BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - REVIEW ARTICLE

Neurotransmitter receptor heteromers in neurodegenerativediseases and neural plasticity

Rafael Franco

Received: 23 July 2008 / Accepted: 20 October 2008 / Published online: 11 November 2008

� Springer-Verlag 2008

Abstract Metabotropic receptors for neurotransmitters

on the plasma membrane of neurons are forming homo-

hetero- dimers and even homo- or hetero-oligomers. Neu-

rotransmission has been studied assuming that these

G-protein-coupled receptors were monomers. Then, on

considering receptor dimers, we are entering a new era for

the understanding how neurotransmitter receptors decode

signals originating at the nervous system. At the moment it

is becoming clear that receptor homo and hetero-oligomers

provide signaling diversity, help to understand synaptic

plasticity and open new therapeutic potential as targets for

neurodegenerative and neuropsychiatric diseases.

Keywords GPCR � Heteromer � Trimer � Oligomer �Neurotransmission � Metabotropic receptors �Heteromer fingerprint

Introduction

G-protein-coupled receptors (GPCRs) constitute a super-

family of proteins consisting of seven helices with

interconnecting loops; they are also known as 7 trans-

membrane (7TM), heptaspanning or heptahelical receptors.

It is generally accepted that some members of class C

GPCRs, which include metabotropic glutamate and taste

receptors function as homodimers and/or heterodimers

(Kunishima et al. 2000; Nelson et al. 2001). Those recep-

tors contain a long N terminal extracellular portion, which

for metabotropic glutamate receptors can be purified and

crystallized and appear as dimers in the solved 3D crystal

structure. Recently it has been possible to obtain crystals of

a member of the rhodopsin class A family of GPCRs, the ß2

adrenergic, and it appears to be monomeric (Rasmussen

et al. 2007; Rosenbaum et al. 2007). This finding is

probably due to the use of detergents during receptor

purification and the special conditions of crystallization. In

fact, there is strong evidence indicating that members of

this superfamily that were suspected to be monomeric

membrane receptors are expressed on the plasma mem-

brane as homo/heterodimers or multimers. Knowing the

details of how GPCRs interact (Ciruela et al. 2004), and the

stoichiometry and geometry of the oligomeric complexes

(including non-receptor scaffolding proteins: see Saura

et al. 1996; Burgueno et al. 2003; Sarrio et al. 2000) will be

instrumental to improve our understanding on how neuro-

transmission is taking place.

Suspecting the existence of receptor heteromers

The hypothesis on the existence of intramembrane recep-

tor–receptor interactions was introduced in the early 1980s

based on radioligand binding studies in membrane prepa-

rations from brain tissue (Agnati et al. 1980, 1982; Fuxe

et al. 1981). The first demonstration of GPCR homodimers

was achieved with ß-adrenergic and muscarinic receptors

R. Franco (&)

Department of Biochemistry and Molecular Biology,

and Centro de Investigacion Biomedica en Red sobre

Enfermedades Neurodegenerativas (CIBERNED),

Institut d’Investigacio Biomedica August Pi i Sunyer

(IDIBAPS), University of Barcelona, Barcelona, Spain

e-mail: rfranco@unav.es

URL: http://www.cima.es

Present Address:R. Franco

CIMA Centro de Investigacion Medica Aplicada,

Universidad de Navarra, Avda Pio XII 55,

31008 Pamplona, Spain

123

J Neural Transm (2009) 116:983–987

DOI 10.1007/s00702-008-0148-y

(Fraser and Venter 1982; Avissar et al. 1983). Maggio et al.

(1993) gave the first evidence for the ability of two GPCRs

to heteromerize by using coexpression of chimeras of M3

muscarinic and a2C-adrenergic receptor. Whereas isolated

chimeric constructs were not able to bind ligands, coex-

pression of two complementary chimeric receptors led to

the appearance of binding of agonists for both M3 and a2C

receptors. Subsequently, coexpression of receptors in the

same cell or tissue and detection of colocalization by

confocal microscopy supported the view that GPCR were

forming heteromers.

Coimmunoprecipitation followed by Western blotting

has been widely used to study protein–protein interactions.

