Báo cáo khoa học: The impact of G-protein-coupled receptor hetero-oligomerization on function and pharmacology pptx

MINIREVIEW The impact of G-protein-coupled receptor hetero-oligomerization on function and pharmacology Roberto Maggio 1 , Francesca Novi 1 , Marco Scarselli 2 and Giovanni U. Corsini 1 1 Department of Neurosciences, University of Pisa, Italy 2 National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, USA G-protein coupled receptors (GPCRs) constitute the largest family of seven-transmembrane receptors. Their evolutionary success is due to their extreme versatility in binding a variety of signaling molecules such as hor- mones and neurotransmitters. The ubiquitous distribu- tion in the human body, along with the capacity to regulate virtually all known physiological processes, has made this family of receptors the most important target for drug research [1]. According to the classical view of hormone–recep- tor interaction, a hormone binds to one receptor protein and, in turn, the hormone–receptor complex activates the effector pathway. A large body of evi- dence has led us to question this classical view of hormone–receptor interaction, for it is now widely accepted that GPCRs may exist as either homo- dimers or even higher-order homo-oligomers, besides being capable of interacting with distantly related receptor subtypes to form hetero-oligomers (reviewed in [2,3]). The huge interest generated by this phenomenon among biologists in the last 10 years has led many groups to study the mechanism(s) by which GPCR dimerization occurs. This has contributed to the dem- onstration that many, if not all, GPCRs can form homo-oligomers and hetero-oligomers, but it has also generated much pharmacological and functional evi- dence that is difficult to reconcile with a unique mechan- istic model of GPCR dimerization. A key problem that still remains controversial is how dimerization affects G-protein coupling. Although GPCR homo-oligo- merization can be accounted for by a simple receptor ⁄ G-protein stoichiometry, GPCR hetero-oligomerization raises the problem of how two different receptors can influence the coupling of each other and determine the ultimate function of the complex. Keywords bivalent ligand; G-protein; mitogen-activated protein kinase (MAPK); oligomerization; b-arrestin Correspondence R. Maggio, Department of Neurosciences, University of Pisa, Via Roma 55, 56100 Pisa, Italy Fax: +39 050 2218717 Tel: +39 050 2218707 E-mail: r.maggio@drugs.med.unipi.it (Received 16 February 2005, revised 7 April 2005, accepted 21 April 2005) doi:10.1111/j.1742-4658.2005.04729.x Although highly controversial just a few years ago, the idea that G-pro- tein-coupled receptors (GPCRs) may undergo homo-oligomerization or hetero-oligomerization has recently gained considerable attention. The recognition that GPCRs may exhibit either dimeric or oligomeric structures is based on a number of different biochemical and biophysical approaches. Although much effort has been spent to demonstrate the mechanism(s) by which GPCRs interact with each other, the physiological relevance of this phenomenon remains elusive. An additional source of uncertainty stems from the realization that homo-oligomerization and hetero-oligomerization of GPCRs may affect receptor binding and activity in different ways, depending on the type of interacting receptors. In this brief review, the functional and pharmacological effects of the hetero-oligomerization of GPCR on binding and cell signaling are critically analyzed. Abbreviations GPCR, G-protein-coupled receptor; LTB 4 , leukotriene B 4 ; MAPK, mitogen-activated protein kinase. FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS 2939 Effect of hetero-oligomerization on G-protein coupling and function To make the issue even more complicated, hetero-oligo- merization has been shown to occur between pairs of receptors that couple with either the same G-protein or different G-proteins. On the assumption that each receptor in the hetero-oligomer may bind only to a sin- gle G-protein, it follows that its coupling selectivity in the complex should, in large part, be conserved. As a matter of fact, several reports that deal with receptor hetero-oligomerization indicate that stimulation of one receptor in cotransfected cells is often sufficient to activate a G-protein, leaving their coupling efficacy unchanged. For instance, b 2 -adrenergic receptors, which are coupled with stimulatory