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Pharmacology & Therapeutics 118 (2008) 359–371 Contents lists available at ScienceDirect Pharmacology & Therapeutics j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h a r m t h e r a Associate editor: A. Christopoulos G protein-coupled receptor dimers: Functional consequences, disease states and drug targets Matthew B. Dalrymple, Kevin D.G. Pfleger ⁎, Karin A. Eidne Laboratory for Molecular Endocrinology - GPCRs, Western Australian Institute for Medical Research (WAIMR) and Centre for Medical Research, University of Western Australia, Nedlands, Perth, WA 6009, Australia A R T I C L E I N F O A B S T R A C T Keywords: GPCR Dimerisation Disease Drug targets Allosterism Abbreviations: A R, Adenosine receptor subtype 2a; AT R, Angiotensin-II receptor subtype 1; β AR, 1 2a Beta-2 adrenergic receptor; B R, Bradykinin-2 2 receptor; BRET, Bioluminescence resonance 2 energy transfer; Ca , Calcium ions; CaSR, 2+ Calcium-sensing receptor; CB1, Cannabinoid receptor subtype 1; CRLR, Calcitoninreceptor-like receptor; D R, Dopamine-2 receptor; DOP, Delta-opioid receptor; ER, 2 Endoplasmic reticulum; ERK, Extracellular signal-regulated kinases; ECD, Extracellular domain; 5-HT, 5-hydroxytryptamine (serotonin); FRET, Fluorescence resonance energy transfer; GABA Rs, Gamma-aminobutyric acid receptors; GnRHR, GonaB dotrophin-releasing hormone receptor; GPCRs, G protein-coupled receptors; HH, Hypogonadotropic hypogonadism; KOP, Kappaopioid receptor; MAPKs, Mitogen-activated protein kinases; mGluRs, Metabotropic glutamate receptors; MOP, Mu-opioid receptor; ORs, Olfactory receptors; OxR1, Orexin receptor subtype 1; RAMPs, Receptor activity-modifying proteins; WT, Wild type. With an ever-expanding need for reliable therapeutic agents that are highly effective and exhibit minimal deleterious side effects, a greater understanding of the mechanisms underlying G protein-coupled receptor (GPCR) regulation is fundamental. GPCRs comprise more than 30% of all therapeutic drug targets and it is likely that this will only increase as more orphan GPCRs are identified. The past decade has seen a dramatic shift in the prevailing concept of how GPCRs function, in particular the growing acceptance that GPCRs are capable of interacting with one another at a molecular level to form complexes, with significantly different pharmacological properties to their monomeric selves. While the ability of like-receptors to associate and form homodimers raises some interesting mechanistic issues, the possibility that unlike-receptors could heterodimerise in certain tissue types, producing a functionally unique signalling complex that binds specific ligands, provides an invaluable opportunity to refine and redefine pharmacological interventions with greater specificity and efficacy. © 2008 Elsevier Inc. All rights reserved. Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . G protein-coupled receptor homo/heterodimerisation 2.1. Agonist-induced dimerisation . . . . . . . . 2.2. Constitutive dimerisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 0 0 360 0 360 0 361 ⁎ Corresponding author. Western Australian Institute for Medical Research (WAIMR), B-Block, QEII Medical Centre, Hospital Ave, Perth, Western Australia, 6009, Australia. Tel.: +61 8 9346 3591; fax: +61 8 9346 1818. E-mail address: kpfl[email protected] (K.D.G. Pfleger). 0163-7258/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2008.03.004 360 M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371 3. 4. Role of dimerisation in G protein-coupled receptor synthesis and maturation Functional relevance of G protein-coupled receptor heterodimerisation . . . 4.1. Chemokine receptors. . . . . . . . . . . . . . . . . . . . . . . . 4.2. Adenosine-2a and metabotropic glutamate-5 receptors . . . . . . . 4.3. Opioid receptors. . . . . . . . . . . . . . . . . . . . . . . . . . 5. Pharmacological rescue of misrouted G protein-coupled receptors . . . . . . 6. Allosteric modulation of G protein-coupled receptor activity . . . . . . . . 6.1. Receptor activity-modifying proteins (RAMPs) . . . . . . . . . . . . 6.2. Dimerisation-mediated G protein-coupled receptor allosterism . . . 7. Dimerisation and disease pathogenesis. . . . . . . . . . . . . . . . . . . 7.1. Preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Parkinson's disease . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Hypogonadotropic hypogonadism. . . . . . . . . . . . . . . . . . 7.4. Schizophrenia and psychosis . . . . . . . . . . . . . . . . . . . . 8. Heterodimer-specific drugs . . . . . . . . . . . . . . . . . . . . . . . . 9. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction GPCRs are heptahelical membrane proteins capable of transducing extracellular signals via heterotrimeric G proteins that elicit downstream responses through second-messenger generation. While GPCRs are classified into several different families (for a review see Bockaert & Pin, 1999), all share the same basic structure consisting of an extracellular N-terminus, 3 intracellular and extracellular loops, 7 transmembrane domains that are highly conserved and a cytoplasmic C-terminus. Despite their architectural homology, GPCRs are activated by a vast array of stimuli, including light photons, odorants, neurotransmitters, calcium ions (Ca2+), peptides and hormones. Analysis of the nematode Caenorhabditis elegans genome indicated that as many as 5% of all genes code for GPCRs (Bargmann, 1998) and it has been suggested that this could correlate with up to 5000 GPCRs in mammals (Marchese et al., 1999). More recent studies of sequences from the human genome project have shown that the total number of genes encoding GPCRs exceeds 950, with at least half of these comprised of taste and odorant receptors (Takeda et al., 2002). In spite of the concerted efforts of numerous research groups, the only GPCRs thus far to have a 3-dimensional crystal structure defined are the Family A receptors rhodopsin (Palczewski et al., 2000) and the beta-2 adrenergic receptor (Rasmussen et al., 2007). Members belonging to Family B GPCRs lack the distinctive DRY motif (AspArg-Tyr) that characterises most Family A GPCRs, exhibit a large extracellular N-terminus that contains numerous disulfide bridges, and includes the corticotrophin-releasing factor and growth hormone-releasing hormone receptors. Family C GPCRs include the metabotropic glutamate receptors (mGluRs), the γ-aminobutyric acid receptors (GABABRs) and calcium-sensing receptor (CaSR), that again exhibit structural properties distinct from those of the Family A GPCRs (see Parmentier et al., 2002), most notable of which are a shortened third intracellular loop and a large, bi-lobed N-terminus that is thought to contain the ligand-binding domain. The original GPCR signalling paradigm proposed a 1:1:1 stoichiometry, with a monomeric GPCR binding a single, specific ligand and initiating a downstream signalling cascade via a single heterotrimeric G protein subunit. While this made logical sense, it is now believed that this model alone cannot explain the complexity of receptor signalling. The notion of GPCR dimerisation was first conceived more than 20 years ago, with researchers utilising a bivalent antibody with a gonadotrophin-releasing hormone (GnRH) antagonist attached at each end. Their data implied that this ‘conjugated’ antagonist might be capable of acting as an agonist that promoted “microaggregates” of GnRH receptor (GnRHR) leading to biphasic regulation of receptor surface expression (Conn et al., 1982a, 1982b). Another early study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 0 362 0 362 0 363 0 363 0 364 0 0 366 366 0 0 366 0 366 367 0 367 0 0 367 0 368 0 368 368 0 0 369 0 369 indicated that GPCRs were capable of forming functional complexes, using a series of chimeric adrenergic/muscarinic receptors to illustrate functional rescue of receptor signalling upon coexpression by transcomplementation of mutant receptors (Maggio et al., 1993). Regardless of these early indications that GPCRs could interact at a molecular level, constituting a new hierarchy of receptor signalling, it was only towards the end of the millennium that acceptance of the concept began to expand. In fact, direct physical evidence for GPCR dimerisation only became available in 2003, following atomic-force microscopy analysis of the rhodopsin receptor that clearly showed neat rows of rhodopsin dimers arranged within retinal disc membranes (Fotiadis et al., 2003; Fig. 1). The term dimer is often used interchangeably with oligomer in the literature, as current methodologies are often unable to differentiate the two. Consequently, for the sake of avoiding confusion during this review, the term ‘dimer’ will be used to refer to any receptor complex consisting of more than one GPCR. The tremendous growth of knowledge regarding GPCR dimerisation over the past few years has been reviewed extensively in a number of recent articles (Bouvier, 2001; Dean et al., 2001; Kroeger et al., 2003; Bai, 2004; Milligan, 2004; Terrillon & Bouvier, 2004; Milligan, 2006). 