However, coimmunoprecipitation of amphiphilic protein

membrane molecules, such as GPCRs, requires membrane

solubilization, thus raising the possibility that the observed

complexes are solubilization artefacts. To overcome this

technical limitation, bioluminescence and fluorescence

resonance energy transfer (BRET and FRET, respectively),

which are powerful biophysical techniques, were imple-

mented. FRET and BRET have allowed demonstration of

receptor–receptor heteromerization in the natural environ-

ment of the living cell. Recently, variations or combination

of these biophysical techniques has allowed detecting tri-

mers (Carriba et al. 2008) and dimers of dimers (Maurel

et al. 2008).

Detecting receptor heteromers in natural tissues:

the heteromer fingerprint

To use FRET for showing the occurrence of heteromers in

natural tissues specific anti-receptor antibodies having

attached fluorescent proteins must be used. This approach,

however, has technical limitations. Another approach is

being developed in which receptors fused to fluorescent

proteins are expressed as transgenes, being possible to

perform FRET in samples from the transgenic animal. It

should be noted that the knockout methodology is not a

method of choice to identify dimers. This is an important

limitation arising from the fact that a given protein may be

forming in the same or in different tissues not only one but

multiple heteromers. Then a lack of function in a knockout

animal may consequence of disruption of not one but

several heteromers. If any, knockouts may be useful as

negative controls of heteromer formation. Studies per-

formed by Maggio et al. (1999) showed that M2 and M3

muscarinic receptor subtypes can cross-interact with each

other and form a new pharmacological heteromeric

receptor, which would display a distinctive ligand binding

profile. This distinctive pharmacological characteristic is

indeed invaluable to detect heteromers in native tissues.

Ciruela et al. (2006) and Ferre et al. (2007) coined the term

‘‘heteromer fingerprint’’, which means that a specific fea-

ture of the heteromer is just needed to prove its existence in

natural tissues. This fingerprint is a specific feature of the

heteromer that is just needed to prove its existence in

natural tissues from animals without any genetic manipu-

lation. Finding a heteromeric fingerprint in a natural tissue

is the most direct evidence to prove the existence of het-

eromers in vivo. When looking for heteromer fingerprints

there are different possibilities, particularly fingerprint at

the ligand binding and at the signaling level. One of the

most common heteromer fingerprints at the ligand binding

level is shown by changes in the binding pattern caused by

binding to the partner receptor. Quite often the affinity of

agonists to one receptor varies when the partner receptor is

occupied by ligand. One of the best studied examples is the

ability of adenosine A2A receptor agonists to modify the

affinity of dopamine D2 agonists in the A2A–D2 receptor

heteromer (Ferre et al. 1991, 2007). A nice example of

heteromer fingerprint detected at the signaling level is

provided by the selective Gq–protein coupling of the

D1–D2 receptor heteromer. Whereas D1 and D2 receptor

expressed as homomers are coupled to Gs and Gi proteins,

respectively, activation of the heteromer leads to a

Gq-mediated calcium signaling (Lee et al. 2004; Rashid

et al. 2007).

New models for receptor dimers

Colquhoun (1973) and Thron (1973) pioneered studies that

led to the subsequent development of models to understand

the operation of neurotransmitter/hormone receptors (De

Lean et al. 1980; Costa and Herz 1989; Onaran et al. 1993;

Samama et al. 1993; Franco et al. 1996; Weiss et al. 1996a,

b, c; Hall 2000; Lorenzen et al. 2002). Those models

consider receptors as monomers and are modifications of

del Castillo and Katz (1957) model of nicotinic acetyl-

choline receptor activation.

Recently two similar models based on receptor dimers

have been devised (Albizu et al. 2006 and references

therein; Franco et al. 2005, 2006). They propose a ‘‘two-

state’’ dimer model based only in dimeric species. One of

its advantages is the usefulness to obtain parameters

‘‘specific’’ of the dimer. Interestingly, the development of

Casado et al. (2007) has demonstrated that a model of

constitutive dimers it is the simplest to explain both simple

and complex binding data. Taking into account the dimeric

structure of G-protein-coupled receptors, the interpretation

of the biphasic binding isotherms (non linear Scatchard

plots) is that the first ligand to the dimer modifies the

equilibrium parameters of binding of the second ligand

molecule to the dimer. If the affinity increases ‘‘positive

cooperativity’’ is detected and if it decreases ‘‘negative

984 R. Franco

123

cooperativity’’ is found. The main advantage of the

development performed by Casado et al. (2007) is the

possibility to obtain dimer-specific parameters (two affinity

constants and a dimer cooperativity index) from experi-

mental binding data.

