- Email: [email protected]
Heterodimerization of G-protein-coupled receptors in the CNS
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Heterodimerization of G-protein-coupled receptors in the CNS Fiona H Marshall Over the last year the combinations of G-protein-coupled receptors that are known to form heterodimeric complexes has rapidly increased. For example, dopamine receptors can dimerize with both somatostatin and adenosine receptors. These studies have been aided by improved technologies to monitor protein/protein interactions in living cells. Crosstalk at the level of the receptors might explain some of the known physiological interactions of these neurotransmitter systems and also provide new approaches for therapeutic intervention. Addresses Molecular Pharmacology Department, GlaxoWellcome Research and Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK; e-mail: [email protected] Current Opinion in Pharmacology 2001, 1:40–44 1471-4892/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations BRET bioluminescence resonance energy transfer CNS central nervous system GABA γ-aminobutyric acid GPCR G-protein-coupled receptor EGFR epidermal growth factor receptor FRET fluorescence resonance energy transfer SSTR somatostatin receptors TM transmembrane domain
Introduction The G-protein-coupled receptors (GPCRs) play a central role in cell–cell communication in the central nervous system (CNS). Through activation by a diverse range of neurotransmitter ligands, the receptors regulate the activity of effector molecules including ion channels, enzymes and transcription factors. Sequencing of the human genome has indicated the presence of around 500 non-sensory GPCRs of which greater than half are still to be paired with their cognate ligand. The GPCRs have a proven track record in drug discovery, with >40% of marketed drugs having activity mediated through this family of proteins. The expansion of the target class through bio-informatics is likely to yield substantial numbers of new drug targets for the future. Several mathematical models have been proposed to describe the activation of GPCRs and their coupling to G proteins; however, these models have generally assumed a stoichiometry of 1:1:1 with ligand, receptor and G protein. Over the past 10 years evidence has accumulated to suggest that GPCRs, like many other cell surface receptors [1], function as dimers or larger oligomers, and that this interaction is a fundamental and essential component of receptor signalling. More surprisingly, recent findings suggest that GPCRs can form heterodimers not only with closely related receptor subtypes but also with more distant GPCRs and even members of other protein classes.
Examples of receptors within the CNS that have been shown to dimerize are reviewed here. The implications of heterodimerization for our understanding of receptor signalling, their pharmacology and how they can be further exploited for drug discovery is discussed.
Homodimerization The evidence for GPCR homodimerization is now compelling [2,3]. Indeed, it seems likely that receptor dimers are the natural conformation for GPCRs and that most, if not all, members of this superfamily exist or can exist as dimers. Data to support this comes from a wide variety of sources. Molecular species corresponding to dimers can be clearly visualised following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and in some cases appear to be the major form of the protein. Physical interaction has been demonstrated by co-immunoprecipitation. More conclusive evidence comes from coexpression of mutant or chimeric receptors, which are non-functional when expressed alone but which generate binding and function when complementary chimeras that comprise the full seven transmembrane domains (TMs) of each receptor are coexpressed [4]. Further evidence comes from the fact that disruption of dimers with transmembrane peptides interferes with receptor activation [5]. More recently, biophysical studies with fluorescence resonance energy transfer (FRET) [6], sometimes combined with photobleaching FRET [7••] or bioluminescence resonance energy transfer (BRET) [8••] have enabled the study of dimerization in living cells. Computational analysis of receptor sequences has led to the proposal that transmembrane helices 5 and 6 form a major interface in GPCR dimers and that this is a common feature of all GPCRs. A simple model for dimerization is one of the contact dimer where two 7 TM helical bundles are adjacent to each other and in contact through helices 5 and 6. An alternative model that helps to explain the functional rescue of mutant receptors through dimerization [4] is the domain-swapped dimer whereby transmembrane domains 1–5 from one protein combine with TMs 6 and 7 of the other protein in the dimer to form the binding pockets [9,10•] (Figure 1). The key role of helices 5 and 6 is supported by studies showing inhibition of dimerization by peptides corresponding to TM helix 6 [5]. Whereas the concept of receptor dimerization for GPCRs is now well established, the formation, regulation and role of dimers in receptor signalling and trafficking is still not well understood. Several papers suggest that dimerization might be promoted by agonist activation of receptors and some have even considered that the dimer could represent the socalled high affinity or R* state of the receptor. More recent data suggest that dimers may be the basal form of receptors
Heterodimerization of G-protein-coupled receptors in the CNS Marshall
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Figure 1 Two models have been proposed to describe the formation of GPCR dimers: the domainswapped dimer model and the contact dimer model [9,10]. Domain-swapped dimers can be used to explain the data that functionally inactive receptors caused by mutation or by making receptor chimeras can be ‘rescued’ by coexpression with receptors or chimeras containing complementary sequences [4]. Contact dimers might be the most likely structure for heterodimer formation. (Reproduced by permission of Christopher A Reynolds).
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and that agonist binding may instead alter the overall conformation of the dimer. Co-immunoprecipitation of many receptors can occur in the absence of agonists [11••] and a BRET signal for the β2 receptor can also be measured in the absence of agonist stimulation [8••]. Furthermore, receptor dimerization may actually occur in the endoplasmic reticulum, and for some receptors may be required for correct folding and trafficking of the receptors to the cell surface [12,13••]. Although the majority of data suggest that dimers are the most common oligomeric form it should be recognised that larger multimeric units are also possible.
