G Protein-coupled Receptors Dimers Research Articles

Dimerization in GPCR mobility and signaling

Many types of cell surface as well as intracellular DNA-binding receptors exist and function as dimers; formation of homodimers or heterodimers appears to not only provide molecular mechanisms for agonist-induced activation but also increase specificity of ligand recognition and versatility of downstream signaling. G-protein-coupled receptors (GPCRs) were long thought to be an exception, but in recent years a lot of evidence has accumulated that GPCRs also can form dimers, even though it is far from certain when and where they actually do so under physiological conditions. Dimerization of GPCRs does not generally seem to be required for ligand recognition or signaling. However, dimerization may serve to affect receptor mobility at the cell surface and in intracellular trafficking, and may be involved in and affect their signaling functions.

Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation

A major obstacle to understanding the functional importance of dimerization between Class A G protein-coupled receptors (GPCRs) has been the methodological limitation in achieving control of the identity of the components comprising the signaling unit. We have developed a functional complementation assay that enables such control and illustrate it for the human dopamine D2 receptor. The minimal signaling unit, two receptors and a single G protein, is maximally activated by agonist binding to a single protomer, which suggests an asymmetrical activated dimer. Inverse agonist binding to the second protomer enhances signaling, whereas agonist binding to the second protomer blunts signaling. Ligand-independent constitutive activation of the second protomer also inhibits signaling. Thus, GPCR dimer function can be modulated by the activity state of the second protomer, which for a heterodimer may be altered in pathological states. Our novel methodology also makes possible the characterization of signaling from a defined heterodimer unit.

Useful pharmacological parameters for G-protein-coupled receptor homodimers obtained from competition experiments. Agonist–antagonist binding modulation

Many G-protein-coupled receptors (GPCRs) are expressed on the plasma membrane as dimers. Since drug binding data are currently fitted using equations developed for monomeric receptors, the interpretation of the pharmacological data are equivocal in many cases. As reported here, GPCR dimer models account for changes in competition curve shape as a function of the radioligand concentration used, something that cannot be explained by monomeric receptor models. Macroscopic equilibrium dissociation constants for the agonist and homotropic cooperativity index reflecting the intramolecular communication within the dopamine D 1 or adenosine A 2A receptor homodimer as well as hybrid equilibrium dissociation constant, which reflects the antagonist/agonist modulation may be calculated by fitting binding data from antagonist/agonist competition experiments to equations developed from dimer receptor models. Comparing fitting the data by assuming a classical monomeric receptor model or a dimer model, it is shown that dimer receptor models provide more clues useful in drug discovery than monomer-based models.

Bivalent Ligands as Specific Pharmacological Tools for G Protein-Coupled Receptor Dimers

G protein-coupled receptors (GPCRs) are major drug targets and are organized in dimeric/oligomeric complexes. These dimers may be composed of identical (homodimer) or different (heterodimer) receptors. GPCR dimerization provides new opportunities for drug design. Different strategies have been developed to specifically target GPCR dimers. Bivalent ligands, which are composed of two functional pharmacophores linked by a spacer, are among the most promising strategies. Due to the constitutive nature of GPCR dimers, bivalent ligands are expected in most cases to bind to and stabilize preexisting dimers rather then to promote ligand-induced dimerization. Most studies on GPCR dimerization were conducted so far in heterologous expression systems. Due the development of heterodimer-specific tools such as bivalent ligands, dimerization has now been confirmed for an increasing number of receptors in native tissues. In this review, we will discuss general considerations for the design and synthesis of bivalent ligands and present the functional in vitro and in vivo properties of reported bivalent ligands.

Physiological relevance of GPCR oligomerization and its impact on drug discovery

The potentially large functional and physiological diversity of G-protein coupled receptor (GPCR) dimers has generated a great deal of excitement about the opportunity that dimerization provides for enabling novel drug discovery. The discovery of physiologically relevant GPCR dimers suggests that new drug targets for diseases such as schizophrenia and pre-eclampsia can be developed by targeting dimers. Most of the previous work on GPCR dimers made use of the overexpression of differentially tagged GPCRs in heterologous cell systems. Current emphasis on the development of physiologically relevant cell systems that endogenously express the appropriate combination of GPCR dimers and accessory proteins is leading to dramatic increases in our understanding of GPCR dimers. These and other new tools such as GPCR-specific antibodies will be required to develop GPCR dimer specific drugs. Given that ligands are available for only a small percentage of the large number of potentially druggable GPCRs, the use of GPCR dimers might provide the necessary targets to increase the breadth and depth of receptors available for therapeutic interventions.

