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30 Ruth, P. et al. (1989) Primary structure of the β-subunit of the DHP-sensitive calcium channel from skeletal muscle. Science 245, 1115–1118 31 Varadi, G. et al. (1991) Acceleration of activation and inactivation by the β-subunit of the skeletal muscle calcium channel. Nature 11, 159–162 32 Pragnell, M. et al. (1991) Cloning and tissuespecific expression of the brain calcium channel β-subunit. FEBS Lett. 291, 253–258 33 Powers, P.A. et al. (1992) Skeletal muscle and brain isoforms of a β-subunit of human voltagedependent calcium channels are encoded by a single gene. J. Biol. Chem. 267, 22967–22972 34 Gregg, R.G. et al. (1996) Absence of the β-subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the α1-subunit and eliminates excitation–contraction coupling. Proc. Natl. Acad. Sci. U. S. A. 93, 13961–13966 35 Strube, C. et al. (1996) Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor β1-subunit. Biophys. J. 71, 2531–2543 36 Scott, V.E. et al. (1996) β-subunit heterogeneity in N-type Ca2+ channels. J. Biol. Chem. 271, 3207–3212

37 Namkung, Y. et al. (1998) Targeted disruption of the Ca2+ channel β3-subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons. Proc. Natl. Acad. Sci. U. S. A. 95, 12010–12015 38 Singer, D. et al. (1991) The roles of the subunits in the function of the calcium channel. Science 253, 553–557 39 Wei, X.Y. et al. (1991) Heterologous regulation of the cardiac Ca2+ channel α1-subunit by skeletal muscle β and γ-subunits. Implications for the structure of cardiac L-type Ca2+ channels. J. Biol. Chem. 266, 21943–21947 40 Freise, D. et al. (2000) Absence of the γ-subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties. J. Biol. Chem. 275, 14476–14481 41 Letts, V.A. et al. (1998) The mouse stargazer gene encodes a neuronal Ca2+ channel γ-subunit. Nat. Genet. 19, 340–347 42 Chen, L. et al. (2000) Stargazing regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408, 936–943 43 Bito, H. et al. (1996) CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87, 1203–1214

44 Deisseroth, K. et al. (1998) Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. Nature 392, 198–202 45 Molkentin, J.D. et al. (1998) A calcineurindependent transcriptional pathway for cardiac hypertrophy. Cell 93, 215–228 46 Graef, I.A. et al. (1999) L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons. Nature 401, 703–708 47 Crabtree, G.R. (1999) Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 96, 611–614 48 Halfon, M.S. et al. (2000) Ras pathway specificity is determined by the integration of multiple signal-activated and tissuerestricted transcription factors. Cell 103, 63–74 49 Flores, G.V. et al. (2000) Combinatorial signaling in the specification of unique cell fates. Cell 103, 75–85 50 Xu, C. et al. (2000) Overlapping activators and repressors delimit transcriptional response to receptor tyrosine kinase signals in the Drosophila eye. Cell 103, 87–97

Heterodimerization of G-proteincoupled receptors: pharmacology, signaling and trafficking Lakshmi A. Devi Although classical models predict that G-protein-coupled receptors (GPCRs) function as monomers, several recent studies acknowledge that GPCRs exist as dimeric or oligomeric complexes. In addition to homodimers, heterodimers between members of the GPCR family (both closely and distantly related) have been reported. In some cases heterodimerization is required for efficient agonist binding and signaling, and in others heterodimerization appears to lead to the generation of novel binding sites. In this article, the techniques used to study GPCR heterodimers, and the ‘novel pharmacology’ and functional implications resulting from heterodimerization will be discussed.