G-proteins, and d-opioid and j-opioid receptors, which are coupled with inhibitory G-proteins, are both known to form hetero- meric complexes, but hetero-oligomerization in this case does not significantly alter their ligand-binding capacity or coupling properties [4]. Likewise, adenylate cyclase stimulation by G s -coupled dopamine D 1 recep- tors and adenylate cyclase inhibition by the G i -coupled dopamine D 2 receptors are not altered in cells coex- pressing both receptors, even though they may form hetero-oligomers [5]. The same phenomenon has been shown to occur in hetero-oligomers formed by G i -cou- pled and G q -coupled receptors [6] and by G s -coupled and G q -coupled receptors [7]. One of the limitations implied in these experiments is the actual impossibility of establishing with certainty how many receptors undergo hetero-oligomerization compared with those that give rise to homo-oligomeric complexes or even remain in a monomeric form. Under these conditions, if the molar ratio between hetero-oligomers and homo- oligomers (or monomers) falls below a certain thresh- old, the effect of hetero-oligomerization would remain undetected, and the occurrence of any functional change in the target cells would be difficult to ascertain experimentally. At odds with the above examples are other reports describing how changes in function or coupling efficacy may simply result from the stimula- tion of one or both receptors of the hetero-oligomer. For example, coexpression of dopamine D 2 and somato- statin SSTR 5 receptors results in synergistic inhibition of adenylate cyclase [8]. Similarly, coexpression of angiotensin I and bradykinin B 2 receptors in HEK-293 cells increases the efficacy and potency of angiotensin II, but it also reduces the ability of bradykinin to sti- mulate inositol phosphate production [9]. In fibro- blasts, pretreatment of A 1 and D 1 receptors with both adenosine and dopamine agonists, but not with either of them separately, has been shown to reduce the signaling efficacy of D 1 receptors on subsequent stimu- lation [10]. In COS-7 cells cotransfected with dopamine D 2 and D 3 receptors, highly selective D 3 agonists inhi- bit adenylate cyclase, but remain ineffective in cells transfected with D 3 alone [11,12]. However, the role played by hetero-oligomerization in each of these func- tional changes remains speculative, as the same effects may also be induced by cross-talk between the signa- ling pathways, downstream of receptor activation. The acquisition of new coupling selectivity by coex- pressed receptors is perhaps one of the most intri- guing aspects of GPCR hetero-oligomerization. Three major studies have shown this phenomenon clearly: (a) l-receptors and d-receptors that changed their coup- ling selectivity from pertussis-sensitive G i ⁄ G o -proteins to pertussis-insensitive (probably G z ) proteins, in transi- ently cotransfected COS-7 cells [13]; (b) chemokine CCR 2b and CCR 5 receptors that gained coupling selec- tivity for G 11 -protein in cotransfected HEK-293 cells [14]; (c) dopamine D 1 and D 2 receptors that gained coupling selectivity for G q -proteins in transiently cotransfected COS-7 cells [5]. In the last instance, if each dopamine receptor is stimulated separately with select- ive agonists, their coupling selectivity for G i and G s is not altered, whereas simultaneous dopamine stimulation of both receptors results in the activation of G q . This observation can be taken to mean that cells may gain a new coupling selectivity when the two components of the receptor hetero-oligomer are activated simulta- neously. Assuming that each receptor in the hetero-oligomer can bind only to single G-proteins, then any new coup- ling selectivity gained by the hetero-oligomer is likely to depend on a newly acquired spatial rearrangement of the intracellular domain(s) that binds to these G-proteins. This conclusion is not unexpected, as seve- ral studies have shown that receptors that activate spe- cific G-proteins can be induced to expose distinct intracellular domains if stimulated by different agonists [15]. The conformational changes that result from receptor–receptor interactions may in fact cause vari- ation in the exposure of certain intracellular domains and, in doing so, alter the specificity of their inter- action. Another possible explanation of this change in coupling selectivity comes from recent work with receptor homodimers. Using a combination of mass