Adenosine-dopamine and adenosine-glutamate receptor

dimers in neuroprotection and Parkinson’s disease

Parkinson’s is a well-characterized disease caused by

striatal neurodegeneration. The lack of dopaminergic

neurons and of dopamine itself causes motor deficits. A

functional interaction between the dopaminergic and ad-

enosinergic systems has been detailed described. In fact

the neuromodulator adenosine in both direct and indirect

projecting striatal pathways is able to counteract the

effects of dopamine. In the rat hemiparkinsonian model it

was demonstrated that agonists of adenosine receptors

were able to worsen the effects of the neurodegeneration

whereas adenosine receptor antagonists reversed those

effects. These results suggest that A2A receptor could be

target for Parkinson’s disease. At present there are dif-

ferent clinical trials in which different synthetic A2A

receptor antagonists are under evaluation. Although a

direct proof is difficult it is suspected that the real targets

for Parkinson’s are dopamine-receptor-containing dimers

(Franco et al. 2000; Ferre et al. 2007).

While lacking direct evidence for dimers as targets for

neurodegenerative and/or neuropsychiatric diseases what it

is true is that antiparkinsonian drugs are targeting dimers/

oligomers. The main therapeutic drug for Parkinson’s dis-

ease, L-DOPA (which is converted into dopamine), is

targeting dopamine receptor dimers as there must be few

monomers expressed on the plasma membrane of neurons.

Furthermore, adenosine-dopamine receptor heteromers

have been identified, and therefore L-DOPA is targeting

dopamine receptors in a variety of dopamine-receptor-

containing heteromers. Dopamine-receptor-containing

dimers include at least D1–D2 (Lee et al. 2004; Rashid et al.

2007) D1–D3 (Marcellino et al. 2008), adenosine-dopamine

A1–D1 (Gines et al. 2000), adenosine-dopamine A2–D2

(Hillion et al. 2002) and cannabinoid-dopamine CB1–D2

(Kearn et al. 2005). A recent report indicates the existence

of trimers of cannabinoid CB1, dopamine D2 and adenosine

A2A receptors (Carriba et al. 2008). As commented above

A2A receptor antagonists are under evaluation for the ther-

apy of Parkinson’s disease. Therefore these compounds are

targeting at least these reported A2A-receptor-containing

heteromers: A1/A2A (Ciruela et al. 2006), dopamine-aden-

osine A2–D2 (Hillion et al. 2002) cannabinoid-adenosine

CB1–A2A (Carriba et al. 2007) and the trimeric CB1–D2–

A2A (Carriba et al. 2008). In summary, alternative

approaches to drug screening based on dopamine receptor

monomers must be explored. Among them new alternatives

include the search for heteromer-selective drugs as the

dopamine-receptor-heteromer-selective drug reported by

Rashid et al. 2007, and dual drugs able to interact

simultaneously with adenosine and dopamine receptor

heteromers (Ventura et al., data in preparation).

Excitoxicity caused by exacerbated levels of extracel-

lular glutamate is behind a number of alterations in the

central nervous system. The role of ionotropic and

metabotropic glutamate receptors in the neurotransmitter-

mediated toxic effects is still controversial. On the other

hand, glutamate could also modulate the well-known

function of adenosine as neuroprotective factor (Dunwiddie

1985). Then it was hypothesized that targeting of gluta-

mate-adenosine mGlu1/A1 receptor heteromers could be

beneficial in situations of enhanced neuronal activity, in

which potentiation of postsynaptic adenosine A1 receptor

limits evoked depolarization. This phenomenon would

result in reduced activation of voltage-dependent Ca2? and

NMDA receptor channels, through which Ca2? ions enter

into cell bodies (Robbins et al. 1999).

In experiments of NMDA-mediated excitoxicity per-

formed in primary cultures of neurons Ciruela et al. (2001)

reported that exposure to both agonists of adenosine A1 and

metabotropic mGluR1a receptors results in an almost

complete neuroprotection, which was not achieved by any

of the two agonists when used alone. Thus, the spatio-

temporal segregation profile of adenosine/glutamate

release during synaptic activity is of special importance to

achieve a neuroprotective or a neurotoxic effect. The

results do not demonstrate that neuroprotection by the

combination of agonists is mediated by glutamate-adeno-

sine receptor heteromers. However as reasoned above,

potential neuroprotective agents acting on these receptors

would in fact act on receptor heteromers; also dual or

heteromer-selective drugs could (also potentially) be more

efficacious in achieving neuroprotection than drugs tar-

geting a single receptor.

