Gratio V1., Nicole P1., Couvelard A1., Voisin T1., Ogier-Denis E2., Couvineau A1.

1Team « From inflammation to cancer in digestive diseases » and 2Team « Intestinal inflammation » from INSERM UMR1149, Centre de Recherche sur l’inflammation (CRI), Faculte de Medecine Site Bichat, Université Paris Diderot, 75018 Paris, France.


FRIDAY 6th Session 4- Drug-Discovery and Genetic appraoches to address GPCR function

Lecture 13

A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents

Timo Müller

Helmholtz Zentrum München and Technische Universität München (Germany)



Lecture 14

Stabilized GPCRs Enable Biophysics and Structure Based Drug Discovery

Gregg SIEGAL

ZoBio Einsteinweg 55, Leiden, NL

Fragment Based Drug Discovery (FBDD) has been successfully applied to an array of soluble pharmaceutical targets. Biophysics based drug discovery can have many potential advantages when applied to membrane proteins including the ability to drug peptide/protein activated receptors and to discover modulators with novel biological activity. ZoBio has used biophysical methods to find both orthosteric and allosteric ligands to StaR stabilized GPCRs in an earlier collaboration with Heptares. More recently we have initiated a collaboration with Prof. Pluckthün (University of Zürich, CH) to apply biophysical techniques for drug discovery on stabilized GPCRs using his CHESS technology. CHESS GPCRs have several possible advantages including: they can be produced in E. coli, they are not locked in 1 conformation and they couple to the G-protein.

So far we have immobilized a version of the neurotensin 1 receptor (NTS1), used TINS (Target Immobilized NMR Screening) to identify fragment hits and Biacore to validate them. As with the StaR receptors, this approach finds both allosteric and orthosteric GPCR modulators in a single screen. However, the next challenge will be to determine the structure of the complex of weekly bound fragment hits to NTR1. In general it has been difficult to use X-ray crystallography to observe these week complexes. We have begun to apply paramagnetic NMR techniques to determine the binding site and structure of our ligands to NTR1. Here I will discuss previous successes, ongoing work on NTR1 and plans to develop efficient NMR methods to elucidate 3D structures of GPCR-ligand complexes.





Lecture 15

Genetic models for the study of RF-amide receptors

F. Simonin.

Biotechnology and Cellular Signalling, UMR7242 CNRS-Uds. 300 bvd Sébastien Brant, 67412 Illkirch.

Chronic pain is a common health issue that remains difficult to treat. Opiates represent the standard treatment used in clinic but their chronic use leads to the development of several adverse side effects including hyperalgesia (enhancement of the pain perception) and tolerance (decrease of the analgesic effects over time). It has been proposed that stimulation of opioid receptors triggers activation of anti-opioid systems that in turns produce hyperalgesia thus diminishing the net analgesic effect of the opioid agonist (tolerance). This process has been evidenced in vivo both in rats and in man where acute and prolonged opioid treatments induce a long lasting hyperalgesia. Anti-opioid receptor blockers could therefore represent a promising strategy for limiting the development of pain hypersensitivity and analgesic tolerance associated with chronic opiates treatments. Several systems have been shown to display anti-opioid properties including RF-amide neuropeptides family and their receptors. These peptides have an Arg-Phe-NH2 motif at their C-terminal and act through 5 specific GPCRs (G protein coupled receptors). However, both genetic and pharmacological tools are missing for several of these receptors, which severely limits their study. Recently, we generated knockout mice for the different RF-amide receptors and studied their phenotype in different models of opioid-induced hyperalgesia and persistent pain. We identified GPR103a as well as NPFF1R and NPFF2R as critical players in the modulation of opioid action. Moreover, we identified and characterized a GPR103 selective antagonist and showed that co-administration of this compound with opiates can efficiently prevent the development of hyperalgesia and analgesic tolerance induced by chronic morphine administration. Altogether, our data clearly point to GPR103 and its endogenous ligand 26RFa as a novel anti-opioid system and indicate that compounds selectively targeting this receptor could represent interesting therapeutic tools to improve opiates action particularly in chronic treatments.





Lecture 16 Therapeutic potential of orphan GPCR

Ralf JOCKERS

Institut Cochin, Paris, Inserm U1016, CNRS 8104, Université Paris Descartes

Seven transmembrane-spanning proteins (7TM), also called G protein-coupled receptors (GPCRs), are among the most versatile and evolutionary successful protein families. Out of the 400 non-odorant members identified in the human genome, approximately 100 remain orphans that have not been matched with an endogenous ligand. Apart from the classical deorphanization strategies, several alternative strategies provided recent new insights into the function of these proteins. Among those are the phenotypic characterization of organisms silenced or overexpressing orphan 7TM proteins, the search for constitutive receptor activity and formation of protein complexes including 7TM proteins as well as the development of synthetic, surrogate, orthosteric and allosteric ligands. Once the function and a HTS-compatible readout of orphan 7TM proteins defined, they can enter the drug development process and become new therapeutic targets.






P59 Regulation of energy metabolism by Endospanin 1, a negative regulator of the leptin receptor.

Arturo Roca-Rivada (1), Timothy Schwartz (1), Christophe Magnan (2), Raphaël Denis (2), Johanna Auriau (1), Stéphanie Migrenne-Li (2), Ralf Jockers (1), Julie Dam (1)

(1) Institut Cochin, INSERM U1016, CNRS 8104, Université Paris Descartes, France (2) Univ Paris

Diderot, Sorbonne Paris Cité, CNRS UMR 8251, France




P65 Searching for ligands targeting a GPCR homodimeric interface

Slynko I (1), Banerjee P (2), Ruat M 2) and Rognan D(1)

(1) Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS-Université de Strasbourg, F-67400 ILLKIRCH; (2) UMR9197 CNRS/Paris-Sud University, F-91198 Gif-Yvette, France


POSTERS

P01 Cognitive impairment induced by delta9-tetrahydrocannabinol occurs through heteromers between cannabinoid CB1 and serotonin 5-HT2A receptors

Estefanía Moreno,3,4, Xavier Viñals,1,2, Laurence Lanfumey5, Arnau Cordomí6, Antoni Pastor2, Rafael de La Torre2, Paola Gasperini3,4,7, Botta J7, Gemma Navarro3,4, Lesley A Howell7, Leonardo Pardo6, Vicent Casadó3,4, Carme Lluís3,4, Enric I. Canela3,4, Peter J. McCormick#,*,3,4,7, Rafael Maldonado#1, Patricia Robledo#,1,2

1Neuropharmacology Laboratory, University Pompeu Fabra, Barcelona, Spain. 2Human Pharmacology and Clinical Neurosciences Research Group, IMIM-Hospital del Mar Medical Research Institute, Barcelona, Spain 3Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas. 4Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Spain. 5CPN, INSERM UMR S894, Université Paris Descartes, UMR S894, Paris, France. 6Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 7 School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ. These authors contributed equally to this work # These authors equally supervised this work Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC, such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but not its acute hypolocomotor, hypothermic, anxiogenic and antinociceptive effects. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that co-stimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.


POSTERS

P02 Two Negative Allosteric Modulators (NAMs) Display Biased Activities on FSH-Induced ß-arrestin Recruitment at the FSHR

Mohammed Akli Ayoub (1), Gwenhael Jégot (1), Anne Poupon (1), Pascale Crépieux (1), Nathalie Langonné-Gallay (1), Sonia-Maria Poli (2), James A. Dias (3), and Eric Reiter (1)

(1) BIOS group, UMR7247 PRC, Nouzilly, France . (2) Addex Therapeutics, Geneva, Switzerland. (3) Department of Biomedical Sciences, State University of New York, Albany, NY USA Abstract body: Two compounds from Addex Pharmaceuticals (i.e.: ADX68692 and ADX68693) are small molecules with close molecular structures. In previous studies, these compounds have been reported as Negative Allosteric Modulators (NAMs) at the follicle-stimulating hormone receptor (FSHR)(Dias et al., Biol Reprod 90(1):19, 2014). Indeed, while ADX68692 inhibited FSH-induced cAMP, progesterone and estradiol production in rat granulosa cells, ADX68693 exhibited biased antagonism by inhibiting only cAMP and progesterone production but not estradiol. In this study, we attempted to dissect the molecular mechanisms behind such a biased allosteric modulation of the FSHR in vitro using HEK293 cells. Thus, we tested the hypothesis that the compounds, by allosterically modifying the FSHR conformation(s), may induce a biased antagonism between Gαs and β-arrestins-dependent signaling compared to FSH alone. Together, our data point to the fact that ADX68692/93 behave as negative allosteric modulators on hFSHR with interesting differences with respect to β-arrestin recruitment. The biased antagonistic effects observed are in line with in vivo studies made in Dias’s group. Whether or not the differential effects reported on Estradiol and Progesterone production in granulosa cells could be linked to the biased β-arrestin signaling will necessitate further investigations.


POSTERS

P03 Effect of orphan GPR88 on delta opioid signaling in vitro and in vivo

Pellissier LP1, Gandia J1, Meirsman AC2, Diaz J3, Clesse D4,. Kieffer BL2, Le Merrer J1,2*, Becker JAJ1,2*

1UMR PRC INRA - CNRS - Université de Tours - IFCE, Equipe « Déficit de récompense GPCR et sociabilité », 37380 NOUZILLY, France. 2Département de Médecine Translationnelle et Neurogénétique, IGBMC, INSERM U-964, CNRS UMR-7104, Université de Strasbourg, Illkirch, France 3Centre de Psychiatrie et Neurosciences, INSERM UMR-894 - Université Paris Descartes, Paris, France 4Département de Neurobiologie des rythmes, Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR-3212, Université de Strasbourg, Strasbourg, France. * Equal contribution GPR88 is an orphan G protein coupled receptor (GPCR) highly expressed in the striatum. The receptor is also expressed in other brain areas and here we examined whether GPR88 function extends beyond striatal-mediated responses. Using a Gpr88 knockout mouse, we demonstrate that GPR88 is implicated in a large repertoire of behavioral responses that engage motor activity, spatial learning and emotional processing. Our data also reveal functional interaction between GPR88 and delta opioid receptor (DOR) as well as other striatal GPCRs.


POSTERS

P04 Native and stabilized membrane proteins for drug discovery: CALIXAR approach

Emmanuel DEJEAN (1), Elodie MANDON (1), Sébastien IGONET (1), Julien DAUVERGNE (1), Vincent CORVEST (1), Thomas IWEMA (1), Morgane AGEZ (1), Daniel LEAMAN (2), Michael ZWICK (2) & Anass JAWHARI (1)

1) CALIXAR, 60 avenue Rockefeller, 69008 Lyon, France ; www.calixar.com. // 2) The Scripps Research Institute, La Jolla, California Membrane proteins represents up to 70% of therapeutic targets that are involved in different key cellular processes and signaling events. Surprisingly, less than 1% of protein structures in the Protein Data Bank correspond to membrane proteins. Those targets are very often instable which represents a bottleneck for their production in homogenous and stable state suitable for structural studies, antibody development and HTS ligand screening. CALIXAR has developed a native approach consisting on native isolation of therapeutic targets from the expression, solubilization/stabilisation, and purification to functional as well as structural characterization. Instead of modifying the protein (by truncations and mutations) to adapt to the environment, we modify the chemical environment to adapt to the protein, using innovative proprietary detergents/ surfactants compounds and combination of compounds to help solubilize and stabilize in the same time. The starting mater ial can be either endogenous (Organs, Primary cells, bacteria, and viruses), or recombinant (E.coli, Yeast, insect cells, CHO and HEK cells). Here we will describe examples of highly druggable targets that were produced in their most native state without any single mutation, truncation or fusion and that could exhibit striking thermo-stability increase and homogeneity improvement, while keeping their functional features. Indeed, more than 30°C thermostability improvement was obtained for the human transporter BCRP while keeping its native sequence and modifying only the chemical environment. We show striking stability improvement of 7TM proteins: bacteriorhodopsin and wild type and non-truncated adenosine receptor for which crystals could be obtained and antibodies were derived. Conditions of functional stabilization of the trimer of HIV envelop protein were also found which open doors to future structural investigations and conformational antibody development of native HIV envelop. Taken together this data show that despite the diversity of membrane protein classes and the variety of the biological starting materials, CALIXAR approach is successfully applied in customize manner to produce high quality proteins for pharmaceutical, biotech and academic partners as well as in the context of internal researches (CALIXAR’s pipeline) on CNS disorders, cancer and infectious diseases. We are convinced that this work will pave a way to more secure discovery programs and therefore to better drugs in the near future.


POSTERS

P05 Loss of Morphine Reward and Dependence in Mice Lacking G Protein–Coupled Receptor Kinase 5

Andrea Kliewer1, Laura Gluck1, Anastasia Loktev1, Lionel Moulédous2, Catherine Mollereau2, Ping-Yee Law3, and Stefan Schulz1

1Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Germany; 2Institut de Pharmacologie et de Biologie Structurale, CNRS/Université de Toulouse, Toulouse Cedex, France; 3Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota. Background: The clinical benefits of opioid drugs are counteracted by the development of tolerance and addiction. We provide in vivo evidence for the involvement of G protein–coupled receptor kinases (GRKs) in opioid dependence in addition to their roles in agonistselective mu-opioid receptor (MOR) phosphorylation. Methods: In vivo MOR phosphorylation was examined by immunoprecipitation and nanoflow liquid chromatography–tandem mass spectrometry analysis. Using the hot-plate and conditioned place preference test, we investigated opioid-related antinociception and reward effects in mice lacking GRK3 or GRK5. Results: Etonitazene and fentanyl stimulated the in vivo phosphorylation of multiple carboxyl-terminal phosphate acceptor sites, including threonine 370, serine 375, and threonine 379, which was predominantly mediated by GRK3. By contrast, morphine promoted a selective phosphorylation of serine 375 that was predominantly mediated by GRK5. In contrast to GRK3 knockout mice, GRK5 knockout mice exhibited reduced antinociceptive responses after morphine administration and developed morphine tolerance similar to wild-type mice but fewer signs of physical dependence. Also, morphine was ineffective in inducing conditioned place preference in GRK5 knockout mice, whereas cocaine conditioned place preference was retained. However, the reward properties of morphine were evident in knock-in mice expressing a phosphorylation-deficient S375A mutation of the MOR. Conclusions: These findings show for the first time that MOR phosphorylation is regulated by agonist-selective recruitment of distinct GRK isoforms that influence different opioid-related behaviors. Modulation of GRK5 function could serve as a new approach for preventing addiction to opioids, while maintaining the analgesic properties of opioid drugs at an effective level. Support by Deutsche Forschungsgemeinschaft Grant Nos. SCHU924/11-2 and SCHU924/15-1 and National Institute on Drug Abuse Grant No. 1R01DA031442-01A1.


POSTERS

P06 NMR Structure and Dynamics of the Agonist Peptides Bound to Opioid Receptors

Casey O’Connor1,2, Guillaume Ferré3, Nathalie Doncescu3, Georges Czaplicki3, Pascal Demange3,

Raymond C. Stevens1, Kurt Wüthrich2, Alain Milon2,3

1USC, CA USA 2 TSRI, CA USA 3 CNRS and Université de Toulouse-UPS, France

The structure of the dynorphin(1-13) peptide bound to the human kappa opioid receptor (KOR) has

been determined by liquid state NMR spectroscopy. 1H and 15N chemical shift variations indicated

that free and bound peptide is in fast exchange in solutions containing 1 mM dynorphin and 0.01 mM

KOR. Radioligand binding indicated an intermediate affinity interaction, with a Kd of ~200 nM.

Transferred nuclear Overhauser enhancement (trNOE) spectroscopy was used to determine the

structure of bound dynorphin. The N-terminal opioid signature, YGGF, was observed to be flexibly

disordered, the central part of the peptide from L5 to R9 forms a helical turn, and the C-terminal

segment from P10 to K13 is flexibly disordered in the bound state. An ensemble of receptor-peptide

conformations was identified with molecular dynamics simulations. The insights from combining

molecular modeling with NMR provide an initial framework for understanding multi-step activation

of a G protein-coupled receptor (GPCR) by its cognate peptide ligand.

Current objective is to extend these results to other opiate receptors, and to characterize the high

affinity states and the receptor's dynamics using stable labelled receptor expressed in E. coli.


POSTERS

P07 Agonist-selective multi-site phosphorylation of the μ-opioid receptor regulates β-arrestin 1/2 recruitment

Elke Miess and Stefan Schulz

Department of Pharmacology and Toxicology, Jena University Hospital - Friedrich Schiller University Jena, Germany Opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor (MOR) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence. There is substantial evidence suggesting that G protein-coupled receptor kinases (GRKs) and β-arrestins play key roles in regulating MOR signaling and responsiveness. Following phosphorylation by GRKs, β-arrestins bind to phosphorylated MORs, which prevents further interactions between the receptor and G proteins even in the continued presence of agonist resulting in diminished G protein-mediated signaling. We have previously shown that agonist-induced phosphorylation of MOR occurs at a conserved 10-residue sequence, 370TREHPSTANT379, in the carboxyl-terminal cytoplasmic tail. Morphine induces a selective phosphorylation of serine375 (S375) in the middle of this sequence that is predominantly catalyzed by G protein-coupled receptor kinase 5 (GRK5). By contrast, high-efficacy opioids not only induce phosphorylation of S375 but also drive higher-order phosphorylation on the flanking residues threonine370 (T370), threonine376 (T376), and threonine379 (T379) in a hierarchical phosphorylation cascade that specifically requires GRK2/3 isoforms. To investigate this mechanism further, we have adapted a β-galactosidase complementation assay for β-arrestin1 and β-arrestin2. Using this assay, we were able to show that activation of MOR by high-efficacy agonists such as DAMGO results in recruitment of both β-arrestin1 and β-arrestin2, whereas activation by low-efficacy agonists such as morphine results only in detectable recruitment of β-arrestin2 but not β-arrestin1. The morphine-induced β-arrestin recruitment was strongly enhanced by overexpression of GRK2 or GRK3. Conversely, siRNA knock down of GRK2 or GRK3 strongly inhibits DAMGO-induced β-arrestin recruitment. Mutation of S375 to alanine strongly inhibited β-arrestin recruitment. However, mutation of all 11 carboxyl-terminal serine and threonine residues of MOR was required to completely abolish interaction with β-arrestin1 and β-arrestin2.


POSTERS

P08 Activation mechanism of GABAB receptor

Jianfeng LIU

Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China G-protein coupled receptors (GPCRs) Class C represent a distant group among the large family of GPCRs. This class includes the receptors for the main neurotransmitters, glutamate and gamma-aminobutyric acid (GABA), and the receptors for Ca2+, some taste and pheromone molecules, as well as some orphan receptors. Like any other GPCRs, these receptors possess a heptahelical domain (HD) involved in heterotrimeric G-protein activation, but most of them also have a large extracellular domain (VFT) responsible for agonist recognition and binding. These receptors are dimers, either homo or heterodimers. GABAB receptor was the first heteromeric G-protein coupled receptor (GPCR) identified. Indeed, both GB1 and GB2 subunits appear necessary to get a functional GABAB receptor. We have demonstrated that the interactions between VFT domain of both GB1 and GB2 were important for receptor activation. We then found that the GABAB receptor induced activation of ERK1/2/CREB and protected neurons from apoptosis by trans-activating IGF-1R. We have also demonstrated that GABAB receptor activation is modulated by the dynamic protein-protein interactions between receptors and its downstream signal proteins such as FAK1 and Rap1.


POSTERS

P09 Use of High Throughput Electric Field Stimulation (EFS) Coupled with Intracellular Ca2+ Kinetics Measurements on iPSC-Derived Cardiomyocytes

Sunao Hisada1, Natsumi Kato1, Masanobu Matsubara1, Takuji Kataoka1, Ralf Kettenhofen2 and Felix von Haniel2

1Hamamatsu Photonics K.K. Systems Division, Hamamatsu City, Shizuoka Pref, Japan & 2Axiogenesis AG, Nattermannallee 1, 50829 Cologne, Germany Presented by JM D’Angelo Hamamatsu has developed a 96-channel electrode array system that is mounted on the FDSS/μCELL. It adds electric field stimulations (EFS) to all 96 wells in a microplate simultaneously while fluorescence/luminescence signals are monitored. We measured oscillations of intracellular Ca2+ concentration, which occurs along with the beating of the cells, with a calcium sensitive fluorescent dye in human iPSC-derived cardiomyocytes (Cor.4U, Axiogenesis AG). We observed that the Ca2+ oscillation in cardiomyocytes was synchronized to the electric stimulation, which indicates that the EFS system is able to pace the beating of cardiomyocytes. We also observed that the Ca2+ oscillations in the presence of some ion channel blockers were modulated by adding EFS. Use of EFS coupled with intracellular Ca2+ kinetics measurements, in the high-throughput manner, should be useful in in vitro assessment of cardiac toxicity of pharmacological compounds using cardiomyocytes, in particular in toxicity screening at early stages of drug development, as well as in cardiomyocyte research.


POSTERS

P10 Conformational ensembles sampled by the chemokine receptor CXCR4 in active and

inactive states

Bruck Taddese (1), Hélène Castel (2), Patrice Rodien (1) and M. Chabbert (1)

(1) UMR CNRS 6214 – INSERM 1083, University of Angers, France; ( 2) INSERM U982, University of

Rouen, France

The chemokine receptor CXCR4 is a G protein-coupled receptor (GPCR) involved in chemotaxis as

well as numerous physiological processes and pathologies, such as cancer metastasis, inflammatory

diseases and AIDS. The crystal structures of CXCR4 in complex with antagonists provide a static view

of the inactive state. However, proteins are inherently dynamic systems and GPCRs are best

described in terms of conformational ensembles. The receptors sample diverse distinct

conformations, each of which leading to different downstream functions and influenced by binding

of different ligands. Knowledge of these conformations and of reactional paths between them is

instrumental in developing drugs that exploit distinct states leading to different downstream

functions.

Here, we investigate CXCR4 conformational ensembles gathered from molecular dynamics (MD)

simulations and the major activation/deactivation transitions typically taking place on the micro to

millisecond timescale. Accelerated MD is an efficient method to enhance conformational sampling

and reduce the computational time necessary to observe such transitions by several orders of

magnitude. Comparison of movements in active and inactive CXCR4 conformations sampled by

accelerated MD provides insights into the distinctive conformational flexibility of these states. In

addition, the inactivation of the active CXCR4 model highlights the time-dependent correlated

conformational changes of side chains surrounding functional GPCR microswitch residues that trigger

the transition from active to inactive state of CXCR4.


POSTERS

P11 BRET2 assay using the FDSS/μCELL: monitoring cellsurface-receptor internalization and intracellular trafficking Cellular

Frédéric Finana1, Luc De Vries1 and Jean-Marc d’Angelo2.

1Cellular and Molecular Biology, Pierre Fabre Research Institute, Castres France and 2Hamamatsu Photonics Europe GmbH After the publication of application note n°23 describing BRET1 technology and based on a study published in 2011, Pierre Fabre Research Institute recently implemented the more sensitive BRET2 technology to the FDSSµCell system for monitoring the internalization of the human dopaminergic D2s receptor, a prototypic G protein-coupled receptor (GPCR). The result of this work will be present.


POSTERS

P12 Anti-inflammatory properties of the neuropeptide orexins in ulcerative colitis: a new

promising therapeutical molecule

Messal N1., Fernandez N2., Bergere M1., Pedruzzi E2., Gratio V1., Nicole P1., Couvelard A1., Voisin T1.,

Ogier-Denis E2., Couvineau A1.