Lakshmi A. Devi Dept of Pharmacology, New York University School of Medicine, New York, NY, USA. e-mail: lakshmi.devi@ med.nyu.edu

The function of most, if not all, cells in the body is regulated by plasma membrane receptors. The vast majority of these receptors belong to the superfamily of G-protein-coupled receptors (GPCRs), which at current estimates account for ~1% of the genes present in a mammalian genome. Agonists or antagonists of GPCRs, in addition to agents that interfere with cellular pathways regulated by these receptors, are widely used in drug therapy. All GPCRs share a common tertiary structure consisting of seven transmembrane (TM) helices http://tips.trends.com

linked by three alternating intracellular and extracellular domains, with an extracellular N-terminus and a cytoplasmic C-terminus. GPCRs interact with heterotrimeric guanine-nucleotidebinding regulatory proteins (G proteins), which then interact with effector systems and regulate various intracellular processes (for an animation of agonistinduced activation of GPCRs, see http://archive. bmn.com/supp/tips/tips2210a.html). Based on sequence similarity, GPCRs can be classified into three major receptor families1. Family A (rhodopsin, β2-adrenoceptor-like) is the largest family of GPCRs. These receptors are characterized by several conserved residues in their TM helices and a palmitoylated cysteine in the C-terminal tail. Family B [glucagon, vasoactive intestinal peptide (VIP), calcitonin receptorlike] is a relatively small group that is characterized by the presence of a large N-terminal domain that contains several well-conserved cysteine residues. Family C (metabotropic neurotransmitter, calcium-sensing receptor-like) is characterized by a very long N-terminal domain that appears to be sufficient for ligand binding.

0165-6147/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(00)01799-5

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Models that describe the interaction of GPCRs with their G proteins are generally based on the assumption that receptors exist as monomers and couple to G proteins in a 1:1 ratio. However, these classical models of receptor–G-protein coupling might be oversimplified. Several recent studies have reported interactions between GPCRs of all three families2,3. These interactions could involve an association between identical proteins (homomers) or non-identical proteins (heteromers) and between two monomers (to form dimers) or multiple monomers (to form oligomers). Thus, a given GPCR could exist as monomers, homo- or hetero-dimers, or homo- or hetero-oligomers. Because a distinction between dimers and oligomers cannot easily be made using current techniques and because dimers represent the smallest of oligomeric units, complexes that result from GPCR interactions will be referred to as ‘dimers’ and the phenomenon will be referred to as ‘dimerization’ in this review.

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Table 1. Techniques to study G-protein-coupled receptor (GPCR) heteromerizationa Receptor dimers

Technique

Biochemical techniques Immunoprecipitation GABAB(1a)–GABAB(2) KOP–DOP Immunoprecipitation MOP–DOP Immunoprecipitation 5-HT1B–5-HT1D Immunoprecipitation β2-adrenoceptor–DOP Immunoprecipitation β2-adrenoceptor–KOP Immunoprecipitation sst2A–sst3 Immunoprecipitation A1–mglu1 Immunoprecipitation AT1–B2 Crosslinking and western blot

Refs

18–22 7 24,25 36 28 28 26 29 27

Biophysical techniques β2-adrenoceptor–DOP BRET sst1–sst5 pbFRET D2–sst5 pbFRET

13 14 15

Functional complementation techniques α2-adrenoceptor–M3 Chimeric mutants sst1–sst5 Deletion mutants

17 14

aAbbreviations:

Methods used to study GPCR dimers

A significant number of studies have suggested the presence of GPCR dimers4–6. Because the results from these studies could also be explained by alternative interpretations, the existence of GPCR dimers and their pharmacological relevance remained obscure until recently. The availability of GPCR cDNAs facilitated studies to examine GPCR dimerization directly using biochemical, biophysical and functional complementation techniques. Biochemical techniques

Differential epitope tagging and selective immunoprecipitation have been used extensively to provide biochemical evidence for the presence of GPCR dimers (Table 1). Using this technique, the two GPCRs under investigation are each tagged with a distinct epitope and expressed in heterologous cells (that do not normally express these receptors). Antibodies to one epitope are used to immuno-isolate the receptor-containing complex, and the associating receptor in the complex is visualized using antibodies to the second epitope. A major concern with this assay is the nonspecific aggregates that are formed during the extraction and under the immuno-isolation conditions. A variety of reagents (e.g. crosslinkers, disulfide reducing agents, capping agents and combinations of detergents) have been employed to address this concern7–9. A good test for artefactual aggregation is to subject a mixture of cells that each express only one of the two epitope-tagged receptors to identical immuno-isolation procedures as that of cells coexpressing these receptors. If dimers are not observed in the case of mixed cells it would imply that the dimers are not artefacts of the isolation procedure7. Controls such as these have been used to provide biochemical evidence for dimerization between members of closely, in addition to distantly, related families of GPCRs (Table 1). http://tips.trends.com

