MINIREVIEW Methods to monitor the quaternary structure of G protein-coupled receptors Graeme Milligan1 and Michel Bouvier2 Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, UK Biochemistry, Universite de Montreal, Quebec, Canada Keywords dimerization; functional reconstitution; G protein coupled receptor; immunoprecipitation; resonance energy transfer Correspondence G Milligan, Davidson Building, University of Glasgow, Glasgow, G12 8QQ Scotland, UK Fax: +44 141330 4620 Tel: +44 141330 5557 E-mail: g.milligan@bio.gla.ac.uk (Received 16 February 2005, accepted April 2005) A wide range of approaches has been applied to examine the quaternary structure of G protein-coupled receptors, the basis of such protein–protein interactions and how such interactions might modulate the pharmacology and function of these receptors These include coimmunoprecipitation, various adaptations of resonance energy transfer techniques, functional complementation studies and the analysis of ligand-binding data Each of the available techniques has limitations that restrict interpretation of the data However, taken together, they provide a coherent body of evidence indicating that many, if not all, G protein-coupled receptors exist and function as dimer ⁄ oligomers Herein we assess the widely applied techniques and discuss the relative benefits and limitations of these approaches doi:10.1111/j.1742-4658.2005.04731.x Introduction In recent times, the view that G protein-coupled receptors (GPCRs) are single polypeptides that function as isolated monomers has been challenged, and largely supplanted, by results consistent with the existence of GPCRs as dimers or higher-order oligomers Data in support of GPCRs being able to form dimers or oligomers had been scattered throughout the literature [1] However, at least in part because expression of a single GPCR cDNA in heterologous cell systems generally resulted in production of a ligand-binding site with the expected pharmacology and the capacity to activate G proteins and hence initiate signal transduction, little thought was given to potential quaternary structure This is despite the fact that dimerization of proteins, as a route to function, is one of the most common themes in biology [2] This had been well established previously for other classes of transmembrane receptors Key to a broad appreciation of the potential of dimerization for GPCR function was the demonstration that the c-aminobutyric acid (GABA)B-receptor, long recognized as a GPCR, was an obligate heterodimer [3,4] Even though the GABABR1 polypeptide is a seven transmembrane protein able to bind both GABA and a GABAB receptor antagonist [5], when expressed alone this polypeptide is not transported to the cell surface, does not have the expected characteristics and affinity for binding of agonists and cannot activate G protein signalling Cell surface delivery and function requires the coexpression of a second, closely related seven transmembrane polypeptide, the GABABR2, which although unable to bind GABAB receptor ligands, allows cell surface delivery of the GABABR1 The heterodimeric complex then functions with pharmacology akin to the native receptor Subse- Abbreviations BRET, bioluminescence resonance energy transfer; DOP, delta opioid peptide; eYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer; GPCRs, G protein-coupled receptors; G(C,Y)FP, green (cyan, yellow) fluorescent protein; NEM, N-ethyl maleimide 2914 FEBS Journal 272 (2005) 2914–2925 ª 2005 FEBS G Milligan and M Bouvier quently, it has become clear that sweet and umami taste responses are also generated by obligate heterodimer pairs of T1 taste receptors [6] and both homoand heterodimerization of other family C receptors is firmly established This topic is covered in the accompanying review by Pin and colleagues [7] Analysis of the importance of quaternary structure for function of the rhodopsin-like family A GPCRs has been more recalcitrant to analysis, as examples of obligate heterodimerization akin to the GABAB receptor have not been noted, although there are reports, for example, of the requirement for heterodimeric interactions to allow cell surface delivery of certain a1-adrenoceptor subtypes [8] and olfactory receptors [9] Nevertheless, it is now generally accepted that family A GPCRs can exist as dimers and ⁄ or higher order oligomers [10–12] A growing database on the formation of GPCR dimers prior to membrane delivery [13–15] and the potential that GPCRs can bind to single heterotrimeric G proteins as dimers [16] is certainly consistent with such a receptor dimer model However, the importance of dimerization for receptor function and regulation in physiological systems remains to be demonstrated unambiguously A significant number of recent reviews have covered these topics [11,12,17] and the purpose of the current piece is to critically evaluate the approaches used to explore dimerization of class A GPCRs Coimmunoprecipitation The ability to differentially epitope-tag GPCRs has been central to the widespread use of coimmunoprecipitation following coexpression of two forms of the same GPCR in heterologous expression systems Initially Hebert et al [18] coexpressed c-myc- and HA-tagged forms of the b2-adrenoceptor in insect Sf9 cells and demonstrated interaction between the two forms of the receptor as both were present in immunoprecipitates generated using either anti-HA or anti-c-myc Igs These studies also provided a series of key controls (a) in a lack of