1Team « From inflammation to cancer in digestive diseases » and 2Team « Intestinal inflammation »

from INSERM UMR1149, Centre de Recherche sur l’inflammation (CRI), Faculte de Medecine Site

Bichat, Université Paris Diderot, 75018 Paris, France.

Orexins (orexin-A and orexin-B) are hypothalamic peptides involved in the sleep/wake control which

interact with two GPCR sub-types, OX1R and OX2R. We have demonstrated that OX1R was highly

expressed in digestive cancer cell lines derived from colon, pancreas and liver cancer (1). Our

immunohistochemistry studies indicate that OX1R was also detected in 100 % of human epithelia of

Inflammatory Bowel Disease (IBD) samples including Crohn’s disease (21 samples) and Ulcerative

Colitis (UC). To determine the role of OX1R in UC, we have investigated the role of Orexin A (OxA) on

acute inflammation in two mice models including chemically mice treated with Dextran Sulfate

Sodium (DSS) and EXCY2 mice genetically invalidated for IL10 and Nox1 genes (IL10-/- and Nox1-/-)

which develop spontaneous UC-like disease (2). The daily intraperitoneal injection of OxA (1

µmole/kg) in orally DSS-treated mice ameliorates the Disease Activity Index scored by measuring

weight, leng th of colon, diarrhea and the presence of blood in the stool and also histologic aspect of

colon epithelium. The analysis of cytokinic profile revealed that OxA reduces the secretion of “pro-

inflammatory” cytokines such as TNFα, IL6, IL8 homolog and IL1B in colon extracts of DSS-induced

colitis mice. In contrast, OxA has no effect on INFγ, IL10, and IL12 cytokine secretion in colon

extracts. The recent development of a new spontaneous UC-like mouse model EXCY2 mice, is ideally

suited to study the OxA effect on spontaneous colitis. Indeed, EXCY2 mice develop a spontaneous

colitis at 6/7 weeks of age with an upwards gradient from the rectum and reproduce all molecular

characteristics seen in UC including, loss of goblet cells, deregulation of ER stress, protective effect of

tobacco…Two intraperitoneal injection of OxA (1 µmole/kg) by week during 21 days alleviated severe

colitis in 10 week-old EXCY2 mice, OxA-treated EXCY2 mice exhibited a normal colonic mucosa (gener

al crypts aspect, crypt size, presence of goblet cells, absence of immune cells infiltration) as

compared to vehicle treated EXCY2 mice. These data indicate that 1) OxA could exert an original anti-

inflammatory properties in DSS-treated mouse model; 2) OxA strongly protects from spontaneous

colitis developed in EXCY2 mice model and trigger mucosal healing certainly by controlling the

different pathways involved in the onset of UC. In conclusion, the system orexins/OX1R may

represent an innovative and effective target in the treatment of UC.

1-Voisin et al., Cancer research 2011, 71:3341-51

2-Treton et al, PLOS One 2014, 9:e101669


POSTERS

P13 Antibodies against G Protein Coupled Receptors

Franck Talmont (1), Jean-Marie Zajac (1), Catherine Mollereau (1), Lionel Mouledous (1)

1CNRS, Institut de Pharmacologie et de Biologie Structurale (IPBS), UMR 5089 CNRS/Université Paul Sabatier, 205 route de Narbonne, BP 64182, F-31077 Toulouse Cedex 4, France Antibodies specific for GPCRs are powerful tools which are particularly helpful to analyze the anatomical localization or to describe biochemical and biological properties of GPCRs. They can be used as screening antibodies for the detection of up- or down regulation of receptors during pathological processes. The antibodies can be used as detecting antibodies, to reveal cell surface expression of GPCRs in vitro, either on living cells or on membrane extracts, as well as in situ on fixed tissue sections. Specific anti-GPCR antibodies may also be helpful to specifically purify receptors, to characterize receptor dimers and to identify their protein partners or to help GPCR stabilization for crystallography. In addition, depending on their agonistic or antagonistic properties, they may be instrumental in triggering or neutralizing receptor mediated effects in vivo and thus they can be used as therapeutic antibodies.


POSTERS

P14 Plasmodium falciparum proteins involved in infected erythrocytes cytoadherence to

chemokine CX3CL1

Patricia Hermand, Liliane Cicéron, Cédric Pionneau #, Catherine Vaquero, Christophe Combadière,

Pierre Buffet and Philippe Deterre

CIMI-Paris INSERM, U 1135 Sorbonne Universités, UPMC Université Paris 06, UMRS 1135 CNRS, ERL

8255

Malaria caused by Plasmodium falciparum is associated with cytoadherence of infected red blood

cells (iRBC) to endothelial cells. Numerous host molecules have been involved in cytoadherence,

including the adhesive chemokine CX3CL1. Most of the identified parasite ligands are from the

multigenic and hypervariable Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1)

family which makes them poor targets for the development of a broadly protective vaccine. Using

proteomics, we have identified two 25-kDa parasite proteins with adhesive properties for CX3CL1,

called CBP for CX3CL1 Binding Proteins. CBPs are coded by single-copy genes with no polymorphic

variation, do not show any homology with other P. falciparum gene products, and have no orthologs

in other Plasmodium species. Both CBP-1 and CBP-2 contain a signal peptide, a host cell targeting

motif and two transmembrane domains. Specific antibodies raised against epitopes from the

predicted extracellular domains of each CBP efficiently stain the surface of RBC infected with

trophozoites or schizonts, which is a strong indication of CBP expression at the surface of iRBC. These

anti-CBP antibodies partially neutralize iRBC adherence to CX3CL1. This adherence is similarly

inhibited in the presence of peptides from the CBP extracellular domains, while irrelevant peptides

had no such effect. CBP1 and CBP2 are new P. falciparum ligands for the human chemokine CX3CL1.

The identification of this non-polymorphic and novel family of P. falciparum virulence factors

provides a new avenue for innovative vaccination approaches.


POSTERS

P15 THE PARKINSON'S DISEASE-ASSOCIATED RECEPTOR GPR37 OLIGOMERIZES WITH THE ADENOSINE A2A RECEPTOR: RELEVANCE IN MOTOR CONTROL

Jorge Gandía (1) (2), Xavier Morató (2), Igor Stagljar (3), Víctor Fernández-Dueñas (2) & Francisco Ciruela (2)

(1) Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique UMR-0085, Centre National de la Recherche Scientifique UMR-7247, Université François Rabelais de Tours, Nouzilly, France. (2) Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L’Hospitalet de Llobregat, 08907 Barcelona, Spain. (3)Department of Biochemistry, Donnelly Centre, University of Toronto, Toronto, Ontario, Canada The orphan G protein-coupled receptor GPR37 (also known as prosaposin receptor or parkin-associated endothelin-like receptor) has been linked to neurological conditions such as Parkinson’s disease (PD), autism and mood disorders. By means of a MYTH (membrane yeast two-hybrid)-based screening, a potential GPR37 interactor identified was the adenosine A2A receptor, whose antagonists have been long investigated as a therapeutic strategy in the treatment of PD. Here, we study more thoroughly the formation of this proposed heterodimer, both at the physical and functional level. In HEK293 cells, we have demonstrated that when they are co-expressed, GPR37 and A2AR co-localize, co-immunoprecipitate and, more importantly, they yield a strong and saturable (i.e. specific) BRET signal. Furthermore, the presence of GPR37 markedly altered the functionality of A2AR, both in its plasma membrane expression and in its signaling pathway. Meanwhile, experiments performed in wild-type (WT) and GPR37-/- mice also revealed the GPR37-A2AR interaction, both in striatal membrane co-immunoprecipitations and in tests assessing general motor skills and tremor. The use of A2AR antagonists in these tests revealed a differentiated response of A2AR depending on the presence (WT) or absence (GPR37-/-) of the orphan receptor. Thus, the global data obtained have allowed us to confirm the formation of the heterodimer GPR37-A2AR and associate it to a potential important role in motor control.


POSTERS

P16 The cardioprotective angiotensin 1-7 peptide acts as a natural biased ligand at the angiotensin II type 1 receptor

Segolène Galandrin*, Colette Denis*, Cedric Boularan*, Du N’Guyen*,†, Jean-Michel Senard*,†, Céline Galés

* Institut des Maladies Métaboliques et Cardiovasculaires, Institut National de la Santé et de la Recherche Médicale, U1048, Université Toulouse III Paul Sabatier, F-31432 Toulouse, France. † Service de Pharmacologie Clinique, Faculté de médecine, Centre Hospitalier Universitaire de Toulouse, F-31432 Toulouse, France. The concept of ligand-biased signaling and its therapeutic benefit in cardioprotection have been extensively explored for the angiotensin II type 1 receptor (AT1-R) but so far have been restricted to synthetic ligands. As yet, it is unknown whether physiological peptides of the renin-angiotensin-aldosterone system (RAAS) also exhibit functional selectivity at AT1-R. To explore this possibility, we systematically determined the efficacies and potencies of four RAAS peptides (angiotensin II, III, IV and 1-7) on the direct activation of G proteins and β-arrestin2, the two main receptor-proximal effectors, in AT1-R-expressing HEK293T cells. We found that, like the prototypical reference AngII AT1-R-agonist, AngIII and AngIV act as full agonists for the activation of similar G proteins although AngIV exhibits a weaker potency. Interestingly, Ang1-7 is unable to promote G proteins activation but behaves as a competitive antagonist for both the AngII/Gi and /Gq pathways. Conversely, all four

RAAS peptides act as agonists on the AT1-R/-arrestin2 axis but display biased activities relative to

AngII as indicated by their differences in potency, AT1-R/-arrestin2 conformation and AT1-R/-arrestin2 intracellular routing. Collectively, these data demonstrate that biased agonism is a natural process that could fine-tune the physiology of the RAAS. More importantly, we reveal that Ang1-7, a known Mas-receptor-specific ligand, also behaves as an AT1-R biased agonist, promoting selective β-arrestin activation while blocking the detrimental AngII/AT1-R/Gq axis. This original pharmacological profile of Ang1-7 at the AT1-R, which mirrors the synthetic and therapeutic AT1-R biased agonists, could in part contribute to its cardiovascular benefits.


POSTERS

P17 Ghrelin receptor antagonists: SAR study on trisubstituted 1,2,4-triazoles

Séverine Denoyelle, Mathieu Maingot, Celine M’Kadmi, Marjorie Damian, Sophie Mary, Didier Gagne, Jacky Marie, Jean-Louis Banères, Jean Martinez, Jean-Alain Fehrentz

Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS - Université Montpellier, Faculté de Pharmacie, 15 avenue Charles Flahault, 34093 Montpellier Cedex 5, France Ghrelin, the natural ligand of the Growth Hormone Secretagogue Receptor type 1a (GHS-R1a), has been intensively studied for its various biological activities such as the stimulation of both growth hormone release and food intake. Ghrelin also regulates the metabolic balance by decreasing fat use, affecting body weight and adiposity. Thus, ghrelin functions as an orexigenic hormone and it is of interest to find agonists and antagonists of its receptor. In our effort to find new ghrelin receptor ligands, we explored families of peptidomimetics based on a decorated 1,2,4-triazole scaffold that led to potent agonists and antagonists of GHS-R1a.(1-3) These compounds were synthesized from (D)tryptophan residue and present in their final structure only one asymmetric carbon atom, whose configuration was controlled during the synthetic process. One of our leading compound, JMV 2959, was found to be a potent GHS-R1a antagonist exhibiting a large range of interesting properties such as the suppression of food intake induced by ghrelin or by fasting and the decrease of fat mass accumulation.(4) In order to improve the pharmacokinetic profile of this compound, we explored structural modifications on its N-terminal part by replacing the glycine residue with various substituents and found out a very potent antagonist comprised of a picolinic moiety. We then extended our SAR study by first modulating the pyridinyl ring(5) and then by introducing a second asymmetric carbon with a controlled configuration. In this study, we report on the consequences of these structural modifications on both the affinity and the biological activity of the new ligands targetting the ghrelin receptor.

(1) Demange, L.; Boeglin, D.; Moulin, A.; Mousseaux, D.; Ryan, J.; Berge, G.; Gagne, D.; Heitz, A.; Perrissoud, D.; Locatelli, V.; Torsello, A.; Galleyrand, J. C.; Fehrentz, J. A.; Martinez, J. Journal of Medicinal Chemistry 2007, 50, 1939. (2) Moulin, A.; Demange, L.; Berge, G.; Gagne, D.; Ryan, J.; Mousseaux, D.; Heitz, A.; Perrissoud, D.; Locatelli, V.; Torsello, A.; Galleyrand, J. C.; Fehrentz, J. A.; Martinez, J. Journal of Medicinal Chemistry 2007, 50, 5790. (3) Moulin, A.; Demange, L.; Ryan, J.; Mousseaux, D.; Sanchez, P.; Berge, G.; Gagne, D.; Perrissoud, D.; Locatelli, V.; Torsello, A.; Galleyrand, J. C.; Fehrentz, J. A.; Martinez, J. Journal of Medicinal Chemistry 2008, 51, 689. (4) Salome, N.; Hansson, C.; Taube, M.; Gustafsson-Ericson, L.; Egecioglu, E.; Karlsson-Lindahl, L.; Fehrentz, J. A.; Martinez, J.; Perrissoud, D.; Dickson, S. L. Journal of Neuroendocrinology 2009, 21, 777. (5) Blayo, A.-L.; Maingot, M.; Aicher, B.; M'Kadmi, C.; Schmidt, P.; Müller, G.; Teifel, M.; Günther, E.; Gagne, D.; Denoyelle, S.; Martinez, J.; Fehrentz, J.-A. Bioorg. Med. Chem. Lett. 2015, 25, 20.


POSTERS

P18 Neuropeptide Y receptor mediates activation of ERK1/2 via transactivation of the IGF receptor

Sandra Lecat 1*, Lazare Belemnaba 2, Jean-Luc Galzi 1, and Bernard Bucher2

1 GPCRs, pain and inflammation team, UMR7242, CNRS-University of Strasbourg, LabEx Medalis 300 Bvd Sébastien Brant, 67412, Illkirch CS10413, France 2 UMR 7213, CNRS/Université de Strasbourg, Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, 74 route du Rhin, CS 60024, 67401 Illkirch, France Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y1 receptors unable to recruit β-arrestins (Ouédraogo et al. 2008), can still activate ERK signalling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead depend ent on Gβ/γ and associated signalling pathways that activate PI3-kinase. Although inhibition of the Epidermal-Growth Factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of Insulin Growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This Gβ/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signalling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that β-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required.

Lecat S, Belemnaba L, Galzi JL, Bucher B. (2015) Neuropeptide Y receptor mediates activation of ERK1/2 via transactivation of the IGF receptor. Cell Signal. 27(7):1297-304. Ouédraogo M*, Lecat S*, Rochdi MD, Hachet-Haas M, Matthes H, Gicquiaux H, Verrier S, Gaire M, Glasser N, Mély Y, Takeda K, Bouvier M, Galzi JL and Bucher B (2008) Distinct motifs of neuropeptide Y receptors differentially regulate trafficking and desensitization Traffic 9:305–324 *equal contribution


POSTERS

P19 Impact of membrane lipid composition on biophysical properties of the dopaminergic D2

receptor

Marie-Lise Jobin (1), Véronique Desmedt-Peyrusse (1), Jonathan A. Javitch (2), Isabel D. Alves (3),

Pierre Trifilieff (1)

Affiliations: (1) NutriNeuro, INRA UMR 1286, 146 rue Léo Saignat, 33000 Bordeaux, France (2) New

York State Psychiatric Institute, Columbia University, New York, USA (3) CBMN, CNRS UMR 5248,

Univ. Bordeaux, Allée Geoffroy St Hilaire, 33600 Pessac, France

Various psychiatric disorders are accompanied by alterations of dopaminergic D2 receptor (D2R)-

dependent signaling and numerous pharmacological treatments for psychiatric disorders target D2R.

The D2R is a 7-transmembrane receptor that strongly interacts with membrane lipids, however, the

implication of membrane lipid environment on the D2R properties has been overlooked. Yet, the

brain is highly enriched in lipids and in polyunsaturated fatty acids (PUFAs) in particular, which are

known to impact the properties of membranes as well as the activity of transmembrane proteins.

Recent data obtained in the lab show that developmental n-3 PUFA deficiency in mice alters D2R

signaling in the ventral striatum, in parallel with deficits in motivation, which highly depends on the

activity of D2R in the ventral striatum. These data suggest that membrane content alteration in PUFA

impact the functionality of the D2R.

The project aims at unraveling the impact of membrane lipid composition on D2R conformation and

pharmacological properties through biophysical studies in both membrane fragments and membrane

models. We developed a D2R Fluorescence Anisotropy (FA) assay using a selective fluorescent

antagonist of the D2R to characterize its ligand binding properties. Membrane fragments isolation

from D2R expressing-cell lines has been performed and the impact of PUFAs incorporated in

membranes on D2R ligand affinity has been investigated. Our data show that ligand binding affinities

was affected by PUFAs and this depending on the type of PUFAs incorporated.


POSTERS

P20 Relationship between heterotrimeric G proteins and beta-adrenergic ligand efficiency

ONFROY Lauriane (1), SEGUELAS Marie-Hélène (1), HEYMES Christophe (1), SENARD Jean-Michel (1), GALES Céline (1)

(1) UMR 1048, Institut des Maladies Métaboliques et cardiovasculaires, INSERM, Université Toulouse III Paul Sabatier, Toulouse, France Beta-adrenergic receptors antagonists (beta-blockers) have a beneficial effect on survival in human heart failure (HF). However, among all available beta-blockers, only some have been shown to be effective in the treatment of HF. One potential hypothesis for explaining such diversity among beta-blockers could rely on their inappropriate classification as antagonists. Indeed, several groups have shown now that beta-blockers exhibit “biased-agonist” activity toward G-protein coupled receptor (GPCR). Basically, the “Biased-agonism” concept in GPCR pharmacology states that efficiency of a ligand depends on the couple Receptor/Effector targeted. It is now accepted that human diseases are subjected to large modifications in the expression level of a lot of proteins, including heterotrimeric G proteins. These proteins represent the first effector, and the only effector common to all GPCR families, rapidly activated in response to the binding of an agonist to the receptor. Thus, we hypothesized that the differences in clinical efficacy among beta-blockers could, in part, result in their biased-agonists properties, secondary to G protein expression level changes occurring in cardiomyocytes during HF. In the present work, we showed that, in cardiomyocytes isolated from mice exhibiting HF, expression levels of genes encoding for all G alpha protein subunits are prone to large deregulations. We initiated a study to evaluate a possible link between G alpha subunits expression level and efficacy of different ligands including beta-blockers and classical synthetic and physiological beta-adrenergic agonists. Using new specific and sensitive probes measuring directly G protein activation, based on Bioluminescence Resonance Energy Transfer (BRET), we demonstrated that the efficacy of both beta-agonists and antagonists highly depends on the G alpha subunit expression level. Moreover, in term of molecular mechanisms, we showed that G alpha expression level influences ligand efficacy by dictating location of both G protein subunit and beta-adrenergic receptor within specific plasma membrane microdomains. Altogether, these results provide new basis for the screen of GPCR ligand efficacy.


POSTERS

P21 Molecular modelling, design, and synthesis of new NTS2-selective neurotensin analogues

Roberto Fanelli(1), Nicolas Floquet(1), Mélanie Vivancos(2), Élie Besserer-Offroy(2), Jean-Michel Longpré(2), Jean Martinez(1), Philippe Sarret(2), Florine Cavelier(1)*

(1) Institut des Biomolécules Max Mousseron, IBMM, UMR-5247, CNRS, Université Montpellier, ENSCM, Place Eugène Bataillon, 34095 Montpellier cedex 5, France. (2) Department pf pharmacology and physiology, Faculty of medicine and health sciences, Université de Sherbrooke, Canada Neurotensin (NT) is a tridecapeptide which was first isolated from bovine hypothalamus.(a) Over the last decade, neurotensin receptors NTS1 and NTS2 were shown to participate in the antinociceptive action of NT when injected directly into the brain. NT also induces other physiological effects, such as hypothermia and hypotension. Structure-activity relationship studies showed that the C-terminal fragment of NT, so called NT[8-13], is the minimal bioactive sequence. In order to improve activity and stability, we recently developed several NT[8-13] analogues using different approaches including unnatural amino acid incorporation,(b) peptide bond modification, and cyclisation.(c) Among the NT receptor subtypes, Sarret et al(d) showed that selectivity toward NTS2 is fundamental to exert analgesic effect without unwanted effects such as hypothermia and hypotension. The interaction between neurotensin and its receptor NTS1 has been clarified by the crystallization of the receptor with its ligand NT[8-13].(e) Based on the differences between NT receptors NTS1 and NTS2 with molecular modeling studies, we observed that Tyr11 residue is located in a pocket bearing a basic residue (Arg) in NTS1 and an acid residue (Glu) in NTS2. Therefore, we designed new NT analogues replacing Tyr11 with an amino acid bearing a basic function on the side chain to increase selectivity toward NTS2. Here, we present molecular modeling studies for the rational design of these new analogues as well as their synthesis and preliminary biological results.

(a) Carraway R. E., Leeman S. E., J. Biol. Chem. 1973, 248, 6854. (b) Vivet B., Cavelier F., Martinez J., Eur. J. Org. Chem. 2000, 5, 807. (c) Bredeloux P., Cavelier F., Dubuc I., Vivet B., Costentin J., Martinez J., J. Med. Chem. 2008, 6, 1610. (d) Roussy G., Dansereau M. A., Baudisson S., Ezzoubaa F., Belleville K., Beaudet N., Martinez J., Richelson E., Sarret P., Mol Pain. 2009, 5, 38. (e) White J. F, et al. Structure Nature, 2012, 490, 508.


POSTERS

P22 Investigation of Group-III mGlu receptors with antagonists as probes

Claudia Desole (1), Delphine Rigault (1), Isabelle McCort (1), Francine Acher (1)

Affiliations: (1) Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR8601-CNRS, Université Paris Descartes Metabotropic glutamate receptors (mGluRs) belong to class C of G-Protein Coupled Receptors (GPCRs) which is characterized by a large amino terminal domain that folds in two lobes connected by a flexible hinge and named Venus Flytrap domain (VFT). Glutamate, the major excitatory neurotransmitter in the brain, binds in the cleft close to the hinge. The VFT domain requires to be closed for receptor activation (1). Competitive antagonists bind at the glutamate site but prevent this closure (1). Most of them are glutamate analogues that hold a bulky substituent at the alpha carbon (e.g. LY341495) (2). We have discovered a series of phosphinic amino acids that are group-III mGluR selective agonists (3,4). Introducing various substituents on the alpha carbon should turn these agonists into antagonists while keeping their selectivity. According to the type of the added moiety and the affinity of the ligand, several probes may be obtained (tritium labeled, fluorescent, MR imaging or PET). The synthesis of these ligands will be presented.

(1) A.-S. Bessis, P. Rondard, F. Gaven, I. Brabet, N. Triballeau, L. Prézeau, F. C. Acher, J. P. Pin. PNAS, 2002, 99, 11097−11102. (2) P. L. Ornstein, T.J.Bleisch, M. B. Arnold, R. A. Wright, B. G. Johnson, D. D. Schoepp. J. Med. Chem., 1998, 41, 346−357. (3) C. Selvam, N. Oueslati, I. A. Lemasson, I. Brabet, D. Rigault, T. Courtiol, S. Cesarini, N. Triballeau, H.-O. Bertrand, C. Goudet, J.-P. Pin, F. C. Acher. J. Med. Chem., 2010, 53, 2797−2813. (4) C. Goudet, B. Vilar, T. Courtiol, T. Deltheil, T. Bessiron, I. Brabet, N. Oueslati, D. Rigault, H.-O. Bertrand, H. McLean, H. Daniel, M. Amalric, F. C. Acher, J.-P. Pin. FASEB J., 2012, 26, 1682−1693.