A1, adenosine type 1 receptor; AT1, angiotensin type 1 receptor; B2, bradykinin type 2 receptor; BRET, bioluminescence resonance energy transfer; D2, dopamine type 2 receptor; DOP, delta opioid peptide receptor; KOP, kappa opioid peptide receptor; M3, muscarinic acetylcholine type 3 receptor; mglu1, metabotropic glutamate type 1 receptor; MOP, mu opioid peptide receptor; pbFRET, photobleaching fluorescence resonance energy transfer; sst2A, somatostatin subtype 2A receptor.

Biophysical techniques

The question of whether GPCR dimers can be detected in living cells has been addressed using biophysical techniques such as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET)10–16. These techniques measure the transfer of energy from a donor molecule to an acceptor molecule when they are less than 50–100 Å apart10–16. In FRET, the acceptor fluorophore is excited with the light emitted by the donor fluorophore, which in turn is excited by an external light source; variants of green fluorescent protein (GFP) fused to the C-termini of GPCRs are generally used as donor and acceptor fluorophores10,11. In BRET, the acceptor (GFP) is excited by the light generated by luciferase activity12,13. Dimerization at the cell surface has been examined by BRET using hydrophilic agonists12,13 or by modified FRET using fluorescent antibodies directed to the extracellular region of the receptors14,15. A limitation of energy transfer techniques is that a distinction between the agonist-mediated increases in the level of dimers and the agonist-induced changes in receptor conformation cannot be made. Another concern is that the observed dimerization in heterologous cells could be due to overexpression of receptors in these cells. This concern has been addressed by expressing receptors at near endogenous levels; under these conditions dimerization has been observed using both biochemical and biophysical techniques11–16. Thus, it is likely that GPCR dimers occur in endogenous cells; however, it remains to be seen whether these energy transfer techniques could be used to detect dimerization in vivo. Advances

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in the biophysical techniques and the development of ligands labeled with distinct fluorophores that allow FRET analyses in tissues will be required to facilitate future investigations of GPCR dimers in vivo. Functional complementation techniques

A third technique used to study GPCR dimerization is functional complementation. In a provocative set of studies, Wess’s group17 addressed the physical interaction between GPCRs of two distinct subfamilies. These authors generated α2c-adrenoceptor and muscarinic acetylcholine M3 receptor chimeras [α2c(TMI–V)–M3(TMVI–VII) and M3(TMI–V)–α2c(TMVI–VII)], which were generated by exchanging TM domains VI and VII (Ref. 17). When transfected separately, these chimeras were unable to bind to their selective ligands17. However, when coexpressed, the receptors gained ligand binding and signaling properties. These studies provided the initial evidence for a physical interaction leading to changes in receptor pharmacology17. Functional complementation has also been used to examine interactions between somatostatin sst1 and sst5 receptors14. The wild-type sst1 receptor, which does not bind the ligand SMS-(201–995), was coexpressed with a C-terminal deletion mutant of the sst5 receptor, which binds this agonist but does not induce signaling14. Treatment of these coexpressing cells with SMS-(201–995) resulted in a dose-dependent decrease in the concentration of cAMP, which indicates that the agonist, by binding to sst5 receptors, is able to induce signaling that is mediated by the sst1 receptor present within the complex14. It appears that this interaction also plays a role in somatostatin receptor trafficking14. The sst1 receptor does not undergo agonist-mediated internalization whereas the sst5 receptor does. When sst1 and sst5 receptors were coexpressed, selective activation of sst5 receptors resulted in a decrease in the number of cell-surface sst1 receptors whereas selective activation of the sst1 receptor resulted in an increase in the number of cell-surface sst5 receptors14. These results underscore the importance of physical interactions in the modulation of receptor trafficking. A limitation of the complementation technique is that it does not directly demonstrate physical interaction but rather the interaction is inferred from functional complementation. As discussed above, each of the techniques (biochemical, biophysical and complementation) that are used to analyze GPCR dimers have strengths and limitations; a combination of these techniques needs to be used to delineate the properties of these dimers. Interactions between closely related members