2. G protein-coupled receptor homo/heterodimerisation A facet of GPCR dimerisation research that remains contentious is whether GPCRs inherently exist in dimeric complexes, or whether dimers are induced by the presence or absence of receptor ligands. 2.1. Agonist-induced dimerisation Several studies utilising bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) analysis of GPCR dimerisation, have shown that the extent of dimerisation may be dependent on ligand activation or significantly modulated in the presence of ligand (Angers et al., 2000; Cheng & Miller, 2001; Kroeger et al., 2001). FRET studies of GnRHR dimerisation found that the addition of agonist but not antagonist led to a significant increase in the FRET signal intensity (Cornea et al., 2001), indicative of a ligand-specific, protein–protein interaction that was not observed in the absence of agonist (Horvat et al., 2001). The reverse may also hold true in some instances, with the addition of ligand resulting in the apparent dissociation of receptor dimers and a loss of BRET signal (Cheng & Miller, 2001). However, due to some of the inherent limitations of resonance energy transfer methods, interpretation of results from such assays requires careful consideration. A number of recent reviews address some of the issues that must be considered when using such techniques M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371 361 Fig. 1. Organisation and topography of the cytoplasmic surface of rhodopsin. (a) Topograph obtained using atomic-force microscopy, showing the paracrystalline arrangement of rhodopsin dimers in the native disc membrane. Inset, arcs in the calculated powder diffraction pattern reflect the regular arrangement of rhodopsin in the membrane. (b) Angularly averaged powder diffraction pattern, showing peaks at (8.4 nm)−1, (4.2 nm)−1 and (3.8 nm)−1. (c) Magnification of a region of the topograph in a, showing rows of rhodopsin dimers, as well as individual dimers (inside dashed ellipse), presumably broken away from one of the rows, and occasional rhodopsin monomers (arrowheads). The rhodopsin molecules protrude from the lipid bilayer by 1.4 ± 0.2 nm (n = 111). The topograph in (c) is shown in relief, tilted by 5°. Vertical brightness ranges: 1.6 nm (a and c). Scale bars: a, 50 nm; inset, (5 nm)−1; c, 15 nm (from Fotiadis et al., 2003 with permission, © 2003 Nature). (Bouvier, 2001; Kroeger et al., 2003; Pfleger & Eidne, 2006). Given that both BRET and FRET are proximity-based methodologies, any change in the structural conformation of the receptor(s) of interest could result in movement of the donor and/or acceptor proteins that in turn leads to alterations in the signal observed. Therefore, due diligence is required when interpreting data when variation in resonance energy transfer is detected (relative to control measurements). While an increase in BRET signal may be indicative of an increase in protein–protein interactions within a sample, it is possible that the change in signal stems from conformational changes in the proteins of interest, which subsequently lead to the donor–acceptor molecules being brought into greater proximity and/or a more favourable relative orientation. 2.2. Constitutive dimerisation It has been shown for a number of Family C GPCRs that the endogenous receptor unit consists of either homo- or heterodimers, which are required for receptor function and/or expression (Jones et al., 1998; Bai et al., 1999; Tateyama et al., 2004). Co-immunoprecipitation studies of wild-type and epitope-tagged metabotropic glutamate receptor 5 (mGluR5) revealed that the receptors existed as covalent dimers linked by disulfide bridges within the N-terminus (Romano et al., 1996). Similar findings were also shown for the mGluR1a, mGluR2/3 and mGluR4 receptor subtypes. Subsequent co-immunoprecipitation work revealed that while the disulfide bridges were essential for covalent mGluR5 dimerisation, truncated receptor mutants with mutated cysteine residues were still capable of forming dimers, presumably through non-covalent interactions (Romano et al., 2001). Several years ago, BRET was utilised to indicate the existence of constitutive β-2 adrenergic receptor (β2AR) homodimers (Angers et al., 2000), with evidence for the specificity of this interaction provided by coexpression of β2AR with another distantly related GPCR that produced no BRET signal. Since this study, a number of papers have used BRET to monitor constitutive dimer formation between GPCRs (reviewed by Pfleger & Eidne, 2005), including the opioid and cannabinoid receptors (Rios et al., 2006). BRET saturation experiments have been used in an effort to demonstrate the specificity of heterodimer interactions. By maintaining a constant amount of donor protein and coexpressing an increasing amount of acceptor protein, a non-specific interaction theoretically produces a linear relationship between BRET signal and the amount of acceptor protein relative to the donor protein. Conversely a specific dimer interaction exhibits a different profile, with the BRET signal reaching a plateau irrespective of ever-increasing amounts of acceptor protein (Mercier et al., 2002; Milligan et al., 2005). While this technique provides a useful means of assessing the specificity of receptor dimerisation, it is not without its critics (James et al., 2006). However, the authors of BRET studies have continually emphasised the need for strict controls to be employed in such assays, in order to minimise erroneous interpretation of results (Bouvier et al., 2007). Again, the inclusion of appropriate (negative) controls in these BRET experiments is of paramount importance when assessing the specificity of receptor interactions. Issues such as receptor expression levels and possible conformational limitations of certain BRET-tagged GPCR constructs, which could hinder or limit efficient RET between proteins of interest, must be considered when designing such assays (Pfleger & Eidne, 2006; Pfleger et al., 2006). There are indications that many, if not most, GPCRs are trafficked to the plasma membrane in a dimeric complex (Bulenger et al., 2005) and that their interaction is not directly influenced by the presence or absence of a particular ligand. Why then should we be interested in compounds that specifically target heterodimers? The reason is that numerous examples already exist in the literature which demonstrate that particular GPCR heterodimeric complexes exhibit markedly different pharmacological profiles when compared to those of the monomeric/homodimeric forms of the receptor following agonist challenge (AbdAlla et al., 2000; Gomes et al., 2000; Hilairet et al., 2003; Gomes et al., 2004; El-Asmar et al., 2005; Breit et al., 2006; Ellis et al., 2006; Rios et al., 2006). Thus, while binding of ligand in most instances has no effect upon GPCR dimers, the dimeric state of the receptors themselves can impact upon the signalling pathways elicited by the binding of ligand. Furthermore, as will be discussed subsequently, the potential to generate cell-permeable pharmaceutical compounds capable of acting as ‘pharmacochaperones’ may provide a therapeutic avenue for the treatment of deleterious conditions stemming from dimerisation deficits. Such cases are often the result of inadequate receptor export from the endoplasmic reticulum (ER) to the plasma membrane, due to a dominant-negative effect of mutant receptor upon wild-type (WT) receptor trafficking. 