POSTERS

P23 A NEW TARGET FOR ALZHEIMER’S DISEASE: THE GLUTAMATE NMDA-DOPAMINE D1-

HISTAMINE H3 RECEPTOR HETEROMER

Mar Rodríguez-Ruiz (1,2), Estefanía Moreno (1,2), David Moreno-Delgado (1,2), Ester Aso (1,3), Isidre

Ferrer (1,3), Josefa Mallol (1,2), Antoni Cortés (1,2), Carme Lluís (1,2), Rafael Franco (1,2), Enric. I.

Canela (1,2), Vicent Casadó (1,2)

(1) Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (2)

Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Spain

(3) Institute of Neuropathology, Bellvitge University Hospital, University of Barcelona

Alzheimer’s disease (AD) is a common neurodegenerative disorder causing progressive memory loss

and cognitive dysfunction. The cortex and hippocampus are two major brain regions implicated in AD

and are crucial for cognition. In AD, neurodegeneration is not limited to a specific neurotransmitter

system and many systems are involved including glutamatergic neurotransmission mainly through

NMDA receptors and dopaminergic neurotransmission. Dopamine D1 receptors (D1R) controlling

learning and memory are targets for AD treatment. Recently, we discovered that D1R and H3R form

heteromers with specific biochemical properties, e.g. binding of H3R ligands inhibits D1R function. In

addition, D1R physically interact and modulate NMDA receptors. Nevertheless, it is not known

whether relationships between NMDA, D1R and H3R function exist in brain areas affected in AD. By

bioluminescence resonance energy transfer (BRET) experiments in transfected HEK cells, we

observed that both D1R and H3R form heteromers with NMDA subunit 1A in the presence of subunit

2B. D1R-H3R-NMDA heterotrimers were observed by BRET with fluorescence complementation

assays. Later, we studied the expression and functionality of D1R-H3R-NMDA heterotrimers in the

brain cortex of two different animal models, i.e. male Sprague-Dawley rats and a mouse model of AD

and the corresponding wild type (WT) littermates. This mouse model is the double-transgenic

amyloid precursor protein/presenilin 1 (APP/PS1) mice that express a chimeric mouse/human APP

bearing the Swedish mutation (Mo/HuAPP695swe) and a mutant human PS1-dE9 both causative of

familial Alzheimer’s disease. The expression of D1R-H3R-NMDA heterotrimers in WT rat brain cortical

slices was determined by co-immunoprecipitation and proximity ligation assays (PLA) using specific

antibodies. A similar expression was observed in the AD mouse model. According to the heteromer

expression, H3R agonists, via negative cross-talk, and H3R antagonists, via cross-antagonism,

decreased D1R agonist signaling determined by ERK1/2 phosphorylation and also counteracted the

cell death induced by over-stimulation of D1R. Both D1R and H3R antagonists are also able to

prevent the NMDA agonists-induced cell death suggesting a complex interaction between D1R-H3R

heteromers and NMDA receptors. Therefore, the H3R ligands are able to block the cell death induced

by overstimulation of either D1R or NMDA in cortical slices of both WT and AD model mice. All these

results point out that D1R-H3R-NMDA heteromers are expressed in brain cortex where H3R ligands

act as a “molecular brake” for D1R and NMDA signaling. Because heteromers are present in the

cortex of a mouse model of AD and NMDA- or D1R-induced cell death is reduced by H3R ligands,

these heterotrimers might be molecular targets to reduce neurodegeneration.


POSTERS

P24 Identification and characterization of a novel GPR103 antagonist

GM Le Coz1, V Utard1, I Bertin1, M Schmitt2, JJ Bourguignon2, F Bihel2, F Simonin1

1UMR7242, Biotechnologie et Signalisation Cellulaire, Illkirch. 2UMR7200, Laboratoire d’Innovation Thérapeutique, Illkirch Chronic pain is a common health issue that remains difficult to treat. Opiates represent the standard treatment used in clinic but their chronic use leads to the development of several adverse side effects including hyperalgesia (enhancement of the pain perception) and tolerance (decrease of the analgesic effects over time). It has been proposed that stimulation of opioid receptors triggers activation of anti-opioid systems that in turns produce hyperalgesia thus diminishing the net analgesic effect of the opioid agonist (tolerance). This process has been evidenced in vivo both in rats and in man where acute and prolonged opioid treatments induce a long lasting hyperalgesia. Anti-opioid receptor antagonists could therefore represent a promising strategy for limiting the development of pain hypersensitivity and analgesic tolerance associated with chronic opiates treatments. Several systems have been shown to display anti-opioid properties including RF-amide neuropeptides family and their receptors. These peptides have an Arg-Phe-NH2 motif at their C-terminal and act through 5 specific GPCRs (G protein coupled receptors). However, pharmacological tools, particularly antagonists, are missing for several of these receptors including the receptor of 26RFa GPR103. In collaboration with a team of chemists, we therefore decided to identify a GPR103 small ligand capable of antagonizing the effect of 26 RFa both in vitro and in vivo. In a first step, we screened a chemical library of RF-amide derivatives (± 2000 molecules) on recombinant human GPR103 receptor stably expressed in CHO cells. From this screening procedure we found several molecules that display significant affinity for GPR103. We further characterized the in vitro pharmacological profile of these hits and identify RF10 that display a good affinity.


POSTERS

P25 Progress in the determination of the human arginine-vasopressin V2 receptor structure

Gérald Gaibelet, Hélène Orcel, Bernard Mouillac, Sébastien Granier

Institut de Génomique Fonctionnelle, 141 Rue de la Cardonille, 34094 Montpellier,

cedex 05, France

The renal arginine-vasopressin V2 receptor (V2R) plays a pivotal role in the body water balance

through the control of diuresis. Its dysregulation is responsible for two conformational diseases (cNDI

and NSIAD)* and is involved in an autosomal dominant polycystic kidney disease (ADPKD). These

different pathologies are all linked to the unbalance of the different cellular V2R signaling pathways.

Therefore, development of biased ligands able to selectively stimulate or inhibit the different

signaling pathways of the V2R is of crucial interest. Resolution of its three-dimensional structure will

help to define the structural mechanism of ligand recognition and activation and to design more

specific drugs.

To develop a highly crystallizable V2R, we engineered different modified versions of the receptor in

fusion with several partners (T4 lysozyme, cytochrome b562RIL, Pyrococcus abysii glycogen synthase,

rubredoxin...) expressed in SF9 insect cell line. Conformational stabilization of V2R by mutagenesis

combined with innovative pharmacological tools (small irreversible ligands, venom toxins,

nanobodies) complete our strategy. LCP (lipid cubic phase) approach is developed in the lab to

achieve efficient crystallogenesis of the purified detergent-solubilized receptor.

The aim of this work is to determine and to compare the three-dimensional structure of the V2R in

complex bound to different ligands (antagonists, agonists, biased ligands) or in complex with G

proteins.

*congenital Nephrogenic Diabetes insipidus (cNDI) and Nephrogenic Syndrome of Inappropriate

Antidiuresis (NSIAD)


POSTERS

P26 ERK1/2-mediated regulation of GPCR trafficking and signaling via β-arrestin phosphorylation.

Justine Paradis (1), Stevenson Ly (1), Élodie Tepaz (1), Jacob Galan (1), Alexandre Beautrait (1), Mark Scott (2), Hervé Enslen (2), Stefano Marullo (2), Philippe Roux (1), Michel Bouvier (1)

(1) Institute for Research in Immunology and Cancer, Université de Montréal (2) Institut Cochin, Université Paris Descartes, France. Mitogen-activated protein kinases (MAPK) are activated in response to G protein-coupled receptors (GPCRs) stimulation and play essential roles regulating cellular processes downstream of these receptors. However, very little is known on the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell surface expression of many GPCRs as a result of their intracellular sequestration. This subcellular redistribution of the receptors resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G protein activation and second messenger generation as well as blunted GPCR kinases (GRK) and βarrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs th at can interact with βarrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not the prostaglandin F receptor that cannot interact with βarrestin, implicating this scaffolding protein in the receptors’ subcellular redistribution. Complementation experiments in mouse embryonic fibroblast (MEFs) lacking βarrestins combined with in vitro kinase assays revealed that βarrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This novel regulatory mechanism was observed following constitutive activation as well as receptor tyrosine kinase- or GPCR-mediated activation of ERK1/2, suggesting that it is a central node in the tonic regulation of cell responsiveness to GPCR stimulation, acting both as an effector and a negative regulator.


POSTERS

P27 The mGlu7 receptor: a new target in the therapy of cocaine and opiates addiction?

Zuzana Hajasova (1), Corinne Canestrelli (1), Delphine Rigault (2), Isabelle Brabet (3), Cyril Goudet (3),

Francine Acher (2), Florence Noble (1), Nicolas Marie (1)

(1) CNRS ERL 3649 « Neuroplasticité et thérapies des addictions » / Inserm UMR-S 1124 / Université

Paris Descartes (2) CNRS UMR 8601 « Laboratoire de Chimie et Biochimie Pharmacologiques et

Toxicologiques » / Université Paris Descartes (3) CNRS UMR 5203 « Institut de Génomique

Fonctionnelle » / Inserm U 1191 / Université de Montpellier

Addiction to drugs such as cocaine and opiates (morphine and its derivatives), is chronic psychiatric

illness mainly characterized by seeking and compulsive consumption of psychoactive substances. It is

a major health issue since it results in high morbidity and mortality. Whereas no specific treatment is

available for cocaine addiction, the opiate addicts can benefit from opioid replacement therapy but

its use does not fully prevent relapse which remains the main problem in the treatment of addiction.

There is therefore a need to find new therapeutic targets to prevent relapse. Accumulating evidences

showed that the glutamatergic system is involved in drug addiction. Indeed, relapse is accompanied

by an increase in glutamate release in brain structures involved in drug addiction, suggesting that

inhibiting this release could block relapse. This could be achieved by stimulating presynaptic

glutamatergic receptors such as the mGlu7 (a group III metabotropic glutamate receptor). The

purpose of this work was to show that the mGlu7 receptor could be a valuable target in the

treatment of opiate and cocaine addiction. To realize this project we used LSP2-9166 (a mixed

mGlu4/mGlu7 agonist and the most powerful mGlu7 orthosteric agonist synthesized so far) in two

models of drug addiction in mice: behavioral sensitization (by measuring locomotor activity) and

conditioned place preference. Since comorbidities such as anxiety or depression are common among

addicts, we tested the effect of LSP2-9166 on these two behaviors with the light/dark box test and

the forced swimming test, respectively. We also measured the effect of LSP2-9166 on spontaneous

locomotor activity and hedonic state by measuring preference for sucrose. We showed that LSP2-

9166 blocks the expression of behavioral sensitization to morphine and conditioned place preference

to cocaine and morphine. These effects are neither due to a blockade of spontaneous locomotor

activity nor anhedonia of animals. Whereas LSP2-9166 has an anxiolytic effect at low dose, an

anxiogenic effect is observed at high doses. Finally, the LSP2-9166 has no effect in the forced

swimming test. All these results suggest that mGlu7 might be a valuable target in the treatment of

drug addiction. However, its role needs to be further clarified especially by delineating the respective

role of mGlu4 and mGlu7 in the effects of LSP2-9166 using selective antagonists to each receptor

subtype.


POSTERS

P28 Agonist-induced NOP receptor phosphorylation revealed by phosphosite-specific antibodies

Anika Mann1, Gloria Brunori2, Lawrence Toll2, Nurulain T. Zaveri3 and Stefan Schulz1

1Department of Pharmacology and Toxicology, Jena University Hospital, Friedrich-Schiller-University Jena, Germany 2Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA 3Astrea Therapeutics, Mountain View, CA, USA The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is the most recently discovered and least characterized member in the opioid receptor family (MOR, KOR and DOR). NOP receptors are widely distributed across tissues and modulate several physiological processes by its endogenous ligand Nociceptin. The NOP receptor is a potential target for the development of ligands with therapeutic use in several pathophysiological states (chronic and neuropathic pain). Consequently, there is increasing interest in understanding the molecular regulation of NOP receptor. For the other three opioid receptor types it has been established that carboxyl-terminal serine residues are phosphorylated by G protein-coupled receptor kinases (GRK). Recently, we generated phosphosite-specific antibodies directed against pS351 and pT362/pS363 and a phosphorylation-independent antibody, to investigate agonist-induced NOP receptor phosphorylation.


POSTERS

P29 The GTPase-activating Protein-related Domain of neurofibromin regulates MC1R-mediated pigmentation in human melanocytes

W Deraredj Nadim, S Hassanaly, C Kieda, H Bénédetti, C Grillon and S Morisset-Lopez

Centre de Biophysique Moléculaire, CNRS UPR4301 affiliated to the University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France The melanocortin 1 receptor (MC1R) is a G-protein coupled receptor which plays a major role in controlling melanogenesis. A large body of evidence indicates that GPCRs are part of large protein complexes that are critical for their signal transduction properties. Among proteins which may affect MC1R signalling, neurofibromin (Nf1), a GTPase activating protein (GAP) for Ras, is of special interest as it regulates adenylyl cyclase activity and ERK signalling, two pathways involved in MC1R signalling. Moreover, mutations in this gene encoding Nf1 are responsible for neurofibromatosis type I, a disease inducing hyperpigmented flat skin lesions. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer experiments we demonstrated a physical interaction of Nf1 with MC1R and that the GAP Related domain (GAP) of Nf1 directly and constitutively interacts with MC1R in melanocytes. In Nf1 deficient cells, α-MSH was shown to enhance cAMP accumulation with a stronger efficacy compared to control cells, suggesting that Nf1 negatively regulates MC1R signalling. Subsequently, using a combination of pharmacologic and genetic strategies, we established that the GAP activity of Nf1 is important to control MC1R-operated signalling and, consequently, melanogenic enzyme expression and melanin production. This study demonstrates a new degree in the regulation of MC1R activity which can be of interest in understanding and curing skin pigmentation disorders.


POSTERS

P30 Characterization of Homodimerization and Heterodimerization Capacity of Loss-Of-

Function Melatonin Receptor Type 2 (MT2) Mutants Linked to Type 2 Diabetes

LAHUNA Olivier, HARB Zeinab, JOURNE Anne-Sophie, KARAMITRI Angeliki, JOCKERS Ralf

INSERM U1016, Institut COCHIN Paris (France)

Type 2 diabetes (T2D) is growing so rapidly worldwide that scientists are in search of causes others

than increased food intake and poor physical activity to explain this recent increase in T2D. In 2009

genome-wide association studies (GWAS) identified two SNPs associated with increased fasting

plasma glucose and T2D that are located close or within the MTNR1B gene coding the melatonin

receptor type 2 (MT2). These studies suggested for the first time a link between melatonin and

glucose homeostasis. Later we identified forty rare MTNR1B variants by sequencing the MTNR1B

gene in cohorts of non-diabetic subjects and T2D patients. Among them four very rare mutations

(A42P, L60R, P95L, Y308S) abolished the binding of [I125]-melatonin to MT2. These four mutated

MT2 are constitutively inactive in the absence or presence of hormone. Genotyping the four total

loss-of-function MTNR1B mutations showed a strong and significant effect on increased T2D risk.

One characteristic of the melatonin receptors of type 1 (MT1) and type 2 (MT2) is their capacity to

form MT1/MT1 or MT2/MT2 homomers and MT1/MT2 heteromers. MT1/MT2 heteromers were

shown to be physiologically important in the regulation of light sensitivity of the mouse retina.

Although the A42P, L60R, P95L and Y308S mutations abolish MT2 dependent signaling pathways,

they don’t affect cell surface expression in HEK293 cells as mutated receptors are expressed equally

well as the MT2 wild-type. Knowing this we characterized the homodimerization and

heterodimerization capacity of the four mutated receptors (MT2-mut) with the MT1 and MT2 wild-

type. We performed co-immunoprecipitation and BRET (Bioluminescence Resonance Energy transfer)

experiments to solve this question. With both techniques we were able to detect formation of MT2-

wt/MT2-mut homomers and MT1-wt/MT2-mut heteromers. The BRET technique enables us to

determine for each mutated receptor a BRET50 value corresponding to 50% of the maximum level of

interaction. Comparison of BRET50 values suggests that the P95L and Y308S mutations increase the

capacity of MT2 to interact with the MT1-wt and MT2-wt. Thus we show that natural mutations

linked to T2D abolish the MT2 functions in terms of signaling pathways but don’t affect its traffic and

dimerization proprieties in HEK293 cells.


POSTERS

P31 β-arrestin-dependent transcriptome & proteome: a comparative study.

LANGONNÉ-GALLAY N(1), VANDERMOERE F(2), BOURQUARD T(1), CASSIER E(2), GAUTHIER C(1), BOULO T(1), POUPON A(1), CREPIEUX P(1), MARIN P(2), REITER E(1).

(1)BIOS group, UMR0085, INRA, F37380, Nouzilly, France. [email protected]. (2)Neuroproteomics and Signaling of Neurological and Psychiatric Disorders, UMR CNRS 5203, INSERM U1191, Université de Montpellier, F34094, Montpellier, France. Introduction : G protein-coupled receptors (GPCRs) are transmembrane molecules devoted to the integration of extracellular stimuli and their transduction into appropriate cellular outcomes. To do so, they regulate both intracellular signaling pathways and gene expression. Among the partners involved in this transduction, β-arrestins have emerged over the last 20 years as key players of GPCR functions, along with G proteins. However, relatively little is known about the mechanisms allowing them to regulate cellular signaling networks leading to gene transcription. To address this issue, we performed large-scale transcriptomic (gene expression arrays) and phosphoproteomic (SILAC isotopic labeling, phosphopeptide enrichment and high-resolution mass spectrometry) analyses comparing 3 different GPCRs in order to: i) identify genes/proteins regulated by β-arrestins; ii) determine which targets are receptor specific and which (if any) are generic, and iii) reconstruct β-arrestin-dependent networks. Material and Methods : 5HT2C-, 5HT4a- and FSH-receptors were transiently expressed in a common background of HEK293 cells and endogenous β-arrestins were silenced using siRNA. Three days after transfection, cells were stimulated with cognate agonist (1µM 5HT for 5HT2C-R, 10 µM 5HT for 5HT4a-R and 3.3 nM hFSH for FSH-R). Total RNAs were purified from cells depleted or not of β-arrestin 1 or 2 and stimulated for 6h. Transcriptomic analyses were carried out using Agilent microarrays. SILAC experiments were carried out on cells depleted or not of both β-arrestin isoforms, and stimulated for 10 min with vehicle or agonist. Results : The phosphoproteomic analysis revealed that β-arrestin-regulated phosphorylation events represented about 20% of agonist-dependent phosphoproteome (i.e.:106/410 (5HT2C-R), 125/469 (5HT4a-R) and 80/370 (FSH-R) identified proteins). A high proportion of these proteins were common to the 3 receptors. However, redundancy was much reduced at the transcriptomic level, suggesting a high receptor-specificity of the signaling pathways leading to gene transcription. For 5HT2C-R, 5HT4a-R and FSH-R, 654 (of 1909), 360 (of 1266) & 586 (of 1778) agonist-dependent genes were differentially regulated after β-arrestins depletion, respectively. Interestingly, FSH-R and 5HT2C-R, both described to activate ERK via β-arrestins, shared more β-arrestin-dependent genes, compared with 5HT4a-R, suggesting that both receptors trigger some common mechanisms. Conversely, FSH-R and 5HT4a-R, 2 Gαs-coupled receptors, showed more common regulated genes in the G-dependent group than with the 5HT2C-R, a Gq-coupled receptor. Data analysis using Ingenuity® Pathway Analysis allowed us to build potential β-arrestin-dependent direct networks from SILAC data, for each receptor. Within these networks, we focused on transcription regulators and found that they were highly connected to corresponding β-arrestin-dependent genes lists. We checked the specificity of these connections by challenging, for each receptor, 30 independent random lists of transcription regulators for their connectivity with β-arrestin-dependent genes, which turned to be significantly lower. Collectively, this study allowed to identify the molecular network underlying β-arrestin-dependent signaling and transcription for each studied GPCR. Moreover, crossing results from 3 different GPCRs revealed a minimal "β-arrestin signature", potentially indicative of conserved β-arrestin-dependent cellular mechanisms.



POSTERS

P32 Neuroprotection induced by 5-HT4R agonists in a mouse model of Alzheimer's disease:

cerebrovascular analysis

Patrizia Giannoni (1, 2, 3), Margarita Arango (1, 2, 3), Ines Das Neves (1, 2, 3) Kevin Baranger (4),

Santiago Rivera (4), Freddy Jeanneteau (1, 2, 3), Nicola Marchi (1, 2, 3) and Sylvie Claeysen (1, 2, 3)

(1) CNRS, UMR-5203, Institut de Génomique Fonctionnelle, 34000 Montpellier, France (2) Inserm,

U1191, 34000 Montpellier, France (3) Université de Montpellier, UMR-5203, 34000 Montpellier,

France (4) Aix-Marseille Univ, NICN, UMR 7259, 13344 Marseille, France

We have demonstrated that chronic treatments with 5-HT4 receptor agonists are able to prevent the

cognitive deficits in a genetic mouse model of Alzheimer's disease (5XFAD). Chronic early

administration of the reference 5-HT4R agonist RS 67333 reduced amyloid load in the brain and

consequently amyloid plaque number. Moreover, the cerebral inflammation (astrogliosis and

microgliosis) was markedly decreased (Giannoni P., 2013).

In a further work, we have performed a longitudinal analysis of the vascular-pericyte changes that

occurs in 5XFAD mice, describing the progressive blood brain barrier damages (Giannoni P.,

submitted).

In the current study, we investigated if chronic administration of RS 67333 to young 5XFAD mice is

able to prevent the amyloid-induced cerebrovascular damages. Amyloid plaque formation and vessel

integrity were tracked by recurrent 2-photon live imaging, using methoxy-X04 and FITC-dextran

staining, respectively. Cognitive performances of the mice have been assessed by novel object

recognition test. Regular thioflavin S staining of the plaques and post mortem brain slice

immunohistochemistry have been realized.

The final conclusions will be presented at the GDR 3545 -2015.


POSTERS

P33 Pharmacological characterization of NPFFR1/GPR147 antagonists for the study of its role in the central control of reproduction

(1)Raphaëlle Quillet, (1)Armand Drieu la Rochelle, (2)Frédéric Bihel, (2)Martine Schmitt, (2)Jean-Jacques Bourguignon, (1)Brigitte Ilien, (1)Frédéric Simonin

(1)Laboratoire de Biotechnologie et signalisation cellulaire UMR7242, CNRS/Université de Strasbourg, Illkirch, (2)Laboratoire d’innovation thérapeutique UMR7200, CNRS/Université de Strasbourg, Illkirch. Control of reproduction is driven by GnRH (Gonadotropin Releasing Hormone), which represents the main positive regulator of the reproductive system. Downstream, GnRH is able to stimulate synthesis and secretion of gonadotropins LH (Luteinizing Hormone) and FSH (Follicle-Stimulating Hormone), which will act on peripheral reproductive organs as ovaries. RF-amide peptides, characterized by their Arg-Phe-NH2 C-terminal, and their G-protein coupled receptors have been demonstrated to be involved in the central control of reproduction. Notably, Kisspeptin/GPR54 system represents a potent activator of reproductive axis, stimulating GnRH neurons. On the other side, RF-amide related peptides (RFRPs)/NPFFR1 system seems to emerge as an inhibitor of reproduction in different species, including mammals. Especially, an implication of this system has been described in seasonal animal models as hamsters. However, the study of the involvement of RFRPs/NPFFR1 system in reproduction is severely limited by the absence of pharmacological tools, particularly antagonists that selectively block NPFFR1 and not the other RF-amide receptors including NPFFR2 subtype and GPR54. In collaboration with a team of medicinal chemists from Faculty of Pharmacy (Illkirch), we screened in binding assay with NPFFR1 and NPFFR2 receptor subtypes a large focused chemical library of compounds (> 2000 molecules) that mimic the RF-amide sequence, which is critically involved in the binding of RF-amide peptides to their receptors. From this screening we identified several derivatives that display nanomolar affinity on NPFFR1 and very weak binding potency on the other RF-amides. NPFFR1 and NPFFR2 are Gi coupled receptors, which prevents the accumulation of cAMP induced by Gs protein activation. We thus look at the effect of the identified compounds on inhibition of the cAMP accumulation in HEK293 cells stably expressing both receptors and GloSensor in order to evaluate their antagonist or agonist nature. From this study we have identified the best compound that efficiently block, in a dose-dependent manner, the inhibition of cAMP accumulation induced by activation of NPFFR1 but not NPFFR2. Altogether, our results indicate that we have identified potent and selective antagonists of NPFFR1 that will be very helpful to elucidate the role of RFRPs/NPFFR1 system in the central control of reproduction in different animal models as well as in other physiological functions that are modulated by RF-amide peptides and their receptors including pain and feeding behaviour.


