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Meeting Highlights
10.1517/14728222.11.9.1251 © 2007 Informa UK Ltd ISSN 1472-8222 1251
1. General overview
2. Allosteric regulation of
G-protein-coupled receptors
3. Post-G-protein-coupled
receptor components
4. G-protein-coupled receptors
in drug development
5. Expert opinion and conclusions
British Pharmacology Society 2nd Focused Meeting on Cell Signalling: GPCRs have sunny days in springtime Leicester15 – 17 April, 2007, Leicester, UK
Paul A Insel UCSD, Departments of Pharmacology & Medicine, La Jolla, CA 92093-0636, USA
The British Pharmacology Society recently held its 2 nd Focused meeting on Cell Signalling in April 2007 at which a number of leading investigators from throughout the world presented findings that emphasized cellular and molecular aspects of G-protein-coupled receptor (GPCR) biology. Although the presentations highlighted both in vitro and in vivo studies, there was renewed and increased attention paid to systems with physiological (or near-physiological) levels of receptor expression. Mechanisms involved in regulating receptor expression, receptor recognition and activation by different classes of drugs, and in the interaction of G-protein-coupled receptor with other molecular entities, including heterotrimeric G proteins, were major themes of the presentations. Several speakers emphasized the challenges of drug discovery in relation to G-protein-coupled receptor, as well as their continuing importance in pharmacology and therapeutics.
Keywords: allosteric regulation, β-arrestin, chemokine receptor, glutamate receptor, G-protein-coupled receptor, receptor desensitization
Expert Opin. Ther. Targets (2007) 11(9):1251-1253
1. General overview
Approximately 150 attendees came to the University of Leicester for two unusually warm and sunny days, both weather-wise and scientifically, to participate in the 2 nd Focused Meeting on Cell Signalling, organized by John Challis and Andrew Tobin and sponsored by the British Pharmacology Society. This meeting focused on G-protein-coupled receptors (GPCRs) with presentations that discussed signaling, regulation and drug discovery. GPCRs are widely recognized to be the largest membrane receptor superfamily in various genomes, including that of humans, and thus have been an important focus for research by pharmacologists and scientists from other disciplines as well. In addition, GPCRs represent the targets to which the largest number of existing drugs are directed and accordingly continue to attract considerable interest from those working in the pharmaceutical industry. Therefore, it was not surprising that attendance at this meeting included a combination of investigators from both the academic and industrial venues. The main emphasis of the 14 invited lectures and 79 poster presentations was on cellular and molecular mechanisms related to GPCR. A synopsis of some of the key points noted in the invited lectures is given in the following sections.
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British Pharmacology Society 2nd Focused Meeting on Cell Signalling: GPCRs have sunny days in springtime Leicester
1252 Expert Opin. Ther. Targets (2007) 11(9)
2. Allosteric regulation of G-protein-coupled receptors
Dr A Christopoulos (Monash Univ., Australia; ‘ Allosteric modulation of G protein-coupled receptors: Expanding the pharmacological toolbox ’ ), began the meeting by asking: How does allosterism occur in response to ligands in various GPCR? Dr Christopoulos attempted to answer this question by exploring both methodologic and conceptual aspects of allosterism. He contrasted orthosteric (competitive, mutually exclusive) binding sites, with allosteric (modulatory) sites with which the orthosteric sites are conformationally linked. He noted that the detection of allosteric regulation can be assay-dependent, perhaps reflecting differing aspects of GPCR biology (e.g., receptor binding, signaling and regulation) that are altered by allosteric modulators. Allosteric sites occur at both extracellular and transmembrane domains with differing locations in different GPCRs. Receptor dissociation studies appear to provide the most definitive approach for characterizing certain types of allosterism, but as Dr Christopoulos noted, there may be an ‘ allosteric smorgasbord ’ with a wide range of allosteric effects as a consequence of the allosteric and orthosteric ligands used for particular studies.
Dr J Conn (Vanderbilt Univ., USA; ‘ Allosteric modulators of GPCR as a novel approach for treatment of CNS disorders ’ ) emphasized the occurrence of allosterism in metabotropic glutamate receptors (mGluR), especially in mGluR5 (NMDA) receptors. A key focus was whether such modulators might provide a therapeutic approach, especially for the treatment of schizophrenia. Dr Conn and his colleagues have developed an assay to measure the ability of allosteric potentiators (agonists) to stabilize an active conformation of the mGluR5 receptor. Use of this assay has facilitated the identification of allosteric potentiators, antagonists and neutral ligands; such agents potentially have a better therapeutic index than available agents that target mGluR5 receptors. A theme that Dr Conn (and other speakers) emphasized is the importance of assessing drug response in experimental systems that have naturally occurring levels of receptor expression rather than the over expressed systems that are often employed in studies of drug action. Using a novel screen that they developed to find allosteric agents active at mGluR5 receptors, Dr Conn and colleagues screened > 160,000 compounds and identified > 1000 as ‘ candidate allosteric modulators ’ .