cross-reaction between the antibodies and the alternately tagged form of the receptor and (b) in a lack of coimmunoprecipitation of the HA-tagged b2-adrenoceptor when coexpressed with a c-myc-tagged form of the M2 muscarinic acetylcholine receptor These studies also noted the resistance to monomerization during SDS ⁄ PAGE of at least a proportion of the coimmunoprecipitated receptors; this is a commonly observed feature in many such studies Despite such apparent receptor dimerization (and indeed multimerization) being detected following SDS ⁄ PAGE resolution of simple membrane preparations of transfected cells and even native tissues [19], detection of SDS-resistant FEBS Journal 272 (2005) 2914–2925 ª 2005 FEBS GPCR quaternary structure Fig Differentially epitope-tagged forms of the a1b-adrenoceptor have to be coexpressed to be coimmunoprecipitated N-terminally Flag- or c-myc-tagged forms of the hamster a1b-adrenoceptor were expressed individually (flag, myc) or coexpressed (flag + myc) in HEK293 cells Samples expressing each form of the receptor were also mixed (mix) Lysates of cells were resolved by SDS ⁄ PAGE and immunoblotted to detect expression of each form, or the samples were immunoprecipitated using anti-Flag Ig, resolved by SDS ⁄ PAGE and then immunoblotted to detect c-myc immunoreactivity Coimmunoprecipitation was only observed from samples coexpressing the two forms Data are modified from [35] higher-order structures was reminiscent of protein aggregation, a well-appreciated potential artefact resulting from membrane protein solubilization Therefore, the hydrophobicity of the transmembrane domains of GPCRs raised concerns that apparent coimmunoprecipitation might reflect little more than nonspecific aggregation following detergent extraction of proteins from cells and membranes This resulted in widespread incorporation of mixed samples into coimmunoprecipitation studies to counter this concern In such experiments detergent extracts of cells or membranes, each expressing only one of the tagged GPCRs, are combined prior to the immunoprecipitation step This is generally considered a sound means to eliminate nonspecific aggregation as a potential explanation of spurious coimmunoprecipitation (Fig 1) Coimmunoprecipitation has been of particular use in efforts to identify heterodimeric interactions between GPCRs [20] However, coimmunoprecipitation studies such as that of Salim et al [21] intimated widespread interactions between coexpressed GPCRs and, assuming that interaction between different GPCRs is not a purely stochastic process, raised further questions about the use of coimmunoprecipitation in the absence of further supporting evidence of protein–protein inter2915 GPCR quaternary structure actions A key issue that remains overlooked in many published coimmunoprecipitation studies is whether the samples are fully solubilized prior to the immunoprecipitation step In many studies, centrifugation after detergent extraction is limited to a short period in a bench top microcentrifuge, with force in the region of 15 000 g This is far too limited to ensure production of a fully soluble fraction and it is suggested that, as in many other protocols, centrifugation should be for at least 60 and > 100 000 g As an alternative, passage of the detergent-generated extract through a 0.22 lm filter would ensure removal of membrane fragments that might contain both coexpressed forms of a GPCR that are not actually physically in contact The addition of alkylating agents such as NEM and iodoacetamide in the lysis and solubilization buffers is also recommended in order to prevent the formation of spurious intermolecular disulfide bonds that could be favoured by the oxidizing condition associated with cell lysis Notwithstanding these caveats, there is no doubt that coimmunoprecipitation remains as the starting point and, indeed at the heart, of many GPCR dimerization studies Indeed, despite the general paucity of specific and high affinity anti-GPCR sera, carefully controlled coimmunoprecipitation studies remain the most practical means to explore, in particular, GPCR heterodimerization in native tissues and can provide key information to support GPCR dimerization ⁄ oligomerization Coimmunoprecipitation of GPCRs, subsequent to cross-linking with cell-impermeant cross-linking agents has also provided a useful means to detect GPCR dimers ⁄ oligomers in the plasma membrane The introduction of cysteine residues into specific locations in transmembrane domains of class A GPCRs, that then allow cross-linking by bi-functional reagents, is an extension of the basic cross-linking and immunoprecipitation strategy but has the added advantage of assisting identification of potential protein–protein interaction sites in GPCR dimers [22,23] and determining whether ligand-binding modulates the organizational structure of a GPCR dimer ⁄ oligomer Although the concept of coimmunoprecipitation can be extended easily to studies that employ cotransfection with three or more forms of the same GPCR, each incorporating a different tag, this has only been employed to date in a limited manner In one such example, where c-myc-, FLAG- and HA-tagged forms of the M2 muscarinic receptor were coexpressed in insect Sf9 cells, Park and Wells [24] were able to immunoprecipitate complexes containing all three epitope tags, hence providing evidence for complexes containing at least three GPCR monomers 2916 G Milligan and M Bouvier Resonance energy transfer studies