3. Role of dimerisation in G protein-coupled receptor synthesis and maturation The pathogenesis of some disorders can involve dominantnegative effects of receptor mutants that prevent signalling of normal 362 M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371 receptors by dimeric inhibition of receptor trafficking. Therefore, an intriguing concept to emerge from this is the possibility that various disease states and disorders may be addressed more effectively by appreciating the role of particular dimeric interactions. In a number of instances, human diseases caused by genetic mutations result from non-functional GPCRs that have an incorrect quaternary structure and are not properly trafficked to the plasma membrane (for reviews see Kim & Arvan, 1998; Morello et al., 2000). These conditions, which are sometimes referred to as ER storage diseases (Callea et al., 1992), often involve a dominant-negative receptor mutant that effectively prevents surface expression of functional WT receptors, reducing potential binding sites and ablating downstream signalling mechanisms as a consequence. The notion that some receptors require dimerisation in order to be correctly targeted to the plasma membrane is not new (for a recent review see Prinster et al., 2005), with a series of studies during the mid 90s indicating this was the case for the GABAB receptor (GABABR). Several groups demonstrated that the GABABR1 subtype was poorly expressed at the cell surface in heterologous systems with little signalling activity, but coexpression with a second subtype (GABABR2) resulted in a fully functional heterodimer that was expressed at the plasma membrane (Couve et al., 1998; Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998). It was later revealed that membrane expression of GABABR1 is prevented by an ER retention motif located on the C-terminus of the receptor and that interaction with the C-terminus of GABABR2 masks this signal, allowing proper trafficking of the functional receptor to the cell membrane (Margeta-Mitrovic et al., 2000). It was also shown that while heterodimerisation was crucial for functional GABABR targeting to the plasma membrane, only intracellular loops of the GABABR2 subtype were required for functional coupling of the heterodimer to the G protein (Margeta-Mitrovic et al., 2001). Although the coiled-coil interface between the C-termini of receptor monomers is crucial for cell surface expression of functional GABABRs, mutagenesis studies utilising C-terminally truncated GABABRs revealed that this domain was not required for heterodimerisation of receptors per se (Pagano et al., 2001). Findings from co-immunoprecipitation studies of vasopressin and oxytocin receptor dimerisation indicated that immature receptors located in the ER could form dimers. This led the authors to suggest that the receptors may form obligate dimers during biosynthesis (Terrillon et al., 2003). A recent study of olfactory receptors (ORs), in particular the mouse 71 (M71) OR, found that in a heterologous system this OR exhibited very poor surface expression that was dramatically improved when β2AR was coexpressed (Hague et al., 2004). The interaction was specific, as increased surface expression of the M71 OR did not occur when coexpressed with other adrenergic receptors. These results imply that dimerisation of ORs may be necessary for successful trafficking of some receptors to the cell membrane. Indeed, co-immunoprecipitation was originally used to show that β2AR monomers interacted with one another, and that a synthetic peptide comprising 20 amino acid residues from transmembrane VI of the receptor substantially inhibited the number of β2AR dimers detected, reducing the ability of the receptor to generate second messengers (Hebert et al., 1996). This indicated that transmembrane VI of the receptor was crucial for protein–protein interactions. Subsequent studies with a series of β2ARs with mutations in the transmembrane VI domain showed that they reduced dimerisation between WT receptors (Salahpour et al., 2004). β2AR mutants with an ER retention motif within the C-terminus acted in a dominantnegative fashion when coexpressed with WT receptors, preventing trafficking to the plasma membrane. This indicated that dimerisation of β2ARs is necessary for receptor export to the plasma membrane. Further analysis of a signalling-impaired, constitutively desensitised β2AR mutant found that coexpression with WT β2AR had a dominantpositive effect, with heterodimerisation resulting in functional complementation of the mutant receptor (Hebert et al., 1998). 4. Functional relevance of G protein-coupled receptor heterodimerisation Recently, it has become apparent that different GPCR subtypes can interact with each other on the cell surface in a variety of complexes with the potential for multiple permutations. The consequences of these interactions are not well-understood, or exploited, but are predicted to have a major impact on drug discovery (George et al., 2000). GPCRs have proven to be very tractable drug targets and therefore are of great interest to the pharmaceutical industry. The mere notion that two previously unrelated GPCRs (either in terms of familial classification or receptor subtype) could directly interact with one another, or impact upon the other in a downstream fashion, creates the proverbial “Pandora's box” with respect to the possibilities of drug specificity and efficacy. Selected examples of functionally relevant GPCR dimers are shown in Table 1 and are discussed below. Examples of potential mechanisms for regulating receptor complexes that have implications for disease pathogenesis, such as allosterism and pharmacochaperones, will be discussed subsequently. 4.1. Chemokine receptors The role chemokine receptors play in determining susceptibility to HIV infection raises the possibility of developing therapeutic agents that target these GPCRs. Coexpression of either CCR3 or CCR5 with the CD4 glycoprotein facilitates HIV infection in previously resistant celllines (Choe et al., 1996). While CD4 was essential for HIV entry and chemokine receptors acted strictly as positive co-factors for this process, it could be shown that another chemokine receptor, CXCR4, functioned as the primary receptor for infection by HIV-2 in CD4negative cells (Endres et al., 1996). Subsequent studies have shown that chemokine receptors are capable of forming both homo- and heterodimers. Using a number of different techniques, including BRET, homo- and heterodimerisation has been demonstrated for the CCR5 and CCR2b receptors (Mellado Table 1 Key examples of functionally relevant GPCR heterodimers Heterodimer Functional relevance Reference(s) GABABR1 + GABABR2 Correct trafficking of GABABR1 to plasma membrane β2AR + M71 OR T1R2 + T1R3 Correct trafficking of M71 OR to the plasma membrane Sweet taste perception Kaupmann et al., 1998 White et al., 1998 Jones et al., 1998 Margeta-Mitrovic et al., 2000 Hague et al., 2004 T1R1 + T1R3 AT1aR + B2R MOP + DOP Umami taste perception (amino acids L-glutamate, L-aspartate) Hypertension/preeclampsia Enhanced morphine analgesia KOP + DOP A2aR + D2R Tissue-specific analgesia Parkinson's disease A2aR + mGluR5 Drug dependence and addiction 5-HT2a + mGluR2 Psychosis and schizophrenia CCR2 + CXCR4 CCR2 + CCR5 HIV resistance Nelson et al., 2001 Zhao et al., 2003 Nelson et al., 2002 Zhao et al., 2003 AbdAlla et al., 2000, 2001 Gomes et al., 2000, 2004 George et al., 2000 Daniels et al., 2005 Waldhoer et al., 2005 Ferre et al., 1991 Kanda et al., 1998 Canals et al., 2003 Fuxe et al., 2003, 2005 Pinna et al., 2005 Ferre et al., 1999, 2002 Nishi et al., 2003 Kachroo et al., 2005 Adams et al., 2007 Aghajanian & Marek, 2000 Scruggs et al., 2003 Gonzalez-Maeso et al., 2007, 2008 Mellado et al., 1999 Rodriguez-Frade et al., 2004 M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371 et al., 2001; Huttenrauch et al., 2002; Issafras et al., 2002). The functional consequences of heterodimerisation between these receptor subtypes are somewhat unclear, although interactions between the CCR5 and CCR2 receptors caused an increased sensitivity of these receptors to chemokine exposure (Mellado et al., 2001). This is in contrast to other studies analysing CCR5/CCR2b heterodimerisation, which found negative binding cooperativity between the receptor subunits for their respective ligands, suggesting that the heterodimer is only capable of binding a single ligand at a time (El-Asmar et al., 2005). BRET was also utilised in a study that showed that the CXCR4 receptor was unable to heterodimerise with CCR5, but forms constitutive receptor clusters (oligomers) consisting of two or more CXCR4 receptor monomers (Babcock et al., 2003). There remains some conjecture in this regard as earlier studies have shown that dimerisation of CXCR4 is not constitutive but rather inducible in the presence of a specific chemokine, SDF-1α (Vila-Coro et al., 1999). The development of agents that can modulate the nature of these protein– protein interactions may thus provide a means of preventing HIV infection, or an alternate means of treating patients with HIV. In particular, the use of allosteric molecules that do not compete with endogenous ligands for their binding site may provide the most specific means of targeting deleterious signalling complexes without the side effects seen with agonist or antagonist treatments. Assays utilising a monoclonal antibody raised against CCR5, which did not compete for the ligand-binding site nor initiate signalling, were able to illustrate that by inducing dimerisation of CCR5 the antibody was capable of preventing HIV infection both in vitro and in vivo (Vila-Coro et al., 2000). Another research group subsequently determined that binding of the HIV-1 envelope glycoprotein to an allosteric site on CCR5 inhibits binding of the endogenous ligand to the receptor (Staudinger et al., 2001). The allosteric nature of chemokine receptor interactions has been further demonstrated by a recent study of CCR2 and CCR5 heterodimerisation, which found negative binding cooperativity between dimer subunits as a result of allosteric interactions (Springael et al., 2006). A small molecule inhibitor, named Repertaxin, was recently shown to bind the chemokine receptors CXCR1 and CXCR2 allosterically, and in doing so prevented receptor signalling by modifying the conformation of the receptor (Bertini et al., 2004). Similarly, two allosteric modulators of CXCR4 have been described, RSVM and ASLW, neither of which compete for the orthosteric binding site but exhibit partial- and super-agonist characteristics respectively (Sachpatzidis et al., 2003). These forms of intervention present a novel, and as yet relatively untapped source of future therapies. 4.2. Adenosine-2a and metabotropic glutamate-5 receptors The adenosine-2a receptor (A2aR) and metabotropic glutamate-5 receptor (mGluR5) are both expressed by γ-aminobutyric acid (GABA) striatal neurons (Schiffmann et al., 1991; Fink et al., 1992; Testa et al., 1995; Ferre et al., 1999, 2002), a region of the brain that has been implicated in reward-seeking behaviour commonly associated with drugs of addiction (Adams et al., 2007). Following a report that indicated that treatment of alcohol-conditioned mice with either adenosine-1 receptor (A1R) or A2aR-specific agonists reduced alcohol withdrawal symptoms (Kaplan et al., 1999), several studies have attempted to elucidate the underlying molecular mechanisms. Using membrane preparations from rat striatal neurons, treatment with either A2aR or mGluR5 specific agonists decreased the affinity of dopamine for dopamine-2 receptor (D2R), and co-administration of these compounds caused a synergistic decline in dopamine binding (Ferre et al., 1999). Confocal microscopy and co-immunoprecipitation were used to demonstrate that A2aR and mGluR5, when coexpressed in HEK293 cells, were colocalised at the plasma membrane and that simultaneous treatment of these receptors with respective agonists led to a synergistic increase in ERK phosphorylation (Ferre et al., 363 2002). Furthermore, these results were replicated in an in vivo rat model, and A2aR and mGluR5 were colocalised in rat striatal membrane preparations. This led the authors to speculate that a functional interaction between A2aR and mGluR5 is necessary to overcome the inhibitory effect of dopamine upon A2aR signalling. Another study utilising mouse striatum found that phosphorylation of the dopaminergic-related protein DARPP-32 by agonist-occupied mGluR5, is dependent upon activation of A2aR by endogenous adenosine (Nishi et al., 2003). Again, a synergistic effect was seen when samples were simultaneously treated with A2aR and mGluR5 agonists with respect to DARPP-32 phosphorylation, and related to the level of ERK signalling. Subsequent studies that have focussed upon the role of these two GPCRs in the pathogenesis of behaviours associated with drug abuse, such as heightened reward-seeking (drug dependence), tolerance, withdrawal and relapse, produced some intriguing insights into the nature of the physiological mechanisms underlying these associated disorders. Using both rat and mouse models, several research groups showed that treatment of animals with the mGluR5 antagonist MPEP (or the highly selective mGluR5 antagonist MTEP) not only reduced the reward-seeking behaviour correlated with substances of abuse, including alcohol (Arolfo et al., 2004; Backstrom et al., 2004; Schroeder et al., 2005; Besheer et al., 2006; Adams et al., 2007), cocaine (Tessari et al., 2004; Kenny et al., 2005) and nicotine (Tessari et al., 2004), but also minimised the occurrence of substance tolerance, withdrawal symptoms and relapse. Similarly, blockade of A2aR by specific antagonists also led to a decline in symptoms associated with substance abuse (Arolfo et al., 2004; Yao et al., 2006), with an analogous effect seen using an A2aR knockout model (Soria et al., 2006). Consequently, studies have investigated the potential for A2aR and mGluR5 to interact as a heterodimeric unit, and the ramifications this may have upon the previously discussed observations in relation to alcohol abuse. Combined, selective antagonism of both receptors at sub-threshold doses reduced alcohol consumption and prevented subsequent relapse following re-presentation of cues previously associated with alcohol availability (Adams et al., 2007; Fig. 2). These effects were not seen in animals treated with an A1R-specific antagonist in combination with either an A2aR or mGluR5 antagonist, suggesting that diminished alcohol consumption and susceptibility to relapse are mediated by interactions between A2aR and mGluR5. Another study found that apparent heterodimerisation between A1R and A2aR subtypes enables the precise regulation of glutamatergic signalling by reducing the affinity of A1R for agonists, which is dependent upon the relative concentration of endogenous adenosine (Ciruela et al., 2006). In addition, research exploring the potential role of A2aR and mGluR5 heterodimerisation in Parkinson's disease found that the anti-Parkinsonian effects of mGluR5 antagonists (increased motor control) were potentiated by an A2aR antagonist (Kachroo et al., 2005). Furthermore, the ability of mGluR5 antagonist to enhance locomotion was dependent upon the presence of a functional A2aR. Interestingly, the absence of D2R expression also abrogated the therapeutic effects of mGluR5 antagonism. Thus, the earlier proposition that multimeric complexes consisting of D2R, A2aR and mGluR5 protomers could be present in striatal neurons seems feasible (Fuxe et al., 2003), and may provide a fascinating target for future therapies aimed at alleviating both the symptoms of Parkinson's disease and the behavioural defects stemming from substance abuse. The role of A2aR/D2R heterodimerisation in the therapeutic intervention of Parkinson's disease will be discussed shortly. 4.3. Opioid receptors The opioid receptors are an important target for analgesia. Coimmunoprecipitation studies showed that the kappa-opioid receptor (KOP) and delta-opioid receptor (DOP) form dimeric complexes when 364 M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371 Fig. 2. Effects of A2aR antagonist (SCH 58261) and mGluR5 antagonist (MTEP) on operant ethanol self-administration in alcohol-preferring (iP) rats (n = 10). Black bars represent ethanol responses; white bars represent water responses; ⁎ denotes significantly different to vehicle. (a) Ethanol self-administration was not significantly altered by SCH 58261 at 1.0 mg/kg i.p., nor MTEP at 0.25 mg/kg i.p. SCH 58261 at 2.0 mg/kg i.p. significantly reduced responding for ethanol (p b 0.001) without altering responding for water. When co-administered, SCH 58261 and MTEP significantly reduced responding for ethanol without affecting responding for water. Veh, Vehicle; M0.25, MTEP (0.25 mg/ kg i.p.); S1 and S2, SCH 58261 at 1.0 and 2.0 mg/kg; S0.5M0.25, SCH 58261 (0.5 mg/kg i.p.) and MTEP (0.25 mg/kg). (b) The ethanol-lever time-course shows SCH 58261 at 2.0 mg/kg i.p. significantly reduced responding in the 5 min (p = 0.009) and 20 min (p = 0.013) bins. SCH 58261 (0.5 mg/kg) and MTEP (0.25 mg/kg i.p.) significantly attenuated responding for the first 10 min (p b 0.001). ■, Vehicle; ▼, SCH 58261 (2 mg/kg); ◊, SCH 58261 (0.5 mg/kg i.p.) and MTEP (0.25 mg/kg). (c) SCH 58261 at 0.5 mg/kg and MTEP