Heterodimerization between related receptor subtypes The first GPCR clearly demonstrated to be a heterodimer was the γ-aminobutyric acid (GABA)B receptor [14–18]. This complex differs from other GPCR heterodimers described to date in that each partner in the dimer, made up of GABAB(1) and GABAB(2), is non-functional when expressed alone. GABAB(1) contains an endoplasmic reticulum retention motif that prevents cell surface expression [13]. This motif is masked through an interaction with the related protein GABAB(2), allowing the complex to be expressed at the cell surface as a functional receptor capable of coupling to effector systems. Interestingly, removal of the retention motif on GABAB(1) allows cell surface expression but does not generate a functional receptor
suggesting that GABAB(2) not only acts as a chaperone to GABAB(1) but also plays a crucial role in coupling the receptor to G proteins and downstream effectors. Before the discovery of the GABAB heterodimer there had been much speculation in the field of opioid receptors that heterodimers between different receptors might exist. Three opioid receptor subtypes were clearly defined by receptor cloning; however, additional pharmacological subtypes were observed in certain systems [19] that could be explained by heterodimerization between the three cloned receptors. Further evidence came from the observations that in transgenic mice where a single receptor was knocked-out, effects on the pharmacology of other related opioid receptors were observed. For example in the MOR knockout changes were observed in analgesic responses to the highly selective δ agonist DPDPE (cyclic[D-penicillamine2,D-peniciallamine5]enkephalin). Clear evidence for heterodimerization between κ and δ opioid receptors was finally published by Jordan and Devi [11••] who showed that differentially epitope-tagged forms of κ receptors would co-immunoprecipitate δ, but not µ, receptors. In addition, heterodimerization of these receptors had important consequences for receptor internalization and pharmacology. Agonist-induced internalization of δ receptors by etorphine was reduced when the receptors were coexpressed with κ receptors. They also reported that
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selective κ and δ receptor agonists had reduced affinity for the heterodimeric receptor, but that a combination of selective agonists could act synergistically to activate mitogen-activated protein kinase phosphorylation. More recently, George et al. [20•] have demonstrated heterodimerization between µ and δ receptors. Again these studies showed that heterodimerization of receptors lead to a decreased affinity for selective agonists, whilst some endogenous peptides such a leu-enkephalin had higher affinity. Agonist activation is thought to lead to changes in receptor conformation, which results in the exposure of intracellular sites that activate specific G proteins. Receptor dimerization itself or agonist activation of receptor dimers might generate altered conformations with novel sites for G-protein activation. This may be seen as either an enhanced activation of G proteins or a difference in G-protein specificity. For example, heterodimerization of µ and δ receptors allows inhibition of adenylate cyclase in the presence of pertussis toxin, which does not occur with either receptor expressed alone. This is probably the result of the heterodimer being able to couple to pertussistoxin-insensitive G proteins, such as Gz. Taken together, these data suggest that a novel ligand-binding pocket and G-protein interface are formed within the heterodimer. There are now more and more examples of closely related GPCR receptors that are able to form heterodimers and this may indeed by a common property among receptor subtypes which are co-localised in the same cell. Another example is the serotonin 5-HT1D and 5-HT1B receptors. These two closely related receptors are coexpressed in some regions of the CNS. Interestingly, studies by Xie et al. [21•] show that although each of the receptors forms homodimers when expressed alone, when the receptors are coexpressed heterodimers, but not homodimers, are formed. The preference for heterodimeric complex formation has also been observed for both GABAB and opioid receptors. Somatostatin receptors (SSTR) form homodimers and heterodimers [22•]. Interestingly heterodimerization is specific between certain subtypes within the SSTR family and not a general phenomena. For example, SSTR5 can heterodimerize with SSTR1 but not SSTR4.
Heterodimerization between distinct GPCRs Heterodimerization also occurs between more distantly related GPCRs. The first clear example of this was the report that SSTR5 could heterodimerize with dopamine D2 receptors [7••]. This interaction is of particular interest in light of the well-documented physiological interactions of these two neurotransmitters. Both receptor types are expressed in high concentrations in the striatum and limbic structures and both transmitters interact to regulate locomotor activity. In the study by Rocheville et al. [22•], photo-bleaching FRET was used to study the interaction of the two receptors. This suggested that no pre-formed heterodimers existed in the absence of agonist and that treatment with either dopamine or somatostatin-14 resulted
in an increased FRET, indicative of dimer formation. Synergy was observed for the heterodimer in coupling to G proteins and to adenylate cyclase. More recently dopamine receptors, in this case the D1 subtype, have been shown to dimerize with adenosine A1 receptors [23••]. As with dopamine and somatostatin there is extensive literature on the interactions of these two transmitters in behavioural and biochemical studies. However, in this case the interaction appears to be one of antagonism rather than synergy, and this is backed up by studies of heterodimer signalling. In fibroblast cells co-transfected with A1 and D1 receptors, D1 signalling through Gs was reduced by pre-treatment with a mixture of A1 and D1 agonists but not by treatment with either one alone. As well as forming heterodimers, the receptors are able to form co-aggregates or clusters and there appears to be a complex relationship between the formation of dimers and co-aggregates. There are also several examples of receptors outside the CNS forming heterodimers, including angiotensin AT1 with bradykinin B2 in smooth muscle [24•] as well as the protease-activated receptors PAR3 and PAR4 in mouse platelets [25].