Receptor dimers and synaptic plasticity

The readout of synaptic plasticity is the spatio/temporal and

quantitative expression of neurotransmitter active receptors

in neurons, especially around synapses and in dendritic

spines. A number of factors affect traffic, cell surface

expression and desensitization of receptors. More often than

not receptor heteromer formation results in qualitatively or

qualitatively ‘‘different’’ receptor traffic, expression and

desensitization. As an example the D1 receptor, which is

almost unable to desensitize when expressed in a heterol-

ogous system, desensitizes easily when coexpressed with a

Neurotransmitter receptor heteromers in neurodegenerative diseases and neural plasticity 985

123

receptor with which it forms heteromers. Thus, in heterol-

ogous cells expressing adenosine-dopamine A1–D1 receptor

heteromers pre-treatment with the D1R agonist SKF-38393

for 30–120 min does not alter the agonist-induced increase

in cAMP accumulation. In contrast, a significant reduction

of the SKF-38393-induced cAMP accumulation is found

after combined pre-treatment with both the dopamine and

adenosine receptor agonists (Gines et al. 2000). Similarly,

the down-regulation of cell surface dopamine and/or

adenosine receptors may markedly vary depending upon the

expression of receptor heteromers and the treatment given,

i.e., whether cells are treated with either an adenosine or a

dopamine receptor agonist or their combination. Interest-

ingly enough, one agonist may induce the internalization of

the heteromer (Gines et al. 2000). The traffic of adenosine

A2A and D2 receptors also depend on heteromer formation

and type of treatment (Agnati et al. 2005; Torvinen et al.

2005). Overall, adenosine and dopamine receptor-mediated

transmission, receptor heteromerization, and receptor traffic

(including cell surface clustering) are phenomena related to

each other in a complex manner (Franco et al. 2000).

As a summary of the above-reported findings concerning

receptor dimers/oligomers it is becoming clear that the

physiological relevance of dimers/oligomers is to provide

signalling diversity. In the central nervous system a given

neuron will sense a given neurotransmitter depending on the

heteromer, i.e., different cells expressing different hetero-

mers will respond differently to the same neurotransmitter:

dopamine, glutamate, etc. Taking advantage of the same

example given above, dopamine will lead to cAMP

increases if the target neuron expresses D1 receptors, to

cAMP decreases if the target neuron expresses D2 receptors

and to increases in intracellular Ca2? if D1–D2 heteromers

are expressed. It is foreseeable a huge variety of dimers/

trimers/tetramers arising by combining the dozens of

receptor subtypes existing for the different neurotransmit-

ters. For this reason synaptic plasticity is not a question of

expressing (qualitatively and/or quantitatively) different

receptor monomers in different circumstances but of having

different receptor heterooligomers in different circum-

stances. To advance in the understanding of how synaptic

plasticity takes place will require a clear understanding of

how heteromer formation and expression is regulated.

References

Agnati LF, Fuxe K, Zini I, Lenzi P, Hokfelt T (1980) Aspects on

receptor regulation and isoreceptor identification. Med Biol

58:182–187

Agnati LF, Fuxe K, Zoli M, Rondanini C, Ogren SO (1982) New

vistas on synaptic plasticity: the receptor mosaic hypothesis.