In addition to what Dr Conn described, other speakers discussed the development of new methodologies to assess GPCRs: S Hill (Univ. of Nottingham, UK ‘ New fluorescent approaches to study the diffusional characteristics of GPCR complexes in microdomains of single living cells ’ ), H Vogel (Lausanne, ‘ Imaging GPCR at the cell surface ’ ), J Wess (NIH, USA ‘ Molecular aspects of GPCR activation ’ ) and G Milligan (Univ. of Glasgow, UK “ How ligands regulate the function of GPCR [without binding to them] ” ). In some cases these
methodologies involved studies of receptors themselves but in others, the approaches assessed components involved in signaling and regulation of GPCR, such as G proteins, β -arrestins, and G protein receptor kinases (GRKs). Linking his talk to those of others that discussed allosteric effects, Dr Milligan proposed the term ‘ off target allosterism ’ to describe the regulation of GPCR other than the ones to which ligands bind (at orthosteric sites) through interactions as heterodimeric GPCR partners.
3. Post-G-protein-coupled receptor components
Several speakers discussed post-GPCR components involved in receptor action or receptor regulation. Dr J Pitcher (Univ. College London, ‘ Nuclear G protein-coupled receptor kinases ’ ) provided new data regarding nuclear localization of GRKs (in particular GRK ’ s 4, -5 and -6, Class 3 GRKs), which phosphorylate agonist-occupied GPCR. The presence of a nuclear localization signal (NLS) in the primary sequence of those GRKs appears to determine this localization, but these sequences also contain nuclear export signals (NES), which may facilitate GRK export from the nucleus. In the case of GRK 5, nuclear export appears to occur via the interaction of calmodulin with a corresponding binding site; nuclear GRK5 can bind and regulate the activity of histone deacetylase 3 (HDAC3), thereby potentially altering gene and protein expression.
Other speakers also discussed the regulation of phosphory-lation of GPCRs as a determinant of cellular expression and localization: G Henderson (Univ. of Bristol, UK, ‘ Protein kinase C and µ -opioid receptor regulation ’ ), whose focus was on phosphorylation and desensitization of opioid receptors and M Houslay (Univ. of Glasgow, UK, ‘ Scaffolding of β -arrestin and cAMP phosphodiesterase-4 in compartmentalized signaling through the β 2-adrenoceptor,), who emphasized β -arrestin/phosphodiesterase 4D5 signaling complexes that create cAMP signaling microdomains.
Several speakers discussed the organization of GPCR signaling complexes in microdomains other than β -arrestin/clathrin, such as in lipid rafts/caveolae: S Hill, P Insel (UCSD, USA, ‘ Caveolae/caveolins and GPCR signaling in health and disease ’ ), H Vogel, J Lopez-Barneo (Univ. of Seville, Spain; ‘ A new metabotropic function of voltage-dependent calcium channels ’ ) and M Caron (Duke Univ., ‘ Novel GPCR signaling paradigms in animal models ’ ).
Dr Caron emphasized dopamine receptor signaling, especially in the CNS and discussed evidence for both a classical cAMP/G protein-dependent mechanism and also, a signaling pathway via Akt and glycogen synthase kinase 3 (GSK3). The latter mechanism may be a target for psycho-pharmacological agents, including lithium, which is used to treat mania and increases Akt and GSK3 phosphorylation. The formation of a complex that includes β -arrestin2, Akt and the protein phosphatase PP2A regulates this
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Insel
Expert Opin. Ther. Targets (2007) 11(9) 1253
phosphorylation via the dephosphorylation of GSK3 by PP2A, thereby slowing synaptic transmission. Lithium has two actions: down regulating the interaction of β -arrestin2, Akt and PP2A and directly inhibiting GSK3. The results suggest that other drugs might alter signaling and regulation by nonclassical, cAMP/G protein-independent pathways.
4. G-protein-coupled receptors in drug development
A number of speakers presented ideas and results related to drug development. The most pertinent and detailed was that of T Wells (Serono, Geneva, ‘ Targeting chemokine receptors as a therapeutic strategy ’ ), who described some of the challenges involved in developing drugs for chemokine receptors. Dr Wells emphasized the need to move the discovery/validation process into the in vivo setting as quickly as possible, especially because of differences noted in data obtained with in vitro versus in vivo studies of chemokine receptors. Even murine in vivo models have had poor predictive value (in terms of both efficacy and toxicity) for humans. It has been difficult to identify small molecule antagonists for chemokine receptors. Alternative approaches have been tried, including modified agonists that block chemokine receptor recycling and chemokine binding proteins, such as glycosaminoglycans that bind chemokines. Use of a chemokine-containing chip has revealed that saliva from ticks has chemokine binding
proteins, including a protein that binds multiple chemokines with high affinity (pM).
5. Expert opinion and conclusions
This meeting provided an excellent snapshot of the landscape of work related to approaches presently being used for the study of GPCR with insights of interest to scientists from both academia and industry. Noteworthy, was the desirability to assess native systems (i.e., not genetically engineered or at least engineered to physiologic levels of receptor expression) and to conduct studies, especially as part of the drug discovery/validation process, in experimental animal models. The development of newer and better methods, especially broad screening approaches and certain biophysical techniques that can provide new insights into cellular and molecular aspects of GPCR biology as well as help in the identification of new hits (i.e., candidate drugs) were also major themes at this meeting. Future work is likely to see further use and application of such methods for the study of GPCR and for drug discovery and development. Of particular note was the continuing and growing interest in allosteric modulators as alternatives to orthosteric compounds for studying GPCR and ultimately for therapeutically targeting allosteric sites. Thus, the sunny days in Leicester suggested that sunny days lie ahead for work on GPCR and, ultimately, in the development of new (and better) drugs that target these receptors.
Affi liationPaul A Insel Professor, University of California at San Diego, Departments of Pharmacology & Medicine, La Jolla, CA 92093-0636, USA E-mail: [email protected]
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