spectrometry after chemical cross-linking and neutron scattering in solution, Baneres & Parello [16] have been able to establish unambiguously that only one G-pro- tein trimer binds to a leukotriene B 4 (LTB 4 ) receptor BLT1 dimer (2·BLT1.LTB 4 ) so as to form a stoichio- metrically defined (2·BLT1.LTB 4 )Ga i2 b 1 c 2 pentameric assembly. They suggested that receptor dimerization Function and pharmacology of hetero-oligomers R. Maggio et al. 2940 FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS may play a crucial role in transducing the LTB 4 - induced signal. Similar conclusions were drawn in a recent paper by Chinault et al. [17], who demonstrated that yeast oligomeric a-factor receptors function in concert to activate G-proteins. Further support for the 2 : 1 receptor ⁄ G-protein coupling stoichiometry comes from experiments performed with receptor fragments. Single transmembrane regions of b 2 or dopamine D 2 receptors prevent dimerization and stop functioning when cotransfected with their cognate wild-type recep- tors, indicating that disruption of their dimeric com- plex impedes these receptors to couple with G-proteins [18,19]. If these results prove valid for heterodimers as well, they could explain how heterodimerization affects receptor coupling selectivity. Whereas homodimers provide pairs of identical intracellular domains, het- erodimers have unique combinations of intracellular domains. This could confer a different coupling effi- ciency and selectivity on the heterodimers compared with that expressible by the homodimers of their respective receptors. Hetero-oligomerization affects b-arrestin coupling and internalization GPCR activation promotes recruitment of b-arrestin to the receptor site. This leads to signal termination by blocking G-protein interaction and it triggers receptor internalization by endocytosis. A large amount of evidence has now accumulated indicating that hetero- oligomerization influences b-arrestin binding and receptor internalization. This is clearly described in the excellent paper by Terrillon et al. [20]. V1a and V2 vasopressin receptors are internalized by way of the b-arrestin-dependent process. However, whereas V1a receptors are rapidly recycled to the plasma membrane after dissociation from b-arrestin, V2 receptors do not dissociate from b-arrestin and consequently accumulate in the endosomes. In their paper, Terrillon et al. [20] demonstrated that, in cotransfected HEK cells, V1a and V2 receptors are endocytosed as stable hetero-olig- omers. Upon activation with nonselective agonists, the V1a ⁄ V2 hetero-oligomer follows the endocytic ⁄ recyc- ling pathway of the V2 receptor up to the endosomes. Conversely, the hetero-oligomer is targeted to the endo- cytic ⁄ recycling pathway of V1a receptor if activated with a selective V1a agonist. In the latter case, the hetero-oligomer is rapidly recycled to the plasma mem- brane. This work clearly indicates that it is the identity of the activated promoter within the hetero-oligomer that determines the fate of the internalized receptors. Other examples of the reciprocal influence of receptors in the internalization process are the adrenergic a 1a and a 1b receptors [21], neurokinin NK 1 and l opioid receptors [22], and the b 2 -adrenergic and d-opioid receptors [4]. In all these studies, selective stimulation of a single receptor component of the hetero-oligomer is sufficient to cause internalization of the entire complex. As discussed in the previous section, a critical issue in assessing the effects of hetero-oligomerization is to establish the extent by which GPCRs tend to hetero- oligomerize. To account for the above results one would have to suppose that a large fraction of the receptors expressed in the plasma membrane are already in a hetero-oligomeric form. However, this is made unlikely by the observation that j-opioid receptors exhibit a higher propensity to form homo-oligomers than b 2 -adrenergic and j-opioid receptors to form hetero-oligomers [23]. Based on this observation, it may be reasonable to think that, in cells coexpressing two receptors, most of them are in a homo-oligomeric form. In spite of this indication, however, internalizat- ion of b 2 -adrenergic receptors, as induced by isopro- terenol, is impeded in the presence of j-opioid receptors [4]. To explain these puzzling data, j-opioid and b 2 -receptor homo-dimers could be assumed to be part of a larger hetero-oligomeric array such as even