Med Biol 60:183–190

Agnati LF, Fuxe K, Torvinen M, Genedani S, Franco R, Watson S,

Nussdorfer GG, Leo G, Guidolini D (2005) New methods to

evaluate colocalization of fluorophores in immunocytochemical

preparations as exemplified by a study on A2A and D2 receptors in

Chinese hamster ovary cells. J Histochem Cytochem 53:941–953

Albizu L, Balestre M-N, Breton C, Pin J-P, Manning M, Mouillac B,

Barberis C, Durroux T (2006) Probing the existence of G-protein-

coupled receptor dimers by positive and negative ligand-

dependent cooperative binding. Mol Phamacol 70:1783–1791

Avissar S, Amitai G, Sokolovsky M (1983) Oligomeric structure of

muscarinic receptors is shown by photoaffinity labeling: subunit

assembly may explain high- and low-affinity agonist states. Proc

Natl Acad Sci USA 80:156–159

Burgueno J, Blake DJ, Benson MA, Tinsley CL, Esapa CT, Canela

EI, Penela P, Mallol J, Mayor F Jr, Lluis C, Franco R, Ciruela F

(2003) The adenosine A(2A) receptor interacts with the actin-

binding protein alpha-actinin. J Biol Chem 278:37545–37552

Carriba P, Ortiz O, Patkar K, Justinova Z, Stroik J, Themann A,

Muller C, Woods AS, Hope BT, Ciruela F, Casado V, Canela EI,

Lluis C, Goldberg SR, Moratalla R, Franco R, Ferre S (2007)

Striatal adenosine A(2A) and cannabinoid CB(1) receptors form

functional heteromeric complexes that mediate the motor effects

of cannabinoids. NeuropsychoPharmacology 32:2249–2259

Carriba P, Navarro G, Ciruela F, Ferre S, Casado V, Agnati L, Cortes

A, Mallol J, Fuxe K, Canela EI, Lluis C, Franco R (2008)

Detection of heteromerization of more than two proteins by

sequential BRET-FRET. Nat Methods 5:727–733

Casado V, Cortes A, Ciruela F, Mallol J, Ferre S, Lluis C, Canela EI,

Franco R (2007) Old and new ways to calculate the affinity of

agonists and antagonists interacting with G-protein-coupled

monomeric and dimeric receptors: the receptor-dimer coopera-

tivity index. Pharmacol Ther 16:343–354

Ciruela F, Escriche M, Burgueno J, Angulo E, Casado V, Soloviev

MM, Canela EI, Mallol J, Chan WY, Lluis C, McIlhinney RA,

Franco R (2001) Metabotropic glutamate 1alpha and adenosine

A1 receptors assemble into functionally interacting complexes.

J Biol Chem 276:18345–18351

Ciruela F, Burgueno J, Casado V, Canals M, Marcellino D, Goldberg

SR, BAder M, Fuxe K, Agnati LF, Lluis C, Franco R, Ferre S,

Woods AS (2004) Combining mass spectrometry and pull-down

techniques for the study of receptor heteromerization. Direct

epitope–epitope electrostatic interactions between adenosine

A(2A) and dopamine D-2 receptors. Anal Chem 76:5354–5363

Ciruela F, Casado V, Rodrigues RJ, Lujan R, Burgueno J, Canals M,

Borycz J, Rebola N, Goldberg SR, Mallol J, Cortes A, Canela EI,

Lopez-Gimenez JF, Milligan G, Lluis C, Cunha RA, Ferre S,

Franco R (2006) Presynaptic control of striatal glutamatergic

neurotransmission by adenosine A1–A2A receptor heteromers.

J Neurosci 26:2080–2087

Colquhoun D (1973) The relationship between classical and cooper-

ative models for drug action. In: Rang HP (ed) A symposium on

drug receptors. University Park Press, Baltimore, pp 149–182

Costa T, Herz A (1989) Antagonists with negative intrinsic activity at

delta opioid receptors coupled to GTP-binding proteins. Proc

Natl Acad Sci USA 86:7321–7325

Del Castillo J, Katz B (1957) Interaction at end-plate receptors

between different choline derivatives. Proc R Soc Ser B

146:369–381

De Lean A, Stadel JM, Lefkowitz RJ (1980) A ternary complex

model explains the agonist-specific binding properties of the

adenylate cyclase-coupled beta-adrenergic receptor. J Biol Chem

255:7108–7117

Dunwiddie TV (1985) The physiological role of adenosine in the

central nervous system. Int Rev Neurobiol 27:63–139

Ferre S, von Euler G, Johansson B, Fredholm BB, Fuxe K (1991)

Stimulation of high-affinity adenosine A2 receptors decreases

986 R. Franco

123

the affinity of dopamine D2 receptors in rat striatal membranes.