POSTERS

P34 Phosphoproteomics of 5-HT2A/mGlu2 heteromers: toward new insights into the mechanism of action of hallucinogens and antipsychotics

Samy Murat(1-3) , Samah Karaki(1-3), Carine Becamel(1-3) , Clotilde Mannoury la Cour(4) , Mark J. Millan(4) , Philippe Rondard (1-3) , Laurent Prezeau(1-3) , Joel Bockaert(1-3) , Philippe Marin(1-3*) , Franck Vandermoere(1-3*)

(1) CNRS, UMR-5203, Institut de Genomique Fonctionnelle, Montpellier; (2) INSERM, U1191, Montpellier; (3) Universite Montpellier, F-34094 Montpellier, France ; (4) Institut de Recherches Servier, Croissy-sur-Seine, France. The serotonin 5-HT2A receptor is the primary target of psychedelic hallucinogens such as LSD, mescaline and psilocybin (agonists), which reproduce some of the core symptoms of schizophrenia and of second-generation antipsychotics such as clozapine, olanzapine and risperidone (antagonists or inverse agonists). Recent findings demonstrate that 5-HT2A receptors form heteromers with metabotropic glutamate mGlu2 receptors, another target of last-generation antipsychotics (agonists or positive allosteric modulators). The association of both receptors has profound consequences on their pharmacology and signal transduction properties as well as on the behavioural effects of drugs that bind to either 5-HT2A receptors or mGlu2 receptors. For instance, 5-HT2A receptor/mGlu2 heteromer formation is essential for the expression of psychotropic-like effects of hallucinogens and imbalanced activity and coupling properties of 5-HT2A and mGlu2 receptors within the heterocomplex might be one of the molecular substrates for a susceptibility to schizophrenia. To get further insight into the mechanism of action of drugs acting at 5-HT2A/mGlu2 heteromers, we explored their impact upon the phosphorylation pattern of each receptor by high-resolution mass spectrometry. We show that hallucinogenic 5-HT2A receptor agonists (LSD, DOI) but not non-hallucinogenic 5-HT2A receptor agonists promote 5-HT2A receptor phosphorylation at Ser280 located in the i3 loop, a region important for receptor desensitization, both in HEK-293 cells and in mice prefrontal cortex. Correspondingly, Ser280 phosphorylation was responsible for the lower capacity of hallucinogens to promote receptor desensitization and internalization, compared with non-hallucinogenic agonists. Conversely, several phosphorylated residues were identified in the C-terminal domain of mGlu2 receptors co-expressed with 5-HT2A receptors in HEK-293 cells. Glutamate treatment increased the phosphorylation state of some of these residues, an effect prevented by the co-application of the synthetic hallucinogen DOI, which alone did not affect mGlu2 phosphorylation. Collectively, these findings reveal novel molecular substrates that might underlie the behavioural effects of drugs acting at each subunit of 5-HT2A/mGlu2 heteromers.


POSTERS

P35 b-arrestins directly interact with PKA, promoting its translocation to the nucleus and form a transcriptionally active complex with CREB upon stimulation of Gs-coupled GPCRs.

Landomiel Flavie, Tranchant Thibaud, Bourquard Thomas, Langonné Nathalie, Gauthier Christophe, Boulo Thomas, Bigot Yves, Akli-Ayoub Mohammed, Poupon Anne, Crépieux Pascale et Reiter Eric

INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France ; CNRS, UMR6175, F-37380 Nouzilly, France ; Université François Rabelais, F-37041 Tours, France. Beta-arrestins are known for their ability to inhibit the functional coupling between G proteins and their cognate GPCRs, thereby dampening second messenger accumulation in a process classically

referred to as homologous desensitization. In addition, -arrestins are involved in GPCR trafficking (i.e.: internalization-resensitization) and act as G protein-independent signal transducers, prompting the assembly of signaling complexes, their recruitment to activated GPCRs and ultimately their signaling.

Here we report that -arrestins exert more complex and subtle regulatory actions than previously thought on the cAMP/PKA/CREB pathway activated by Gs-coupled GPCRs. We found that agonist-induced cAMP production was increased in β-arrestin-depleted cells whereas, counterintuitively,

CRE-driven transcription was profoundly impaired in -arrestin-depleted cells compared to control. To further investigate this apparent paradox, we used vasopressin V2 receptor as a model Gs-

coupled GPCR. We demonstrated that -arrestin depletion increased PKA activity in the cytosol but

decreased it in the nucleus, suggesting that -arrestins contribute to the nuclear translocation of active PKA. Besides, we found that b-arrestins directly interact with PKA catalytic subunit and CREB in

a mutually exclusive manner. Interestingly, we observed that -arrestin-CREB interaction led to the formation of a transcriptionally active complex in an agonist-dependent manner. Indeed, by electromobility shift assay, co-incubation of nuclear extracts and a CRE probe with β-arrestin antibody induced a supershift in contrast to nuclear extracts co-incubated with control IgG. Collectively, our data suggest that, beyond the classical paradigm, b-arrestins exert a bimodal action on the cAMP/PKA/CREB pathway. On the one hand, they counteract cAMP accumulation and PKA activation in the cytosol while on the other hand, they promote PKA catalytic subunit translocation to the nucleus and interact with CREB to form a transcriptionally active complex.


POSTERS

P36 GABAB receptor cell surface export is controlled by an endoplasmic reticulum gatekeeper

Stéphane Doly1,2,3*, Hamasseh Shirvani1,2,3*, Gabriel Gäta1,2,3,4, Frank J. Meye5, Michel-Boris Emerit3,6, Hervé Enslen1,2,3, Lamia Achour1,2,3, Liliana Pardo-Lopez1,2,3,§, Seung-Kwon Yang3,6, Vincent Armand3,6, Robert Gardette3,6, Bruno Giros7,8, Martin Gassmann9, Bernhard Bettler9, Manuel Mameli5, Michèle Darmon3,6, Stefano Marullo

1: INSERM, U1016, Institut Cochin, Paris, France 2: CNRS, UMR8104, Paris, France 3: Université Paris Descartes, Sorbonne Paris Cité, France 4 : Université Paris Diderot, Sorbonne Paris Cité, France 5. Institut du Fer a Moulin, INSERM UMR-S 839, Université Pierre et Marie Curie, Paris France. 6 : INSERM U894, Centre de Psychiatrie et Neurosciences, Paris, France 7: INSERM U952, CNRS UMR 7224, Université Pierre et Marie Curie, Physiopathologie des Maladies du Système nerveux Central, Paris, France 8. Douglas Hospital Research Center, Department of Psychiatry, McGill University, Montreal, QC, Canada 9. Department of Biomedicine, University of Basel, Basel, Switzerland Endoplasmic reticulum (ER) release and cell surface export of many G protein-coupled receptors (GPCRs), are specifically regulated. For GABAB receptors of GABA, the major mammalian inhibitory neurotransmitter, the ligand-binding GB1 subunit is maintained in the ER by unknown mechanisms in the absence of hetero-dimerization with the GB2 subunit. We report that GB1 retention is regulated by a specific gatekeeper, PRAF2. This ER resident transmembrane protein binds to GB1, preventing its progression in the biosynthetic pathway. GB1 release occurs upon competitive displacement from PRAF2 by GB2. PRAF2 concentration, relative to that of GB1 and GB2, tightly controls cell surface receptor density and controls GABAB function in neurons. Its experimental perturbation in vivo caused marked hyperactivity disorders in mice. These data reveal an unanticipated major impact of specific ER gate-keepers on GPCR function and identify PRAF2 as a new molecular target with therapeutic potential for psychiatric and neurological diseases involving GABAB function.


POSTERS

P37 Regulation of the translatome by a GPCR, the follicle-stimulating hormone receptor, in primary rat Sertoli cells

Aurélie Tréfier* (1), Kelly León* (1), Thomas Bourquard* (1), Astrid Musnier (1), Julia Morales (2), Nathalie Langonné-Gallay (1), Eric Reiter (1), Florian Guillou (3), Anne Poupon (1), Pascale Crépieux (1).* Equal contribution

(1) Biology and Bioinformatics of Signaling Systems (BIOS) group, INRA, UMR85, Physiology of Reproduction and Behaviors (PRC), CNRS, UMR7247, François Rabelais University, F37041, France. (2) Sorbonne University, UPMC University Paris 06, UMR 8227, Integrative Biology of Marine Models, Translation Cell Cycle and Development, Station Biologique de Roscoff, Roscoff cedex, France. CNRS, UMR 8227, Integrative Biology of Marine Models, Translation Cell Cycle and Development, Biological Station of Roscoff, Roscoff cedex, France. (3) Genomic Plasticity and Phenotypic Expression (PGEP) group, INRA, UMR85, Physiology of Reproduction and Behaviors (PRC), CNRS, UMR7247, François Rabelais University, F37041, France. G protein-coupled receptors (GPCR) play an important role in gene transcription control, but little is known about their role in RNA translation. Translational regulatory mechanisms allow the cell to respond in a few minutes to subtle changes in the extracellular milieu. For example, in response to subtle variations of the follicle-stimulating hormone (FSH) input (e.g.: glycosylated isoform, variations in hormonal concentration or number of FSHR), regulation occurring at the translation level could serve to fine-tune Sertoli cell (SC) protein content. In male, FSH controls SC secretory activity, which is essential for the maturation of the sperm germline, by associating to a GPCR, the FSHR. Although the proteome induced by FSH is not available yet, a number of studies report that a limited, yet significant amount of proteins, is neosynthesized after one hour of FSH stimulation whereas transcriptomic analyses show that hundreds of genes are up or down regulated in response to six hours of FSH stimulation . This suggests that there is a regulation node between the modulation of gene expression through transcription and protein neosynthesis through translation. Our hypothesis is that FSH may regulate the translation of preexisting mRNA pools in SC. In line with this hypothesis, our group has previously shown that FSH targets mRNA-specific translation of c-fos and vegf1. To extend this work and assess to what extent the whole translatome is affected by FSH, we combined polysome profiling3 by sucrose density gradient fractionation with RNA-seq analysis4 in rat primary SC exposed to FSH for one hour. Comparison of normalized polysomal fractions in stimulated vs non-stimulated cells allowed us to identify about four hundreds mRNA whose translation goes up or down in response to FSH. Ingenuity pathway analysis (IPA) highlights that several components of the same functional network of proteins might be co-translated in response to FSH. Validation with the corresponding mRNA in normalized polysomal fractions is ongoing. To our knowledge, this study presents the first GPCR-dependent translatome. We expect that it will eventually provide in sight on the molecular mechanisms whereby this class of receptor physiologically controls translation. 1 Musnier and Leon, 2012. Mol.Endocrinol, 26(4):669-680. 2 McLean Dj, 2002. Mol.Endocrinol, 16(12):2780-2792. 3 Aneichyk et al., 2013. BMC Genomics, 1471-2164-14-844. 4 Schott at al., 2014. PLOS Genetics, 10(6):e1004368.


POSTERS

P38 Carboxyl terminal tail of GPR50 interacts with General transcription factor TFII-I and modulates nuclear functions

Raise Ahmad1, Anissa Sidibe1, Stefanie Wojciech1, Avais Daulat1, Marine Luka1, Anne-Sophie Journé1, Jean-Luc Guillaume1, Philippe Delagrange2 and Ralf Jockers1

1Functional Pharmacology and Pathophysiology of Membrane Receptors" Department of Endocrinology, Metabolism and Diabetes (EMD) INSTITUT COCHIN INSERM U1016, CNRS 8104, Université Paris Descartes 22 Rue Méchain, 75014 PARIS 2 Institut de Recherches SERVIER, Suresnes, France. GPR50, an orphan G protein-coupled receptor (GPCR), has recently been shown to play a role in modulating melatonin receptor function (Levoye et al, Embo J, 2006) and glucocorticoid receptor signalling (Li et al. PLoS ONE, 2011) through its one of the longest carboxyl terminal tail. However, despite these available reports the underlying mechanisms and the functional consequences of GPR50 signalling remain largely unknown. To address these questions, we used HEK-293T cells transfected either with the full length GPR50 WT or the ΔTTGH variant, or GPR50 without Carboxyl terminal (ΔC-tail) or the C-tail alone. Western blot analysis revealed constitutive proteolytic cleavage of the carboxyl terminal part of both full length variants. Following cell fractionation, we observed nuclear translocation of both GPR50 variants as well that of C-tail alone. Similar results were obtained in DMH neurons expressing endogenous GPR50. Further by using in-vitro cleavage assay we found Calpain to be responsible for GPR50 C-tail cleavage and this effect was abrogated in presence of a specific Calpain inhibitor. It has been reported that replacing residues by phosphomimetic amino acids (aspartic acid; glutamic acid) before and after proline residue could enhance nuclear translocation (Chuderland et al, Mol. Cell, 2008). We used the same approach for the C-tail of GPR50 and mutated its two potential phosphomimetic residues (aspartic acid) to alanine (APA mutant). Following transfection in HEK-293 cells we observed decreased nuclear translocation of “APA” in comparison to WT C-tail which raises the possibility for GPR50 to translocate into the nucleus in an NLS-independent manner as reported previously for ERKs, MEKs (Yoon and Seger, Growth Factors, 2006), and SMADs (Massagueet al, Genes Dev. 2005). In order to decipher the functional relevance of the nuclear translocation of the C-tail of GPR50 we identified the general transcription factor TFII-I as its potential interacting partner by using the Tandem-affinity-purification (TAP) method. The interaction was confirmed in transiently transfected HEK cells as well as in brain tissue of GPR50 WT mouse by co-immunoprecipitation. Further, the interaction was observed in both cytosol and nuclear fractions of overexpressed HEK cells. These result further complemented by Immunofluorescent microscopy where GPR50 full length as well as C-tail exclusively co-localized with TFII-I in the cytosol and nucleus. In the same line, we also observed a significant increase in TFII-I-dependent transcriptional activity (c-fos promoter activation) in the presence of GPR50. Taken together, we suggest that Calpain could be responsible for the proteolytic cleavage of the C-tail of GPR50 possibly followed by NLS independent nuclear translocation of GPR50. Translocation of GPR50 C-tail promotes the transcription of c-fos in a TFII-I-dependent manner which signifies the physiological relevance of this translocation. This represents an original signal transduction mode for GPCRs that does not rely on the activation of classical intracellular signalling pathways and generation of second messengers.

Chuderland D, Konson A, Seger R. Identification and characterization of a general nuclear translocation signal in signaling proteins. Mol Cell 2008 31(6):850-61. Levoye A, Dam J, Ayoub MA, Guillaume JL, Couturier C, Delagrange P, Jockers R (2006) The orphan GPR50 receptor specifically inhibits MT(1) melatonin receptor function through heterodimerization. EMBO J 25(13): 3012-3023. Li J, Hand LE, Meng QJ, Loudon AS, Bechtold DA (2011) GPR50 interacts with TIP60 to modulate glucocorticoid receptor signalling. PLoS One 6(8): e23725. Massague J, Seoane J, Wotton D. (2005) Smad transcription factors. Genes Dev. 19(23):2783-810. Yoon S1, Seger R. (2006) The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24(1):21-44


POSTERS

P39 The heterotrimeric G protein homo-dimerization

Poirot Mathias Charles (1), Floquet Nicolas (2), Senard Jean-Michel (1), Gales Céline (1)

(1) INSERM U1048/I2MC Eq8 ; (2) IBMM UMR5247

G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors. They

participate in the regulation of many physiological functions and are the target of approximately 40%

of currently marketed drugs. The heterotrimeric G protein complex, made up of alpha (α), beta (β)

and gamma (γ) subunits, is the common primary intracellular effectors for all GPCRs families and

ensure the relay of the signal between extracellular stimulus and intracellular signalization. However,

the structural and molecular bases of GPCRs activation still remain unclear.

The purpose of our study is to validate a 3D modeling, based on X-ray crystallography, that propose a

G proteins homo-dimerization for a GPCR monomer, unlike the ratio 1:1 generally described in the

literature. To answer that we used different BRET1 probes developed and validated in our laboratory

allowing the detection of tight protein-protein interactions. We used the β2-adrenergic receptor (β2-

AR) and its cognate Gαsβ1γ2 protein as a study’s model. The dimerization between the G proteins

was measured in HEK293T cells co-expressing Gαs-Rluc and Gαs-eYFP labeled in different positions in

the presence of untagged receptor and stimulated or not by the agonist .


POSTERS

P40 Are LH and hCG naturally biased for β-arrestin recruitment at the LHCGR?

Laura Riccetti (1), Mohammed Akli Ayoub (2), Danielle Klett (2), Yves Combarnous (2), Livio Casarini (1,3), Manuela Simoni (1,3,4), Eric Reiter (2)

(1) Unit of Endocrinology, Department of Biomedicine, Metabolism and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy (2) Biology and Bioinformatics of Signaling Systems (BIOS) group, INRA, UMR85, Physiology of Reproduction and Behaviors (PRC), CNRS, UMR7247, François Rabelais University, F37041, France (3) Center for Genome Research, University of Modena and Reggio Emilia, Modena, Italy (4) Azienda USL, Modena, Italy The lutropin/choriogonadotropin receptor (LHCGR) belongs to the super-family of the G-protein coupled receptors (GPCRs) and mediates the activity of the luteinizing hormone (LH) and human chorionic gonadotropin (hCG) in the control of sexual development and fertility. For many years, LH and hCG were considered biologically equivalent because of their structural similarities and their binding to the same receptor. However, accumulating evidences suggest that LHCGR is able to differentiate the binding of these two gonadotropins triggering differentially downstream intracellular signaling and trafficking. Aim: We investigated the signaling and trafficking properties of LHCGR upon activation by rhLH (Luveris®, Merck Serono) and rhCG (Ovidrelle®, Merck Serono), focusing on Gs protein-dependent

signaling (cAMP production) and -arrestin 2 recruitment. Methods: Bioluminescence resonance energy transfer (BRET) technology was used in order to analyze LHCGR activity after rhCG/rhLH binding. For this, human embryonic kidney 293 cells (HEK293) were transiently transfected with the plasmids coding for hLHCGR and the appropriate

BRET biosensors in order to assess quantitatively cAMP response and -arrestin 2 recruitment. Dose-response and real-time kinetic experiments were performed in live cells. Results: In dose-response experiments, upon rhCG stimulation, LHCGR is able to promote a 30-fold more potent cAMP response compared to rhLH stimulation (EC50: 12,91±0,171 pM; and 378,6 ±

0,0181 pM, n=4, respectively). rhCG is also more potent in inducing -arrestin 2 recruitment, with a 4-fold increase comparing to rhLH activity (EC50: 13,17 ±0,177 nM; and 57,3 ± 0,071 nM, n=4, respectively). Our study also showed the left-warded shift (about 3 logs) of cAMP curves (EC50 in picomolar range) compared to ß-arrestin 2 curves (EC50 in nanomolar range), confirming our recent data and indicating the high efficiency of the two gonadotropins for the canonical Gs/cAMP pathway. Interestingly, rhLH promoted partial ß-arrestin 2 response compared to rhCG while both hormones fully activated Gs/cAMP pathway. Kinetics experiments showed no significant differences between rhCG and rhLH for both cAMP response and ß-arrestin 2 recruitment. Conclusion: Our study reports that rhLH and rhCG differentially activate LHCGR in terms of cAMP response and ß-arrestin 2 recruitment. This supports the notion that LHCGR can discriminate the binding of the two gonadotropins thereby triggering different intracellular responses. To better dissect the differential activity of hCG and hLH, the internalization of the receptor, its G protein coupling and steroid production will be examined. Further investigations in this field may be useful in order to better understand the impact of such in vitro-determined biological activities on the use of these hormones in clinical practice.


POSTERS

P41 STRUCTURAL STUDIES ON THE PURIFIED CXCR4

Vinesh JUGNARAIN, Edward MITCHELL, Trevor FORSYTH, Micheal HAERTLEIN, Sandra CORTES, Bruno

TILLIER

ESRF/ILL/SYNTHELIS

Le projet de recherche s’articule autour de deux axes principaux : la purification de CXCR4 sous

forme de protéoliposomes et la détermination structurale, à savoir la caractérisation de la forme, de

la taille et des interactions, de CXCR4 sous forme soluble et / ou de proteoliposome par l’utilisation

de la diffusion des rayons X aux petits angles (SAXS) et par l’analyse sous une source de neutrons. Un

second aspect crucial de l’etude represente la mise au point d’un protocole pour la determination de

la structure de RCPGs sous forme soluble par SAXS et sous forme de protéoliposome par analyse sous

une source de neutrons. L’etude sera realisee à partir de la proteine CxCR4 telle que produite dans la

première partie du projet. La protéine CxCR4 pourra également être produite sous forme deutérée

par la société SYNTHELIS. La protéine CxCR4 sera analysée, après purification (chromatographie), par

SAXS et sous une source de neutrons, afin d’en determiner la structure et comparer les donnees avec

celles dejà obtenues par d’autres groupes de recherche sur cette proteine. Un travail important

devra être effectué afin de séparer le signal de diffusion attribué aux micelles de détergents du signal

spécifique de la protéine CxCR4 en utilisant le marquage par deuterium. Selon les résultats obtenus

pour la purification de la protéine lors de la première étape du projet, les analyses structurales de

CxCR4 pourront être complétées par une approche cristallographique sous rayons X.


POSTERS

P42 Optopharmacological control of endogenous G protein-coupled receptors

C. Goudet a,b,c, C. Zussy a,b,c, S. Pittolo d, X. Gómez-Santacana d,e,f, K. Eckelt d,g, X. Rovira a,b,c, J. Dalton f, J.-P. Pin a,b,c, A. Llobet g, J. Giraldo f, P. Gorostiza d,h,i and A. Llebaria e

a Institut de Génomique Fonctionnelle, CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Université de Montpellier, F-34000 Montpellier, France b INSERM, U661, F-34000 Montpellier, France c Université de Montpellier 1 & 2, F-34000 Montpellier, France d Institute for Bioengineering of Catalonia (IBEC) e Laboratory of Medicinal Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC) f Laboratory of Systems Pharmacology and Bioinformatics, Institut de Neurociències and Unitat de Bioestadística, Universitat Autònoma de Barcelona Autonomous University of Barcelona Barcelona, 08193 Bellaterra, Spain (UAB) g Network Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) h Bellvitge Biomedical Research Institute (IDIBELL) i Catalan Institution for Research and Advanced Studies (ICREA) Optopharmacology is a very promising technology to tackle pathophysiological processes. Contrary to optogenetics which requires the use of transgenes, optopharmacology allows for the targeting of endogenous receptors in their native tissues through the use of photoswitchable ligands that can be switched ON and OFF with light. This method provides fine spatial and temporal control of drug activity and offers unique opportunities for in vivo investigation. Herein we report the development and characterization of what is, to our knowledge, the first photoswitchable allosteric modulator of a G protein–coupled receptor. Alloswitch-1 is a selective negative allosteric modulator for the metabotropic glutamate receptor mGlu5 which enables the optical control of endogenous mGlu5 receptors, both in vitro and in vivo.