at 0.25 mg/kg i.p. significantly increased (p b 0.001) the latency from the session start until the first reinforced ethanol reward (from Adams et al., 2007 with permission, © 2007 International Journal of Neuropsychopharmacology). In contrast, other researchers used BRET to demonstrate a specific interaction between the MOP and KOP subtypes (Wang et al., 2005). Previous immunocytochemical studies showed that these subtypes were coexpressed in rat spinal tissue (Wessendorf & Dooyema, 2001) suggesting the in vivo relevance of the receptor association. Other studies illustrated that DOP and MOP were able to form heterodimers (Gomes et al., 2000). Binding of selective DOP agonists to DOP increased the binding affinity of MOP agonists and vice versa, suggesting positive binding cooperativity may occur between receptor monomers comprising this heterodimer. Additionally, treatment with a combination of DOP- and MOP-specific agonists caused synergistic potentiation of heterodimer signalling with a significant increase in the ability of the receptor to couple to downstream effector mechanisms. An investigation of MOP/DOP heterodimers by BRET analysis showed that dimerisation was independent of agonist challenge and treatment with a DOP antagonist led to a substantial increase in MOPmediated morphine analgesia (Gomes et al., 2004). More recently, BRET analysis of cannabinoid receptor 1 (CB1) showed specific interactions with opioid receptors (mu, delta and kappa), independent of ligand, but no dimerisation with CCR5 (Rios et al., 2006). Of particular interest was the observation that binding of agonist to one receptor of the heterodimer pair (CB1) resulted in a significant reduction in the signalling efficacy of the second receptor (opioid) when exposed to its endogenous ligand. This effect was reciprocal and indicates that in certain tissue types the CB1 and opioid receptors may be able to allosterically modulate the actions of one another, although to what end is still unclear. Evidence for MOP/DOP heterodimers, and observations that cotreatment of cells with a DOP antagonist caused enhanced binding affinity and potency of a selective mu-agonist at MOP (Gomes et al., 2000), led to a succession of investigations into this relationship. The MOP/DOP heterodimer was found to be a unique signalling complex with a novel binding pocket and alternate G protein coupling, distinct from either receptor monomer (George et al., 2002). BRET analyses reaffirmed these findings and further suggested that cotreatment with a DOP-specific antagonist augmented the analgesic properties of morphine acting via MOP (Gomes et al., 2004). Finally, the discovery of an agonist that is specific to an opioidreceptor heterodimer is particularly interesting. Waldhoer et al. (2005) showed that an analgesic agonist, 6′-GNTI, selectively activated DOP/KOP heterodimers in a tissue-specific manner (Fig. 3). The formation of a unique signalling complex was further illustrated by altered binding affinities of antagonists for their respective receptor monomers when compared to the heterodimer profile. This raises the possibility that GPCR heterodimers can be specifically targeted without activating receptor monomers (or other dimers), thereby avoiding potential side effects due to activation of alternate opioid signalling pathways (Gupta et al., 2006). The side effects of morphine and other opiates can be minimised when administered intrathecally (Fairbanks, 2003), and previous studies have demonstrated the coexpression of DOP and KOP in spinal neurons (Wessendorf & Dooyema, 2001), so there is considerable potential for compounds such as 6′-GNTI to be utilised in future pain management strategies that are efficacious and exhibit negligible side effects. 5. Pharmacological rescue of misrouted G protein-coupled receptors coexpressed in a heterologous system (Jordan & Devi, 1999). This interaction resulted in altered receptor pharmacology and was specific, as KOP did not dimerise with the mu-opioid receptor (MOP). The finding agreed with computational modelling of opioidreceptor dimerisation, which predicted that there were no residues between these two receptors capable of forming an interface (Filizola et al., 2002). A multitude of factors influence the correct trafficking of GPCRs from the ER to the cell membrane, including motifs in the C-terminus that act as an ER retention signal, GTPases that target receptors to transport proteins (for review see Duvernay et al., 2005) and RAMPs that promote the trafficking of receptors such as the CaSR from the ER to the Golgi apparatus for receptor maturation (Bouschet et al., 2005). M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371 365 Fig. 3. KOP/DOP heterodimers are specifically targeted by 6′-GNTI. (a) Coexpression of opioid-receptor types in HEK-293 cells was visualised by immunofluorescent staining of cells stably expressing HA-tagged DOP (DOP) and FLAG-tagged KOP (KOP), with antibodies directed to the respective epitope tags (scale bar = 10 μm). (b) Ratio of opioid-receptor heterodimers versus homodimers by serial immunoprecipitation (IP). Cells coexpressing both HA-DOP (HADOP) and FLAG-KOP (FKOP), or each individually was lysed and the receptors were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were immunoblotted (IB) with anti-HA antibody to detect KOP/DOP heterodimers (lanes 5 and 7). After the FLAG immunoprecipitation (F), the lysates were immunoprecipitated with anti-HA antibody and immunoblotted for HA to detect the DOP homodimers/monomers remaining after immunoprecipitation of the heterodimers. Compare lanes 5 (heterodimers) and 6 (homodimers/monomers). (c) 6′-GNTI-induced Ca2+ release in HEK-293 cells expressing one or two opioid-receptor types. Agonist-mediated intracellular Ca2+ release was measured in cells expressing the chimeric G protein Δ6-Gqi4-myr (200 ng for every 40,000 cells) and MOP (Δ), DOP (□), or KOP (○) alone or MOP/DOP (■), KOP/MOP (▲), or KOP/DOP (●). Intracellular Ca2+ release was measured in a Flex apparatus (Molecular Devices), where relative light units (RLU) = maximum Ca2+ peak / cell number × 1000. Shown are representative curves carried out in duplicate (n = 4). (Inset) Structure of 6′-GNTI. (d) Effects of receptor type-selective antagonists on 6′-GNTI-induced Ca2+ release in cells expressing the KOP/DOP heterodimer. Cells expressing the KOP/DOP heterodimer were preincubated for 30 min with increasing doses of DOP-selective antagonist NTI (○) or KOP-selective antagonist NorBNI (●) and stimulated with 100 nM 6′-GNTI. Agonist-induced Ca2+ release was assessed as described in (c). Data are mean ± SEM measured in duplicates. (e and f) Effect of 6′-GNTI (e) and KOP- or DOP-selective antagonists, (f) on competition for [3H] diprenorphine binding to cells expressing KOP/DOP heterodimers. Whole-cell competition binding experiments were performed on cells stably expressing the KOP/DOP heterodimers. Cells were incubated with 1.5 nM [3H] diprenorphine and increasing amounts of 6′-GNTI (■) (e), NorBNI (●) (f), or NTI (○) (f). Shown are representative curves (n = 4) carried out in duplicate. Note that error bars in (e) are too small to be visualised (from Waldhoer et al., 2005 with permission, © 2005 PNAS). Thus, a number of potential approaches could be used to develop pharmaceutical intervention strategies, based upon the utilisation of such cellular mechanisms, in order to treat conditions related to the incorrect trafficking of GPCRs. Hypogonadotropic hypogonadism (HH) is a disease model involving dysfunctional receptor trafficking. Fourteen naturally occurring mutations have been described for GnRHR that result in HH of varying severity, a disorder characterised by a lack of development at puberty and the subsequent absence of secondary sexual development (Conn et al., 2002). Such a disease aetiology can be overcome through the use of ‘pharmacochaperones’ engineered to correct the quaternary structure of misfolded proteins which enable proper export of the receptor to the plasma membrane and functional rescue of the receptor (Leanos-Miranda et al., 2002). Design criteria for pharmacochaperones include: specificity for the receptor of interest, ability of the molecule to reach the target (cell permeability) and the ability to dissociate from the receptor or at least not compete for the same (orthosteric) binding site as the endogenous ligand (Conn et al., 2002). Studies utilising the ‘pharmacochaperone’ antagonist IN3, which targets human GnRHR (Janovick et al., 2002; Leanos-Miranda et al., 2002; Janovick et al., 2003; Leanos-Miranda et al., 2003, 2005), have clearly demonstrated that this cell-permeable compound alleviates the dominant-negative effect of mutant receptors upon the trafficking of WT GnRHR. Thus therapeutic strategies do not need to directly modulate receptor activity, but can act by modulating GPCR expression at the plasma membrane. Such approaches are based upon the dominant-negative effects of the dimerisation of mutant receptors with WT receptors that underlie the disease state. This may not always be the case, since one particular point mutation in the 366 M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371 human GnRHR displayed a lack of ligand-binding specificity or second-messenger signalling, but little if any difference was observed between WT and mutant receptors in cell surface expression (Pralong et al., 1999). This would suggest that the lack of ligand binding was not due to a lack of cell surface receptor expression, but rather an inability of mutant receptors to couple to downstream signalling machinery. 