Fluorescence resonance energy transfer The green fluorescent protein (GFP) from Victoria aequoria has found widespread use in cell biology to monitor the expression and distribution of proteins tagged with this polypeptide Mutation of this protein has produced a range of variants with altered spectral characteristics, and pairs of these are able to function as fluorescence resonance energy transfer (FRET) partners The most widely used pairing is a cyan fluorescent protein (CFP) as energy donor and a yellow fluorescent protein (YFP) as an energy acceptor, but other pairings are also practical Obvious attractions in the use of FRET for studies of GPCR dimerization ⁄ oligomerization include the capacity to monitor protein–protein interactions in intact living cells both in cell populations and single cells It can be combined with cell imaging and photo-bleaching protocols to examine the cellular location of such interactions in specific subcellular compartments [25–27] Photobleaching of the energy acceptor construct also provides an important control to demonstrate that FRET-signals actually reflect energy transfer Photobleaching of the acceptor should result in increased fluorescence output from the energy donor as well as destruction of signal from the acceptor as no energy transfer can then occur This is a particularly important control as direct excitation of the energy acceptor by the exogenous light source in sensitization FRET experiments can generate background signals that can be mistakenly interpreted as real FRET Also, the spectral overlap between the donor and acceptor emission spectra needs to be properly monitored and controlled to determine the legitimate FRET signal These are not trivial issues as the contribution of the background signals can vary considerably depending on the relative expression levels of the FRET partners and the band pass width of the fluorescence excitation and emission detection systems Although some wild-type GFPs have the tendency to oligomerize, a characteristic that could promote artefactual oligomerization of the proteins attached to them, variants such as CFP and YFP that have lower affinity for one another, have been used successfully to monitor the quaternary structure of several GPCRs FRET between CFP and YFP covalently fused to distinct GPCRs should occur only when the FRET partners are brought within 100 A of each other; a distance that falls within the dimensions anticipated for GPCR dimers ⁄ oligomers [28] The occurrence of FRET between CFP- and YFP-fused GPCRs has FEBS Journal 272 (2005) 2914–2925 ª 2005 FEBS G Milligan and M Bouvier therefore been interpreted as strong evidence for the existence of dimers or oligomers in living cells As with all approaches based on Forsters’ principles of resonă ance energy transfer, the efciency of transfer is determined both by the orientation of the energy transfer partners and by the distance between them [28] The fact that the efficiency of transfer varies with the 6th-power of the distance between the fluorophores presents both opportunities and limitations for the analysis of data (Fig 2) For instance, although the FRET assays can be extremely sensitive detectors of conformational changes that can reflect ligand-promoted alteration in GPCR quaternary structure, the extent of the D FRET observed for a given change is highly dependent on the initial distance between the energy donor and acceptor For example, if constitutive dimerization of two GPCR FRET-tagged constructs brings the energy transfer partners into very close proximity, the energy transfer efficiency should be close to maximal and thus, a ligand-induced alteration in distance between the energy-transfer partners would be poorly reported (Fig 2) In contrast, if the initial distance between energy donor and acceptor is within the region of the FRET R0 (i.e.: the distance between the donor and acceptor leading to 50% of the maximal transfer efficiency) where the Denergy transfer ⁄ Ddistance is maximal, the same conformational change would lead to large DFRET (Fig 2) A range of fluorescence-based techniques, including intra- and inter- Fig Variation in resonance energy transfer efficiency as a function of distance between energy donor and acceptor Relationship between the energy transfer efficiency (Y-axis) and the distance between the energy donor and acceptor (X-axis) The distance is expressed as the ratio of the distance ‘r’ separating the donor and the acceptor over the distance resulting in 50% of the maximal efficacy (R0) As indicated by the Forster equation, the efciency of ă transfer varies inversely with the 6th-power of the distance The extreme steepness of the relationship results in large changes in RET for relatively small modifications in distance around the R0 In contrast, large changes in the distance may not result in any RET change when the distance is much smaller than the R0 FEBS Journal 272 (2005) 2914–2925 ª 2005 FEBS GPCR quaternary structure molecular FRET, have been used to demonstrate conformational changes in GPCR monomers upon agonist binding [29,30] and recent studies have expanded this to show that an agonist bound protomer of the leukotriene B4, BLT1 receptor produces a confirmation change in the partner GPCR of the homodimer [31] It follows that reports of agonist-induced alterations in GPCR quaternary structure based only on resonance energy transfer studies may simply reflect very small conformational alterations in the GPCR that are