The strongest evidence supporting an interaction between GPCRs that leads to modulation of receptor function has come from studies using the GABAB receptor, a member of GPCR family C (Refs 18–22). Efforts to clone the cDNA encoding the GABAB receptor resulted in the isolation of ‘GABAB(1a)’, which had a seven-TM topology. GABAB(1a) could not account http://tips.trends.com

for the functional activity of the native GABAB receptor. This led to an intense search and subsequent identification of a protein that would bind and functionally complement GABAB(1a). This protein, designated ‘GABAB(2)’, exhibited significant amino acid sequence homology to GABAB(1a), including the seven-TM topology. These proteins exhibited extensive colocalization in the CNS and their coexpression was found to be required for their surface expression. The two proteins could be co-precipitated using antisera directed against endogenous GABAB(1a) or GABAB(2) proteins, which supports the presence of heterodimers in vivo. The possibility that heterodimerization of these non-functional receptors leads to the generation of a functional receptor was tested by coexpression of GABAB(1a) with GABAB(2) (Refs 18–22). A crucial physiological effect mediated by native GABAB receptors is the activation of outward K+ currents through the opening of inwardly rectifying K+ channels. Coexpression of GABAB(1a) and GABAB(2) proteins resulted in a robust increase in K+ conductance via activation of the K+ channel in a pertussis-toxin-sensitive manner and an apparent shift (10–30-fold) in the affinity for agonists. These results provide strong support for a physical interaction between GABAB(1a) and GABAB(2) to generate functional GABAB receptors. There are several examples of significant pharmacological changes following coexpression of receptors (Table 2). As discussed above, GABAB receptors gain their ability to bind agonists with high affinity only when both receptor subtypes are coexpressed, which demonstrates that a functional site is formed from the interaction of both receptors. Similarly, interactions between muscarinic acetylcholine M2 and M3 receptors have been shown to lead to a new binding site with unique pharmacology23. Studies using opioid peptide and somatostatin receptors have also shown that receptor interactions can alter their pharmacological properties substantially7,14,24–26. For example, in cells that express both kappa opioid peptide (KOP) and delta opioid peptide (DOP) receptors, neither of these receptors exhibited high affinity for their respective selective ligands although they exhibited high-affinity binding to less selective ligands7. When exposed to a combination of two ligands, one that was selective for KOP receptors and one that was selective for DOP receptors, both receptors were able to bind the ligands with high affinity, which suggests that these receptors, when coexpressed, cooperatively bind selective ligands7. Interactions between mu opioid peptide (MOP) and DOP receptors also lead to altered pharmacology24,25. In cells expressing MOP and DOP receptors, both receptors exhibited a substantial decrease in affinity for several of their respective selective ligands24 and treatment of these cells with a combination of selective ligands led to a substantial increase in the number of binding sites25. Specifically, a DOP receptor

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Table 2. Modulation of function by G-protein-coupled receptor (GPCR) heteromerization Receptor dimers

Modulation of function

Closely related members of family A GABAB(1a)–GABAB(2) Restores agonist binding and functional coupling; required for surface expression KOP–DOP Decrease in selective agonist affinity and potency; a combination of ligands restores agonist affinity and potency; decrease in agonist-mediated endocytosis MOP–DOP Decrease in agonist affinity; switch to pertussis-toxininsensitive G proteins; increase in agonist potency with a combination of ligands sst1–sst5 Increase in agonist potency and efficacy; change in agonist-mediated endocytosis sst2A–sst3 Decrease in selective agonist affinity and potency; change in agonist-mediated endocytosis M2–M3 Change in selective agonist affinity Distantly related members of family A α2-adrenoceptor–M3 Restores high-affinity agonist binding sst5–D2 Increase in agonist affinity and potency with a combination of ligands AT1–B2 Increase in agonist efficacy and potency; switch to dynamin-dependent endocytosis DOP–β2-adrenoceptor Increase in agonist-mediated endocytosis KOP–β2-adrenoceptor Decrease in agonist-mediated endocytosis; decrease in MAPK signaling Members of family A and family C A1–mglu1 Alteration of signaling