Heterodimerization of GPCRs and other proteins Heterodimerization of GPCRs with other membrane proteins has also been reported and adds another layer of complexity to the functioning of GPCRs in the membrane. The ligand specificity and pharmacology of members of the secretin family of GPCRs is determined by the interaction with a family of 1TM proteins called RAMPs (receptor activity modifying proteins) [26,27]. Another 1TM protein, calcyon, alters the coupling of dopamine D1 receptors from Gs to Gq [28]. GPCRs can also heterodimerize with other signalling proteins and this may provide a novel route to enable GPCRs to activate or inhibit other signal transduction pathways in the absence of G-protein coupling. The C-terminal tail of the dopamine D5 receptors has a direct physical association with the second intracellular loop of the GABAA γ2 receptor subunit, part of a ligand-gated chloride channel [29••]. Association of GABAA receptors with D5 receptors is dependent on coactivation of the two receptors. Once formed there is a codirectional inhibition of GABAA and D5 receptor function. This is likely to be of physiological relevance as injection of a mini-gene encoding the D5 C-terminal tail, which inhibited receptor interaction, also blocked D5 agonist inhibition of GABAA receptor-mediated miniature inhibitory postsynaptic currents (peak amplitude) in cultured hippocampal neurones. More recently a direct interaction has been found between a GPCR and a receptor tyrosine kinase. The β2 adrenergic receptor can induce activation of the epidermal growth factor receptor (EGFR) leading to EGFR dimerization and autophosphorylation. This process involves the formation of a multireceptor complex containing both the β2 receptor and the EGFR [30].
Heterodimerization of G-protein-coupled receptors in the CNS Marshall
Implications for pharmacology and drug discovery There is clear evidence that heterodimerization of GPCRs has profound effects on the pharmacology of natural ligands and that this is likely to be of physiological relevance. Crosstalk between receptors may underlie certain disease pathologies and, in doing so, provide an alternative approach for drug intervention. For example, in the treatment of Parkinson’s disease it may be of benefit to consider drugs directed at adenosine receptors rather than dopamine receptors [23••]. Heterodimerization, however, may also lead to unwanted side effects; for example, the SSTR agonist octreotide can cause side effects associated with modification of the dopaminergic system and these may be a result of effects of SSTR–dopamine heterodimers. The challenge for the pharmaceutical industry will be to understand the implication of receptor heterodimerization to the identification of new compounds acting at GPCRs and the optimisation of these compounds into drug candidates. Current screens for GPCRs usually express a single receptor subtype in a recombinant cell line. Such an assay system will of course prevent heterodimerization of the receptor that may occur in vivo. As a result, the pharmacology of the cell line may be quite distinct from the pharmacology of the receptor in its native state. It may be possible to harness heterodimerization for improved drug discovery. The heterodimeric receptor provides a target in its own right and it may be possible to develop compounds that are selective for the heterodimer over the single receptor components. One approach is to develop dimeric ligands that would coactivate the dimer or generate small molecules that could specifically disrupt dimer formation. In the case of the GABAB receptor compounds selective for the GABAB(2) subunit would be expected to have a very different profile in vivo to agonists at the GABAB(1) subunit due to the differential effects of endogenous GABA at the two subunits. Knowledge of heterodimerization needs to be applied to functional genomic strategies that are being used to study novel GPCRs. Receptor knockouts are a useful approach to identify the role of orphan GPCRs in physiology and disease. As discussed above for the opioid receptors, analysis of knockouts of a single receptor gene might have significant effects on other receptors with which that protein dimerizes. Many groups are currently searching for orphan GPCR ligands, primarily using single receptor systems. It is possible that some orphan receptors will not function in their own right but only exist as heterodimers with other receptors.
Conclusions The colocalization of receptors on individual cells within the CNS has been recognised for many years; however, the knowledge that these receptors may physically interact and, in doing so, alter their function is only starting to
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emerge. Dimerization has effects on receptor trafficking, G-protein coupling and thereby receptor signalling as well as receptor pharmacology. It is likely that over the next few years many other examples of receptor heterodimers will be identified, a process that will be enhanced by developing technologies in the study of protein/protein interactions. These discoveries might help us elucidate whether there is a common underlying mechanism for dimerization of GPCRs, how this is regulated by agonist activation, and also what role this plays in the subsequent signalling and internalization of receptors. In the future we may see new compounds designed to target receptor heterodimers.
Acknowledgements I thank Christopher Reynolds and Steve Foord for their comments on the manuscript and I also thank Christopher Reynolds for providing me with the Figure. I thank Jane Pelling for her excellent administrative assistance.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Heldin CH: Dimerization of cell surface receptors in signal transduction. Cell 1995, 80:213-223.
2.
Hebert TE, Bouvier M: Structural and functional aspects of G protein-coupled receptor oligomerization. Biochem Cell Biol 1998, 76:1-11.
3.
Salahpour A, Angers S, Bouvier M: Functional significance of oligomerization of G-protein coupled receptors. Trends Endocrinol Metab 2000, 11:163-168.
4.
Maggio R, Vogel Z, Wess J: Coexpression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular ‘cross-talk’ between G-protein-linked receptors. Proc Natl Acad Sci USA 1993, 90:3103-3107.
5.
Hebert TE, Moffett S, Morello JP, Loisel TP, Bichet DG, Barret C, Bouvier M: A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J Biol Chem 1996, 271:16384-16392.
6.
Overton MC, Blumer KJ: G protein coupled receptor function as oligomers in vivo. Curr Biol 2000, 10:341-344.
7. ••
Rocheville M, Lange DC, Kumar U, Patel SC, Patel RC, Patel YC: Receptors for dopamine and somatostatin: formation of heterooligomers with enhanced functional activity. Science 2000, 288:154-157. This paper was the first published example of heterodimerization between two distinct classes of GPCR. The paper uses the powerful technique of photo-bleaching FRET to study the dimerization process. 8. ••
Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M: Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 2000, 97:3684-3689. This paper describes the use of BRET to study receptor dimerization. 9.
Gouldson PR, Snell CR, Bywater RP, Higgs C, Reynolds CA: Domain swapping in G-protein coupled receptor dimers. Protein Eng 1998, 11:1181-1193.