amplified by the high sensitivity of the resonance energy transfer responses to distance and orientation [32] (Fig 2) Thus, a large increase or decrease in FRET values observed upon ligand-binding cannot be interpreted, as is too often the case, as an indication of ligand-promoted dimer formation or dissociation The occurrence of FRET between fluorophoretagged GPCRs could result both from the formation of dimers or of higher oligomeric structures Unfortunately, the currently available FRET techniques not permit one to distinguish easily between these two possibilities However, recent studies employing atomic force microscopy to visualize the organizational structure of rhodopsin in murine rod outer segment discs have shown higher order arrays of GPCRs [33,34] and analysis of the results of GPCR transmembrane interface mapping for both the complement C5a receptor [23] and the a1b-adrenoceptor [35] have suggested ways in which these GPCRs may organize into higher order structures Although not yet reported in relation to GPCR quaternary structure, three component FRET systems based on serial energy transfer from cyan to yellow to red fluorescent proteins are available [36] These and related systems may be invaluable to explore the potential of higher-order GPCR oligomerization They are, however, likely to suffer from low sensitivity and thus, at least in the short term, the need to express rather high levels of each component may raise concerns in relation to the specificity of interactions observed Quantitation of the fraction of a GPCR existing as monomer vs dimer or higher-order oligomer remains a challenging task Efforts to assess this for homodimers of neuropeptide Y receptor subtypes were based on FRET efficiency of appropriately tagged GPCRs compared to the FRET signal produced by a positive control generated by fusing together the two FRET partner fluorescent proteins [37] A similar attempt was made for the b2-adrenoceptor homodimer using another resonance energy transfer approach known as bioluminescence resonance energy transfer (BRET, see below) and using theoretical maximal transfer values as the 100% dimer reference [38] How2917 GPCR quaternary structure G Milligan and M Bouvier ever, the values obtained, which were in the region of 20–40% as dimers for the first study and greater than 85% in the dimeric form for the latter, required a range of assumptions, including the fact that the receptors existed as dimers and not larger oligomers Therefore, such estimates must be viewed as extremely uncertain and require further experimental confirmation As indicated above, pictures of the structural organization of rhodopsin in the disks from rod outer segments from mice suggest a much higher level of dimerization ⁄ oligomerization [33] However, the high concentration of rhodopsin in rods may impose order and structural organization that is not required for other class A GPCRs Attempts to explore this by employing discs from heterozygote rhodopsin knockout mice simply resulted in rod outer segments with reduced volume rather than an alteration in the pattern of interactions between rhodopsin monomers [34] [3H]antagonist binding studies [43] As this receptor displays a high level of constitutive, agonist-independent internalization and recycling [44,45], the FRET data are certainly consistent with at least a fraction of the population of this receptor existing as a dimer ⁄ oligomer at all stages in its life history The major potential caveats for time-resolved FRET studies relates to the obligatory use of antibodies Appropriate controls need to be performed to make sure that oligomerization of the partners is not promoted by the bivalent nature of the antibodies In studies assessing the effect of ligand-binding on the quaternary structure of the receptors, the possibility that the antibodies could inhibit binding to the receptor also needs to be controlled Finally, the presence of an antibody between the fluorophores and the proteins of interest, increases the uncertainty concerning the minimum and maximum FRET permissive distance Time-resolved FRET Bioluminescence resonance energy transfer The capacity to image FRET signals in single cells and in specific cellular compartments, including the plasma membrane, provides a means to confirm interactions between GPCR monomers at the surface of individual cells [25–27] A distinct FRET-based approach to monitor interactions between GPCRs at the cell surface in cell populations is based on time-resolved FRET using anti-epitope tag Igs labelled with suitable FRET acceptor and donors [39] N-terminally epitope-tagged forms of GPCRs should only be accessible to such antibodies in intact cells if they have been delivered successfully and inserted into the plasma membrane The use of antibodies labelled with Europium chelates as energy donor allows the use of ‘time-resolved’ protocols, where short-lived fluorescence (up to some 50 ls) derived from excitation of endogenous fluorophores is allowed to decay before the long lived time-resolved FRET signal is monitored over the ensuing 100– 200 ls This generates improved signal-to-noise ratios Although employed in a limited number of studies to date [40–43], limiting signals to GPCRs at the cell surface using this approach has particular advantages for analysis of GPCRs expressed in heterologous systems where a significant fraction of the expressed GPCR(s) can often be shown to be inside the cell, presumably within the endoplasmic reticulum