Interactions between distantly related members Refs 18–22 7

24,25

14 26 23 17 15 27 28 28

29

aAbbreviations:

A1, adenosine type 1 receptor; AT1, angiotensin type 1 receptor; B2, bradykinin type 2 receptor; D2, dopamine type 2 receptor; DOP, delta opioid peptide receptor; KOP, kappa opioid peptide receptor; M2, muscarinic acetylcholine type 2 receptor; MAPK, mitogen-activated protein kinase; mglu1, metabotropic glutamate type 1 receptor; MOP, mu opioid peptide receptor; sst1, somatostatin subtype 1 receptor.

antagonist was able to increase the level of MOP receptor agonist-mediated signaling significantly in cells that express both receptors25. This increase was also observed in spinal cord membranes (where both MOP and DOP receptors are known to be colocalized), but not in membranes from mice that lack DOP receptors (I. Gomes et al., unpublished). Taken together, these results suggest that the synergistic binding of MOP and DOP receptor ligands leads to a significant increase in MOP receptor function and both receptors are required to mediate this effect. Studies using somatostatin sst3 and sst2A receptors have shown that coexpression of these receptors leads to differential effects on their ligand binding and signaling properties26. In cells that express both receptors the affinity for a selective sst3 receptor ligand was decreased 100-fold whereas the affinity for a selective sst2A receptor ligand remained unchanged26. Furthermore, there was no significant sst3-receptorinduced signaling in these cells whereas the signaling by sst2A receptors remained intact26. Thus, although in some cases the physical interaction between GPCRs leads to functional activation18–22 or enhanced functional activity14,24,25, in other cases26 such an interaction appears to lead to functional inactivation. The development of heterodimer selective ligands should facilitate further studies to delineate the novel pharmacology of these receptors. http://tips.trends.com

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The first set of studies to show an interaction between distantly related members of GPCR family A was reported by Rocheville and colleagues15. These authors used a photobleaching FRET (pbFRET) technique to examine the ability of the sst5 receptor to interact with the dopamine D2 receptor, and showed that exposure to either a selective sst5 receptor agonist or a selective D2 receptor agonist resulted in an increase in the level of dimers, which suggests that activation of one of the receptors in the complex was sufficient to mediate dimerization15. Ligand binding studies revealed that the sst5 and D2 receptors, in combination, exhibit a higher affinity for a combination of selective sst5 receptor agonists and selective D2 receptor agonists compared with a single agonist alone15. Furthermore, the combination of agonists resulted in an increase in the efficacy of signaling15. D2 receptors and sst5 receptors colocalize to the same neuronal subgroups in the striatum15; therefore, physical interactions between these two receptors is likely to be relevant physiologically. In an elegant set of studies, Quitterer’s group showed that the angiotensin II AT1 receptor, which couples to Gαi, can interact physically with the bradykinin B2 receptor, which couples to Gαq (Ref. 27). This interaction resulted in increased activation of both Gαq and Gαi proteins. Because angiotensin II is a vasopressor (i.e. increases vascular contractility and blood pressure) and bradykinin is a vasodepressor (i.e. decreases vascular contractility and blood pressure and thus serves as a functional antagonist of angiotensin II), interaction between the AT1 receptor and the B2 receptor would be physiologically important in the modulation of blood pressure. Quitterer and colleagues found that in cells coexpressing both receptors, the potency and efficacy of angiotensin II was increased and the potency and efficacy of bradykinin was decreased26. To test whether an AT1–B2 receptor interaction occurs in vivo, the effects of selective reduction in the level of B2 receptors (by antisense oligonucleotides) on AT1-receptor-mediated signaling was examined in smooth muscle cells that endogenously express both receptors27. A reduction in the number of B2 receptors resulted in a significant decrease in the angiotensinII-stimulated increase in intracellular Ca2+ without changes in the level of AT1 receptor protein, which supports an interaction between AT1 receptors and B2 receptors that could modulate angiotensin-IImediated signaling in vivo27. The endocytic pathway involved in receptor trafficking was also altered in cells coexpressing AT1–B2 receptor dimers. When expressed individually, AT1 and B2 receptors undergo agonist-mediated internalization via the dynaminindependent pathway. However, when coexpressed, both receptors underwent agonist-mediated internalization via the dynamin-dependent pathway because internalization of these receptors was blocked by a dominant-negative mutant of dynamin27.