6. Allosteric modulation of G protein-coupled receptor activity The ability of compounds, other than endogenous ligands, to bind GPCRs and alter their functional capability is a facet of GPCR research that is rapidly gaining impetus. Rather than binding to the receptor within the natural binding pocket (orthosteric site), allosteric compounds are capable of interacting with GPCRs at alternate sites that do not compete with natural ligands for receptor binding, modifying receptor activity through a number of mechanisms (Christopoulos & Kenakin, 2002; May et al., 2004; Durroux, 2005; Presland, 2005; May et al., 2007). 6.1. Receptor activity-modifying proteins (RAMPs) The importance of RAMPs in GPCR functionality was first established in a study of the calcitonin-receptor-like receptor (CRLR), which revealed that the pharmacological profile of this receptor was determined by its association with a family of novel proteins termed RAMPs (McLatchie et al., 1998). When dimerised with RAMP1, CRLR functions as a calcitonin-gene-related peptide receptor, however, if associated with either RAMP2 or RAMP3, CRLR behaves as an adrenomedullin receptor. Studies of the calcitonin receptor show that receptor affinity for amylin is modified by dimerisation with particular RAMP subtypes (Hay et al., 2004). The specificity of these interactions was further illustrated by measuring receptor/RAMP binding affinities for several different agonists and antagonists in order to pharmacologically distinguish receptor complexes (Hay et al., 2005). The pharmacology of CRLR is partially affected by the manner in which the receptor is glycosylated and its associated RAMP subtype (McLatchie et al., 1998). Thus, the ability of different RAMPs to associate with certain GPCRs and effectively form distinct heterodimeric protein complexes that exhibit vastly divergent pharmacological properties, provides strong support for the rationale that such effects could be observed in heterodimeric structures comprised of different GPCR subtypes. The possibility then that a single GPCR could exhibit a multitude of pharmacological profiles depending upon the GPCR subtype with which it is associated is plausible, and has significance for the design of novel drugs. 6.2. Dimerisation-mediated G protein-coupled receptor allosterism Although the role of RAMPs in altering the activity of GPCRs has been briefly discussed, several other examples of allosteric modulation have been reported for GPCRs. Aside from chemical compounds and small peptides, it has been proposed that GPCR dimerisation can also facilitate modified receptor activity. Allosteric modification has been shown between the CB1 and the orexin receptor (Hilairet et al., 2003). When stably transfected into CHO cells, these GPCRs can exist as heterodimers at the plasma membrane in the absence of ligand, but exhibit dramatically different pharmacological properties to their monomeric selves when exposed to ligands. When coexpressed with the CB1, hypersensitisation of the orexin receptor (subtype 1; OxR1) was observed in the presence of its endogenous ligand, with a 100fold increase in orexin potency in respect to the activity of ERK1/2. In addition, coexpression of human OxR1 and CB1 in HEK293 cells resulted in receptor heterodimers that failed to traffic correctly to the plasma membrane, in contrast to expression of receptors alone (Ellis et al., 2006). Treatment with either OxR1 or CB1 antagonist restored expression of both receptors at the cell surface, and reciprocally antagonised the other, unoccupied receptor's ability to activate MAPKs following treatment with respective agonist. This effect was only seen when OxR1 and CB1 were coexpressed in HEK293 cells. Family C GPCRs, including mGluRs, GABABRs and the CaSR exhibit allosterism, although the means by which this is accomplished is diverse. While it has been shown by a number of studies that the proper trafficking of the GABABR1 subtype to the plasma membrane requires dimerisation with the GABABR2 subtype (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998), a GABABR1 mutant that is capable of trafficking to the plasma membrane in the absence of GABABR2 was unable to activate downstream signalling pathways in spite of ligand binding to the receptor (Margeta-Mitrovic et al., 2000; Pagano et al., 2001). These results indicate that interactions between different domains of Family C GPCR subtypes (particularly the N-terminal) are necessary for effective coupling to heterotrimeric G proteins and subsequent production of second messengers (Pin et al., 2005). This was further demonstrated by a series of chimeric receptors comprised of alternately paired extracellular domains (ECD) and heptahelical domains of the two receptor subtypes (Galvez et al., 2001). While the ECD of GABABR1 bound ligand and the heptahelical domain of GABABR2 was necessary for G protein coupling and specificity, the paramount determinant of a functional receptor was the heterodimeric pairing of respective domains from each receptor subtype. Not only does such complementation lead to an active GABAB receptor, the binding affinity of ligand to the GABABR1 subtype is allosterically enhanced by the presence of the GABABR2 ECD. An interesting perspective of this concept relates to the prevalence of orphan GPCRs considered in light of GPCR dimerisation and the possible implications this has on the understanding of GPCR ligand binding and activation (Levoye et al., 2006b). Although classically defined as a GPCR for which no known ligand has been identified, “orphan” receptors could also be a GPCR monomer in a dimeric complex that does not bind ligand. An example of such an “orphan” receptor would be the GABABR2 subtype, which does not bind ligand but does increase the affinity of GABABR1 for GABA by heterodimerisation and allosteric modulation (Galvez et al., 2001; Kniazeff et al., 2002; Pin et al., 2004). The T1R3 taste receptor forms obligate heterodimers with either T1R2 or T1R1 subtypes, but does not bind ligands (Nelson et al., 2001, 2002; Zhao et al., 2003; Xu et al., 2004). Further evidence from studies involving mammalian taste receptors show that detection of specific tastes is determined by the particular combination of T1-family receptor subtypes comprising different heterodimers (Nelson et al., 2001; Zhao et al., 2003; Cui et al., 2006), indicating endogenous heterodimerisation was crucial to the functional specificity of certain GPCRs. These observations could have a profound impact upon the food industry, with the future development of artificial flavours such as sweeteners and umami (amino acid) tastes based upon the particular heterodimeric taste-receptor complex the compound is capable of activating. Similarly, the recently discovered GPR50 receptor can, when dimerised with the melatonin receptor subtype 1 (MT1R), alter the affinity of MT1R for endogenous ligands (Levoye et al., 2006a). Thus, a number of so-called “orphan” GPCRs may in fact play a crucial role in the functional capacity of certain heterodimeric complexes, and in turn could theoretically be targeted as a potential avenue of intervention for disorders associated with non-orphan GPCRs. 