or as protein that has failed to pass cellular quality control mechanisms and is destined for degradation Time-resolved FRET pairings have also been used in membrane fractions generated by sucrose density sedimentation to detect dimers ⁄ oligomers of a1a-adrenoceptors in all membrane fractions containing this GPCR as measured by Conceptually similar to FRET, except that energy is donated to a fluorescent protein energy acceptor by luciferase-mediated oxidation of a substrate, bioluminescence resonance energy transfer (BRET) has become almost as popular an approach as FRET The various benefits of BRET compared to FRET, particularly the lack of requirement of a light source to excite the energy donor, have been discussed previously in more specialized articles [28] In particular, we should note that the lack of potential direct excitation of the energy acceptor that can occur in FRET experiments (see above) greatly simplifies controlling for the background signals However, a clear limitation of BRET is that currently it is not sufficiently sensitive to allow high resolution, single cell imaging and analysis and therefore cannot usefully report on the subcellular location of the signal Although single cell BRET signals resulting from the homodimerization of the b2-adrenoceptor and melatonin MT1 and MT2 homodimers could be detected [32], obtaining subcellular resolution will require the development of more sensitive cameras or of luminescence donors with higher light output In initial BRET studies, the energy transfer pairing was Renilla luciferase and enhanced YFP (eYFP) with h-coelenterazine acting as luciferase substrate However, the overlap between the spectrum generated via this enzymic activity of luciferase and the emission of light from eYFP subsequent to energy transfer is substantial, resulting in a relatively high background signal that needs to be carefully controlled As a means to improve this, the Renilla luciferase substrate DeepBlueC, which results in emis- 2918 FEBS Journal 272 (2005) 2914–2925 ª 2005 FEBS G Milligan and M Bouvier sion of light that is substantially blue-shifted in comparison to the oxidation of h-coelenterazine, has been used The wavelength of the light emitted by the oxidation of DeepBlueC is not well aligned for energy transfer to eYFP but is suited for energy transfer to the modified fluorescent protein GFP2 and the output from GFP2 is relatively well resolved from the Renilla luciferase ⁄ DeepBlueC oxidation spectrum [46] (Fig 3) This results in improved signal-to-noise ratios when employing BRET2 The major limitation of BRET2 is the poor quantum efficiency associated with oxidation of DeepBlueC resulting in much lower absolute signals that thus necessitates higher GPCR expression levels With both their limitations and advantages, BRET1 or BRET2 can be best suited to study specific proteins of interest Taking advantage of the distinct spectral characteristics of the two BRET generations in the same experiment, Perroy et al [47] were able to monitor the interaction between three partners simultaneously in the same cell population Such combinations between Fig Spectral properties of the energy donor and acceptor for the two generations of BRET Schematic representation of the emission spectra for Renilla luciferase (Rluc) using coelenterazine H (BRET1) or DeepBlue Coelenterazine (BRET2) as substrates and of the overlap between the emission spectrum of Rluc and the excitation spectrum of the fluorescent energy acceptors YFP for BRET1 and GFP2 for BRET2 The overlap is more complete for BRET2 than BRET1 favouring a better transfer Also shown are the emission spectra of GFP2 and YFP A better separation from the RLuc emission is obtained with GFP2 in BRET2 leading to a better signalto-noise ratio Not shown on this figure is the fact that the output of light is much smaller for DeepBlue Coelenterazine than for coelenterazine H leading to a lower detection sensitivity for BRET2 than BRET1 [46] FEBS Journal 272 (2005) 2914–2925 ª 2005 FEBS GPCR quaternary structure different types of resonance energy transfer will certainly be increasingly used to examine the stoichiometry of GPCR complexes Following the initial report employing BRET that examined interactions between Renilla luciferase- and eYFP-tagged forms of the b2-adrenoceptor [48], BRET has been used, to date, predominantly in ‘single point’ assays In these, single amounts of Renilla luciferase and fluorescent protein-tagged forms of a single GPCR, to study homodimerization ⁄ oligomerization, or pairs of GPCRs, to study potential heterodimerization ⁄ oligomerization are cotransfected into host cells However, as with coimmunoprecipitation and FRET studies, the intensity of the signal obtained does not provide a measure of absolute, or even relative, interaction affinities between the GPCRs linked to the BRET partners, because the extent of BRET signal is dependent upon both distance between the energy donor and acceptor and their orientation The most significant advance in this area since then has been the introduction of ‘saturation’ BRET by Mercier and colleagues [38] Taking advantage of the possibility of expressing differing amounts of energy donor and acceptor-linked GPCRs in heterologous cells, these studies demonstrated that an asymptotic approach to a maximal BRET signal was obtained with increasing energy acceptor ⁄ energy donor ratios Saturation curves so generated resemble ligand ⁄ GPCR binding curves and, as such, the ratio of