Proc Natl Acad Sci USA 88:7238–7241

Ferre S, Ciruela F, Woods AS, Lluis C, Franco R (2007) Functional

relevance of neurotransmitter receptor heteromers in the central

nervous system. Trends Neurosc 30:440–446

Franco R, Casado V, Ciruela F, Mallol J, Lluis C, Canela EI (1996)

The cluster-arranged cooperative model: a model that accounts

for the kinetics of binding to adenosine receptors. Biochemistry

35:3007–3015

Franco R, Ferre S, Agnati LF, Torvinen M, Gines S, Hillion J, Casado

V, Lledo PM, Zoli Z, Lluis C, Fuxe K (2000) Evidence for

adenosine/dopamine receptor interactions: indications for hetero-

merization. Neuropsychopharmacology 23:S50–S59

Franco R, Casado V, Mallol J, Ferre S, Fuxe K, Cortes A, Ciruela F,

Lluis C, Canela EI (2005) Dimer-based model for heptaspanning

membrane receptors. Trends Biochem Sci 30:360–366

Franco R, Casado V, Mallol J, Ferrada C, Ferre S, Fuxe K, Cortes A,

Ciruela F, Lluis C, Canela EI (2006) The two-state dimer

receptor model: a general model for receptor dimers. Mol

Pharmacol 69:1905–1912

Fraser CM, Venter JC (1982) The size of the mammalian lung beta

2-adrenergic receptor as determined by target size analysis

and immunoaffinity chromatography. Biochem Biophys Res

Commun 109:21–29

Fuxe K, Agnati LF, Benfenati F, Cimmino M, Algeri S, Hokfelt T,

Mutt V (1981) Modulation by cholecystokinins of 3H-spiroper-

idol binding in rat striatum: evidence for increased affinity and

reduction in the number of binding sites. Acta Physiol Scand

113:567–569

Gines S, Hillion J, Torvinen M, Le Crom S, Casado V, Canela EI,

Rondin S, Lew JY, Watson S, Zoli M, Agnati LF, Verniera P,

Lluis C, Ferre S, Fuxe K, Franco R (2000) Dopamine D1 and

adenosine A1 receptors form functionally interacting hetero-

meric complexes. Proc Natl Acad Sci USA 97:8606–8611

Hall DA (2000) Modeling the functional effects of allosteric

modulators at pharmacological receptors: an extension of the

two-state model of receptor activation. Mol Pharmacol 58:1412–

1423

Hillion J, Canals M, Torvinen M, Casado V, Scott R, Terasmaa A,

Hansson A, Watson S, Olah ME, Mallol J, Canela EI, Zoli M,

Agnati LF, Ibanez CF, Lluis C, Franco R, Ferre S, Fuxe K (2002)

Coaggregation, cointernalization, and codesensitization of aden-

osine A2A receptors and dopamine D2 receptors. J Biol Chem

277:18091–18097

Kearn CS, Blake-Palmer K, Daniel R, Mackie K, Glass M (2005)