POSTERS

P43 Thermostabilisation of a Negative Allosteric Modulator-bound conformation of the human

metabotropic mGlu5 receptor

Karine Rottier, Romain Marcellin, Nasrallah Chady and Guillaume Lebon

Institut de Génomique Fonctionnelle, CNRS-UMR5203, INSERM U1191, Université de Montpellier,

141 Rue de la Cardonille, 34094 Montpellier Cedex 05.

Solving structure of GPCRs is a challenging task. Innovative strategies in protein engineering such as

the identification of thermostabilising point-mutations, have led to the structure determination of

several GPCRs structure, including human adenosine A2A receptor, turkey β1 adrenergic receptor

and rat neurotensin receptor NTSR1, bound either to antagonist and inverse agonist or to agonists

[1]. As a first step towards determining the structure of inactive conformation of the human mGlu5

receptor dimer bound to a Negative Allosteric Modulator (NAM) MPEP, we screened for

thermostabilising single point mutation. Here, we show how alanine scanning mutagenesis coupled

to a selection strategy can be used to identify single point mutation for improved thermostability of

inactive MPEP-bound conformation of the dimeric mGlu5 receptor. Assembly of thermostabilising

mutations significantly improved the thermal stability of the detergent-solubilised mGlu5 receptor

bound to MPEP. The thermostabilised mutant shows stability in several detergents. Expression and

purification of thermostable mGlu5 receptor are in progress.


POSTERS

P44 A strategy to discover decoy chemokine ligands with an anti-inflammatory activity

Dayana Abboud1, François Daubeuf2, Quoc Tuan Do3, Valérie Utard1, Pascal Villa4, Jacques Haiech2, Dominique Bonnet2, Marcel Hibert2, Philippe Bernard3, Jean-Luc Galzi1#* & Nelly Frossard2#

1Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS/Université de Strasbourg, and Labex Medalis, ESBS, 300 Boulevard Sébastien Brant, 67412 Illkirch, France 2Laboratoire d’Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, and Labex Medalis, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France 3GreenPharma, 3 allée du Titane, 45100 Orléans, France 4PCBIS Plate-forme de Chimie Biologique Intégrative de Strasbourg, UMS 3286 CNRS/Université de Strasbourg, and Labex Medalis, ESBS, 300 Boulevard Sébastien Brant, 67412 Illkirch, France Excessive signaling by chemokines has been associated with chronic inflammation or cancer, thus attracting substantial attention as promising therapeutic targets. Inspired by chemokine-clearing molecules shaped by pathogens to escape the immune system, we designed a generic screening assay to discover chemokine neutralizing molecules (neutraligands) and unambiguously distinguish them from molecules that block the receptor (receptor antagonists). This assay, called TRIC-r, combines time-resolved intracellular calcium recordings with pre-incubation of bioactive compounds either with the chemokine or the receptor-expressing cells. We describe here the identification of high affinity neutraligands of CCL17 and CCL22, two chemokines involved in the Th2-type of lung inflammation. The decoy molecules inhibit in vitro CCL17- or CCL22-induced intracellular calcium responses, CCR4 endocytosis and human T cell migration. In vivo, they inhibit inflammation in a murine model of asthma, in particular the recruitment of eosinophils, dendritic cells and CD4+T cells. Altogether, we developed a successful strategy to discover as new class of pharmacological tools to potently control cell chemotaxis in vitro and in vivo.


POSTERS

P45 Monitoring YAP activity in colon cancer cells from patients using a rapid and easy-to-use

innovative HTRF® YAP Assay

L Prézeau (abc), C Volabc, O Lechat (abc), M Vereecke (abc), F Charrier-Savournin (d), T Roux (d), P

Mensat (d), E Dupuis (d), JP Pin (abc), E Trinquet (d), J Pannequin (abc)

(a) CNRS, UMR5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France; (b) INSERM,

U1191, F-34000 Montpellier, France; (c) Université de Montpellier, F-34000 Montpellier, France; (d)

Cisbio bioassays, F-30200 Codolet, France

The Hippo pathway is described to be widely involved in physiological (organ growth and tissue

renewal) and pathological (cancer) processes. Thus, accurate assessing of the activity of this pathway

is crucial for better understanding its roles, but also for performing screening of drugs, conditions

and strategies that would help to control it.

The Hippo pathway involves a cascade of phosphorylation events, as activation of the Mst1/2 and

LATS1/2 kinases leads to the phosphorylation of YAP and TAZ pro-proliferation transcription co-

factors, which are then switched off, retained in the cytosol or degraded.

Thus, besides few YAP/TAZ activity gene reporter assays, detecting the phosphorylation of the Hippo

pathway proteins has been the way to monitor their activity, mostly using biochemical western

blotting, which remains long and fastidious. There was then a crucial need for easy-to-use tools to

perform high-scale analysis and screening.

In this context, we are engineering innovative HTRF® assays to quantify Hippo pathway component

expression and activity suitable for rapid and reliable biological analysis and high scale screening.

HTRF® technique is based on Forster Resonance Energy Transfer (FRET) observed between

compatible donor and acceptor fluorophores, such that activation of the donor leads to acceptor

fluorescence when they are in close proximity (nm). We have recently generated powerful HTRF®

YAP expression and YAP-S127 phosphorylation assays, and used them routinely for assessing the

expression and activity of YAP in different classical cell lines as well as colon cancer cells freshly

established from patients.


POSTERS

P46 Chemotactic G-protein coupled receptors control cell migration by suppressing

autophagosome biogenesis

Coly Pierre-Michaël (1), Perzo Nicolas (1), Le Joncour Vadim (1), Lecointre Céline (1), Shouft Marie-

Thérèse, Tonon Marie-Christine (1), Wurtz Olivier (1), Gandolfo Pierrick (1), Castel Hélène (1), and

Morin Fabrice (1)

(1) Inserm U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation,

DC2N, Astrocyte and Vascular Niche, Biomedical Research Institute (IRIB), University of Rouen,

76821, France.

Chemotactic migration is a fundamental behavior of cells and its regulation is particularly relevant in

physiological processes such as organogenesis and angiogenesis, as well as in pathological processes

such as tumor metastasis. The majority of chemotactic stimuli bind to cell surface receptors that

belong to the G-protein-coupled receptor (GPCR) superfamily. Although the autophagy machinery

has been shown to play a role in cell migration, its mode of regulation by chemotactic GPCR remains

largely unexplored. We found that ligand-induced activation of two chemotactic GPCRs, the

chemokine receptor CXCR4 and the urotensin receptor UT, triggers a marked reduction in the

biogenesis of autophagosomes, in both HEK-293 cells and U87 glioblastoma cells. Chemotactic GPCRs

exert their anti-autophagic effects by preventing the targeting of Atg16L protein to clathrin-coated

preautophagic vesicles forming from the plasma membrane. We further demonstrated that CXCR4 or

UT-induced inhibition of autophagy favors the formation of adhesions complexes to the extracellular

matrix and is required for chemotactic migration. This leads us to propose that compartmentalized

inhibition of autophagy by chemotactic GPCRs allows the maintenance of adequate pools of

phospholipids and cell-adhesion proteins at the migration front. Altogether our findings reveal a new

link between GPCR signaling and the autophagy machinery and may help to envisage therapeutic

strategies in pathological processes such as cancer and cell invasion.


POSTERS

P47 Expression and purification of the human metabotropic Glutamate Receptor 5

Chady Nasrallah, Karine Rottier, Gaetan Bellot & Guillaume Lebon

Institut de Genomique Fonctionnelle, UMR 5203 CNRS – U 1191 INSERM – Univ. Montpellier, 141 rue

de la Cardonille, Montpellier, France.

Metabotropic glutamate receptors (mGlus) are class C G protein-coupled receptors that modulate

the synaptic transmission throughout the central nervous system (CNS). The therapeutic spectrum of

mGlu5 in neurological disorders such as Parkinson’s disease, Fragile X, depression and schizophrenia

[1-3] makes this receptor an attractive drug target and highlights the need for understanding at the

molecular level its mechanism of activation and its allosteric modulation. Expressed as constitutive

homodimers at the cell surface, these receptors are activated when bound to L-glutamate, the

primary excitatory neurotransmitter of the CNS, within an orthosteric binding site located in the

extracellular domain (ECD) [4]. The ECD is composed of an extracellular bilobate Venus flytrap

domain (VFT) and linked via a cysteine rich domain (CRD) to the transmembrane domain (7TM),

responsible for transducing signals to intracellular partners. The 7TM domain harbours an alloster

ic binding site that potentiates glutamate activity when bound to positive allosteric modulators

(PAMs) or inhibits its activity when bound to negative allosteric modulators (NAMs) [5]. We are

aiming to express and purify human C-terminal truncated version of mGlu5 receptor to homogeneity.

Here, we will expose the strategies exploited to express and purify the receptor in its inactive

conformation when bound to a NAM 2-Methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP).

Insect cells were used as a host expression system for mGlu5 and infected using recombinant

baculovirus. We assessed the effects of peptide signals; tags positions; insect cells lines and the

presence of fusion protein (EGFP) on the receptor expression yield. Following this strategy, we were

able to increase expression and to select the optimal construct for insect cells expression system.

Extraction and solubilisation conditions were developped using suitable mixture of detergent and

cholesterol. Thermostabilising mutations were furthermore introduced into the final construct to

facilitate expression, purification and crystallization of the inactive MPEP-bound conformation of the

human mGlu5 receptors. 1. Moghaddam, B., Targeting metabotropic glutamate receptors for treatment of the cognitive symptoms of schizophrenia.

Psychopharmacology (Berl), 2004. 174(1): p. 39-44.

2. Pilc, A., et al., Mood disorders: regulation by metabotropic glutamate receptors. Biochem Pharmacol, 2008. 75(5): p. 997-

1006.

3. Conn, P.J., et al., Metabotropic glutamate receptors in the basal ganglia motor circuit. Nat Rev Neurosci, 2005. 6(10): p.

787-98.

4. Pin, J.P., T. Galvez, and L. Prezeau, Evolution, structure, and activation mechanism of family 3/C G-protein-coupled

receptors. Pharmacol Ther, 2003. 98(3): p. 325-54.

5. Nickols, H.H. and P.J. Conn, Development of allosteric modulators of GPCRs for treatment of CNS disorders. Neurobiol

Dis, 2014. 61: p. 55-71.


POSTERS

P48 Physiological interaction of orphan GPR50 with Nogo-A : Role in neurodegenerative diseases

Marine Luka (1), Raise Ahmad (1), Anne-Sophie Journé (1), Sarah Gallet (2), Vincent Prevot (2), Philippe Delagrange (3) and Ralf Jockers (1)

(1) Institut Cochin, Inserm U1016, Paris, France ; (2) Jean-Pierre Aubert Research Center, Inserm U837, Lille, France ; (3) Institut de Recherches SERVIER, Suresnes, France GPR50 is an orphan receptor, also called melatonin-related receptor (MRR), due to its high sequence homology (45%) with melatonin MT1 and MT2 receptors. Despite this close structural relationship GPR50 does not bind melatonin. It has been shown that GPR50 is expressed in both the developmental and adult brain. However the physiological function of GPR50 is still poorly understood. Recent genetic studies provided first hints on potential functions, suggesting that variants of the GPR50 gene are associated with increased risk for psychiatric disorders and modified lipid metabolism. Several studies suggest that GPR50 behaves as a scaffolding protein. In particular its long carboxyl terminal tail may play the role of a platform for other proteins. Described examples are complexes between GPR50 and MT1, MT2, TIP60 and Nogo-A. Here we decided to characterize the interaction between GPR50 and Nogo-A, a potent neurite outgrowth inhibitor. Nogo-A is mainly expressed by oligodendrocytes in the adult CNS and highly expressed by developing and immature neurons in plastic CNS regions such as the cortex, hippocampus and dorsal root ganglia. Recent studies have shown that Nogo-A negatively influences the cytoskeleton and the growth program of neurons. Many studies have reported that the neutralization of Nogo-A or Nogo-A receptor elements restores plasticity, improves axonal growth and enhances behavioral recovery after injury with no obvious side-effects. We studied the physical interaction and colocalization between GPR50 and Nogo-A following overexpression in HEK293-T and in HeLa cells as well as in mouse tissues (median eminence, cortex, tanycytes, brain sections) using different experimental approaches such as co-immunoprecipitation, co-localisation and Proximity Ligation Assay. We were able to confirm the interaction between Nogo-A and GPR50 in vitro and in vivo. The functional consequences of this interaction are currently under investigation by using NG108-15 cells, primary cultures of tanycytes and of Neural Progenitors Cells. The effect on parameters like neurite outgrowth, differentiation, migration and proliferation will be studied. Our preliminary results show that GPR50 may have a negative effect on the neurite length. Taken together our findings provide a new potential link between GPR50 and CNS plasticity and associated disorders.


POSTERS

P49 In vivo characterization of MTR/5-HT2c heteromers in mouse brain

Romain Gerbier (1), Florence Gbahou (1), Delphine Ndiaye-Lobry (1), Philippe Delagrange (2), Ralf

Jockers (1)

(1) Inserm, U1016, Institut Cochin, Paris, France; (2) Institut de Recherches Servier, Suresnes, France

According to the World Health Organization, 350 million people worldwide suffer from depression. In

2010, a novel antidepressant, Agomelatine (Valdoxan), has been introduced on the market. This

compound represents a novel class of antidepressant as it is the first drug to act simultaneously as

both a melatonergic MT1 and MT2 receptor agonist and a 5-HT2C antagonist, all belonging to the G

protein-coupled receptor (GPCR) super-family. Interestingly, the unique activity profile of

Agomelatine depends on both components. However, the molecular mechanism of this cross-talk is

currently unknown.

Increasing evidence shows that GPCR heteromers can be considered as a distinct functional entity.

Indeed, the changes in the ligand binding and/or signalling properties that accompany

heterodimerization could give rise to pharmacological and functional properties different from those

of the corresponding homomers.

We firstly demonstrated the existence of this cross-talk via physical association between MT2 and 5-

HT2C receptors in transfected cells, by co-immunoprecipitation and BRET. Moreover, we showed

that MT2/5-HT2C heteromers activate Gi proteins in a similar manner as MT2 alone and trans-

activate Gq proteins through the 5-HT2C protomer.

Secondly, to study the relevance of MT1/5-HT2C and/or MT2/5-HT2C heteromer signaling in vivo,

we determined the activation of Gi and Gq proteins in mouse brain by measuring [35S]GTPγS

incorporation into immunoprecipitated Gα subunits. Melatonin induced the activation of both Gi and

Gq proteins in cerebellum and hypothalamus. Whereas S20928, a specific MTRs antagonist, inhibited

both, Gi and Gq activation, a specific 5-HT2C inverse agonist, SB243,213, only inhibited Gq activation,

suggesting that melatonin receptor signaling through Gq is dependent on 5-HT2C transactivation.

When using MT1 and MT2 knock-out mice melatonin-induced Gq activation was only seen in MT1 KO

mice. Taken together these data indicate that MT2, and not MT1, functionally interacts with the Gq-

coupled 5-HT2C in mouse cerebellum and hypothalamus. Activation of the Gq/PLC pathway was

further studied by measuring Inositol-1 phosphate (IP1) levels by HTRF in brain structures of

melatonin-treated mice. Melatonin, at a dose of 1.5 mg/kg, increased IP1 levels in mouse cerebellum

and hypothalamus unlike in mouse hippocampus and cortex.

Taken together, our study provides for the first time in vivo evidence for the existence of functional

MT2/5-HT2C heteromers in different mouse brain structures. Interestingly, activation of these

heteromers endows melatonin with the unique property to transactivate the Gq/PLC pathway.

Future studies will clarify whether this unique property is associated to the antidepressant effect of

agomelatine.


POSTERS

P50 Nanobodies and HTRF® technology : A new way for an orphan class B GPCR investigations

M. Da Silva12, E. Dupuis1, F. Maurin1, K. Dumas1, P. Rondard2, J.-P. Pin2, D. Baty, L3. Agnelli3, M. Bas4

1Cisbio Bioassays, Codolet, France 2Institut Génomique Fonctionnelle, CNRS UMR5203, INSERM U1191, Université de Montpellier, Montpellier France 3Antibody therapeutics and immunotargeting Inserm unit, Marseille, France 4Sanofi, Exploratory unit, System biology, Montpellier, France GPR97 receptor is an orphan class B GPCR with a very large multidomain N-terminal extracellular region (~200 amino acids). The physiological roles of this receptor are not well-known but it is thought to play a role in immune function and neurodegenerative diseases. In this collaborative work several nanobodies (single domain antibodies) were developed by the Antibody Therapeutics and Immunotargeting team using a phage display selection. Two specific clones of GPR97 receptors were selected by flow cytometry and validated on transfected cells expressing the human GPR97 receptor which has been fused to SNAP-tag® at N-terminal extremity by Cisbio Bioassays. Then, these nanobodies were coupled with the compatible donor and acceptor of the HTRF® technology and used as tools for the detection of cell surface expression and dimerization of wild-type GPR97 receptors in HTRF® cell-based assays. Furthermore, several agonist and antagonist compounds which have previously been selected by Sanofi in a large scale screening of an internal chemical library were used to characterize the nanobodies. In this poster, we show the results of the characterization of both HTRF®-coupled nanobodies in various cell-based assays like binding and microscopy experiments. Finally, the miniaturization of assay formats (sv 384 well plate) prove that the combination of nanobodies and HTRF technology can be suitable for screening assays. These innovative tools were also used to label GPR97 receptors and highlight constitutive and induced internalization in microscopy assays.


POSTERS

P51 TOWARDS A MECHANISTIC UNDERSTANDING OF THE OPIOID MU RECEPTOR ACTIVATION

BY LIQUID-STATE NMR SPECTROSCOPY

Rémy Sounier (1), Camille Mas (1), Jan Steyaert (2, 3), Toon Laeremans (2, 3), Aashish Manglik (4),

Weijiao Huang (4), Brian Kobilka (4), Héléne Déméné (5) and Sébastien Granier (1).

1) Institut de Genomique Fonctionnelle, CNRS UMR-5203 INSERM U1191, University of Montpellier,

F-34000 Montpellier, France. (2) Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, B-

1050 Brussel, Belgium. (3) Structural Biology Research Center, VIB, Pleinlaan 2, B-1050 Brussel,

Belgium. (4) Department of Molecular and Cellular Physiology, Stanford University School of

Medicine, Stanford, California 94305, USA. (5) Centre de Biochimie Structurale, CNRS UMR 5048-

INSERM 1054- University of Montpellier, 29 rue de Navacelles, 34090 Montpellier Cedex, France.

Opioid receptors (OR), members of the G protein-coupled receptor (GPCR) superfamily, constitute

the major and the most effective target for the treatment of pain[1]. The use of opioid drugs acting

at these receptors is however a leading cause of death by overdose in Europe and North America.

Both beneficial and adverse effects of illicit opioid drugs (opium, heroin) as well as approved

therapeutics (morphine and codeine) are mediated by the activation of the mu-opioid receptor

(µOR).

We recently described the structure of an inactive conformation of the µOR[2]. It provided important

information regarding the binding site of small morphinan antagonists, revealed a largely exposed

binding pocket, and demonstrated key molecular determinants for antagonist binding preferences

for OR. However, much remains to be learned about the mechanisms by which different agonists can

induce distinct levels of Gi protein activation and/or arrestin recruitment upon activation of µOR.

Pharmacological and biophysical studies suggest that this versatility can be achieved through the

structural plasticity of GPCRs3.

In this study, we propose to analyse the conformational landscape of the µOR in distinct

pharmacological conditions using liquid-state NMR spectroscopy by monitoring signals from methyl-

labelled lysines. Assignment of resonances is achieved by a mutagenesis approach. We thus could

analyze the structure and dynamics changes upon binding to different ligands ranging from agonist

to antagonists, as well as upon binding the effector Gs protein and a mimetic nanobody thereof. Our

results show that there is very weak allosteric coupling between the agonist binding pocket and G

protein coupling interface (transmembrane TM 5 and 6). Furthermore, the analysis provides clues on

the successive structural events leading to the full active conformation of moR [4]. A better

knowledge of the structural basis for opioid drug efficacy may lead to new therapeutic approaches

with limited side effects. [1] Melnikova I (2010) Nat. Rev. Drug Discov. 9(8):589-590.

[2] Manglik A, et al. (2012) Nature 485(7398):321-326.

[3] Ghanouni P, et al. (2001)J. Biol. Chem. 276(27):24433-24436.

[4] Sounier, R., et al. (2015) Nature 524, 375-378.


POSTERS

P52 Analysis Of The Influence Of Haplotype On β2-Adrenoceptor Internalisation In Human Stem Cells (HUES7)

Joelle Goulding (1), Alexander Kondrashov (1), Tony Melarangi (1), Amanda Wheal (1), Sarah Mistry (1), Yorick Guerout (1), Chris Denning (1), Stephen Hill (1)

(1) University of Nottingham, Nottingham, UK The importance of the β-adrenoceptor system in cardiac function is well known and drugs targeting this system, such as certain β-blockers and adrenaline, are prescribed extensively to treat heart failure and cardiac arrest respectively (1). There are a number of single nucleotide polymorphisms within the β2-adrenoceptor (β2-AR) of which Gly16Arg and Glu27Gln have been associated with altered mortality in patients receiving β-blockers after acute coronary syndrome (2). Cardiovascular drug discovery has been limited by the model cell lines available to research the role of β2-AR and to evaluate drugs that target this receptor for disease indications. Often cardiomyoctyes from rats are used in research but within these cells the β2-AR displays altered signalling compared to that in humans (3). To address this we have created human embryonic stem cell (HUES7) lines which stably express one of four SNAP-tagged β2-AR haplotypes (varying at sites 16 and 27) and studied the internalisation of these receptors in response to agonist challenge. HUES7 cell lines (4) stably expressing one of 4 N-terminal SNAP tagged β2-AR haplotypes were created (SNAP-β2-AR_R16Q27; R16E27; G16Q27; G16E27). Whole cell radioligand binding was carried out using 3H-CGP 121771 in serum-free DMEM as previously described (4). For internalisation studies, cells were seeded in 96-well black-walled plates in RPMI-B27 medium. Prior to agonist addition, cells were labelled with SNAP-surface® Alexa Fluor® 488 (New England Biolabs) (0.1 µM) for 30 minutes in medium at 37°C. Cells were then washed and incubated in 100 µl HEPES buffered saline solution in the absence or presence of formoterol, salbutamol and salmeterol (1 pM-10 µM) for 1 hr at 37°C. Cells were then fixed, labelled with Hoechst nuclear stain and imaged on the Ultra confocal plate reader (Molecular Devices) the following day. Intracellular receptor was quantified using a granularity analysis algorithm on the MetaXpress software. Radioligand binding confirmed typical β2-AR pharmacology in all 4 cell lines with no differences observed in expression level nor the affinity of 3H-CGP 121771 and unlabelled ligands (propranolol, CGP 121771, ICI 118551, CGP 20712A). Agonist induced internalisation of the SNAP-tagged β2-AR was confirmed in all 4 haplotypes by confocal imaging. Salmeterol and salbutamol induced internalisation displayed partial agonism in comparison to formoterol. Agonist ranking and potency was conserved across the 4 β2-AR haplotypes. In summary, we report the agonist-induced internalisation of SNAP tagged β2-AR in HUES7 cells expressing different haplotypes of the β2-AR.