10. Gouldson PR, Higgs C, Smith RE, Dean MK, Gkoutos GV, • Reynolds CA: Dimerization and domain swapping in G-protein coupled receptors: a computational study. Neuropsychopharmacology 2000, 23:560-577. This interesting paper discusses a number of potential models including domain swapping to explain the formation of receptor dimers. 11. Jordan BA, Devi LA: G protein coupled receptor •• heterodimerization modulates receptor function. Nature 1999, 399:697-700. This long-awaited paper was the first to show that opioid receptors could indeed form heterodimers and that this might explain some of the mismatch between in vivo pharmacology and cloned receptor pharmacology.
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12. Benkirane M, Jin DY, Chun RF, Koup RA, Jeang KT: Mechanism of transdominant inhibition of CCR5-mediated HIV-1 infection by ccr5delta32. J Biol Chem 1997, 272:30603-30606. 13. Mitrovic MM, Jan YN, Jan LY: A trafficking checkpoint controls •• GABAB receptor heterodimerization. Neuron 2000, 27:97-106. This paper describes the role of dimerization in masking an ER retention motif in the GABAB1 subunit. The paper also demonstrates that dimerization must occur in the ER and that GABAB(1) is non-functional in the absence of GABAB(2) even when it is expressed at the cell surface. 14. Jones KA, Borowsky B, Tamm JA, Craig DA, Durkin MM, Dai M, Yao WJ, Johnson M, Gunwaldsen C, Huang LY et al.: GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2. Nature 1998, 396:674-679.
22. Rocheville M, Lange DC, Kumar U, Sasi R, Patel RC, Patel YC: • Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers. J Biol Chem 2000, 275:7862-7869. This paper provides a good example of functional rescue of a mutant receptor through dimerization. 23. Gines S, Hillion J, Torvinen M, Le Crom S, Casado V, Canela EI, •• Rondin S, Lew JY, Watson S, Zoli M et al.: Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. Proc Natl Acad Sci USA 2000, 97:8606-8611. In this example of heterodimerization an additional layer of complexity is added with the finding that receptors can form co-clusters. This paper is also of interest because the relationship between the receptors in the heterodimer appears to be one of antagonism rather than synergy.
15. White JH, Wise A, Main MJ, Green A, Fraser NJ, Disney GH, Barnes AA, Emson P, Foord SM, Marshall FH: Heterodimerization is required for the formation of a functional GABA(B) receptor. Nature 1998, 396:679-682.
24. Abdalla S, Lother H, Quitterer U: AT-receptor heterodimers show • enhanced G-protein activation and altered receptor sequestration. Nature 2000, 407:94-98. Heterodimers between angiotensin and bradykinin receptor in smooth muscle lead to enhanced signalling through G proteins by angiotensin.
16. Kaupmann K, Malitschek B, Schuler V, Heid J, Froestl W, Beck P, Mosbacher J, Bischoff S, Kulik A, Shigemoto R et al.: GABA(B)receptor subtypes assemble into functional heteromeric complexes. Nature 1998, 396:683-687.
25. Nakanishi-Matsui M, Zheng YW, Sulciner DJ, Weiss EJ, Ludeman MJ, Coughlin SR: PAR3 is a cofactor for PAR4 activation by thrombin. Nature 2000, 404:609-613.
17.
Kuner R, Kohr G, Grunewald S, Eisenhardt G, Bach A, Kornau HC: Role of heteromer formation in GABAB receptor function. Science 1999, 283:74-77.
18. Ng GY, Clark J, Coulombe N, Ethier N, Hebert TE, Sullivan R, Kargman S, Chateauneuf A, Tsukamoto N, McDonald T et al.: Identification of a GABAB receptor subunit, gb2, required for functional GABAB receptor activity. J Biol Chem 1999, 274:7607-7610.
26. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM: RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998, 393:333-339. 27.
Christopoulos G, Perry KJ, Morfis M, Tilakaratne N, Gao Y, Fraser NJ, Main MJ, Foord SM, Sexton PM: Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product. Mol Pharmacol 1999, 56:235-242.
19. Kieffer BL: Opioids: first lessons from knockout mice. Trends Pharmacol Sci 1999, 20:19-26.
28. Lezcano N, Mrzljak L, Eubanks S, Levenson R, Goldman-Rakic P, Bergson C. Dual signaling regulated by calcyon, a D1 dopamine receptor interacting protein. Science 2000, 287:1660-1664.
20. George SR, Fan T, Xie Z, Tse R, Tam V, Varghese G, O’Dowd BF: • Oligomerization of µ and δ opioid receptors. Generation of novel functional properties J Biol Chem 2000, 275:26128-26135. Another interesting paper on opioid receptor dimerization — this time between µ and δ receptors. Of signficiance is the fact that some endogenous peptides have a higher affinity for the heterodimer than the individual receptors.
29. Liu F, Wan Q, Pristupa ZB, Yu XM, Wang YT, Niznik HB: Direct •• protein–protein coupling enables cross-talk between dopamine D5 and gamma-aminobutyric acid A receptors. Nature 2000, 403:274-280. This fascinating paper took the heterodimerization of GPCRs one step further by showing interactions with receptors from a completely different class – ligand-gated ion channels.
21. Xie Z, Lee SP, O’Dowd BF, George SR: Serotonin 5-HT1B and • 5-HT1D receptors form homodimers when expressed alone and heterodimers when co-expressed. FEBS Letters 1999, 456:63-67. An interesting example of homo- and heterodimerization between members of the 5-HT receptor family.
30. Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y, Lefkowitz RJ, Luttrell LM: The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J Biol Chem 2000, 275:9572-9580.
Heterodimerization of G-protein-coupled receptors in the CNS Fiona H Marshall Over the last year the combinations of G-protein-coupled receptors that are known to form heterodimeric complexes has rapidly increased. For example, dopamine receptors can dimerize with both somatostatin and adenosine receptors. These studies have been aided by improved technologies to monitor protein/protein interactions in living cells. Crosstalk at the level of the receptors might explain some of the known physiological interactions of these neurotransmitter systems and also provide new approaches for therapeutic intervention. Addresses Molecular Pharmacology Department, GlaxoWellcome Research and Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK; e-mail: [email protected] Current Opinion in Pharmacology 2001, 1:40–44 1471-4892/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations BRET bioluminescence resonance energy transfer CNS central nervous system GABA γ-aminobutyric acid GPCR G-protein-coupled receptor EGFR epidermal growth factor receptor FRET fluorescence resonance energy transfer SSTR somatostatin receptors TM transmembrane domain
Introduction The G-protein-coupled receptors (GPCRs) play a central role in cell–cell communication in the central nervous system (CNS). Through activation by a diverse range of neurotransmitter ligands, the receptors regulate the activity of effector molecules including ion channels, enzymes and transcription factors. Sequencing of the human genome has indicated the presence of around 500 non-sensory GPCRs of which greater than half are still to be paired with their cognate ligand. The GPCRs have a proven track record in drug discovery, with >40% of marketed drugs having activity mediated through this family of proteins. The expansion of the target class through bio-informatics is likely to yield substantial numbers of new drug targets for the future. Several mathematical models have been proposed to describe the activation of GPCRs and their coupling to G proteins; however, these models have generally assumed a stoichiometry of 1:1:1 with ligand, receptor and G protein. Over the past 10 years evidence has accumulated to suggest that GPCRs, like many other cell surface receptors [1], function as dimers or larger oligomers, and that this interaction is a fundamental and essential component of receptor signalling. More surprisingly, recent findings suggest that GPCRs can form heterodimers not only with closely related receptor subtypes but also with more distant GPCRs and even members of other protein classes.
Examples of receptors within the CNS that have been shown to dimerize are reviewed here. The implications of heterodimerization for our understanding of receptor signalling, their pharmacology and how they can be further exploited for drug discovery is discussed.
Homodimerization The evidence for GPCR homodimerization is now compelling [2,3]. Indeed, it seems likely that receptor dimers are the natural conformation for GPCRs and that most, if not all, members of this superfamily exist or can exist as dimers. Data to support this comes from a wide variety of sources. Molecular species corresponding to dimers can be clearly visualised following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and in some cases appear to be the major form of the protein. Physical interaction has been demonstrated by co-immunoprecipitation. More conclusive evidence comes from coexpression of mutant or chimeric receptors, which are non-functional when expressed alone but which generate binding and function when complementary chimeras that comprise the full seven transmembrane domains (TMs) of each receptor are coexpressed [4]. Further evidence comes from the fact that disruption of dimers with transmembrane peptides interferes with receptor activation [5]. More recently, biophysical studies with fluorescence resonance energy transfer (FRET) [6], sometimes combined with photobleaching FRET [7••] or bioluminescence resonance energy transfer (BRET) [8••] have enabled the study of dimerization in living cells. Computational analysis of receptor sequences has led to the proposal that transmembrane helices 5 and 6 form a major interface in GPCR dimers and that this is a common feature of all GPCRs. A simple model for dimerization is one of the contact dimer where two 7 TM helical bundles are adjacent to each other and in contact through helices 5 and 6. An alternative model that helps to explain the functional rescue of mutant receptors through dimerization [4] is the domain-swapped dimer whereby transmembrane domains 1–5 from one protein combine with TMs 6 and 7 of the other protein in the dimer to form the binding pockets [9,10•] (Figure 1). The key role of helices 5 and 6 is supported by studies showing inhibition of dimerization by peptides corresponding to TM helix 6 [5]. Whereas the concept of receptor dimerization for GPCRs is now well established, the formation, regulation and role of dimers in receptor signalling and trafficking is still not well understood. Several papers suggest that dimerization might be promoted by agonist activation of receptors and some have even considered that the dimer could represent the socalled high affinity or R* state of the receptor. More recent data suggest that dimers may be the basal form of receptors
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Figure 1 Two models have been proposed to describe the formation of GPCR dimers: the domainswapped dimer model and the contact dimer model [9,10]. Domain-swapped dimers can be used to explain the data that functionally inactive receptors caused by mutation or by making receptor chimeras can be ‘rescued’ by coexpression with receptors or chimeras containing complementary sequences [4]. Contact dimers might be the most likely structure for heterodimer formation. (Reproduced by permission of Christopher A Reynolds).
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and that agonist binding may instead alter the overall conformation of the dimer. Co-immunoprecipitation of many receptors can occur in the absence of agonists [11••] and a BRET signal for the β2 receptor can also be measured in the absence of agonist stimulation [8••]. Furthermore, receptor dimerization may actually occur in the endoplasmic reticulum, and for some receptors may be required for correct folding and trafficking of the receptors to the cell surface [12,13••]. Although the majority of data suggest that dimers are the most common oligomeric form it should be recognised that larger multimeric units are also possible.