the smallest fraction of this receptor complex could actu- ally affect functioning of the entire cluster. The idea that receptors may co-operate within larger aggregates has been put forward by Park et al. [24] on the basis of radioligand binding to muscarinic M 2 receptors. Muscarinic cholinergic receptors can appear to be more numerous when labeled with [ 3 H]quinuclidinyl- benzilate than with N-[ 3 H]methylscopolamine. Binding at near-saturating concentrations of [ 3 H]quinuclidinyl- benzilate was blocked fully by unlabeled N-methyl- scopolamine, which therefore appeared to inhibit noncompetitively at sites inaccessible to N-[ 3 H]methyl- scopolamine. Both the shortfall in capacity for N-[ 3 H]methylscopolamine and the noncompetitive effect of N-methylscopolamine on [ 3 H]quinuclidinylbenzilate has been described quantitatively in terms of co-opera- tive interactions within a receptor that is at least tetra- valent. Besides their effects on dampening receptor–G-pro- tein coupling and on receptor internalization, b-arres- tin also plays a major role in GPCR activation of mitogen-activated protein kinase (MAPK). In this con- text it may act as an adaptor or scaffolding for recruit- ing signaling molecules into a complex along with the agonist-occupied receptors (for a review see [25]). The first evidence of this effect has been provided by Luttrell et al. [26], who showed that agonist phos- phorylation of b 2 -adrenergic receptors leads to rapid R. Maggio et al. Function and pharmacology of hetero-oligomers FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS 2941 recruitment of b-arrestin-1, carrying the activated receptor c-Src with it. Subsequent reports showed that b-arrestins can also interact directly with component kinases of the ERK1 ⁄ 2 and c-Jun N-terminal kinase 3 MAPK cascades. b-Arrestins have been shown to form complexes with angiotensin II type 1A receptor, cRaf-1 and ERK1 ⁄ 2 [27,28], with protease-activated receptor type 2, Raf-1 and ERK1 ⁄ 2 [29], and with neurokinin-1 receptor, c-Src and ERK1 ⁄ 2 [30]. That hetero-oligomerization may interfere with b-arrestin-mediated signaling is demonstrated by the observation of Lavoie et al. [31] that activation of b-arrestin-mediated ERK1 ⁄ 2 phosphorylation by b 2 -adrenergic receptors is inhibited on coexpression of b 1 -adrenergic receptors. They suggested that hetero- oligomerization between b 1 and b 2 receptors may inhi- bit the agonist-promoted b 2 ability to activate the ERK1 ⁄ 2 signaling pathway. In another paper, Breit et al. [32] showed that hetero-oligomerization of b 3 -adrenergic receptors with b 2 -receptors modified their effect on ERK1 ⁄ 2 phosphorylation. Novi et al. [6,33] showed that a mutant muscarinic M 3 receptor that is incapable of binding to b-arrestin-1 impairs completely the ability of wild-type M 3 receptors to recruit b-arrestin-1 to the plasma membrane and to stimulate ERK1 ⁄ 2 phosphorylation. All these data indicate quite clearly that homo-oligomerization and hetero-oligomerization have a pivotal role in defining the GPCR–b-arrestin specificity, and consequently in determining the receptor fate and b-arrestin-mediated MAPK activation. As discussed above for G-proteins, the mechanism and the stoichiometry by which GPCR and b-arrestin interact is not known. Conventionally, GPCRs and b-arrestins are assumed to interact in a 1 : 1 molar ratio. However, this conventional view should be reconsidered on the basis of more recent GPCR hetero-oligomerization data and be replaced by a more complex model of interaction between the two pro- teins. For example, two b-arrestin molecules may bind to a receptor dimer, and this may in turn cause more efficient sequestration and signaling of the GPCR– b-arrestin complex. Han et al. [34] proposed a mechan- istic model of b-arrestin–receptor interaction in which the initial binding of the first b-arrestin molecule to the receptor is followed by displacement of its terminal C-tail and dimerization with another b-arrestin mole- cule. They speculated that b-arrestin dimerization may help the b-arrestin–receptor complexes to cope with the internalization machinery of the coated pits. How- ever, the possibility that dimerization of b-arrestin also acts as a scaffold for MAPK was left open, given the molecular dimensions of the complexes containing both b-arrestin and MAPK [27,29,30]. Another possi- bility yet to be considered is that a b-arrestin monomer may bind to a receptor dimer so