energy acceptor ⁄ energy donor that generates half-maximal BRET signal can provide a useful measure of the relative interaction affinities of the GPCRs linked to the BRET reagents For example, although measurable BRET signals can be recorded following coexpression of a DOP opioid receptor-Renilla luciferase ⁄ a1A-adrenoceptorGFP2 BRET pair in HEK293 cells that is commensurate with the signals obtained for each of these two GPCR homodimers, saturation BRET studies indicated that the interaction affinity of this heterodimer ⁄ oligomer pairing was some 75-fold lower than for the a1A-adrenoceptor homodimer ⁄ oligomer [43] This suggests that such a heteromeric pairing would be unlikely to have physiological significance, even if interactions can be monitored in heterologous expression systems Although it was noted in early studies that heterodimeric interactions between structurally homologous receptors were of higher affinity than interactions between more distantly related class A GPCRs [46] it is not inevitable that homodimer interactions will be of higher affinity than heterodimeric interactions Indeed, in the studies of Ramsay et al [46] hetero interactions between the closely related KOP and DOP opioid receptors were reported to be at least as high affinity 2919 GPCR quaternary structure as KOP receptor homo interactions More recent studies are also consistent with melatonin MT1 ⁄ MT2 heterodimers being generated with higher affinity than MT2 receptor homodimers [49] Such saturation BRET studies have also been combined with deliberate variation in expression levels to demonstrate that potential artefacts arising from physical crowding of GPCRs and the production of so called ‘bystander’ BRET (Fig 4) that does not reflect quaternary structure interactions, is only produced with levels of expression in the region of 25 pmolỈmg)1 membrane protein [38] and thus well beyond the range of expression normally used in such studies G Milligan and M Bouvier In the available reports employing saturation BRET, the energy acceptor ⁄ energy donor ratio is generally reported simply as a fluorescence : luminescence ratio However, when well validated GPCR radioligands are available, attempts have been made to generate standard curves of GPCR ligand-binding site number against fluorescence or luminescence signals to allow absolute quantitatation of energy acceptor ⁄ energy donor ratio [38,43] Although luminescence and fluorescence signals increase in a linear fashion with ligandbinding site number, surprisingly, the signal per GPCR binding site has not been equal for different GPCRs [38,43] It is not currently possible to establish the A B 2920 Fig BRET2 levels as a function of energy donor and acceptor concentrations (A) BRET2 saturation performed in cells coexpressing a constant level of a GPCR fused to the energy donor Rluc and increasing concentration of a GPCR fused to the energy acceptor GFP2 The BRET levels are plotted as a function of the GPCR– GFP : GPCR–Rluc ratio In the case of specific dimerization between the two GPCRs, a classical hyperbolic increase in the BRET signal that rapidly saturates is observed BRET 50 represents the GPCR– GFP : GPCR–Rluc ratio leading to 50% of the maximal BRET and reflects the relative affinity of the BRET partners for one another In the case of random collisions between the BRET partners, bystander BRET leads to a quasi-linear relationship that will eventually saturate only at very high GPCR-GFP ⁄ GPCR-Rluc ratios (B) BRET2 measurements performed in cells expressing increasing concentrations of GPCR– Rluc and GPCR–GFP2 while maintaining a constant GPCR–GFP2 : GPCR–Rluc ratio of 1 In the case of specific GPCR dimers, BRET levels are constant for a wide range of total concentration since a constant proportion of GPCR–GFP2 are engaged by a GPCR–Rluc In the case of random collisions between the BRET partners, the bystander BRET should increase as a function of the total receptor concentration for the entire range The increase in BRET observed for very high receptor expression levels > 45 pmolỈmg)1 of protein most likely results from random collisions between GPCR dimers Adapted from [38] FEBS Journal 272 (2005) 2914–2925 ª 2005 FEBS G Milligan and M Bouvier basis for this but it seems surprising that the enzyme activity of Renilla luciferase or, even more so, the fluorescence of variants of GFP should vary markedly depending on which GPCR they are linked to A possible explanation would be that the difference in folding rates between the GFP fluorophore (or luciferase catalytic site) and the binding sites of specific GPCRs varies from one receptor to another It follows that the amount of properly folded binding site ⁄ folded fluorophore would be an intrinsic property of the individual GPCR-fusion construct Alternatively, it is certainly the case that different, but individually well studied, ligands can generate different Bmax values for the same GPCR in the same membrane preparation [50] and thus that the proportion of receptor molecule recognized by the radioligand may differ for different receptors Functional complementation If the coexpression of two nonequivalent and nonfunctional mutants of a GPCR is both able and required to reconstitute receptor ligand-binding and ⁄ or function, this can provide evidence in favour of direct protein–protein interactions and quaternary structure for the active receptor Indeed, coexpression of two forms of the angiotensin AT1 receptor that were unable to bind