Heterodimerization between related receptor subtypes The first GPCR clearly demonstrated to be a heterodimer was the γ-aminobutyric acid (GABA)B receptor [14–18]. This complex differs from other GPCR heterodimers described to date in that each partner in the dimer, made up of GABAB(1) and GABAB(2), is non-functional when expressed alone. GABAB(1) contains an endoplasmic reticulum retention motif that prevents cell surface expression [13]. This motif is masked through an interaction with the related protein GABAB(2), allowing the complex to be expressed at the cell surface as a functional receptor capable of coupling to effector systems. Interestingly, removal of the retention motif on GABAB(1) allows cell surface expression but does not generate a functional receptor
suggesting that GABAB(2) not only acts as a chaperone to GABAB(1) but also plays a crucial role in coupling the receptor to G proteins and downstream effectors. Before the discovery of the GABAB heterodimer there had been much speculation in the field of opioid receptors that heterodimers between different receptors might exist. Three opioid receptor subtypes were clearly defined by receptor cloning; however, additional pharmacological subtypes were observed in certain systems [19] that could be explained by heterodimerization between the three cloned receptors. Further evidence came from the observations that in transgenic mice where a single receptor was knocked-out, effects on the pharmacology of other related opioid receptors were observed. For example in the MOR knockout changes were observed in analgesic responses to the highly selective δ agonist DPDPE (cyclic[D-penicillamine2,D-peniciallamine5]enkephalin). Clear evidence for heterodimerization between κ and δ opioid receptors was finally published by Jordan and Devi [11••] who showed that differentially epitope-tagged forms of κ receptors would co-immunoprecipitate δ, but not µ, receptors. In addition, heterodimerization of these receptors had important consequences for receptor internalization and pharmacology. Agonist-induced internalization of δ receptors by etorphine was reduced when the receptors were coexpressed with κ receptors. They also reported that
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selective κ and δ receptor agonists had reduced affinity for the heterodimeric receptor, but that a combination of selective agonists could act synergistically to activate mitogen-activated protein kinase phosphorylation. More recently, George et al. [20•] have demonstrated heterodimerization between µ and δ receptors. Again these studies showed that heterodimerization of receptors lead to a decreased affinity for selective agonists, whilst some endogenous peptides such a leu-enkephalin had higher affinity. Agonist activation is thought to lead to changes in receptor conformation, which results in the exposure of intracellular sites that activate specific G proteins. Receptor dimerization itself or agonist activation of receptor dimers might generate altered conformations with novel sites for G-protein activation. This may be seen as either an enhanced activation of G proteins or a difference in G-protein specificity. For example, heterodimerization of µ and δ receptors allows inhibition of adenylate cyclase in the presence of pertussis toxin, which does not occur with either receptor expressed alone. This is probably the result of the heterodimer being able to couple to pertussistoxin-insensitive G proteins, such as Gz. Taken together, these data suggest that a novel ligand-binding pocket and G-protein interface are formed within the heterodimer. There are now more and more examples of closely related GPCR receptors that are able to form heterodimers and this may indeed by a common property among receptor subtypes which are co-localised in the same cell. Another example is the serotonin 5-HT1D and 5-HT1B receptors. These two closely related receptors are coexpressed in some regions of the CNS. Interestingly, studies by Xie et al. [21•] show that although each of the receptors forms homodimers when expressed alone, when the receptors are coexpressed heterodimers, but not homodimers, are formed. The preference for heterodimeric complex formation has also been observed for both GABAB and opioid receptors. Somatostatin receptors (SSTR) form homodimers and heterodimers [22•]. Interestingly heterodimerization is specific between certain subtypes within the SSTR family and not a general phenomena. For example, SSTR5 can heterodimerize with SSTR1 but not SSTR4.
Heterodimerization between distinct GPCRs Heterodimerization also occurs between more distantly related GPCRs. The first clear example of this was the report that SSTR5 could heterodimerize with dopamine D2 receptors [7••]. This interaction is of particular interest in light of the well-documented physiological interactions of these two neurotransmitters. Both receptor types are expressed in high concentrations in the striatum and limbic structures and both transmitters interact to regulate locomotor activity. In the study by Rocheville et al. [22•], photo-bleaching FRET was used to study the interaction of the two receptors. This suggested that no pre-formed heterodimers existed in the absence of agonist and that treatment with either dopamine or somatostatin-14 resulted
in an increased FRET, indicative of dimer formation. Synergy was observed for the heterodimer in coupling to G proteins and to adenylate cyclase. More recently dopamine receptors, in this case the D1 subtype, have been shown to dimerize with adenosine A1 receptors [23••]. As with dopamine and somatostatin there is extensive literature on the interactions of these two transmitters in behavioural and biochemical studies. However, in this case the interaction appears to be one of antagonism rather than synergy, and this is backed up by studies of heterodimer signalling. In fibroblast cells co-transfected with A1 and D1 receptors, D1 signalling through Gs was reduced by pre-treatment with a mixture of A1 and D1 agonists but not by treatment with either one alone. As well as forming heterodimers, the receptors are able to form co-aggregates or clusters and there appears to be a complex relationship between the formation of dimers and co-aggregates. There are also several examples of receptors outside the CNS forming heterodimers, including angiotensin AT1 with bradykinin B2 in smooth muscle [24•] as well as the protease-activated receptors PAR3 and PAR4 in mouse platelets [25].