that the resulting receptor combination reinforces the bond strength of the heterodimers. This hypothesis is based on a recent study on the organization of rhodopsin in native plasma membranes [35]. Arrestin, the cognate b-arres- tin of the visual system, has a bipartite structure with two structurally homologous seven-stranded b-sand- wiches forming two putative rhodopsin-binding grooves separated by 3.8 nm [36,37]. This spatial arrangement may mean that the rhodopsin dimer sur- face matches perfectly the arrestin molecule by charge complementarity. A cartoon showing the hypothetical mechanisms of receptor–b-arrestin interaction and ERK1 ⁄ 2 signaling is shown in Fig. 1. Regardless of the mechanism by which b-arrestins bind to GPCRs, the signaling pathway activated by these proteins is another way by which GPCR hetero- oligomerization can influence cell physiology. In view of the fact that MAPK plays a pivotal role in such cell processes as cell growth, division, differentiation and apoptosis, it is likely that, in the near future, the phar- macology of GPCR hetero-oligomers can be exploited to gain control of these cellular events. Pharmacological diversity In the last 6 years, a growing number of receptors have been shown to behave as hetero-oligomers and to exhibit an unexpected level of pharmacological diver- sity. Jordan & Devi [38] presented the first evidence that the pharmacology of interacting receptors is dif- ferent from that of the constituent monomers (or homodimers). They showed that j–d-opioid hetero- oligomers had no significant affinity for either j-selective or d-selective agonists or antagonists in cotransfected cells, even though the hetero-oligomers had a stronger affinity for the partially selective lig- ands. Following this pivotal work, many other researchers have shown how ligand affinity changes on receptor coexpression [39–41]. In all these studies, the extent by which ligand affinity changes is accounted for by a single parameter determined by competition binding analysis. It should be clear that this parameter does not provide a realistic measure of the ligand affin- ity for the hetero-oligomer. At most, it may represent an average measure of all the different affinities that the ligand expresses for the binding site(s) of both het- ero-oligomers and homo-oligomers. Quite often, in the absence of detailed analyses, it is not possible to estab- lish which binding fractions can be attributed to the hetero-oligomer and which to the homo-oligomer(s). Function and pharmacology of hetero-oligomers R. Maggio et al. 2942 FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS Under these circumstances, if two GPCRs exhibit an equivalent propensity to form either homo-oligomer or hetero-oligomer, only 50% of the receptor population would be in the hetero-oligomeric form. This percent- age would be even lower if the tendency to form hetero-oligomers is significantly different. The pharmacological changes that occur within the hetero-oligomers are most likely due to allosteric rear- rangements induced by the interacting receptor mono- mers. Ligand binding to half of the dimer may somehow modify the affinity for the other half. This view is supported by the work of Mesnier & Baneres [42] with the LTB 4 receptor BLT1 homodimer. By studying how fluorescence properties of 5-hydroxy- tryptophan vary, these authors have been able to show that agonist binding to part of the LTB 4 receptor BLT1 homodimers induces conformational changes in the remaining part of the homodimer. Although not generally accepted, another possible explanation for how receptor pharmacology may change, at least among receptor subtypes, is domain swapping [43–45]. According to this model of interaction, two receptors may interact in such a way as to induce rearrangement of their transmembrane domains, and this would even- tually result in the formation of two novel binding sites. So far, domain swapping has been shown to occur only among functionally impaired receptors and never with wild-type receptors. This may be because of the technical complexity of devising experiments to observe the effect of domain swapping when both receptors are functional. The oligomeric nature of GPCRs can be exploited to improve drug specificity by developing dimeric ligands capable of acting as bivalent ligands. The first publica- tion showing the feasibility of constructing a bivalent ligand directed to heterodimeric receptors has come AB C