POSTERS

P53 The accessory protein SET regulates GnRH receptor signaling not only at plasma membrane but also in the nucleus.

Charlotte Avet, Chantal Denoyelle, Joëlle Cohen-Tannoudji, Violaine Simon

Physiologie de l'axe gonadotrope INSERM U1133, Univ Paris Diderot, Sorbonne Paris Cité, Unité Biologie Fonctionnelle et Adaptative (BFA), CNRS UMR 8251 Reproductive function is under the control of the hypothalamic neurohormone Gonadotropin-Releasing Hormone (GnRH), which activates a G-protein coupled receptor (GnRHR) expressed in pituitary gonadotrope cells. We identified the first interacting partner of GnRHR, the proto-oncogene SET (1), and showed that SET induces a signaling switch of GnRHR from calcium to cAMP pathways. Here, we show that in addition to its role on plasma membrane GnRHR signaling, SET inhibits the expression of the two gonadotropin hormones, LH and FSH, which are induced by GnRH. Interestingly, GnRH treatment of gonadotrope cells rapidly decreases SET expression as early as 30 minutes, probably through a post-transcriptional mechanism involving proteasomal and microRNA pathways. Moreover, GnRH treatment induces SET phosphorylation, a post-traductional modification favoring SET exit from the nucleus. Impact of GnRH on SET expression and localization may thus release the inhibitory action exerted by SET in the nucleus on gonadotropins gene expression and this mechanism may explain at least in part GnRH-mediated gonadotropin expression. Altogether, our work shows that the accessory protein SET influences not only GnRHR signaling at plasma membrane but also GnRH induction of gonadotropins gene expression in the nucleus. (1) Avet et al. SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells. J. Biol. Chem., 2013, 288(4): 2641-54.


POSTERS

P54 CCR5 dimerization and export

Jun Jin (1), Fanny Momboisse (1), Thibault Lagache (1), Gaelle Boncompain (2), Florence Niedergang

(3), Franck Perez (2), Fernando Arenzana-Seisdedos(1), Bernard Lagane (1), Esther Kellenberger (4),

Anne Brelot (1)

(1) Institut Pasteur; (2) Institut Curie; (3) Institut Cochin; (4) Université de Strasbourg

It is well established that class C GPCRs form constitutive dimers. However, the structural

organization of class A oligomers and its functional consequences are less understood.

Methodological limitations often make difficult the interpretation of results and a number of

interactions remain controversial. The recent crystallographic structures of class A GPCRs revealed

several homodimerization interfaces, opening the way for new studies on the functional significance

of this process.

In this study, we investigated the organization of CCR5 as homodimers and its function in the export

process. This class A GPCR is the receptor for CCL3, CCL4, CCL5 chemokines and also a CD4-associated

coreceptor required for HIV-1 entry into host cells. It is a suitable therapeutic target to inhibit HIV-1

entry. We first predicted residues of three potential dimer interfaces by computational analysis

based on known dimeric GPCR structures and the structure of CCR5 in its monomeric form. The

defined interfaces involve TM5, TM5/TM6 and TM1/TM2/H8. Through cross-linking experiments we

verified the two interfaces involving TM5. These results confirm the organization of CCR5 as dimer

and indicate different ways to interact. Stability of the interfaces in molecular dynamic simulations of

CCR5 dimers also provides evidence of the existence of two models. Moreover, site-directed

mutagenesis revealed that substitution of appropriate residues within TM5 altered the stability of

dimers, confirming the interfaces. We monitored CCR5 dimer formation by using both a conventional

energy transfer strategy and a new cellular assay based on the synchroneous transport of proteins.

With this latter assay, we also established that CCR5 dimerization both occurs in the endoplasmic

reticulum and plays a major role in its export to the cell surface. Furthermore, we showed a new

property of maraviroc, a CCR5 inhibitor currently used in HIV treatment. It acts as a pharmacological

chaperone by promoting both dimerization and cell surface targeting. Binding of maraviroc likely

modifies TM5 interfaces and stabilizes CCR5 in a new dimer conformation.


POSTERS

P55 Modulation of dopamine transporter activity by melatonin receptors

Alan HEGRON (1,2,6), Abla Benleulmi-Chaachoua (1,2,3), Marine Le Boulch (1,2,3), Kate Sokolina (4),

Victoria Wong (4), Igor Stagljar (4), Philippe Delagrange (5), Michel Bouvier (6,7), Ralf Jockers (1,2,3)

(1): Inserm U1016, Institut Cochin, Paris, France (2): CNRS UMR8104, Paris, France (3): Univ. Paris

Descartes, Sorbonne Paris Cité, Paris, France (4): Donnelly Centre, Department of Biochemistry,

Department of Molecular Genetics, Faculty of Medicine, University of Toronto, 160 College Street,

University of Toronto, ON, Canada, M5S 3E1 (5): IDR SERVIER, Croissy/Seine, France (6): Institut de

Recherche en Immunologie et en Cancérologie, Université de Montréal, Montréal, QC H3C 1J4,

Canada (7): Département de Biochimie et médecine moléculaire, Université de Montréal, Montréal,

QC H3C IJ4, Canada

The dopamine (DA) is a monoamine neurotransmitter transporter for dopamine (DAT) mediating the

re-uptake of DA into presynaptic nerve termini to clear the presynaptic cleft. Melatonin, a

neurohormone, is synthesized and secreted mainly by the pineal gland in a circadian manner and

thus regulating endogenous circadian rhythms and modulating other physiological functions, like the

regulation of DA levels. But how melatonin accomplishes this task is not clear. In mammals two G

protein-coupled receptors, MT1 (encoded by MTNR1A) and MT2 (encoded by MTNR1B) mediate the

actions of melatonin. We show here that coexpression of MT1 and DAT in HEK293 cells inhibit DA

uptake in the absence of melatonin. When both MT1 and MT2 are coexpressed with DAT, the

inhibititory effect of MT1 on DA uptake is abolished. Biochemical characterization shows the

formation of DAT/MTRs signaling complexes that result in decreased DAT cell surface localization.

This effect is independent of post-translational modifications of the N-terminus of DAT, a common

way to regulate DAT activity. Consistently, mice with targeted deletion of MTRs showed markedly

increased DA uptake in synaptosomal preparations of the striatum. In conclusion, the DA re-uptake

capacity of synaptosomes and DAT cell surface expression is regulated by MTRs and indicates a

regulatory role of MTRs on extracellular DA levels that are likely to be submitted to circadian

variation.


POSTERS

P56 Functional analysis of signaling signature of 40 SNPs found in melatonin type 2 receptor:

new candidates involved in type 2 diabetes

Angeliki Karamitri1, Bianca Plouffe2, Min Chen1, Jonathan Michael Gallion3, Olivier Lahuna1, Alan

Hegron1, Jean-Luc Guillaume1, Amélie Bonnefond4, Philippe Froguel4, Olivier Lichtarge3, Michel

Bouvier2 and Ralf Jockers1 1 Institut Cochin, Inserm U1016, CNRS UMR 8104, Université Paris Descartes 2 IRIC-Université de Montréal,

Montreal, Quebec, Canada 3 Baylor College of Medicine, Houston, Texas, U.S.A.

4 CNRS UMR8199 & Institut de

Biologie de Lille, E.G.I.D - FR3508 European Genomics Institute of Diabetes.

Melatonin, a neurohormone mainly secreted by the pineal gland, has been implicated in the regulation of circadian rhythms in mammals and insulin secretion in rodents (Peschke et al. J Pineal Res, 2007). The hormone binds with high affinity two G protein-coupled receptors (GPCRs): MT1 and MT2 which are both coupled to the Gi/cAMP pathway (Jockers et al. Br J Pharmacol, 2008). Further interest in the involvement of melatonin in metabolism came from the discovery of rare, non-synonymous mutations of MT2 receptor in the general population and in type 2 diabetes (T2D) cohorts, of which a significant number of them, with general defects in binding and signaling, were associated with increased type 2 diabetes (T2D) risk (Bonnefond et al. Nature Genetics 2012). This study established a first link between defective melatonin signaling and increased risk for T2D. Given that GPCRs can engage multiple distinct signaling pathways, the objectives of this study were to assess the effects of the mutations on basal and melatonin-stimulated activation of Gαi1, Gαz, β-arrestin recruitment and ERK phosphorylation by use of bioluminescence resonance energy transfer-based assays, protein-complementation assays and alphascreen technology, respectively, in order to determine whether the resulting functional defects are associated with increased risk of T2D. The relative melatonin-induced responses were determined using the operational model and expressed as Δlog(τ/Ka), while the constitutive receptor activation was expressed as normalized difference between wild-type and each SNP. Following the characterization, the data were plotted on radial graphs in order to obtain for each mutant receptor a signaling signature. In addition, evolutionary trace (ET) analysis was undertaken in order to assign the MT2 mutants into clusters based on the phenotypical defects of the tested pathways as well as on the phylogenetic information for the sequence variations of functional site residues. The results indicate that 16 mutants display drastic overall signaling impairment, half of which are exclusively found in the T2D patients or individuals with impaired fasting glucose. Asymmetrical radial graphs indicate that certain MT2 mutants preferentially reduce G protein coupling over β-arrestin recruitment while other mutations have the opposite effect. Moreover, this biased profile is extended to the constitutive and agonist-mediated activities of MT2 mutants since they are not always similarly affected by SNPs. For instance, while no mutation induced a complete loss of constitutive activity, many lead to total loss of melatonin-induced response. Finally, the ET analysis showed that the clustering of signaling signatures is in agreement with the evolutionary analysis and with our observation that signaling impairment is correlated with T2D. The more a given mutation induces a strong signaling impairment, the higher the correlation with T2D and the more the non-mutated residue is conserved through evolution. Conversely, the less a mutation impairs signaling of MT2, the higher the correlation with non-T2D individuals and the less the non-mutated residue is conserved through evolution. Undergoing experiments will determine whether the functional defects identified have an impact on insulin signaling and the circadian clock of the pancreas. Together, our signaling signatures along with the ET analysis of MT2 mutations may help understanding the etiology of T2D and extend the concept of SNP-bias into potential therapeutic interventions that would eventually potentiate a defective response.


POSTERS

P57 Variants of the human V1B vasopressin receptor associated with affective disorders exhibit

modified pharmacological properties

Maxime J Manière, Véra Boulay, Csaba Tomboly1, Manon Kiry2, Xavier Leroy2, Philippe Courtet3,

Gilles Guillon and Maithé Corbani

Institut de Génomique Fonctionnelle, CNRS UMR5203-INSERM U1191, Université de Montpellier,

France 1Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary 2Actelion

Pharmaceutical Ltd, Dpt Drug Discovery, Allschwill, Switzerland. 3CHRU Montpellier, INSERM U1661,

Université de Montpellier, France

Stressful events activate the HPA axis. The release of vasopressin (AVP) and corticoliberin (CRH)

triggers the release of ACTH by the mean of V1B and CRF1 receptors and activate adrenal production

of catecholamines and corticosteroids. Genetic modifications of either element may produce severe

consequences. Three non-synonymous variants of the human V1B receptor gene were found in

patients with phobia, hyperactivity or suicidal behaviors. We explored the pharmacological

properties of these V1B variants (K65N, R364H and G191R), looking for a possible alteration of the

receptor function. In HEK293 cells expressing each variant, we evaluated pharmacological properties

such as ligands affinity, PLC coupling and calcium mobilization, but also association with beta

arrestin, MAP kinase activity and heterodimerization properties since we have previously shown that

the V1B and CRF1 can form heterodimers with synergistic properties.

Our results show that each variant was correctly expressed at the membrane and displayed a similar

low constitutive activity. Ligand affinities showed very few difference with similar binding for the

main natural agonists AVP and oxytocin. However, the capacity of variants K65N and R364H to

accumulate inositol phosphates and to activate MAP kinase under AVP activation was lower as

compared to wt, although Ki were not different. On the contrary, the G191R variant was

overstimulating for both PLC and MAP kinase pathways. The variants were not similar in beta-

arrestin interaction either, with K65N totally impaired and R364H very reduced for this function,

indicating that these receptors could be considered as natural “biased” receptors. Finally, we found

that K65N was also poorly heterodimerizing with CRF1 as measured by BRET50 and BRETmax

modulation. On the contrary, G191R could associate even better with CRF1 than the wt V1B and

produced a significantly higher synergistic effect.

Altogether, these data reveal that V1B receptor variants in phobic and suicidal patients display

modified signaling and dimerization properties. These mutations could participate to a predisposition

to depression and could constitute diagnosis genetic markers in risky patients.

M Manière’s Ph.D Scholarship was granted on a translational CHRU/UM1 program. Research was

funded by the ANR “VASCO”.


POSTERS

P58 A new chapter for Dopastatins – Using a membrane potential assay to evaluate

somatostatin and dopamine receptor signaling in neuroendocrine cell models

Thomas Günther, Michael Culler, Anika Mann, Elke Miess, Falko Nagel, Stefan Schulz

Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena,

Germany

Somatostatin and dopamine receptors (sst/DR) belong to the most important GPCRs insinuated in

neuroendocrine regulation. Their powerful anti-secretory actions are of great clinical value in the

treatment of neuroendocrine-related disorders. Stable somatostatin analogues (SSA) such as

octreotide are successfully used in the long-term treatment for somatostatin receptor 2 (sst2)-

positive neuroendocrine tumors. Bromocriptine and cabergoline are established Dopamine 2

receptor (D2R) agonists for the treatment of prolactinomas. However some tumors escape from a

successful targeted therapy with these drugs. While most pituitary adenomas express both sst2 and

D2R, there is growing evidence for gastro-entero-pancreatic neuroendocrine tumors also co-

expressing both receptors. The chimeric compound BIM23A760 binds with high affinity to both sst2

and D2R (sst2-D2R). The superiority of BIM23A760 versus the combination of octreotide and

cabergoline has already been proven in non-functioning pituitary tumors. Targeting two receptors

with one molecule could be useful to optimize targeted drug therapy to improve the response rate in

octreotide-resistant tumors. The major action of the potent antisecretory effects of SSA and D2R

agonists is driven by Gαi-mediated inhibition of adenylate cyclase. Other mechanisms involved are

the activation of potassium channels and inhibition of calcium channels that stabilize the membrane

potential making the cell more resistant to prosecretory stimuli. Here we present a functional

approach to evaluate models for functional neuroendocrine tumors. We established a fluorescence-

based membrane potential assay to measure ligand-induced activation of ssts and D2R in a

fluorometric imaging plate reader (FLIPR) format in our lab. Using a proprietary non-ion selective

oxonol-dye we evaluated two permanent cell lines for this technique. Among these is one

representative for a pituitary adenoma (AtT-20) and the other for a gastro-entero-pancreatic

metastatic site (BON-1). The ACTH-secreting adeno-pituitary cell line AtT-20 expresses two

somatostatin receptors namely sst2 and sst5. Both receptor subtypes activate endogenously

expressed G-protein inwardly rectifying channels (GIRK channels) via Gβγ-subunits. A cabergoline-

induced hyperpolarization can be detected after heterologous expression of D2R. Interestingly, the

presence of D2Rs in AtT-20 cells strongly facilitated GIRK channel activation elicited by the sst2-D2R

chimeric ligand BIM-23A760, suggesting a synergistic action of sst2 and D2Rs. The BON-1 cell line is

representative for a gastro-entero-pancreatic tumor derived from a metastatic site of the foregut

with pancreatic origin. This human cell line natively expresses sst2 and D2R. We detected unique

time-dependent signal profiles for BIM23120 (sst2-selective) and quinpirole (D2R agonist), which is

indicative for modulation of different effectory systems. Thus, a co-activation of both receptors with

BIM23A760 could facilitate antisecretory effects.


POSTERS

P59 Regulation of energy metabolism by Endospanin 1, a negative regulator of the leptin receptor. Arturo Roca-Rivada (1), Timothy Schwartz (1), Christophe Magnan (2), Raphaël Denis (2), Johanna Auriau (1), Stéphanie Migrenne-Li (2), Ralf Jockers (1), Julie Dam (1) (1) Institut Cochin, INSERM U1016, CNRS 8104, Université Paris Descartes, France (2) Univ Paris Diderot, Sorbonne Paris Cité, CNRS UMR 8251, France Introduction: Endospanin 1 is a transcript generated from the same locus than the leptin receptor (OBR) but does not share any sequence similarity with it. In a previous work, our group silenced endospanin 1 in mouse hypothalamic arcuate nucleus (ARC) showing an improved leptin sensitivity preventing diet-induced obesity (1,2). Finally, we demonstrated that endospanin 1 regulates a post-internalization step of the OB-R endocytic pathway (3). These results opened an important question about the specific role of endospanin 1 in the whole body since it is a highly conserved protein expressed in several tissues such as brain, liver or adipose tissue. To solve this question, we investigated the effects of endospanin 1 deletion in a general Knocked-out (KO) model on energy balance regulation and body weight dynamics. Results: Wild-type (WT) and KO mice showed a similar metabolic profile when they were fed with chow diet or high-fat diet. There were no significant changes between both groups in overall food intake, body weight and several metabolic parameters such as circulating leptin and insulin sensitivity. However, the KO mice showed systematically a specific feeding behavior between the 05.00-07.00am interval with a significant decrease in food intake and respiratory exchange ratio, and an increase in fatty acid oxidation; indicating the existence of a circadian rhythm dysregulation. When we restricted access to food for 48h, the KO mice were capable of maintaining significantly higher glucose levels than the WT mice, suggesting a better energy balance regulation in a caloric-restriction situation. In line with this, caloric content of feces was lower in KO mice suggesting an improved nutrient absorption in the KO animals. Conclusions: Additionally to the data we obtained in our previous studies, the results in Endospanin 1 KO mice suggest that Endospanin1 participates in different metabolic processes linked to circadian rhythm regulation and intestinal nutrient absorption, showing the complexity and importance of Endospanin 1 in energy regulation. 1- Couturier C et al. Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity. (2007). Proc Natl Acad Sci U S A; 104(49):19476-81. 2- Vauthier V et al. Endospanin 1 silencing in the hypothalamic arcuate nucleus contributes to sustained weight loss of high fat diet obese mice. Gene Ther. 2014 Jul;21(7):638-44 3- Séron K et al. Endospanins regulate a postinternalization step of the leptin receptor endocytic pathway. J Biol Chem. 2011 May 20;286(20):17968-81.


POSTERS

P60 Direct interaction and functional coupling between human 5-HT7 receptor and LINGO-1

Anthony Guillemain, Marion Bonel, Elodie Robin, Martine Decoville, Catherine Grillon and Séverine

Morisset-Lopez

Equipe microenvironnement cellulaire et cibles pharmacologiques, Centre de biophysique

moléculaire, CNRS UPR 4301, Orléans, France

LINGO-1 is a single transmembrane protein of 620 amino acids displaying a large extracellular region

consisting in 12 LRR motifs (leucine-rich repeat) and one Immunoglobulin domain. It is a negative

regulator in the nervous system mainly affecting axonal regeneration, neuron survival,

oligodendrocyte differentiation and myelination. Recently, we demonstrated that LINGO-1 can form

homodimers in living cells using a Bioluminescence Resonance Energy Transfer (BRET)-based assay.

We also showed that modulating the conformation of LINGO-1 dimers by using small chemical

compounds affects LINGO-1 downstream signalling. To date no natural ligand of LINGO-1 has been

discovered. In fact, several data suggest that LINGO-1 mediates its function through its interaction

with others receptors. Thus, the molecular mechanisms by which LINGO-1 mediates the inhibition of

the regeneration of neurites and the axonal outgrowth involve its physical interaction with the Nogo-

A receptor (NgR1) and the p75 neurotrophin receptor to form a ternary complex (LINGO-1/NgR/p75).

In addition, effects of LINGO-1 on neuron survival involve its interaction with EGF receptor.

In this study, we decided to find new protein partners of LINGO-1 by using a BRET-based assay as a

platform for screening. Among the tested receptors, the 5-HT7 receptor seemed particularly

interesting due to its strong interaction with LINGO-1 (low BRET50 value). The 5-HT7 receptor is

involved in various disorders (depression, Alzheimer’s disease, Schizophrenia, or pain…). It is

commonly coupled to Gs protein, resulting in 5-HT7 receptor-mediated augmentation of cAMP levels

and, consequently, in the activation of extracellular regulated kinase ½. Using co-

immunoprecipitation experiments, we confirm the specific interaction between the 5-HT7 receptor

and LINGO-1. Functionally, we found that overexpression of LINGO-1 increases the agonist-induced

cAMP accumulation and ERK phosphorylation in HEK-293 cells stably expressing 5-HT7 receptor. In

addition, we demonstrated that LINGO-1 increases the cell surface expression of 5-HT7 receptor,

suggesting that LINGO-1 is involved in the trafficking and/or biogenesis of this receptor. Taken

together, these results suggest that expression and signal transduction of 5HT7 receptor are

regulated by LINGO-1, which might play a role in 5-HT7-mediated signaling in the brain. Further

studies have to be performed in order to explore the functional relevance of this interaction both in

physiological and in pathophysiological conditions.


POSTERS

P61 Vasoactive peptide urotensin II as a new chemokine exhibits migration/adhesion

mesenchymal properties accompanying angiogenesis during glioma development

H. Castel1, P-O Guichet1, C. Lecointre1, V. LeJoncour1, J.E. Joubert1, N. Perzo1, L. Desrues1; R. Leduc2, L.

Prézeau3, A. laquerrière4, F. Morin1 and P. Gandolfo1

1, Inserm U982, DC2N, IRIB, Université de Rouen, 76821 Mont-Saint-Aignan. 2, Department of

Pharmacology, Institut of Pharmacology, Faculté de Médecine et des Sciences de la Santé, Université

de Sherbrooke, Sherbrooke, Québec, Canada, 3, IGF, Institut of Functional Genomic, CNRS UMR

5203, Inserm U661, University of Montpellier 1 and 2, Montpellier, France. 4, Service of

Anatomocytopathology, CHU of Rouen, ERI28 Inserm, IRIB, Rouen, France.

Glioblastoma multiform (GBM), the most aggressive form of gliomas, is particularly resistant to

therapy. We recently established chemoattractive effects of the vasoactive peptide urotensin II (UII)

via activation of its G protein-coupled receptor UT in recombinant cells.

Here we showed a high expression of UII/UT in human GBM, gliosarcoma and a number of carcinoma

compared to oligodendrogliomas or health brain tissue. In GBM, a strong staining in vascular and

peri-necrotic area and a systematic co-expression of UII/UT with SDF1α/CXCR4 were observed. In

GBM cell lyses analyzed via hybridization on protein chips, UII induced significant variation in the

expression of 58 proteins (among 247), and the global analysis (Ingenuity Pathway Analysis software)

predicted activation of the PI3K/AKT and NF-ĸB networks involved in invasiveness and inflammation.

In glioma and endothelial cells, gradient concentrations of UII induced chemoattracting migratory

effects and tube formation. In glioma cell lines, this gradient of UII mainly involved the

G13/Rho/ROCK pathway while partially requiring Gi/o/PI3K components. In contrast, we observed

that homogeneous concentrations of UII blocked cell motility and stimulated cell-matrix adhesions

through a UT/Gi/o signaling cascade, partially involving PI3K. UII also induced relocalization of UT

pre-coupled to Gαi in filipodia and initiated actin stress fibers and integrin-/vinculin-stained focal

points.