7. Dimerisation and disease pathogenesis While evidence correlating receptor dimerisation and pathogenesis of human disease is currently limited, there are indications that aberrant cross-talk between GPCRs can directly impact on health. M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371 7.1. Preeclampsia Studies involving the vasopressor angiotensin II and its cognate GPCR (type I angiotensin-II receptor; AT1R) found that a significant interaction occurred between this receptor and the bradykinin-2 receptor (B2R; AbdAlla et al., 2000). Not only was it shown that heterodimerisation of these receptors was dependent on the relative amount of receptors present, but that the signalling capacity and internalisation profiles of the receptors were altered in conditions where the receptors were coupled. Given the enhanced signalling efficacy of the AT1R when coupled with the B2R, the authors then carried out a study to investigate the potential effects that this may have in vivo. Previous studies suggested that increased expression of AT1R was not correlated with preeclampsia (Masse et al., 1998; Pouliot et al., 1998) and that circulating levels of angiotensin II were not significantly different from control subjects (de Jong et al., 1991). This indicated that hypersensitivity to angiotensin II seen in women with preeclampsia (Abdul-Karim & Assalin, 1961; Oney & Kaulhausen, 1982) may somehow be related to heterodimerisation between AT1R and B2R. Indeed, the study illustrated that preeclampsia was strongly correlated with an increased level of B2R expression, and increased AT1R/B2R heterodimerisation with elevated sensitivity to circulating angiotensin II (AbdAlla et al., 2001). Furthermore, they showed that heterodimerisation rendered the AT1R resistant to free-radical inactivation that exacerbated angiotensin-II sensitivity, establishing the first disease model associated with irregular GPCR heterodimerisation. For a recent review see Shah (2005). 7.2. Parkinson's disease The use of L-DOPA (a dopamine precursor) in the treatment of Parkinson's disease has been documented since the 1970s, with the harmful side effects, particularly the uncontrolled muscle contractions known as dyskinesia, having been described shortly thereafter (for reviews see Sassin, 1975; Nutt, 1990; Blandini, 2003). As a consequence, much research has focussed on the understanding of the molecular mechanisms that underlie this phenomenon, and the development of treatments that are as efficacious as L-DOPA but which lack the undesirable side effects. Evidence for a functional interaction between A2aR and D2R was first shown by Ferre et al. (1991). Studies in membrane preparations of rat striatum indicated that activation of A2aRs by a selective agonist resulted in a significant decrease in the affinity of D2R agonist binding sites (see also Fuxe et al., 2003, 2005) The authors of the original study postulated that cross-talk between receptors may contribute to the physiological responses observed following administration of adenosine antagonists such as caffeine. A novel A2aR antagonist (KW-6002) was examined in primates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced symptoms of Parkinson's disease, to determine whether it could enhance motor control without the side effects of L-DOPA therapy (Kanda et al., 1998). MPTP selectively damages neurons in the substantia nigra, a region of the mid-brain involved in motor control, producing symptoms that are almost identical to those seen in Parkinson's patients. Not only did the A2aR antagonist significantly improve motor disability in a dose-dependent fashion, it did so without causing dyskinesia or hyperactivity. Another study found that primates subjected to chronic L-DOPA treatment developed profound dyskinesia as a result, but also exhibited a significant increase in A2aR mRNA levels (Zeng et al., 2000). Findings with an alternative A2aR antagonist (MSX-3) revealed that by removing the antagonistic effect of A2aRs on D2R function, the activity of D2Rs in the presence of dopamine agonists was potentiated (Stromberg et al., 2000). Combination therapies for Parkinson's disease with L-DOPA and adenosine antagonists have been proposed, with early results suggesting that 367 treatments with an A2aR antagonist to potentiate D2Rs followed by a reduced dose of L-DOPA may alleviate motor control deficits without inducing dyskinesia (reviewed by Pinna et al., 2005). Perhaps the most revealing insight into A2aR and D2R interactions came from a study that utilised dual-label immunofluorescence with confocal microscopy to show colocalisation of these receptors at the cell membrane in human neuroblastoma cells (Hillion et al., 2002). These confocal studies were complemented by co-immunoprecipitation that detected the constitutive formation of heterodimeric complexes between A2a and D2 receptors. They also found that D2Rs, which have been shown to be largely resistant to agonist-induced desensitisation (Ng et al., 1997), exhibited accelerated desensitisation after prolonged exposure to both dopamine and adenosine agonists, possibly due to their co-internalisation with the A2aRs. Further evidence for A2aR/D2R heterodimerisation has been demonstrated utilising BRET, with two separate studies showing a BRET signal between A2aRs and D2Rs in living cells that was not significantly altered in the presence of agonists for either receptor (Canals et al., 2003; Kamiya et al., 2003). 7.3. Hypogonadotropic hypogonadism Polymorphisms identified in the human GnRHR cause hypogonadotropic hypogonadism (HH), resulting from a lack of receptor expression at the cell surface. Numerous studies have shown that mutant receptors, including splice variants and C-terminal truncations, act upon their WT congeners in a dominant-negative fashion. Examples include the vasopressin-2 receptor (Zhu & Wess, 1998), the dopamine D3 receptor (Karpa et al., 2000) and luteinising hormone receptor (Lee et al., 2002). A study of eight naturally occurring mutations in the human GnRHR demonstrated that in virtually all cases the mutant receptor acted in a dominant-negative manner when coexpressed with WT GnRHR, and was capable of inhibiting ligand binding, WT receptor activation and generation of second messengers (Leanos-Miranda et al., 2005). While the authors speculated that this may be due to reduced WT receptor expression (as a result of cotransfection with a second receptor), or competition for relevant G proteins by mutant receptors, they considered this unlikely and concluded that dimerisation between the WT GnRHR and traffickingdeficient mutant receptor most likely led to ablation of WT receptor signalling and expression. Furthermore, and perhaps of greater import, was their discovery that treatment of cells expressing both WT and mutant GnRHR with the ‘pharmacochaperone’ antagonist IN3 was able to completely overcome the signalling and expression inhibition observed for untreated, co-transfected cells. In order to try and unravel the molecular basis for WT GnRHR inhibition by coexpression with GnRHR mutants, another experimental approach was adopted. By genetically fusing a green fluorescent protein (GFP) to the C-terminus of the WT GnRHR (including a spacer consisting of sequence from the catfish WT GnRHR C-tail) a chimeric receptor was generated which could be visualised using confocal microscopy (Brothers et al., 2004). By doing so the authors were able to demonstrate that the dominant-negative effect of mutant GnRHRs on human WT GnRHR signalling (Conn et al., 2002; Leanos-Miranda et al., 2002; Janovick et al., 2003; LeanosMiranda et al., 2003; Ulloa-Aguirre et al., 2004) was a direct result of retention of the WT receptor within the ER. This inhibition of WT GnRHR expression at the plasma membrane caused a significant decrease in the ability of the WT receptor to initiate secondmessenger signalling following agonist treatment. While treatment with the GnRHR antagonist IN3 was able to pharmacologically rescue the functional capacity of WT GnRHR in the presence of two of the mutant receptors tested, the dominant-negative effect of a third mutant receptor (S168R) remained unperturbed. These findings were supported by a more recent study that showed that in the presence of the GnRHR antagonist IN3, pairs of naturally 368 M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371 occurring mutant GnRHRs found in compound heterozygote HH patients could be functionally restored, enabling receptor activation of effector mechanisms and ligand binding in the majority of instances (Leanos-Miranda et al., 2005). However it was noted that the extent of this functional rescue was dependent upon the specific genotype of patients, as an improvement in receptor signalling was not applicable to all receptor pairs when treated with IN3. This would suggest that in some instances the pharmacochaperone is capable of stabilising the structure of mutant receptors sufficiently to overcome ER retention, but that in other cases the structural abnormality/instability of mutant receptors cannot be circumvented through use of such chaperones. 