No effect No effect ERK activation ERK activation ERK activation H HH HH HH H H HH H H HH HH H GPCR β-arrestin β-arrestin β-arrestin β-arrestin β-arrestin β-arrestin β-arrestin GPCR GPCR GPCR GPCR GPCR GPCR GPCR GPCR GPCR GPCR GPCR GPCR GPCR GPCR GPCR GPCR GPCR Fig. 1. Alternative models of GPCR–b-arres- tin interaction. (A) The sequential binding of the ligands to each half of the receptor dimer induces the recruitment of two molecules of b-arrestin and then the activation of ERK. (B) Only one molecule of b-arrestin binds to the ligand-saturated receptor dimer and activates ERK. (C) A dimer of b-arrestin binds all at once to a receptor dimer and activates ERK. Fig. 2. Proposed models of association of bivalent ligands with GPCR hetero-oligomers. (A) Bivalent ligands bind pairs of receptor hetero-dimers. (B) Bivalent ligands bridge two different subtypes of neighboring receptor homodimers. R. Maggio et al. Function and pharmacology of hetero-oligomers FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS 2943 from Saveanu and coworkers [46]. The chimeric agonist they synthesized comprises a somatostatin–dopamine molecule (BIM-23A387) directed against the dopamine D 2 and somatostatin SST 2 receptors. They claimed that this agonist suppresses secretion of both growth hor- mone and prolactin in human pituitary somatotrophic adenoma cells (each cell coexpressing both dopamine D 2 and somatostatin SST 2 receptors) much more powerfully than either of the two pharmacophores given all at once or separately. Portoghese’s group [47] has more recently synthesized the ligand KDN-21, which belongs to a series of bivalent ligands containing d-opioid and j-opioid antagonist pharmacophores attached to variable-length spacers. This compound has been shown to have substantially greater affinity for d-opioid and j-opioid receptors than the univalent ana- logs. Furthermore, this compound had 200-fold higher affinity for cotransfected d-opioid and j-opioid recep- tors than for the same receptors transfected separately and then allowed to interact. To understand the mech- anism by which these bivalent ligands work, the struc- tural organization of GPCR oligomers in the plasma membrane needs to be clarified. The models of bivalent ligand–receptor interaction proposed in Fig. 2 foresee two possible oligomeric organizations for GPCRs. The possibility of developing ligands that are select- ive for hetero-oligomeric GPCRs is the most promising strategy yet for targeting different tissues of the human body. Screening for drugs that would be so selective as to restrict binding to hetero-oligomer receptors in the presence of the corresponding homo-oligomers is the real challenge for scientists working in this field in the near future. With these selective drugs at hands, we will be able to shed new light on the physiological role played by receptor hetero-oligomerization. Acknowledgements We thank Dr Franco Giorgi for advice and helpful discussion. References 1 Lefkowitz RJ (2004) Historical review: a brief history and personal retrospective of seven-transmembrane receptors. Trends Pharmacol 25, 413–422. 2 Agnati LF, Ferre S, Lluis C, Franco R & Fuxe K (2003) Molecular mechanisms and therapeutical implica- tions of intramembrane receptor ⁄ receptor interaction among heptahelical receptors with examples from stri- atopallidal GABA neurons. Pharmacol Rev 55, 509–550. 3 Angers S, Salahpour A & Bouvier M (2002) Dimeriza- tion: an emerging concept for G protein-coupled receptor ontogeny and function. Annu Rev Pharmacol Toxicol 42, 409–435. 4 Jordan BA, Trapaidze N, Gomes I, Nivarthi R & Devi LA (2001) Oligomerization of opioid receptors with beta2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation. Proc Natl Acad Sci USA 98, 343–348. 5 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. 6 Novi F, Scarselli M, Corsini GU & Maggio R (2004) The paired activation of the two components of the muscarinic M3 receptor dimer is required for induction of ERK1 ⁄ 2 phosphorylation. J Biol Chem 279, 7476– 7486. 7 Terrillon S, Durroux T, Mouillac B, Breit A, Ayoub MA, Taulan M, Jockers R, Barberis C & Bouvier M (2003) Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during bio- synthesis. Mol Endocrinol 17, 677–691. 8 Rocheville M, Lange DC, Kumar U, Patel SC, Patel RC & Patel YC (2000) Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Science 288, 154–157. 