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(a)

(b) Model 1

Model 2 Intracellular

ER

ER

Golgi

Golgi

Plasma membrane

Plasma membrane

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Fig. 1. Models proposed for the cellular compartment involved in G-protein-coupled receptor (GPCR) dimer and/or oligomer assembly. In model 1 GPCRs assemble as oligomeric arrays in the endoplasmic reticulum (ER), and are transported to the plasma membrane as oligomers. Agonist treatment has no effect on the level of oligomers. In model 2 GPCRs are synthesized as monomers that are transported to the plasma membrane as monomers. Agonist (green circles) treatment leads to an increase in the level of dimers and/or oligomers.

Two independent groups have examined whether β2-adrenoceptors that couple to the stimulatory G protein Gαs can heterodimerize with opioid receptors that couple to the inhibitory G protein Gαi (Refs 13,28). McVey et al. showed an interaction between β2-adrenoceptors and DOP receptors using the BRET assay13, whereas Jordan et al. provided biochemical evidence for the interaction between β2-adrenoceptors and DOP or KOP receptors28. Interestingly, the coexpressed receptors did not exhibit significant differences in their signaling properties compared with the individually expressed receptors28. However, the endocytic properties of the coexpressed receptors were significantly altered28. β2-Adrenoceptors coexpressed with DOP receptors underwent opioid-mediated endocytosis28. By contrast, β2-adrenoceptors coexpressed with KOP receptors failed to undergo isoproterenol-mediated endocytosis27. This loss of endocytosis was accompanied by a substantial decrease in the isoproterenol-induced phosphorylation of mitogen-activated protein kinases (MAPKs)27. These results, together with the fact that β2-adrenoceptors in cells that express KOP–β2 receptor dimers were not able to activate the MAPK pathway but retained the ability to activate the adenylyl cyclase pathway, suggests that the interaction between these GPCRs differentially affects their signal transduction pathways. http://tips.trends.com

In a recent study, Ciruela and colleagues29 examined the ability of a member of family A (i.e. adenosine A1 receptor) to interact with a member of family C (i.e. metabotropic glutamate mglu1 receptor)29. A1–mglu1 receptor dimers could be isolated from cerebellar neuronal cultures in addition to from heterologous cells transfected with differentially epitope-tagged receptors29. Furthermore, treatment of these cells with a combination of ligands led to a synergistic interaction at the level of signaling both in primary neurons and in heterologous cells29. There appears to be extensive colocalization of these two receptors in primary neurons, which suggests that this interaction could have functional relevance in vivo. From the studies discussed above it is clear that not only distantly related members of the same family (family A) of GPCRs physically interact with each other but members of two different families also interact and this interaction leads to changes in receptor function. GPCR dimer assembly Domains of the receptor involved in dimerization

An examination of the possible domains involved in GPCR interactions has implicated the extracellular, TM and/or C-terminal regions. The interaction could be mediated by covalent (disulfide) and/or noncovalent (ionic, hydrophobic) interactions of the N-terminal, TM and/or intracellular domains. Hydrophobic interactions within the TM domain are thought to provide the proper receptor conformation to facilitate the formation of additional interactions at other domains. An involvement of TM domains in receptor dimerization has been suggested in the case of β2-adrenoceptors and D2 receptors; disulfide bonds are not thought to be required for the dimerization of these receptors30,31. By contrast, disulfide-bonded interactions (in addition to ionic and hydrophobic interactions) are involved in the dimerization of the calcium-sensing receptor32 and the metabotropic glutamate mglu5 receptor33. Recent crystallographic studies with the extracellular domain of the mglu1 receptor have shown that this domain exists as a dimer and a conserved cysteine residue plays an important role in the dimerization of this domain34. Heterodimerization via C-terminal tail interactions has been documented in the case of the GABAB receptor18–22. Both GABAB(1a) and GABAB(2) contain a coiled-coil domain and this has been implicated in the dimerization of these receptors18–22. It is possible that multiple domains are involved in heterodimerization of GPCRs; future studies using crystallographic data will provide information on the mechanism of heterodimerization. Cellular compartment of GPCR dimerization

GPCR dimers could assemble either in the endoplasmic reticulum or at the cell surface (Fig. 1). For example, the receptors could be assembled as

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Acknowledgements I would like to thank Bryen Jordan and Ivone Gomes for critical reading of the manuscript and Clyde Scott for help with the illustrations. This work was supported in part by NIH grant K02 DA-00458.