In vivo, intratumoral injection of UII accelerated GBM growth and necrosis, and stimulated

angiogenesis through metalloprotease activation. UT Antagonists/biased ligands inhibited tumor

growth, angiogenesis and prolonged mice survival. Thus, UII promotes the recruitment of angiogenic

cells, induces cell adhesions and stimulates angiogenesis, all involved in GBM growth.

Supported by Inserm, Haute-Normandie Region, the University of Rouen, Géfluc and the Perene

network. 1, Lecointre, C., Desrues, L., Joubert, J.E., , Perzo, N., Guichet, P.O., Le Joncour, V., Brulé, C., Chabbert, M., Leduc, R.,

Prézeau, L., Laquerrière, A., Proust, F., Gandoldo, P., Morin, F. and Castel, H. : Signaling switch of the urotensin II vasoactive

peptide GPCR : Prototypic chemotaxic mechanism in glioma. Oncogene, doi: 10.1038/onc.2014.433 (2015).

2, Brulé, C.*, Perzo, N.*, Joubert, J.E.*, Sainsily, X., Leduc, R.**, Castel, H.** and Prézeau, L.** : Biased signaling regulates

the pleiotropic effects of the urotensin II receptor to modulate its cellular behaviors. FASEB J. 28:5148-5162 (2014) *, co-

first authors; **, co-last authors


POSTERS

P62 Modulatory effects of the neurotoxic amyloid-beta peptide on membrane receptors

Erika Cecon, Ralf Jockers

Inserm U1016 - Institut Cochin, CNRS UMR 8104, University Paris Descartes, Sorbonne Paris Cité, Paris, France Alzheimer’s disease (AD) is the most common age-related dementia worldwide, affecting more than 35 million people. AD neurodegenerative processes are mainly linked to neurotoxic effects of the amyloid beta (Aβ) peptide, which forms aggregates known as senile plaques and corresponds to the main molecular hallmark observed in the brain of AD patients. Despite decades of research, the etiology of AD is still poorly defined, which limits the development of specific treatments. Accumulating evidences suggest that Aβ can bind to a variety of membrane receptors and thus interfere with cellular metabolism and function. We recently showed that Aβ impairs the proper signaling of melatonin MT1 and MT2 Gi protein-coupled receptors (Cecon et al., FASEB J 2015, 29:2566). Here we aimed to elucidate Aβ modulatory effects on the function of several membrane receptors, as well as to determine specific Aβ binding targets. In HEK293T and neuronal Neuroscreen-1 (NS-1) cell lines, overnight pre-incubation with Aβ (1 μM) led to impaired insulin signaling (10 nM, 5 min), detected at the level of ERK and AKT phosphorylation. Similarly, Aβ affected the intracellular signaling triggered by leptin (20 nM, 5 min) through activation of the OBRb receptor, as well as the signaling of the neurotropic factors NGF (10 ng/mL, 5 min) and BDNF (10 nM, 30 min) through TrkA and TrkB receptors, respectively. On the other hand, the signaling triggered by the addition of fetal bovine serum or by activation of EGF receptors was not affected by Aβ, suggesting its modulatory effect is receptor-specific. The previously shown Aβ-induced impairment of melatonin receptor signaling in transfected HEK293T cells was confirmed in NS-1 cells, expressing endogenous receptors. Interestingly, β-arrestin recruitment to MT1 and MT2 was also affected by Aβ. We show that these effects are likely due to a direct interaction of Aβ with melatonin receptors. A similar interaction was observed with the insulin receptor but not with the β2-adrenergic receptor, an observation that is in agreement with the absence of effect of Aβ on isoproterenol-triggered signaling. In summary, our data show that Aβ interferes with the signaling pathway of several relevant hormones and neurotrophic factors in a receptor-specific manner, displaying distinct binding affinities to its targets. These receptor-dependent modulatory effects might correspond to the first Aβ-induced molecular changes that lead to impaired brain energy metabolism, evolving to neurodegeneration. The detection and characterization of specific Aβ interaction partners is, therefore, highly relevant to the development of new and more efficient treatment of AD patients.


POSTERS

P63 Modulating glutamatergic synapse activity with nanobodies

Scholler Pauline (1,2), Nevoltris Damien (1,3), De Bundel Dimitri (1), Bossi Simon (4), Blanc Emilie (1), Rovira Xavier (1), D. El-Moustaine (1), Dupuis Elodie (2), Valjent Emmanuel (1), Daniel Hervé (4), McLean Heather (4), Baty Daniel (3), Pin Jean-Philippe (1), Rondard Philippe (1)

(1) Institut de Génomique Fonctionnelle, CNRS UMR5203, INSERM U1191, Université de Montpellier, Montpellier, France; (2) Cisbio Bioassays, Codolet, France; (3) CRCM, INSERM UMR1068, CNRS UMR7258, Marseille, France; (4) Laboratoire de Pharmacologie et Biochimie de la Synapse, CNRS UMR8619, Université Paris Sud-XI, Orsay, France Abstract body: The metabotropic glutamate receptors (mGluRs) are glutamate-activated G protein-coupled receptors widely expressed in the central nervous system. The eight mGluR subtypes modulate transmission at many synapses, and are interesting therapeutic targets for the treatment of many neurological and psychiatric diseases. These receptors are dimers with each subunit made of a large extracellular ligand-binding domain where glutamate binds, and a transmembrane domain that contains the binding site for synthetic allosteric modulators and responsible for G protein activation. Developing mGluR subtype-specific modulators is challenging since the glutamate binding pocket is conserved, and the allosteric modulators have the inconvenient to be hydrophobic. In this study, we took advantage of the original structure of the mGluRs to develop new ligands such as camelid single chain antibodies, or nanobodies. We describe the characterization of a high affinity nanobody that activ ates the receptor mGlu2 both in vitro and in vivo. We show that this nanobody is specific of the active conformation of mGlu2, and that it potentiates the activation of the receptor by an agonist. We also show that this nanobody binds in the dimeric interface of extracellular binding domain, compatible with the stabilization of the activate state of the receptor. Interestingly, in brain slices this nanobody can inhibit the pre-synaptic increase in calcium in mossy fiber terminals in the CA3 area, as expected for the activation of mGluR2. Since this area is involved in spatial memory, we further explored the effect of this nanobody in vivo, by injecting them alone, or in combination with DCG-IV (a group-II mGluR agonist), and found that the nanobody potentiated the effect of a low and inactive concentration of DCG-IV in a fear conditioning test. Altogether our results show that nanobodies could be used as new pharmacological agents to modulate the activity of mGluRs in vivo.


POSTERS

P64 Identification and characterization of a new phosphorylation site in GABAB receptor.

Gregory Stewart1, Lenea Nørskov-Lauritsen1, Rostislav Turecek2, Franck Vandermoere1, Martin Gassmann2, Bernhard Bettler2, Jean-Philippe Pin1, Julie Kniazeff1

1-Institut de Génomique Fonctionnelle, CNRSUMR5203, INSERM U1191, Université de Montpellier, Montpellier, France, 2-Department of Biomedicine, University of Basel, Basel, Switzerland. The GABAB receptor, a class C GPCR, is an obligatory heterodimer composed of GABAB1 and GABAB2 subunits. The first bears the agonist binding site while the latter is required for G protein activation. In the last years, this receptor was found to form higher ordered oligomers that modify the G protein-coupling properties. In an attempt to identify new phosphorylation sites of the GABAB receptor with a putative specificity for a given oligomeric state, we performed mass spectrometry analysis. We identified a novel threonine residue that is phosphorylated in GABAB2 C-terminus in all samples. Surprisingly, mutating this residue to Ala alters the G protein coupling efficacy without affecting the receptor expression or internalization. After raising a phospho-specific antibody, we showed that this new site is constitutively phosphorylated both in transfected HEK293 cells and in primary culture. Altogether, this suggests that this new phosphorylation site in the GABAB receptor is likely playing a role in controlling the G protein signaling in neurons. This represents that first report of a GABAB phosphorylation site with such a role.


POSTERS

P65 Searching for ligands targeting a GPCR homodimeric interface Slynko I (1), Banerjee P (2), Ruat M 2) and Rognan D(1) (1) Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS-Université de Strasbourg, F-67400 ILLKIRCH; (2) UMR9197 CNRS/Paris-Sud University, F-91198 Gif-Yvette, France Smoothened (SMO) is a G protein-coupled receptor involved in the signaling of the hedgehog (Hh) pathway. This receptor plays a key role during embryonic development and is up-regulated during the initiation and progression of various kinds of tumors. Altough many conventional SMO antagonists have already been described, even reached the market (vismodegib) and/or are under clinical evaluation for the treatment of diverse cancers (basal cell carcinoma, medulloblastoma, and cancers linked to Hh pathway overactivation), the efficacy of SMO antagonists is rapidly hampered by the occurrence of point mutations targeting the transmembrane binding site. Negative results have been already reported with some of the clinical candidates and two of them (saridegib and TAK-441) have been discontinued. The reasons of these failures include the presence of multiple SMO conformations in the tumors resistant to these inhibitors as well as residue mutations in the binding pocket located at the SMO trans-membrane domain. The key innovation in our project is the targeting of alternative binding sites at the surface of the SMO homodimer which have never been reported to be mutated in tumors. Starting from the X-ray structure of the SMO homodimer, we selected one potentially druggable cavity at the homodimer interface, for a structure-based virtual screening of 5 million commercially available compounds. 66 potential hits were selected from an in-house developed virtual screening flowchart combining cavity-based pharmacophore search and ligand docking. The 66 hits were purchased and evaluated at a unique concentration of 5 microM in two independent assays aimed at measuring the SMO-dependent Shh-induced activation of the Hh pathway: (1): differentiation of the mesenchymal cell line C3H10T1/2; (2) activation of a reporter gene (Gli-dependent luciferase activity) in Shh-light cells. 6 hits were confirmed in both assays to inhibit the activation of the Hh pathway by the Sonic Hedgehog ligand. Site-directed mutagenesis of the targeted interface and competition assays with conventional SMO antagonists ([3H]-MRT-72) are currently developed to ascertain the molecular mechanism of action of these novel non-conventional Hh pathway antagonists.


POSTERS

P66 New structural insights into apelin receptor activation.

Cynthia BISOO, Pierre COUVINEAU, Catherine LLORENS-CORTES, Xavier ITURRIOZ.

Center for Interdisciplinary Research in Biology (CIRB), INSERM U1050/CNRS UMR 7241 - Collège de

France, 75005, Paris, France.

Apelin, is the natural ligand of the orphan seven-transmembrane domain G protein-coupled receptor

APJ, now named the apelin receptor (ApelinR). Apelin is a neuro-vasoactive peptide that has the

ability to decrease vasopressin release in the blood circulation, inducing aqueous diuresis. In addition

apelin decreases arterial blood pressure, improves cardiac contractility and decreases cardiac

loading. Therefore, apelin and its receptor play a prominent role in the control of body fluid and

cardiovascular homeostasis. In order to better understand the mode of activation of the ApelinR, we

performed structure-function studies by site-directed mutagenesis of the human ApelinR. We focus

our interest in residues present in the transmembrane domain III, a domain that has been shown to

be critical for GPCRs activation. We substituted Asn 112 (Asn 3.35), Thr 121 (Thr 3.44) and the DRY motif

(Asp 3.49 and Arg 3.50) by a Ser, an Ala and AAY respectively. The mutation Ala 112 led to a

constitutively activated ApelinR (only for Gai coupling) with a bias towards Gai coupling. The

mutation AAY, as expected, led to an inactive ApelinR, unable to bind radiolabeled pE13F. The

mutant AAY which is not able to recruit ß-arrestin 2 upon apelin treatment; displays a higher basal ß-

arrestin 2 recruitment when compared to wild-type ApelinR. Interestingly, CHO cells stably

expressing AAY mutant displayed an higher basal activation of ERK1/2 when compared with cells

stablt expressing wild-type ApelinR. Finally, the Ala substitution of Thr 121 strongly increased ApelinR

expression suggesting a higher stability of the mutated ApelinR when compared to the wild-type

ApelinR. Altogether, this work brings new structural insights into the mode of apelin receptor

activation and shows that ApelinR shares similar activation mechanism than angiotensin receptor

type 1. These different mutants of ApelinR would represent interesting tools to further explore the

role of the apelin/ApelinR system in vivo in transgenic mice.


POSTERS

P67 Biased Signaling Favoring Gi over β-Arrestin Promoted by an Apelin Fragment Lacking the C-terminal Phenylalanine

Rodrigo Alvear-Perez‡§, Emilie Ceraudo‡§, Cécile Galanth‡§, Eric CarpentierII, Anne-Marie SchoneggeII, Xavier Iturrioz‡§, Michel BouvierII, and Catherine Llorens-Cortes‡§

‡Laboratory of Central Neuropeptides in the Regulation of Body Fluid Homeostasis and Cardiovascular Functions, INSERM U1050/CNRS, UMR 7241, Paris F-75005, France, the §Center for Interdisciplinary Research in Biology, Collège de France, Paris F-75005, France and the IIDepartment of Biochemistry, Institute for Research in Immunology and Cancer, and Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montréal, Québec H3T 1J4, Canada Apelin plays a critical role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or Gi-protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in inducing BP decrease may involve a Gi-independent but β-arrestin-dependent signaling pathway, inducing MAPK activation. For this purpose, we used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates Gi and promotes β-arrestin recruitment to the receptor, K16P had a much reduced ability to promote β-arrestin recruitment while maintaining its Gi activating property, revealing the biased agonist character of K16P. We further showed that both β-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity whereas the K16P-promoted ERK1/2 activity is entirely Gi-dependent. In addition, the PTX-resistant ERK1/2 phosphorylation evoked by K17F was completely blocked by the dominant negative mutant of β-arrestin 2, which lacks its capacity to bind ERK1/2. These findings clearly show that

apelin receptor stimulation by K17F activates a Gi-protein-independent but β-arrestin-dependent MAPK signaling pathway, and that the C-terminal Phe of K17F is required for this effect. We then investigated the ability of K17F and K16P to induce vasorelaxation of glomerular afferent arterioles (AA) preconstricted by Ang II in absence or in presence of Pertussis Toxin (PTX). Treatment of isolated AA with AngII significantly reduced arteriole diameter compared with values measured under baseline conditions. Addition of K17F to preconstricted AA by AngII increased the arteriole diameter, whereas K16P had no significant effect. These results indicated clearly that K17F induced a vasodilatation of AA previously preconstricted by AngII whereas K16P, a G protein-biased agonist displaying a strongly impaired β-arrestin signaling pathway, is ineffective. This suggests a direct link between the biased signaling and the differential vasodilatory response promoted by these apelin fragments. Moreover, the K17F induced-vasodilatory response of AA preconstricted by AngII was not modified in the presence of PTX treatment, suggesting that the activation by K17F of the β-arrestin-dependent ERK1/2 signaling is involved in the K17F-induced vasorelaxation of AA. This study will help to better understand the mechanisms of activation of the apelin receptor and suggests the activation of the β-arrestin-dependent MAPK signaling pathway when K17F decreases arterial BP. This constitutes a first step to further develop biased apelin receptor agonists activating only one signaling pathway to target one biological action.


POSTERS

P68 Targeted nanotherapy of endocrine tumors by magnetic hyperthermia.

Jeanjean Pauline 1, Clerc Pascal 1, El Hajj Diab Darine1, Carrey Julian2, Fourmy Daniel 1, Gigoux

Véronique 1.

1) EA4552 Université Toulouse III, CHU Rangueil, 1 avenue Jean Pouilhes, BP 84225, 31432

TOULOUSE Cedex 4 - [email protected] 2) Laboratoire de Physique et Chimie des Nano-

Objets, CNRS UMR5215-INSA, 135, av. de Rangueil, 31077 Toulouse

Endocrine tumors are usually diagnosed through the use of an imaging technique using a

radiolabeled peptide (somatostatin or Osteoscan) whose receptor is present in 80% of tumors. The

over-expression of the CCK2R which belongs to the seven transmembrane domains receptors family,

also named G protein-coupled receptor (GPCR), has also been demonstrated in human endocrine

tumors. In addition, our team has recently shown that the CCK2R internalizes massively and is

directed with its ligand to lysosomes.

We hypothesize that the overexpression of a GPCR in endocrine tumors compared to normal tissues

and their ability to internalize massively may be used advantageously to develop new diagnostic and

therapeutic approach. The strategy consists in the development of a nano-platform composed by

magnetic nanoparticles vectorized with a GPCR agonist. We have chosen as the first model for the

study: the gastrin ligand of the CCK2R. The first objectives were to optimize the accumulation of

nanoparticles in tumor cells and to eradicate tumor cells by magnetic hyperthermia. This step is

crucial to validate the concept of targeted therapeutic hyperthermia, before resolving the in vivo

studies troubles such as the nanoparticles stealth and the vascular barrier crossing... The second

objective was to study the molecular mechanisms of tumoral cell death induced by magnetic

hyperthermia.

After optimization of the nanoparticles vectorization, the gastrin-vectorized nanoparticles internalize

massively and specifically in endocrine tumor line INR1G9 (glucagon -producing islet cells) stably

expressing the CCK2R; then they accumulate in lysosomes by a mechanism similar to the free ligand.

We showed that the targeting of tumor cells expressing the CCK2R using gastrin-vectorized magnetic

nanoparticles is effective, specific and allows their massive accumulation in lysosomes. We then

established the conditions for the magnetic field to eradicate tumor cells. The application of a

magnetic field of 275 kHz and 40 mT for 2 hours induced reactive oxygene species production,

lysosome membrane permeability with the leakage of lysosomal enzymes in the cytoplasmic

compartment, followed by tumoral cell death. Moreover, we showed that ROS production and

lysosome membrane permeability are detected only 30 minutes after magnetic field application,

demonstrating that they occur at an early stage in the cascade of events leading eventually to cell

death. We are currently seeking to clarify the mechanisms involved in cell death induced by magnetic

field.

Our strategy should be a new diagnostic and therapeutic approach of tumors by using GPCR targeting

and magnetic nanoparticles as contrast agent for imaging and therapeutic agent for magnetic

hyperthermia. The therapeutic possibilities of this project will be to further combine

chemotherapeutic and hyperthermic treatments to induce massive death of endocrine tumor cells.



POSTERS

P69 Fluorescent Turn-On Ligands for Background-Free GPCR Imaging

Iuliia A. Karpenko (1), Mayeul Collot (2), Yves Mély (2), Marcel Hibert (1), Andrey S. Klymchenko (2), Dominique Bonnet (1)

(1) Laboratoire d’Innovation Therapeutique, UMR7200 CNRS/Universite de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France (2) Laboratoire de Biophotonique et Pharmacologie UMR 7213 UdS CNRS Faculté de Pharmacie, Université de Strasbourg 74 route du Rhin, 67401 Illkirch Cedex, France.* Fluorescent turn-on systems are probably the most powerful tools in biological sensing, enabling direct quantification of an analyte without removal of unreacted probe and providing the possibility of in-vivo monitoring of molecular interactions. [1] Especially, turn-on probes will be of high interest to study G protein-coupled receptors (GPCRs), the largest and most diverse group of membrane receptors in eukaryotes, at the surface of living cells. To date, fluorescence anisotropy and Förster Resonance Energy Transfer (FRET) are two well-established fluorescence techniques for GPCR studies without removal of the non-interacted ligand (homogeneous conditions). However, the first method suffers from low sensitivity, while the second requires fluorescent labeling of both ligand and receptor. Therefore, efforts have been made to achieve a turn-on response based exclusively on fluorescent labeling of the ligand. This approach, which is still largely underexplored, involves environment-sensitive dyes [2] that are not fluorescent in aqueous medium but turn on their fluorescence in the hydrophobic environment (so-called fluorogenic dyes). Few GPCR ligands bearing fluorogenic dyes have been reported to date. [3] They suffer from limited efficacy due to non-specific interactions or from the requirement to perform studies on isolated membranes and not living cells. Moreover, they were limited to blue dyes, while red dyes are advantageous for cellular studies due to lower sample photo-damage, light scattering and auto-fluorescence. In the present work, we report the first ligands that turn on their fluorescence in the red [4] and far-red [5] spectral region after binding to the target GPCR in situ on living cells. The first fluorogenic Squaraine dimers with polarity-sensitive folding as bright far-red probes for background-free oxytocin GPCR imaging will be also described. [6] [1] Nadler, A.; Schultz, P. Angew. Chem. Int. Ed. 2013, 52, 2408–2410. [2] G. S. Loving, M. Sainlos, B. Imperiali, Trends Biotechnol. 2010, 28, 73–83. [3] R. Sridharan, J. Zuber, S. M. Connelly, E. Mathew, M. E. Dumont, Biochim. Biophys. Acta 2014, 1838 (1), 15–33. [4] I. A. Karpenko, R. Kreder, C. Valencia, P. Villa, C. Mendre, B. Mouillac, Y. Mély, M. Hibert, D. Bonnet and A. S. Klymchenko Chembiochem, 2014, 15, 359–63. [5] I. A. Karpenko, A. S. Klymchenko, S. Gioria, R. Kreder, I. Shulov, P. Villa, Y. Mély, M. Hibert, D. Bonnet. Chem. Commun., 2015, 51, 2960-2963. [6] I. A. Karpenko, M. Collot, L. Richert, C. Valencia, P. Villa, Y. Mély, M. Hibert, D. Bonnet and A. S. Klymchenko J. Am. Chem. Soc., 2015, 137, 405-412.


POSTERS

P70 The orphan GPR50 regulates TGFß receptor-dependent tumor development in two tumor models

Anne-Sophie Journé 1,2,3, Stefanie Wojciech 1,2,3, Raise Ahmad 1,2,3, Zakia Belaid-Choucair 3,4, Philippe Delagrange 5, Olivier Hermine 3,4, Céline Prunier 6 , Julie Dam 1,2,3, and Ralf Jockers1,2,3*

1 Inserm, U1016, Institut Cochin, Paris, 75014, France 2 CNRS UMR 8104, Paris, France, 75014, France 3 Univ. Paris Descartes, Paris, 75014, France 4 Hôpital Necker, CNRS UMR 8147 Paris, 75015, France 5 Institut de Recherches SERVIER, Suresnes, 92150, France 6 INSERM U893, Centre de Recherche Saint-Antoine, Paris, 75012, France We previously showed that the orphan GPR50 receptor can have ligand-independent properties by forming molecular complexes with other receptors with well-known function. In the complex with the melatonin MT1 receptor, GPR50 inhibits MT1 functions (Levoye et al. Embo J, 2006). More recently, we showed that GPR50 renders the transforming growth factor-ß (TGFß) receptor I (TßRI) constitutively active in a stable molecular complex with TßRI. TGFβ signaling is known to have anti-proliferative effects, thus playing an important role in counteracting cancer development and progression. Here, we evaluated the in vivo consequences of GPR50 on TGFß signaling in two tumor models, a MDA-MB-231 breast cancer xenograft model and the MMTV-Neu model forming spontaneous mammary tumors. We established GPR50-overexpressing TGFβ-responsive MDA-MB-231 breast cancer cells and studied the effect of GPR50 on cell proliferation. These cells were also injected into nude mice in xenograft experiments. Our results indicate a TGFβ-like inhibitory effect of GPR50 on cellular proliferation and tumor development in these models. We then generated a murin model with a targeted deletion of GPR50 in the MMTV-Neu genetical background, which develops spontaneous tumors in the mammary gland epithelium. The absence of GPR50 led to a poor survival prognosis in this model, involving an earlier onset of primary tumors and accelerated tumor growth. Our results describe a previously unappreciated spontaneous mode of TßRI activation and identify GPR50 as a TßRI co-receptor with major impact on TßRI-dependent cancer development.


