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© 2000 American College of NeuropsychopharmacologyPublished by Elsevier Science Inc. 0893-133X/00/$–see front matter655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(00)00154-8

INTRODUCTION

Dimerization of G-Protein Coupled Receptors

Protein-protein interactions govern a large number ofbiological processes. A number of cell surface and nu-clear receptors mediate their actions via dimerization.While this has been well established, the dimerization ofG-protein coupled receptors (GPCRs) (that represents

z

1% of the genes present in a mammalian genome) hasnot been well documented until recently. It was gener-ally assumed that these receptors existed as monomersand coupled to G proteins in a 1:1 stoichiometric ratio.However, these classical models of receptor/G proteincoupling may be oversimplified since a number of stud-ies report not only the presence but also a role for GPCRmultimers in modulating receptor function.

Early evidence for the interaction between GPCRscame from cross-linking experiments, radiation inactiva-tion experiments, and hydrodynamic studies. Analysisof complex binding data from early pharmacologicalstudies provided indirect evidence for the existence ofGPCR dimers. Radiation inactivation (target size analysis),a technique based on the inverse relationship betweenthe dose-dependent inactivation of a macromolecule byionizing radiation and the size of that macromolecule,showed that a functional unit of GPCR is a complex ofgreater molecular mass than the size predicted by dena-turing techniques. Cross-linking studies to visualize thereceptors revealed multiple sizes of proteins. In somecases, agonists selectively bound to the higher molecularweight form, consistent with the notion that the func-tional unit of GPCR is a dimer/oligomer.

The isolation of cDNA clones encoding the receptorshas enabled the critical evaluation of GPCR dimeriza-tion. Antisera against epitope tagged or native recep-tors have been used to visualize the receptor in heterol-ogous cells or in native tissue. Differential epitopetagging of the receptor cDNA followed by selective im-munoprecipitation of the dimer has been used toconfirm the ability of members of the rhodopsin, secre-

tin, and calcium sensing receptor family of GPCRs todimerize.

A number of studies have explored the effect of ago-nists on the level of receptor dimers and found that, insome cases, they stabilize and/or increase the level ofdimers, whereas in others, they have no effect nor de-crease the level of dimers. The function of GPCR dimer-ization/oligomerization explored using peptides thatblock dimerization or mutant receptors that do notdimerize support a role for dimerization in the modula-tion of signal transduction, in addition to influencingagonist affinity and agonist-mediated endocytosis.

Several lines of evidence suggest the existence ofGPCR heterodimers. An early study with chimeric re-ceptors between the alpha2c-adrenergic receptor andm3-muscarinic receptor suggested the possible het-erodimerization between GPCR types. Direct evidencefor GPCR heterodimerization came from studies with

non-functional GABA

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receptors that dimerize to gener-ate a functional receptor. Recent studies have also pro-vided direct evidence for the heterodimerization betweenfully functional GPCRs. These studies have shown thatheterodimers have distinct physical and functional prop-erties and that heterodimerization leads to modulation ofreceptor function by regulating ligand binding proper-ties, signaling, as well as receptor trafficking properties.

Receptor dimerization, brought about by the associa-tion of two monomers, could be mediated by covalent(disulfide) and/or non-covalent interactions; thesecould be association of the extracellular domains, trans-membrane domains, and/or C-terminal tail. Severalstudies with a number of GPCRs have suggested that acombination of the above mentioned interactions be-tween multiple domains occur during dimerization.Two mechanisms have been proposed for GPCR dimer-ization/oligomerization. One involves the association of1:1 stoichiometric molecular complexes of receptors and

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the other involves the swapping of domains betweentwo distinct receptor molecules resulting in a single ordual binding pockets. Further studies are required to de-lineate the molecular mechanism of GPCR dimerization.

Although several lines of evidence suggest that GPCRscan dimerize, it remains to be established whether thisphenomenon is a general characteristic of these receptorsand whether it is essential for receptor function. Also,studies need to be carried out to understand whetherthese receptors dimerize prior to targeting to the plasmamembrane and to what extent GPCRs dimerize

in vivo.

The use of sensitive biophysical techniques such as, fluo-rescence resonance energy transfer (FRET) or biolumines-cence resonance energy transfer (BRET) have been help-ful in answering some of these questions.

Development of tools that will allow selective detec-tion and/or activation of dimers are required to exam-

ine the role of GPCR dimers

in vivo.

A complete under-standing of the mechanisms of heterodimerization andits functional implications has enormous clinical signifi-cance as well as a high impact on GPCR pharmacologysince it represents another mechanism that could mod-ulate receptor function and thus provides a new strat-egy for the development of novel therapeutic drugs.

Lakshmi A. Devi, Ph.D.New York University School of Medicine

Department of PharmacologyNew York, NY

Linda S. Brady, Ph.D.Molecular & Cellular Neuroscience Research

NIMHBethesda, MD