7.4. Schizophrenia and psychosis The complexity of schizophrenia and its related symptoms has led to the development of treatment protocols that, although effective, leave a lot to be desired with respect to efficacy and unwanted side effects. Insight into the potential neuronal mechanisms underlying the psychotic hallucinations associated with the condition came from observations that indoleamine and phenethylamine psychedelics shared a high affinity for a specific subset of serotonin (5-HT) receptors that activated the cerebral cortex (Aghajanian & Marek, 2000). Further analysis of this relationship revealed that the potent hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) activated serotonin 2a (5-HT2a) receptors in the somatosensory cortex (Scruggs et al., 2003). This resulted in increased glutamate release that was blocked by cotreatment with a 5-HT2a receptor antagonist, leading the authors to suggest that 5-HT2a “heteroceptors” mediated the actions of hallucinogens. An elegant study recently published in Nature has provided substantial evidence to support this hypothesis. A collaborative effort amongst several research groups assessed the potential interactions of GPCRs within the cerebral cortex using a combination of BRET, FRET and co-immunoprecipitation methodologies, clearly demonstrating that the 5-HT2a receptor and mGluR2 formed functional heterodimeric complexes with a distinct signalling profile (Gonzalez-Maeso et al., 2008). In addition to the allosteric modulation of endogenous ligand-binding affinities when receptors were dimerised, coexpression of mGluR2 significantly impaired high affinity activation of Gαq/11 by the 5-HT2a receptor while enhancing Gαi activation by the 5-HT2a receptor. This was a fundamental observation as the authors noted the similarities between these findings and the divergent response of pyramidal neurons to hallucinogenic and non-hallucinogenic agonists at the 5-HT2a receptor. While non-hallucinogenic agonists led to c-fos induction requiring Gαq/11-dependent activation of phospholipase C, hallucinogens including DOI induced the early growth response-2 gene (egr-2) that was dependent upon Gαi activation (GonzalezMaeso et al., 2007). It was subsequently shown that induction of the hallucinogen-specific marker egr-2 could be prevented by co-administration of an mGluR2 agonist in cortical cultures, which also abrogated head-twitch behaviour specific to hallucinogens (Gonzalez-Maeso et al., 2008). Finally the authors found elevated expression of the 5-HT2a receptor and diminished levels of mGluR2 expression in post-mortem samples from untreated schizophrenic patients. They concluded that dysregulation of mGluR2 and 5-HT2a receptor expression, and thereby heterodimers comprised of these receptors within cortical tissue, may result in aberrant signalling that predisposes schizophrenic patients to psychosis. 8. Heterodimer-specific drugs There is considerable interest in the design of drugs that specifically target GPCR heterodimers and this area of GPCR research already boasts approaches to determine the structural requirements needed to produce molecules that explicitly target heterodimeric GPCR pairs (for reviews see George et al., 2002; Milligan, 2006). Computer modelling systems (in silico) have been employed to design novel heterodimeric compounds using a homology-based approach. Structural models of GPCR heterodimers are generated based on the known structures of other GPCRs that share a degree of homology with the receptor(s) of interest (Filizola & Weinstein, 2005a, 2005b; Filizola et al., 2006). Given that the only GPCRs for which a complete 3D crystal structure has been determined (by X-ray crystallography and micro-crystallography) are bovine rhodopsin (Palczewski et al., 2000) and β2AR (Rasmussen et al., 2007), the structure of rhodopsin has been the primary basis for the modelling of numerous GPCR structures. Interestingly, the β2AR was crystallised as a monomer (Rasmussen et al., 2007), however, it is unclear if this represents the physiological scenario or if the purification process disrupted dimerisation. There already exist a number of drugs on the market that were designed using structure-based homology modelling, including the anti-impotence drug Viagra® (Lundstrom, 2006, 2007), and the AIDS drugs Agenerase® and Viracept®, whose structures were based on that of the HIV proteinase. De novo structure modelling on the other hand uses first principles to determine receptor structure using the known amino acid sequence of the protein (for recent reviews see Reggio, 2006; Schlyer & Horuk, 2006). One group has predicted the active conformation of rhodopsin bound to retinal, using a de novo model sharing very high homology with the known crystal structure (Vaidehi et al., 2002). Thus, the ability to calculate the 3D structure of different GPCRs may anticipate how two different GPCR subtypes interact, and what physical constraints may be imposed as a result of such associations. This de novo method of receptor modelling could prove extremely useful for predicting the 3D structure of receptors that do not belong to Family A GPCRs. A third technique that has been adopted to design heterodimerspecific compounds uses bivalent molecules. These consist of two different agonists/antagonists, or a combination of both, with amino acid spacers of varying lengths separating the ligands. Since a DOP antagonist enhances the analgesic properties of morphine acting at MOP (Gomes et al., 2004), a bivalent ligand was developed consisting of MOP agonist and DOP antagonist, linked by a spacer (Daniels et al., 2005). This dual agonist/antagonist demonstrated that bivalent compounds can induce analgesia while at the same time minimising tolerance and physical dependence to the morphine-analogue component, and that the effect was primarily determined by the distance between the two pharmacophores (Lenard et al., 2007). Similar efforts have also been undertaken for DOP/KOP heterodimer studies utilising a bivalent DOP/KOP-specific antagonist (Xie et al., 2005), and a bivalent agonist that targets a heterodimer consisting of DOP and a sensory neuron-specific receptor (Breit et al., 2006). This provides an intriguing opportunity to design pharmaceutical compounds that exclusively target GPCR heterodimers, with the size of the spacer between the two pharmacophores designed according to the structural arrangement of the GPCR monomers comprising the protein complex. 9. Concluding remarks There is a rapidly expanding body of evidence indicating that most, if not all, GPCRs are present at the plasma membrane as dimeric complexes, and a growing acceptance that such complexes exhibit distinct pharmacological properties and functional characteristics (for a recent review see Fredholm et al., 2007). Of profound interest is the indication that GPCRs of different types interact with one another at the molecular level, adding yet another level of complexity and opening an entirely new perspective with regard to pharmacological intervention when these receptors are coexpressed in vivo. The concept of GPCR dimerisation provides two major new avenues of drug discovery/development research. These include the potential existence of the heterodimer as a drug target distinct from that provided by monomers/homodimers of individual receptor M.B. Dalrymple et al. / Pharmacology & Therapeutics 118 (2008) 359–371 subtypes, and secondly, the development of drugs capable of targeting each receptor component resulting in synergistic effects. There is increasing evidence that certain disease states arise from the incorrect trafficking of GPCRs from the ER to the plasma membrane. Indeed some mutant receptors appear to have a dominant-negative effect on the WT receptor due to dimerisation. An understanding of this concept will facilitate the design of therapeutic strategies to overcome such dominant-negative effects, including the use of cell-permeable compounds such as ‘pharmacochaperones’. 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