Heterodimerization of GPCRs and other proteins Heterodimerization of GPCRs with other membrane proteins has also been reported and adds another layer of complexity to the functioning of GPCRs in the membrane. The ligand specificity and pharmacology of members of the secretin family of GPCRs is determined by the interaction with a family of 1TM proteins called RAMPs (receptor activity modifying proteins) [26,27]. Another 1TM protein, calcyon, alters the coupling of dopamine D1 receptors from Gs to Gq [28]. GPCRs can also heterodimerize with other signalling proteins and this may provide a novel route to enable GPCRs to activate or inhibit other signal transduction pathways in the absence of G-protein coupling. The C-terminal tail of the dopamine D5 receptors has a direct physical association with the second intracellular loop of the GABAA γ2 receptor subunit, part of a ligand-gated chloride channel [29••]. Association of GABAA receptors with D5 receptors is dependent on coactivation of the two receptors. Once formed there is a codirectional inhibition of GABAA and D5 receptor function. This is likely to be of physiological relevance as injection of a mini-gene encoding the D5 C-terminal tail, which inhibited receptor interaction, also blocked D5 agonist inhibition of GABAA receptor-mediated miniature inhibitory postsynaptic currents (peak amplitude) in cultured hippocampal neurones. More recently a direct interaction has been found between a GPCR and a receptor tyrosine kinase. The β2 adrenergic receptor can induce activation of the epidermal growth factor receptor (EGFR) leading to EGFR dimerization and autophosphorylation. This process involves the formation of a multireceptor complex containing both the β2 receptor and the EGFR [30].
Heterodimerization of G-protein-coupled receptors in the CNS Marshall
Implications for pharmacology and drug discovery There is clear evidence that heterodimerization of GPCRs has profound effects on the pharmacology of natural ligands and that this is likely to be of physiological relevance. Crosstalk between receptors may underlie certain disease pathologies and, in doing so, provide an alternative approach for drug intervention. For example, in the treatment of Parkinson’s disease it may be of benefit to consider drugs directed at adenosine receptors rather than dopamine receptors [23••]. Heterodimerization, however, may also lead to unwanted side effects; for example, the SSTR agonist octreotide can cause side effects associated with modification of the dopaminergic system and these may be a result of effects of SSTR–dopamine heterodimers. The challenge for the pharmaceutical industry will be to understand the implication of receptor heterodimerization to the identification of new compounds acting at GPCRs and the optimisation of these compounds into drug candidates. Current screens for GPCRs usually express a single receptor subtype in a recombinant cell line. Such an assay system will of course prevent heterodimerization of the receptor that may occur in vivo. As a result, the pharmacology of the cell line may be quite distinct from the pharmacology of the receptor in its native state. It may be possible to harness heterodimerization for improved drug discovery. The heterodimeric receptor provides a target in its own right and it may be possible to develop compounds that are selective for the heterodimer over the single receptor components. One approach is to develop dimeric ligands that would coactivate the dimer or generate small molecules that could specifically disrupt dimer formation. In the case of the GABAB receptor compounds selective for the GABAB(2) subunit would be expected to have a very different profile in vivo to agonists at the GABAB(1) subunit due to the differential effects of endogenous GABA at the two subunits. Knowledge of heterodimerization needs to be applied to functional genomic strategies that are being used to study novel GPCRs. Receptor knockouts are a useful approach to identify the role of orphan GPCRs in physiology and disease. As discussed above for the opioid receptors, analysis of knockouts of a single receptor gene might have significant effects on other receptors with which that protein dimerizes. Many groups are currently searching for orphan GPCR ligands, primarily using single receptor systems. It is possible that some orphan receptors will not function in their own right but only exist as heterodimers with other receptors.
Conclusions The colocalization of receptors on individual cells within the CNS has been recognised for many years; however, the knowledge that these receptors may physically interact and, in doing so, alter their function is only starting to
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emerge. Dimerization has effects on receptor trafficking, G-protein coupling and thereby receptor signalling as well as receptor pharmacology. It is likely that over the next few years many other examples of receptor heterodimers will be identified, a process that will be enhanced by developing technologies in the study of protein/protein interactions. These discoveries might help us elucidate whether there is a common underlying mechanism for dimerization of GPCRs, how this is regulated by agonist activation, and also what role this plays in the subsequent signalling and internalization of receptors. In the future we may see new compounds designed to target receptor heterodimers.
Acknowledgements I thank Christopher Reynolds and Steve Foord for their comments on the manuscript and I also thank Christopher Reynolds for providing me with the Figure. I thank Jane Pelling for her excellent administrative assistance.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Heldin CH: Dimerization of cell surface receptors in signal transduction. Cell 1995, 80:213-223.
2.
Hebert TE, Bouvier M: Structural and functional aspects of G protein-coupled receptor oligomerization. Biochem Cell Biol 1998, 76:1-11.
3.
Salahpour A, Angers S, Bouvier M: Functional significance of oligomerization of G-protein coupled receptors. Trends Endocrinol Metab 2000, 11:163-168.
4.
Maggio R, Vogel Z, Wess J: Coexpression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular ‘cross-talk’ between G-protein-linked receptors. Proc Natl Acad Sci USA 1993, 90:3103-3107.
5.
Hebert TE, Moffett S, Morello JP, Loisel TP, Bichet DG, Barret C, Bouvier M: A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J Biol Chem 1996, 271:16384-16392.
6.
Overton MC, Blumer KJ: G protein coupled receptor function as oligomers in vivo. Curr Biol 2000, 10:341-344.
7. ••
Rocheville M, Lange DC, Kumar U, Patel SC, Patel RC, Patel YC: Receptors for dopamine and somatostatin: formation of heterooligomers with enhanced functional activity. Science 2000, 288:154-157. This paper was the first published example of heterodimerization between two distinct classes of GPCR. The paper uses the powerful technique of photo-bleaching FRET to study the dimerization process. 8. ••
Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M: Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 2000, 97:3684-3689. This paper describes the use of BRET to study receptor dimerization. 9.
Gouldson PR, Snell CR, Bywater RP, Higgs C, Reynolds CA: Domain swapping in G-protein coupled receptor dimers. Protein Eng 1998, 11:1181-1193.