9 AbdAlla S, Lother H & Quietterer U (2000) AT 1 -recep- tor heterodimers show enhanced G-protein activation and altered receptor sequestration. Nature 407, 94–98. 10 Gine ´ s S, Hillion J, Torvinen M, LeCrom S, Casado V, Canela E, Rondin S, Lew J, Watson S, Zoli M, et al. (2000) Dopamine D1 and adenosine A1 receptors assemble into functionally interacting heteromeric com- plexes. Proc Natl Acad Sci USA 97, 8606–8611. 11 Scarselli M, Novi F, Schallmach E, Lin R, Baragli A, Colzi A, Griffon N, Corsini GU, Sokoloff P, Levenson R, et al. (2001) D 2 ⁄ D 3 dopamine receptor heterodimers exhibit unique functional properties. J Biol Chem 276, 30308–30314. 12 Maggio R, Scarselli M, Novi F, Millan MJ & Corsini GU (2003) Potent activation of dopamine D3 ⁄ D2 heterodimers by the antiparkinsonian agents, S32504, pramipexole and ropinirole. J Neurochem 87, 631–641. 13 George SR, Fan T, Xie Z, Tse R, Tam V, Varghese G & O’Dowd BF (2000) Oligomerization of l- and d-opi- oid receptors. Generation of novel functional properties. J Biol Chem 275, 26128–26135. 14 Mellado M, Rodriguez-Frade JM, Vila-Coro AJ, Fernandez S, Martin de Ana A, Jones DR, Toran JL & Martinez AC (2001) Chemokine receptor homo- or heterodimerization activates distinct signaling pathways. EMBO J 20, 2497–2507. 15 Allouche S, Polastron J, Hasbi A, Homberger V & Jauzac P (1999) Differential G-protein activation by alkaloid and peptide opioid agonists in the human Function and pharmacology of hetero-oligomers R. Maggio et al. 2944 FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS neuroblastoma cell line SK-N-BE. Biochem J 342, 71–78. 16 Baneres JL & Parello J (2003) Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein. J Mol Biol 329 , 815–829. 17 Chinault SL, Overton MC & Blumer KJ (2004) Sub- units of a yeast oligomeric G protein-coupled receptor are activated independently by agonist but function in concert to activate G protein heterotrimers. J Biol Chem 279, 16091–16100. 18 Hebert TE, Moffett S, Morello JP, Loisel TP, Bichet DG, Barret C & Bouvier M (1996) A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activa- tion. J Biol Chem 271, 16384–16392. 19 George SR, Ng GY, Lee SP, Fan T, Varghese G, Wang C, Deber CM, Seeman P & O’Dowd BF (2003) Block- ade of G protein-coupled receptors and the dopamine transporter by a transmembrane domain peptide: novel strategy for functional inhibition of membrane proteins in vivo. J Pharmacol Exp Ther 307, 481–489. 20 Terrillon S, Barberis C & Bouvier M (2004) Hetero- dimerization of V1a and V2 vasopressin receptors deter- mines the interaction with beta-arrestin and their trafficking patterns. Proc Natl Acad Sci USA 101, 1548–1553. 21 Stanasila L, Perez JB, Vogel H, Cotecchia S, Stanasila L, Perez JB, Vogel H & Cotecchia S (2003) Oligomeri- zation of the alpha 1a- and alpha 1b-adrenergic receptor subtypes. Potential implications in receptor internaliza- tion. J Biol Chem 278, 40239–40251. 22 Pfeiffer M, Kirscht S, Stumm R, Koch T, Wu D, Laugsch M, Schroder H, Hollt V & Schulz S (2003) Heterodimerization of substance P and mu-opioid recep- tors regulates receptor trafficking and resensitization. J Biol Chem 278, 51630–51637. 23 Ramsay D, Kellett E, McVey M, Rees S & Milligan G (2002) Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells moni- tored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences. Biochem J 365, 429–440. 24 Park PS, Sum CS, Pawagi AB & Wells JW (2002) Cooperativity and oligomeric status of cardiac muscari- nic cholinergic receptors. Biochemistry 41 , 5588–5604. 25 Luttrell LM & Lefkowitz RJ (2002) The role of beta- arrestins in the termination and transduction of G-pro- tein-coupled receptor signals. J Cell Sci 115, 455–465. 26 Luttrell LM, Ferguson SSG, Daaka Y, Miller WE, Maudsley S, Della Rocca GJ, Lin FT, Kawakatsu H, Owada K, Luttrell DK, et al. (1999) Beta-arrestin- dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science 283, 655–661. 27 McDonald PH, Chow C-W, Miller WE, Laporte SA, Field ME, Lin F-T, Davis RJ & Lefkowitz RG (2000) Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3. Science 290, 1574–1577. 28 Tohgo A, Pierce KL, Choy EW, Lefkowitz RJ & Luttrell LM (2002) beta-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity but inhibits ERK-mediated transcription following angiotensin AT1a receptor stimulation. J Biol Chem 277, 9429–9436. 29 DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD & Bunnett NW (2000) beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1 ⁄ 2. J Cell Biol 148, 1267–1281. 