angiotensin II or related ligands due to point mutations in either transmembrane III or transmembrane region V restored ligand-binding [51] Such an approach has also been used to explore mechanisms of dimerization Theoretical models of GPCR dimerization include both ‘contact’ and ‘domain-swap’ dimers [52] Class A GPCRs can, at least to some degree, re-assemble from coexpressed fragments The most widely used strategy in this regard has been to split GPCR sequences in two by cleavage within the third intracellular loop Such experiments have resulted in the N-terminus and transmembrane regions I–V and transmembrane regions VI–VII and the C-terminal tail being considered as units that are able to fold independently (reference [53] in this series of reviews) Indeed, generation of chimeric GPCRs based on this general strategy provided some of the most elegant early data in favour of molecular cross-talk between GPCRs [54] Using the histamine H1 receptor as a model, Bakker et al [41] showed that although single point mutations in both transmembrane region III and transmembrane region VI prevented binding of antagonist radioligands (including [3H]mepyramine), coexpression of the two mutants resulted in reconstitution of [3H]mepyramine binding sites with the anticipated pharmacological characteristics Conceptually this should not be possible for a contact dimer in which FEBS Journal 272 (2005) 2914–2925 ª 2005 FEBS GPCR quaternary structure transmembrane domains are not exchanged but simply appose each other Although these studies provided support for domain swapping in the histamine H1 receptor, the Bmax obtained for [3H]mepyramine in the coexpression studies was only a small fraction of that obtained when the wild-type histamine H1 receptor was expressed [41] This may reflect that domain-swap and contact dimers are not mutually exclusive but can coexist, although domain-swapping may be energetically less favourable The energetics of domain-swapping for a src homology domain protein has been discussed recently [55] Domain-swapping may also contribute to GPCR heterodimerization and would be expected, almost as a matter of course, to alter the details of ligand pharmacology This is yet to be explored in significant detail but it is certainly true that, for example, coexpression of pairs of opioid receptor subtypes has been reported to result in the generation of novel pharmacology of ligand-binding and function [56,57] and this is also noted for other GPCR pairs [58] Although these changes could be explained by allosteric interactions between the protomers of a contact dimer they could reflect domainswap dimer interactions In addition to the restoration of ligand-binding, studies that have used pairs of nonfunctional mutants to restore GPCR signalling have produced data consistent with GPCR–GPCR interactions By generating mutants of the luteinizing hormone receptor that were either unable to bind ligand or unable to signal, although able to bind the agonist, Lee et al [59] were able to reconstitute agonist-mediated regulation of cAMP levels following coexpression of the two mutants The luteinizing hormone receptor, as with other GPCRs with related ligands, has an extended N-terminal region involved in ligand-binding As such, Lee et al [59] were able to consider the N-terminal exodomain and the seven transmembrane element endodomain as distinct entities in a manner equivalent to the extracellular and transmembrane elements of class C GPCRs that have allowed elegant chimeric receptor approaches to understand the mechanism of signal transduction through obligate hetero-dimers [7] Interestingly, the pairs of complementary mutants described above have allowed demonstration of signal transduction through both transmembrane bundles when such mutant receptors interact, supporting the notion of transactivation within receptor dimers [60] As a modification of the idea of pairs of exo- and endodomains in a single GPCR, Carrillo et al [61] extended the use of GPCR-G protein fusion proteins [62] to study both homo- and hetero dimerization of GPCRs Mutations were introduced to generate pairs 2921 GPCR quaternary structure with potential complementary function One of the pairs had a mutation in the GPCR element of the fusion that allowed ligand-binding but not G protein activation, whereas the second fusion had a mutation in the G protein element that prevented agonist-mediated guanine nucleotide exchange Although both were nonfunctional when expressed individually, coexpression resulted in reconstitution of agonist-mediated binding of [35S]GTPcS As [3H]antagonist binding was unaltered by the mutations, then assays were performed in conditions in which the total number of GPCR-G protein fusion polypeptides were known and this allowed estimates of the fraction of the constructs that produced functional dimers [61] Intermolecular interactions between the fusion proteins were responsible for the generation of activity, rather than activation of endogenously expressed G proteins, as similar results were produced when the pairs of constructs were expressed in mouse embryo fibroblasts lacking expression of the appropriate G proteins [61] One potential caveat to this approach has been provided by the work of Molinari et al [63] who used a similar strategy to explore functions of fusions between the DOP opioid receptor and Gao These workers reported that the reconstitution of function might simply reflect the higher concentration of G protein provided