10. Gouldson PR, Higgs C, Smith RE, Dean MK, Gkoutos GV, • Reynolds CA: Dimerization and domain swapping in G-protein coupled receptors: a computational study. Neuropsychopharmacology 2000, 23:560-577. This interesting paper discusses a number of potential models including domain swapping to explain the formation of receptor dimers. 11. Jordan BA, Devi LA: G protein coupled receptor •• heterodimerization modulates receptor function. Nature 1999, 399:697-700. This long-awaited paper was the first to show that opioid receptors could indeed form heterodimers and that this might explain some of the mismatch between in vivo pharmacology and cloned receptor pharmacology.
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12. Benkirane M, Jin DY, Chun RF, Koup RA, Jeang KT: Mechanism of transdominant inhibition of CCR5-mediated HIV-1 infection by ccr5delta32. J Biol Chem 1997, 272:30603-30606. 13. Mitrovic MM, Jan YN, Jan LY: A trafficking checkpoint controls •• GABAB receptor heterodimerization. Neuron 2000, 27:97-106. This paper describes the role of dimerization in masking an ER retention motif in the GABAB1 subunit. The paper also demonstrates that dimerization must occur in the ER and that GABAB(1) is non-functional in the absence of GABAB(2) even when it is expressed at the cell surface. 14. Jones KA, Borowsky B, Tamm JA, Craig DA, Durkin MM, Dai M, Yao WJ, Johnson M, Gunwaldsen C, Huang LY et al.: GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2. Nature 1998, 396:674-679.
22. Rocheville M, Lange DC, Kumar U, Sasi R, Patel RC, Patel YC: • Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers. J Biol Chem 2000, 275:7862-7869. This paper provides a good example of functional rescue of a mutant receptor through dimerization. 23. Gines S, Hillion J, Torvinen M, Le Crom S, Casado V, Canela EI, •• Rondin S, Lew JY, Watson S, Zoli M et al.: Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. Proc Natl Acad Sci USA 2000, 97:8606-8611. In this example of heterodimerization an additional layer of complexity is added with the finding that receptors can form co-clusters. This paper is also of interest because the relationship between the receptors in the heterodimer appears to be one of antagonism rather than synergy.
15. White JH, Wise A, Main MJ, Green A, Fraser NJ, Disney GH, Barnes AA, Emson P, Foord SM, Marshall FH: Heterodimerization is required for the formation of a functional GABA(B) receptor. Nature 1998, 396:679-682.
24. Abdalla S, Lother H, Quitterer U: AT-receptor heterodimers show • enhanced G-protein activation and altered receptor sequestration. Nature 2000, 407:94-98. Heterodimers between angiotensin and bradykinin receptor in smooth muscle lead to enhanced signalling through G proteins by angiotensin.
16. Kaupmann K, Malitschek B, Schuler V, Heid J, Froestl W, Beck P, Mosbacher J, Bischoff S, Kulik A, Shigemoto R et al.: GABA(B)receptor subtypes assemble into functional heteromeric complexes. Nature 1998, 396:683-687.
25. Nakanishi-Matsui M, Zheng YW, Sulciner DJ, Weiss EJ, Ludeman MJ, Coughlin SR: PAR3 is a cofactor for PAR4 activation by thrombin. Nature 2000, 404:609-613.
17.
Kuner R, Kohr G, Grunewald S, Eisenhardt G, Bach A, Kornau HC: Role of heteromer formation in GABAB receptor function. Science 1999, 283:74-77.
18. Ng GY, Clark J, Coulombe N, Ethier N, Hebert TE, Sullivan R, Kargman S, Chateauneuf A, Tsukamoto N, McDonald T et al.: Identification of a GABAB receptor subunit, gb2, required for functional GABAB receptor activity. J Biol Chem 1999, 274:7607-7610.
26. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM: RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998, 393:333-339. 27.
Christopoulos G, Perry KJ, Morfis M, Tilakaratne N, Gao Y, Fraser NJ, Main MJ, Foord SM, Sexton PM: Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product. Mol Pharmacol 1999, 56:235-242.
19. Kieffer BL: Opioids: first lessons from knockout mice. Trends Pharmacol Sci 1999, 20:19-26.
28. Lezcano N, Mrzljak L, Eubanks S, Levenson R, Goldman-Rakic P, Bergson C. Dual signaling regulated by calcyon, a D1 dopamine receptor interacting protein. Science 2000, 287:1660-1664.
20. George SR, Fan T, Xie Z, Tse R, Tam V, Varghese G, O’Dowd BF: • Oligomerization of µ and δ opioid receptors. Generation of novel functional properties J Biol Chem 2000, 275:26128-26135. Another interesting paper on opioid receptor dimerization — this time between µ and δ receptors. Of signficiance is the fact that some endogenous peptides have a higher affinity for the heterodimer than the individual receptors.
29. Liu F, Wan Q, Pristupa ZB, Yu XM, Wang YT, Niznik HB: Direct •• protein–protein coupling enables cross-talk between dopamine D5 and gamma-aminobutyric acid A receptors. Nature 2000, 403:274-280. This fascinating paper took the heterodimerization of GPCRs one step further by showing interactions with receptors from a completely different class – ligand-gated ion channels.
21. Xie Z, Lee SP, O’Dowd BF, George SR: Serotonin 5-HT1B and • 5-HT1D receptors form homodimers when expressed alone and heterodimers when co-expressed. FEBS Letters 1999, 456:63-67. An interesting example of homo- and heterodimerization between members of the 5-HT receptor family.
30. Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y, Lefkowitz RJ, Luttrell LM: The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J Biol Chem 2000, 275:9572-9580.