Concurrent stimlation of cannabinoid CB1 and dopamine D2

receptors enhances heterodimer formation: a mechanism for

receptor cross-talk? Mol Pharmacol 67:1697–1704

Kunishima N, Shimada Y, Tsuji Y, Sato T, Yamamoto M, Kumasaka

T, Nakanishi S, Jingami H, Morikawa K (2000) Structural basis

of glutamate recognition by a dimeric metabotropic glutamate

receptor. Nature 407:971–977

Lee SP, So CH, Rashid AJ, Varghese G, Cheng R, Lanca AJ, O’Dowd

BF, George SR (2004) Dopamine D1 and D2 receptor co-

activation generates a novel phospholipase C-mediated calcium

signal. J Biol Chem 279:35671–35678

Lorenzen A, Beukers MW, van der Graaf PH, Lang H, van Muijlwijk-

Koezen J, de Groote M (2002) Modulation of agonist responses

at the A1 adenosine receptor by an irreversible antagonist,

receptor-G protein uncoupling and by the G protein activation

state. Biochem Pharmacol 64:1251–1265

Maggio R, Vogel Z, Wess J (1993) Coexpression studies with mutant

muscarinic/adrenergic receptors provide evidence for intermo-

lecular ‘‘cross-talk’’ between G-protein-linked receptors. Proc

Natl Acad Sci USA 90:3103–3107

Maggio R, Barbier P, Colelli A, Salvadori F, Demontis G, Corsini GU

(1999) G protein-linked receptors: pharmacological evidence for

the formation of heterodimers. J Biol Chem 291:251–257

Marcellino D, Ferre S, Casado V, Cortes A, Le Foll B, Mazzola C,

Drago F, Saur O, Stara H, Soriano A, Barnes C, Goldberg SR,

Lluis C, Fuxe K, Franco R (2008) Identification of dopamine

D1–D3 heteromers: indications for a role of synergistic D1–D3

receptor interactions in the striatum. J Biol Chem 283:26016–

26025

Maurel D, Comps-Agrar L, Brock C, Rives ML, Bourrier E, Ayoub

MA, Bazin H, Tinel N, Durroux T, Prezeau L, Trinquet E, Pin JP

(2008) Cell-surface protein–protein interaction analysis with

time-resolved FRET and snap-tag technologies: application to

GPCR oligomerization. Nat Methods 5:561–567

Nelson G, Hoon MA, Chandrashekar J, Zhang Y, Ryba NJ, Zuker CS

(2001) Mammalian sweet taste receptors. Cell 106:381–390

Onaran HO, Costa T, Rodbard D (1993) Betagamma subunits of

guanine nucleotide-binding proteins and regulation of spontane-

ous receptor activity: thermodynamic model for the interaction

between receptors and guanine nucleotide-binding protein sub-

units. Mol Pharmacol 43:245–256

Rashid AJ, So CH, Kong MM, Furtak T, El-Ghundi M, Cheng R,

O’Dowd BF, George SR (2007) D1–D2 dopamine receptor

heterooligomers with unique pharmacology are coupled to rapid

activation of Gq/11 in the striatum. Proc Natl Acad Sci USA

104:654–659

Rasmussen SGF, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS,

Edwards PC, Burghammer M, Ratnala VRP, Sanishivi R,

Fischetti RF, Schertler GFX, Weis WI, Kobilka BK (2007)

Crustal structure of the human beta2 adrenergic G-protein-

coupled receptor. Nature 450:383–387

Robbins MJ, Ciruela F, Rhodes A, McIlhinney RAJ (1999) Charac-

terization of the dimerization of metabotropic glutamate

receptors using an N-terminal truncation of mGluR1alpha.

J Neurochem 72:2539–2547

Rosenbaum DM, Cherezov D, Hanson MA, Rasmussen SGF, Thian

FS, Kobilka TS, Choi HJ, Yao XJ, Weis WI, Stevens RC,

Kobilka BK (2007) GPCR engineering yields high-resolution

structural insights into beta2 adrenergic receptor function.

Science 318:1266–1273

Samama P, Cotecchia S, Costa T, Lefkowitz RJ (1993) A mutation-

induced activated state of the beta2-adrenergic receptor. Extend-

ing the ternary complex model. J Biol Chem 268:4625–4636

Sarrio S, Casado V, Escriche M, Ciruela F, Mallol J, Canela EI, Lluis

C, Franco R (2000) The heat shock cognate protein hsc73

assembles with A(1) adenosine receptors to form functional

modules in the cell membrane. Mol Cell Biol 20:5164–5174

Saura C, Ciruela F, Casado V, Canela EI, Mallol J, Lluis C, Franco R

(1996) Adenosine deaminase interacts with A(1) adenosine

receptors in pig brain cortical membranes. J Neurochem

66:1675–1682

Thron CD (1973) On the analysis of pharmacological experiments in

terms of an allosteric receptor model. Mol Pharmacol 9:1–9

Torvinen M, Torri C, Tombesi A, Marcellino D, Watson S, Lluis C,

Franco R, Fuxe K, Agnati LF (2005) Trafficking of adenosine

A2A and dopamine D2 receptors. J Mol Neurosci 25:191–200

Weiss JM, Morgan PH, Lutz MW, Kenakin TP (1996a) The cubic

ternary complex receptor-occupancy model I. Model description.

J Theor Biol 178:151–167

Weiss JM, Morgan PH, Lutz MW, Kenakin TP (1996b) The cubic

ternary complex Receptor-occupancy model II. Understanding

apparent affinity. J Theor Biol 178:169–182

Weiss JM, Morgan PH, Lutz MW, Kenakin TP (1996c) The cubic

ternary complex receptor-occupancy model III. Resurrecting

efficacy. J Theor Biol 181:381–397

Neurotransmitter receptor heteromers in neurodegenerative diseases and neural plasticity 987

123