30 DeFea KA, Vaughn ZD, O’Bryan EM, Nishijima D, Dery O & Bunnett NW (2000) The proliferative and antiapoptotic effects of substance P are facilitated by formation of a beta-arrestin-dependent scaffolding com- plex. Proc Natl Acad Sci USA 97, 11086–11091. 31 Lavoie C, Mercier JF, Salahpour A, Umapathy D, Breit A, Villeneuve LR, Zhu WZ, Xiao RP, Lakatta EG, Bouvier M, et al. (2002) Beta 1 ⁄ beta 2-adrenergic recep- tor heterodimerization regulates beta 2-adrenergic recep- tor internalization and ERK signaling efficacy. J Biol Chem 277, 35402–35410. 32 Breit A, Lagace M & Bouvier M (2004) Hetero-oligo- merization between beta2- and beta3-adrenergic recep- tors generates a beta-adrenergic signaling unit with distinct functional properties. J Biol Chem 279, 28756– 28765. 33 Novi F, Stanasila L, Giorgi F, Corsini GU, Cotecchia S & Maggio R (2005) Paired activation of the two compo- nents within muscarinic M3 receptor dimers is required for recruitment of b-arrestin-1 to the plasma membrane. J Biol Chem 280 , 19768–19776. 34 Han M, Gurevich VV, Vishnivetskiy SA, Sigler PB & Schubert C (2001) Crystal structure of beta-arrestin at 1.9 A ˚ : possible mechanism of receptor binding and membrane translocation. Structure 9, 869–880. 35 Liang Y, Fotiadis D, Filipek S, Saperstein DA, Palczewski K & Engel A (2003) Organization of the G protein-coupled receptors rhodopsin and opsin in native membranes. J Biol Chem 278, 21655–21662. 36 Granzin J, Wilden U, Choe HW, Labahn J, Krafft B & Buldt G (1998) X-ray crystal structure of arrestin from bovine rod outer segments. Nature 391, 918–921. 37 Hirsch JA, Schubert C, Gurevich VV & Sigler PB (1999) The 2.8 A ˚ crystal structure of visual arrestin: a model for arrestin’s regulation. Cell 97, 257–269. 38 Jordan BA & Devi LA (1999) G-protein-coupled recep- tor heterodimerization modulates receptor function. Nature 399, 697–700. 39 Cheng ZJ, Harikumar KG, Holicky EL & Miller LJ (2003) Heterodimerization of type A and B R. Maggio et al. Function and pharmacology of hetero-oligomers FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS 2945 cholecystokinin receptors enhance signaling and promote cell growth. J Biol Chem 278, 52972–52979. 40 Xu J, He J, Castleberry AM, Balasubramanian S, Lau AG & Hall RA (2003) Heterodimerization of alpha 2A- and beta 1-adrenergic receptors. J Biol Chem 278, 10770–10777. 41 Grant M, Patel. RC & Kumar U (2004) The role of subtype-specific ligand binding and the C-tail domain in dimer formation of human somatostatin receptors. J Biol Chem 279 , 38636–38643. 42 Mesnier D & Baneres JL (2004) Cooperative conforma- tional changes in a GPCR dimer, the leukotriene B4 receptor BLT1. J Biol Chem September 9 [Epub ahead of print]. 43 Maggio R, Vogel Z & Wess J (1993) Coexpression studies with mutant muscarinic ⁄ adrenergic receptors provide evi- dence for intermolecular ‘cross-talk’ between G-protein- linked receptors. Proc Natl Acad Sci USA 90, 3103–3107. 44 Monnot C, Bihoreau C, Conchon S, Curnow KM, Corvol P & Clauser E (1996) Polar residues in the trans- membrane domains of the type 1 angiotensin II receptor are required for binding and coupling. Reconstitution of the binding site by co-expression of two deficient mutants. J Biol Chem 271, 1507–1513. 45 Bakker RA, Dees G, Carrillo JJ, Booth RG, Lopez- Gimenez JF, Milligan G, Strange PG & Leurs R (2004) Domain swapping in the human histamine H1 receptor. J Pharmacol Exp Ther 311, 131–138. 46 Saveanu A, Lavaque E, Gunz G, Barlier A, Kim S, Taylor JE, Culler MD, Enjalbert A & Jaquet P (2002) Demonstration of enhanced potency of a chimeric somatostatin-dopamine molecule, BIM-23A387, in sup- pressing growth hormone and prolactin secretion from human pituitary somatotroph adenoma cells. J Clin Endocrinol Metab 87, 5545–5552. 47 Bhushan RG, Sharma SK, Xie Z, Daniels DJ & Por- toghese PS (2004) A bivalent ligand (KDN-21) reveals d spinal and j opioid receptors are organized as hetero- dimers that give rise to d1 and j2 phenotypes. Selective targeting of d–j heterodimers. J Med Chem 47, 2969– 2972. Function and pharmacology of hetero-oligomers R. Maggio et al. 2946 FEBS Journal 272 (2005) 2939–2946 ª 2005 FEBS . ways, depending on the type of interacting receptors. In this brief review, the functional and pharmacological effects of the hetero-oligomerization of GPCR on binding. MINIREVIEW The impact of G-protein-coupled receptor hetero-oligomerization on function and pharmacology Roberto Maggio 1 , Francesca Novi 1 , Marco Scarselli 2 and