POSTERS

P71 Sensitive detection of GPCR-mediated Erk1/2 phosphorylations in various cellular models with the HTRF® Advanced Phospho-ERK1/2 (Thr202/Tyr204) kit

Elodie Dupuis1, Angelique Vrastor1, Fabrice Maurin1, Julie Pannequin2, Thomas Roux1 and Eric Trinquet1

1Cisbio Bioassays, Codolet, France 2Institut de Génomique Fonctionnelle, Montpellier, France G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors and represent a high percentage of investigational drug targets because of their involvement in many diseases. GPCRs have two principal signal transduction pathways: the cyclic AMP (cAMP) and the phosphatidylinositol (IP). As a world leader of advanced homogeneous assays (HTRF®) and reagents for GPCR studies, Cisbio Bioassays offers a comprehensive and efficient line of HTRF® assays to study the GPCR activation pathways. This include cAMP and IP-One HTRF® platforms to measure second messenger accumulations as upstream readouts of reference in biochemical, cell-based assays. As several receptors are not optimally coupled through the cAMP or IP pathways, and other effectors are involved in the GPCR signaling pathways, Cisbio Bioassays has developed a new highly sensitive kit to detect modulations of ERK1/2 phosphorylations, illustrating phosphorylated ERK1/2 proteins as downstream readouts of GPCR stimulations. From now on Cisbio Bioassays offers the Advanced Phospho-ERK1/2 (Thr202/Tyr204) kit (ref catalogue 64AERPEH) as an excellent and versatile tool for studying the downstream effect of pharmacological compounds on GPCRs. This new powerful product is the linkage in the HTRF® GPCR product portfolio between the well-known upstream readouts (cAMP, IP) and the protein phosphorylation investigations as downstream readouts. This allows the use of a single sample-saving technology to characterize pharmacological compounds on several readouts. In this poster we demonstrate that Advanced Phospho-ERK1/2 (Thr202/Tyr204) kit enables measurement of ERK1/2 protein phosphorylations in several cellular contexts of varying complexity (from stable cell lines to tumor patient-derived cells) when mediated by overexpressed or endogenous GPCRs. Finally, we remind users of experimental optimizations required to perform ERK1/2 phosphorylation experiments with the Advanced Phospho-ERK1/2 (Thr202/Tyr204) kit for GPCR investigations.


POSTERS

P72 Quantifying Signaling Bias: A Simple Approach to Quantify Functional Selectivity and Agonist Bias

Lakshmi Anantharaman and Neil Charter

DiscoveRx Corporation, Fremont, CA 94538-3142 USA Presented by Andrew Green Interest in GPCR ligand bias has increased in recent years due to evidence that positive and negative aspects of drug activity can be driven by differential pathway signaling. As a result it is possible to develop drugs with reduced side effects that enhance positive effects through favouring one pathway over another. The availability of assays that can measure signaling events such as calcium mobilization, cAMP modulation, arrestin recruitment and receptor internalization provides to ability to characterize compound action in multiple pathways. Here we provide examples of ligand bias and how quantifi cation of receptor potency and effi cacy in different pathways can be used to generate a ligand bias index.


POSTERS

P73 A GIRK-based FLIPR assay for measuring activation of Gi-coupled GPCRs – a versatile technique used for characterization of multi-opioid receptor ligands

Thomas Günther, Pooja Dasgupta, Anika Mann, Elke Miess, Falko Nagel, Stefan Schulz

Jena University Hospital (Germany) Opioids are still the mainstay of modern pain treatment. Most of the clinically established substances primarily exert their effects via the µ-opioid receptor (MOR). However, many side effects such as tolerance, obstipation and respiratory depression limit their therapeutic use. The efficacy of MOR agonists in the treatment of chronic pain is unsatisfactory. In general analgesic effects can be mediated by all four members of the opioid receptor family. The nociception receptor (NOP) is the latest member of the opioid receptor family. There is a rapidly growing interest for the development of novel NOP and combined MOR/NOP agonists. The aim of this development is novel therapeutic agents with improved analgesic characteristics and less classical MOR-mediated side effects. The prototype of this potential class of new analgesics is cebranopadol. Recently, nalfurafine, a potent kappa-opioid receptor (KOR) agonist was granted by japanesse authorities for the treatment of uremic pruritus. Even though KOR agonists are known to mediate dysphoria and hallucinations this has not been reported for nalfurafine. Taking this development into account we present an easy-to-use probe to measure ligand-induced

receptor activation. This assay can be performed in a fluorometric imaging plate reader (FLIPR)

format for extended compound libraries. This technique is based on changes in membrane potential

that are mediated by coupling of all opioid receptors to G-protein inwardly-rectifying potassium

channels (GIRK). Using an easy-to-transfect permanent cell line with a native source of GIRK1/GIRK2

channels, we established an easy-to-perform protocol to evaluate ligand-induced changes in

membrane potential. GIRK1/GIRK2 channels are the most widely expressed GIRK subtypes of the

central nervous system and represent a more naturalistic effector system than artificially transfected

Gqi5 for calcium flux measurements. However, at present there are only few cellular assays that

detect receptor activation of novel compounds without disrupting cells to obtain the second

messenger content. This protocol is a genuine extension for measuring G-protein activation. We first

validated this assay using the standard full agonists DAMGO (MOR), DPDPE (DOR), U-50,488 (KOR)

and Nociceptin (NOP). These pertussis-toxin and tertiapin-q sensitive changes in fluorescence signal

were completely blocked with naloxone for MOR and DOR, partially for KOR and remained

unaffected for NOP. J-113397, a selective NOP-antagonist, abrogated the nociceptin-mediated

change in fluorescence signal. We further extended this protocol for the characterization of

antagonists with various application modes to determine competitive antagonists. This probe is a

valuable tool to characterize novel opioid-ligands on all four opioid receptors and can be scaled up to

even screen large libraries of potential drugs.


POSTERS

P74 Identification of “single domain” antibodies targeting β-arrestin-1 and β-arrestin-2 using phage display approach combined with HTRF® technology

Dubois Fanny (1), Douayry Najim (2), Dupuis Elodie (2), Roux Thomas (2), Chames Patrick (3), Baty Daniel (3), Trinquet Eric (2), Pin Jean-Philippe (1), Rondard Philippe(1)

(1). Institute of Functional Genomics (IGF), CNRS-UMR 5203-INSERM U1191, Montpellier University, Montpellier, France (2). Cisbio Bioassays, Codolet, France (3). Cancer Research Center of Marseille (CRCM), CNRS UMR7258- INSERM UMR1068, Aix-Marseille University, Marseille, France Arrestin family, especially the β-arrestin-1 and -2, has emerged as a key player in G protein-coupled receptors (GPCRs) signalling pathway1, and has been implicated in GPCR desensibilization and recycling. However, tools to study arrestin proteins and to decipher their cellular functions are quite limited. Overgrowing studies focusing2 on these proteins require new technologies, but also development of drugs and monoclonal antibodies targeting specifically the two isoforms of β-arrestins. Thus, in this study, we report the generation of camelid “single domain” antibodies, also called nanobodies, targeting specifically each of the isoforms of β-arrestin. Basically, recombinant GST- β-arrestin-1 and -2 proteins have been produced in E.coli and then injected in llama prior to immunization process. From the bacteria phage library, nanobodies have been selected and screened for each targeted-protein (β-arrestin-1 or -2) using a combination of phage display3 and HTRF®4 approaches. Specific nanobodies have been selected and isolated for each of the isoforms injected. Further characterization processes are forthcoming to isolate high affinity nanobodies that will discriminate β-arrestin-1 versus β-arrestin-2. The most specific nanobodies will be used as tools for two applications: (1) monitor the trafficking of β-arrestins during receptor signalling; (2) block the recruitment of arrestins to the GPCRs.

1. Shenoy SK, et al. Trends Pharmacol Sci. 2011; 32(9):521-33 2. Reiter E, et al. Annu Rev Pharmacol Toxicol. 2012;52:179-97 3. McCafferty J, et al. Nature. 1990;348(6301):552-4 4. Degorce F, et al. Curr Chem Genomics. 2009;3:22-32


NOTES


NOTES


NOTES


EXPOSANTS

AstraZeneca

[email protected]

Berthold

[email protected]

BMG Labtech

[email protected]

Cisbio

[email protected]

DiscoveRx Corporation

[email protected]

Labogene

[email protected]

Mettler Tolédo

TECAN

[email protected]









LOCAL ORGANIZING COMMITTEE

Catherine Mollereau-Manauté

Véronique Gigoux

Lionel Moulédous

Jean-Michel Sénard

Laurence Salomé

Alain Milon

Monique Falières

Daniel Fourmy

Céline Galès

Jean-Marie Zajac


LIST OF PARTICIPANTS

AHMAD Raise, Institut Cochin Paris (France) [email protected] P38

ALVEAR-PEREZ Rodrigo, Collège de France Paris (France) [email protected] P67

AMAR Muriel, CEA Saclay (France) [email protected]

AUDABRAM Cédric, DiscoveRx Corporation (France) [email protected]

AYOUB Mohammed Akli, INRA Tours (France) [email protected] P02

BANÈRES Jean-Louis, IBMM Montpellier (France) [email protected] L05

BECKER Jerome, INRA Tours (France) [email protected]

BHATT Harsheshkumar, IGF Montpellier (France) [email protected]

BONEL Marion, CBM Orléans (France) [email protected]

BONNET Dominique, LIT Université de Strasbourg (France) [email protected] P69

BOSCH Felix, Esteve foundation Barcelona (Spain) [email protected]

BOTTA Joaquin, University of East Anglia Norwich (UK) [email protected] P01

BOULO Thomas, INRA Tours (France) [email protected]

BRAY Lauriane, IPBS Toulouse (France) [email protected]

BRELOT Anne, Institut Pasteur Paris (France) [email protected] P54

BRUNEAU Gilles, INRA Tours (France) [email protected]

CASTEL Hélène, DC2N Rouen (France) [email protected] P61

CECON Erika, Institut Cochin Paris (France) [email protected] P62

CHABBERT Marie, Université d'Angers (France) [email protected] P10

CLAEYSEN Sylvie, IGF Montpellier (France) [email protected] P32

CLERC Pascal, I2MC Toulouse (France) [email protected]

COLY Pierre-Michaël, Université de Rouen (France) [email protected] P46

CORBANI Maithé, IGF Montpellier (France) [email protected] P57

COUVINEAU Pierre, Collège de France Paris (France) [email protected] P66

CREPIEUX Pascale, INRA Tours (France) [email protected]

CUKIER Cyprian, IPBS Toulouse (France) [email protected]




















DA SILVA Mélanie, IGF Montpellier (France) [email protected] P50

DAM Julie, Institut Cochin Paris (France) [email protected]

DAMIAN Marjorie, IBMM Montpellier (France) [email protected]

D'ANGELO Jean-Marc, Hamamatsu Photonics Massy (France) [email protected] P09 P11

DE VRIES Luc, Institut de Recherches Pierre Fabre Castres

(France)

[email protected]

DEMANGE Pascal, IPBS Toulouse (France) [email protected]

DEMENE Hélène, CBS Montpellier (France) [email protected] P51

DENOYELLE Séverine, IBMM Montpellier (France) [email protected] P17

DERAREDJ Wissem, CBM Orléans (France) [email protected] P29

DESOLE Claudia, Université Paris Descartes (France) [email protected] P22

DETERRE Philippe, UPMC Paris (France) [email protected] P14

DIAZ Constantino, Evotec Toulouse (France) [email protected]

DOLY Stéphane, Institut Cochin Paris (France) [email protected] P36

DONCESCU Nathalie, IPBS Toulouse (France) [email protected]

DRIEU LA ROCHELLE Armand, ESBS Université de Strasbourg

(France)

armand.drieu-la-

[email protected]

P33

DROCTOVÉ Laura, CEA Saclay (France) [email protected]

DUBOIS Fanny, IGF Montpellier (France) [email protected] P74

DUMAS Fabrice, IPBS Toulouse (France) [email protected]

DUPUIS Elodie, CISBIO BIOASSAYS Codolet (France) [email protected] P71

DURROUX Thierry, IGF Montpellier (France) [email protected]

ENSLEN Hérvé, Institut Cochin Paris (France) [email protected]

FAGNI Laurent, IGF Montpellier (France) [email protected]

FALIERES Monique, GENOTOUL [email protected]

FEHRENTZ Jean-Alain, IBMM Montpellier (France) [email protected]

FERRE Guillaume, IPBS Toulouse (France) [email protected] P06

FlOQUET Nicolas, IBMM Montpellier (France) [email protected] P21

FOULD Benjamin, Institut de Recherces SERVIER (France) [email protected]














FOURMY Daniel, Université de Toulouse (France) [email protected]

FROSSARD Nelly,LIT Université de Strasbourg (France) [email protected] P44

FUMAGALLI Amos, IGF Montpellier (France) [email protected]

GAIBELET Gérald, IGF Montpellier (France) [email protected] P25

GALANDRIN Ségolène, I2MC Toulouse (France) [email protected] P16

GALES Céline, I2MC Toulouse (France) [email protected]

GALZI Jean-Luc, ESBS Université de Strasbourg (France) [email protected]

GANDIA-SANCHEZ Jorge, INRA Tours (France) [email protected] P15

GAUTIER Frédérique, TECAN (France) Frederique.Gautier-

[email protected]

GBAHOU Florence, Institut Cochin Paris (France) [email protected]

GERBIER Romain, Institut Cochin Paris (France) [email protected] P49

GERVAIS Virginie, IPBS Toulouse (France) [email protected]

GIGOUX Véronique, Université de Toulouse (France) [email protected]

GILLES Nicolas, CEA Saclay (France) [email protected]

GIRAUD Pierre, Metler Tolédo

GOMES Sylvia, I2MC Toulouse (France) [email protected]

GOUDET Cyril, IGF Montpellier (France) [email protected] P42

GOULDING Joelle, University of Nottingham (UK) [email protected] P52

GREEN Andrew, DiscoveRx Corporation P72

GUERY Mélodie, IBMM Montpellier (France) [email protected]

GUILLEMAIN Anthony, CBMM Orléans (France) [email protected] P60

GUILLON Gilles, IGF Montpellier (France) [email protected]

GÜNTHER Thomas, University of Jena (Germany) [email protected] P58

HAJASOVA Zuzana, Université Paris Descartes (France) [email protected] P27

HEGRON Alan, Institut Cochin Paris (France) [email protected] P55

HENRI Pauline, IBMM Montpellier (France) [email protected]

HEUNINCK Joyce, IGF Montpellier (France) [email protected]

HIBERT Marcel, LIT Université de Strasbourg (France) [email protected]














IGONET Sébastien, CALIXAR Lyon (France) [email protected] P04

ISMAIL Sadek, Université de Toulouse (France) [email protected]

ITURRIOZ Xavier, Collège de France Paris (France) [email protected]

JACOBSON Kenneth A., NIH Bethesda (USA) [email protected] L08

JEANJEAN Pauline, I2MC Toulouse (France) [email protected] P68

JOBIN Marie-Lise, INRA Bordeaux (France) [email protected] P19

JOCKERS Ralf, Institut Cochin Paris (France) [email protected] L16

JOULIA Emeline, IPBS Toulouse (France) [email protected]

JOURNE Anne Sophie, Institut Cochin Paris (France) [email protected] P70

JUGNARAIN Vinesh, SYNTHELIS Grenoble (France) [email protected] P41

KARAMITRI Angeliki, Institut Cochin Paris (France) [email protected] P56

KESSLER Pascal, CEA Saclay (France) [email protected]

KLIEWER Andrea, University of Jena (Germany) [email protected] P05

KNIAZEFF Julie, IGF Montpellier (France) [email protected] P64

LACHAIZE Véronique, I2MC ITAV Toulouse (France) [email protected]

LAFFARGUE Muriel, I2MC Toulouse (France) [email protected]

LAGANE Bernard, Institut Pasteur Paris (France) [email protected] L10

LAHUNA Olivier, Institut Cochin Paris (France) [email protected] P30

LAMOTTE Florence, ASTRAZENECA (France) [email protected]

LANDOMIEL Flavie, INRA Tours (France) [email protected] P35

LANGONNE Nathalie, INRA Tours (France) [email protected] P31

LAUDETTE Marion, I2MC Toulouse (France) [email protected]

LE COZ Glenn Marie, ESBS Université de Strasbourg (France) [email protected] P24

LE MERRER Julie, INRA Tours (France) [email protected]

LEBON Guillaume, IGF Montpellier (France) [email protected]

LECAT Sandra, ESBS Université de Strasbourg (France) [email protected] P18

LEPRINCE Jérome, Université de Rouen (France) [email protected]

LIU Jianfeng, Huazhong University (China) [email protected] P08

LOLIGNIER Stéphane, Neuro-Dol Université d'Auvergne [email protected]





















(France)

LUKA Marine, Institut Cochin Paris (France) [email protected] P48

MAITY Amit, IGF Montpellier (France) [email protected]

MANN Anika, University of Jena (Germany) [email protected] P28

MARIE jacky, IBMM Montpellier (France) [email protected]

MARIN Philippe, IGF Montpellier (France) [email protected]

MARQUES Thomas, Berthold (France) [email protected]

MARTIN Frédéric, ASTRAZENECA (France)

MARULLO Stefano, Institut Cochin Paris (France) [email protected]

MARY Sophie, IBMM Montpellier (France) [email protected]

MASRI Bernard, I2MC Toulouse (France) [email protected]

McCORT Isabelle, Université Paris Descartes (France) [email protected]

MENDRE Christiane, IGF Montpellier (France) [email protected]

MESSAL Nassima, CRI Université Paris Diderot (France) [email protected] P12

MIALET-PEREZ Jeanne, I2MC Toulouse (France) [email protected]

MIESS Elke, University of Jena (Germany) [email protected] P07

MILON Alain, IPBS Toulouse (France) [email protected]

MOLLEREAU-MANAUTE Catherine, IBPS Toulouse (France) [email protected]

MOMBOISSE Fanny, Institut Pasteur Paris (France) [email protected]

MOREAU Christophe, IBS Grenoble (France) [email protected]

MORENO Estefania, University of Barcelona (Spain) [email protected] P01

MORISSET-LOPEZ Séverine, CBM Orléans (France) severine.morisset-lopez@cnrs-

orleans.fr

MOUILLAC Bernard, IGF Montpellier (France) [email protected]

MOULEDOUS Lionel, IPBS Toulouse (France) [email protected]

MOURIER Gilles, CEA Saclay (France) [email protected]

MULLER Isabelle, IPBS Toulouse (France) [email protected]

MÜLLER Timo, Helmholtz Zentrum and Universität München

(Germany)

[email protected] L13





















MUNDELL Stuart, University of Bristol (United Kingdom) [email protected] L07

MURAT Samy, IGF Montpellier (France) [email protected] P34

MUSNIER Astrid, INRA Tours (France) [email protected]

NASRALLAH Chady, IGF Montpellier (France) [email protected] P47

NDIAYE-LOBRY Delphine, Institut Cochin Paris (France) [email protected]

ODAERT Benoît, CBMN Bordeaux (France) [email protected]

ONFROY Lauriane, I2MC Toulouse (France) [email protected] P20

ORCEL Hélène, IGF Montpellier (France) [email protected]

PAJOT-AUGY Edith, INRA Jouy-en-Josas (France) [email protected]

PALEA Stefano, Toulouse (France) [email protected]

PARADIS Justine, Institut Cochin Paris (France) [email protected] P26

PAUL Erik, BMG LABTECH Champigny sur Marne (France) [email protected]

PECQUERY Anne, DiscoveRx Corporation

PEDELACQ Jean-Denis, IPBS Toulouse (France) [email protected]

PELLISSIER Lucie, INRA Tours (France) [email protected] P03

PERROY Julie, IGF Montpellier ( France) [email protected] L06

PETAJA-REPO Ulla, University of Oulu (Finland) [email protected] L01

PIN Jean-Philippe, IGF Montpellier (France) [email protected]

POIROT Mathias, I2MC Toulouse (France) [email protected] P39

PONS Véronique, I2MC Toulouse (France) [email protected]

POUPON Anne, INRA Tours (France) [email protected]

PREZEAU Laurent, IGF Montpellier (France) [email protected] P45

QUOYER Julie, Institut Cochin Paris (France) [email protected]

REAT Valérie, IPBS Toulouse (France) [email protected]

REITER Eric, INRA Tours (France) [email protected]

RENAULT Marie, IPBS Toulouse (France) [email protected]

REUBI Jean-Claude, University of Berne (Switzerland) [email protected] L11

RICCETTI Laura, INRA Tours (France) [email protected] P40

RIGAULT Delphine, Université Paris Descartes (France) [email protected]




















ROBIN Philippe, CEA Saclay (France) [email protected]

ROCA-RIVADA Arturo, Institut Cochin Paris (France) [email protected] P59

RODARIE David TECAN (France) [email protected]

RODRIGUEZ Mar, University of Barcelona (Spain) [email protected] P23

ROGNAN Didier, LIT Université de Strasbourg (France) [email protected]

RONDARD Philippe, IGF Montpellier (France) [email protected] P63

ROTTIER Karine, IGF Montpellier (France) [email protected] P43

ROUX Thomas, CISBIO BIOASSAYS Codolet (France) [email protected]

SALOME Laurence, IPBS Toulouse (France) [email protected]

SANZ Guenhael, INRA Jouy en Josas (France) [email protected] L12

SATCHELL Rupert, Sygnaturediscovery Nottingham (UK) a.bortolan-

[email protected]

SAUREL Olivier, IPBS Toulouse (France) [email protected]

SBAI Oualid, CAMINNOV Alès (France) [email protected]

SCHULZ Stefan, University of Jena (Germany) [email protected] P73

SENARD Jean-Michel, I2MC Toulouse (France) [email protected]

SERVENT Denis, CEA Saclay (France) [email protected]

SERVENT Florence, CISBIO BIOASSAYS (France) [email protected]

SIE Pierre/LHERMUSIER Thibault, CHU Toulouse (France) [email protected] L09

SIEGAL Gregg, ZoBio Leidein (The Netherlands) [email protected] L14

SIMON Violaine, BFA Université Paris Diderot (France) [email protected] P53

SIMONIN Frédéric, ESBS Illkirch (France) [email protected] L15

SLYNKO Inna, LIT Université de Strasbourg (France) [email protected] P65

SOULE Julien, IGF Montpellier (France) [email protected]

STANDFUSS Jörg, Paul Scherrer Institute (Switzerland) [email protected] L04

TALMONT Franck, IPBS Toulouse (France) [email protected] P13

TOBIN Andrew, University of Leicester (United Kingdom) [email protected] L02

TRANCHANT Thibaud, ESBS Université de Strasbourg [email protected]

TREFIER Aurélie, INRA Tours (France) [email protected] P37






















TRIFILIEFF Pierre, INRA Bordeaux (France) [email protected]

VALET Philippe, I2MC Toulouse (France) [email protected]

VANDERMOERE Franck, IGF Montpellier (France) [email protected] L03

VINCENS Monique, Institut Cochin Paris (France) [email protected]

VIVAUDOU Michel, IBS Grenoble (France) [email protected]

WELLENSIEK Jan, LaboGene [email protected]

ZAJAC Jean-Marie, IPBS Toulouse (France) [email protected]

ZHOU Zhicheng, Institut Pasteur Paris (France) [email protected]






Documents

Livret complet.pdf