in the membranes by the : GPCR : G protein stoichiometry of the fusion proteins allowing the G protein fused to one receptor to interact with the G protein of another receptor-G protein fusion that may or may not be part of the same oligomer This is certainly an important issue to consider and control However, Carrillo et al [61] showed that providing extra, nonGPCR-linked, G protein through attachment to a truncated GPCR did not permit interaction with the full length fusion proteins, ruling out the possibility that the functional recovery resulted from membrane crowding and random collisions between fusion proteins Ligand-binding studies Classic monophasic ligand-binding isotherms as generally observed for the binding of antagonists is consistent with the ligand-binding to a single class of noninteracting sites As such, cooperative characteristics of the binding of a antagonist is not compatible with such a model and may be used to infer protein–protein interactions between GPCR monomers This model has been most actively explored for the M2 muscarinic acetylcholine receptor At least in certain conditions different 3H-labelled antagonists at this GPCR display significantly different Bmax values [64] Assuming that such effects not reflect trivial 2922 G Milligan and M Bouvier issues such as incorrect measurements of the specific activities of the two ligands, or the binding studies being performed under nonequilibrium conditions, such results are not compatible with the concept of a single population of noninteracting sites Mathematical analysis of this data has been interpreted to suggest that the M2 muscarinic acetylcholine receptor may exist as at least a tetramer and that ligands can bind to the receptor sites in a cooperative manner [64] Related approaches have also been applied to the dopamine D2 receptor expressed in CHO cells Here, when Na+ was eliminated from binding studies, the observed Bmax for [3H]raclopride was markedly lower than for [3H]spiperone, although this was not observed in the presence of Na+ [50] Equally, in the absence of Na+, raclopride appeared to display negative cooperativity on both its own binding and that of [3H]spiperone These results were modelled based on a GPCR dimer Although of considerable interest, these results may reflect altered interactions with other receptor partners such as the G proteins rather than the existence of dimerization This is particularly important considering that compounds considered as antagonists may display distinct inverse efficacy in the presence or absence of Na+ [65] and that ligand-binding studies using radiolabelled inverse agonists may lead to a different apparent Bmax as a function of the spontaneous interaction with G proteins [66] Despite this caveat, as long as they are performed in detail with appropriate controls, such ligand-binding studies offer means to examine GPCR protein–protein interactions in native tissues and without the need to manipulate the sequence of GPCRs to add tags required for detection in other types of assays Conclusions The range of approaches that have been applied to assess the quaternary structure of class A GPCRs is both large and diverse Use of these techniques has led to a general appreciation that GPCRs can certainly exist as dimers and ⁄ or higher order oligomers and that such interactions may be central to delivery of certainly some GPCRs to the cell surface and to their function However, given the nature of each of the widely used approaches it is still possible that observed interactions require the intermediacy and participation of GPCR-interacting accessory or scaffolding proteins Indeed a very recent study [67] has suggested that MOP and DOP opioid receptor heterodimerization may require G protein interactions It is important that a significant range of different approaches is used to explore such topics for any particular GPCR, FEBS Journal 272 (2005) 2914–2925 ª 2005 FEBS G Milligan and M Bouvier because none of those currently in widespread use are entirely convincing in isolation Key questions that require further technical advances include the proportion of any specific GPCR that is present as a monomer ⁄ dimer ⁄ oligomer and the significance of this for function Equally, although methods are being developed and used to explore the relative affinities of interactions between different GPCRs, an issue that is of particular importance to the likely relevance of potential GPCR heterodimers in physiology, these are not in widespread use at this time Also further documentation of the existence of distinct pharmacology and functions for GPCR heterodimers should certainly lead to the development of ligands with selective affinity and function for such heterodimer pairings and thus provide ultimate tools to probe the role of heterodimerization in vivo Acknowledgements Studies on GPCR dimerization in the Milligan and Bouvier labs are supported by the Biotechnology and Biosciences Research Council, the Medical Research Council and the Wellcome Trust to Graeme Milligan and the 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FEBS GPCR quaternary structure Fig Differentially epitope-tagged forms of the a1b-adrenoceptor have to be coexpressed to be coimmunoprecipitated N-terminally Flag- or c-myc-tagged forms of the. .. between the antibodies and the alternately tagged form of the receptor and (b) in a lack of coimmunoprecipitation of the HA-tagged b2-adrenoceptor when coexpressed with a c-myc-tagged form of the