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dimeric or multimeric units in the endoplasmic reticulum and transported to the cell surface as oligomeric arrays; agonist treatment would have no effect on the level of the oligomers. Alternatively, receptors could be synthesized as monomers and transported to the cell surface where they assemble to dimers and/or oligomers in an agonist-dependent fashion. From the evidence gathered thus far it appears that some GPCRs are assembled as oligomers in the endoplasmic reticulum whereas others assemble to oligomers at the cell surface. The GABAB(1a) receptor is poorly expressed at the cell surface in the absence of GABAB(2) and its surface expression is greatly enhanced in the presence of GABAB(2) (Ref. 35). These results suggest that GABAB receptors are assembled as dimers and/or oligomers in the endoplasmic reticulum35. By contrast, results from biophysical studies suggest that other GPCRs assemble as dimer and/or oligomers at the cell surface in an agonist-dependent fashion because there is an increase in the energy transfer signal in

References 1 Gether, U. (2000) Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocrinol. Rev. 21, 90–113 2 Bouvier, M. (2001) Oligomerization of G-proteincoupled transmitter receptors. Nat. Rev. Neurosci. 2, 274–286 3 Gomes, I. et al. (2001) G protein coupled receptor dimerization: implications in modulating receptor function. J. Mol. Med. 79 226–242 4 Birdsall, N.J.M. (1982) Can different receptors interact directly with each other? Trends Neurosci. 5, 138–139 5 Brady, L.S. and Devi, L.A., eds (2000) Dimerization of G-protein coupled receptors. In Neuropsychopharmacology Special Supplement Issue, Elsevier 6 Conn, P.M. et al. (1982) Conversion of a gonadotropin-releasing hormone antagonist to an agonist. Nature 296, 653–655 7 Jordan, B.A. and Devi, L.A. (1999) G-proteincoupled receptor heterodimerization modulates receptor function. Nature 399, 697–700 8 Zeng, F.Y. and Wess, J. (1999) Identification and molecular characterization of m3 muscarinic receptor dimers. J. Biol. Chem. 274, 19487–19497 9 Bai, M. et al. (1998) Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells. J. Biol. Chem. 273, 23605–23610 10 Overton, M.C. and Blumer, K.J. (2000) G-protein-coupled receptors function as oligomers in vivo. Curr. Biol. 10, 341–344 11 Cornea, A. et al. (2001) Gonadotropin releasing hormone microaggregation: rate monitored by fluorescence resonance energy transfer. J. Biol. Chem. 276, 2153–2158 12 Angers, S. et al. (2000) Detection of β2adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. U. S. A. 97, 3684–3689 http://tips.trends.com

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response to agonists11,14,15. However, this increase could also be due to changes in the conformation of preexisting dimers in response to agonists. Thus, the extent of agonist-induced increase in the level of dimers at the cell surface is not clear. Methods to carry out careful quantification and comparison of the strengths of the energy transfer signal are needed to help address this issue. In conclusion, heterodimerization of GPCRs could lead to changes in receptor function by modulating many of their properties (Table 2). The extent of modulation of function by GPCR heterodimerization in vivo is not known. Development of new tools (e.g. fluorescent ligands, dimer-selective ligands and dimer-selective antibodies) in addition to advances in technology (e.g. crystallography, fluorescence microscopy, and spectrometry) should facilitate significant progress in our understanding of the nature of GPCR dimers and their multiple functions, and also provide new strategies for the development of novel drug therapies.

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Chemical name SMS-(201–995): D-Phe-Cys-Phe=D-Tr-Lys-Thr-CysThr-ol