Mutations Affecting the Oligomerization Interface of G-Protein-Coupled Receptors Revealed by a Novel De Novo Protein Design Framework

Specific functional and pharmacological properties have recently been ascribed to G-protein-coupled receptor (GPCR) dimers/oligomers. Because the association of two identical or two distinct GPCR monomers seems to be required to elicit receptor function, it is necessary to understand the exact nature of this interaction. We present here a novel method for de novo protein design and its application to the prediction of mutations that can stabilize or destabilize a GPCR dimer while maintaining the monomer's native fold. To test the efficacy of this new method, the dimer of the single-spanned transmembrane domain of glycophorin A was used as a model system. Experimental data from mutagenesis of the helix-helix interface are compared with computational predictions at that interface, and the model's results are found to be consistent with the experimental findings. A flexible template was developed for the rhodopsin homodimer at atomic resolution and used to predict sets of three and five mutations. The results are found to be consistent across eight case studies, with favored mutations at each position. Mutation sets predicted to be the most disruptive at the dimerization interface are found to be less specific to the flexible template than sets predicted to be less disruptive.

Dimerization and oligomerization of G-protein-coupled receptors: debated structures with established and emerging functions

Dimerization or oligomerization of G-protein-coupled receptors (GPCRs) is a novel concept, which may lead to the reevaluation of the actions of pharmacological ligands, hormones, neurotransmitters, and other mediators acting on GPCRs. Although a large number of data obtained using different biophysical, biochemical and structural methods, and functional approaches argue for dimerization or oligomerization of these receptors, several publications criticized the applied methods and challenged the concept. The aim of this paper is to review the data that support the concept of receptor oligomerization, and the most important arguments against it. We conclude that it will require major methodical improvements to obtain decisive proof, whether GPCRs exist in their native membrane environments as homo- or heterodimeric or oligomeric complexes, in which receptor monomers have stable direct interactions. However, overwhelming amounts of data suggest that many GPCRs exhibit functional properties that require direct or indirect interactions between clustered receptors. Although it is difficult to conclude, about the exact nature of these interactions, dimerization or oligomerization of GPCRs is a useful paradigm for pharmacologists to study properties of receptors, which require functionally important clustering of receptors, such as trafficking of newly synthesized receptors to the cell surface, allosteric modulation of ligand binding, signaling specificity, co-internalization, or cross-inhibition of GPCRs.

Computational studies of Family A and Family B GPCRs

A full picture of the similarities between Family A and Family B GPCRs (G-protein coupled receptors) has been frustrated by the lack of clear homology between the respective sequences. Here, we review previous computational studies on GPCR dimerization in which the putative dimerization interfaces have been analysed using entropy, the ET (evolutionary trace) method and related methods. The results derived from multiple sequence alignments of Family A subfamilies have been mapped on to the rhodopsin crystal structure using standard alignments. Similarly, the results for the Family B alignments have been mapped on to the rhodopsin crystal structure using the 'cold-spot' alignment. For both Family A and Family B GPCRs, the sequence analysis indicates that there are functional sites on essentially all transmembrane helices, consistent with the parallel daisy chain model of GPCR oligomerization in which each GPCR makes interactions with a number of neighbouring GPCRs. The results are not too sensitive to the quality of the alignment. Molecular Dynamics simulations of the activation process within a single transmembrane bundle of the rhodopsin and the beta(2)-adrenergic receptor have been reviewed; the key observation, which is consistent with other computational studies, is that there is a translation and bending of helix 6, which contributes to a significant opening out of the intracellular face of the receptor, as shown in the accompanying movies. The simulations required the application of specific experiment-derived harmonic and half-harmonic distance restraints and so the application of such simulations to Family B GPCRs requires considerable care because of the alignment problem. Thus, in order to address the alignment problem, we have exploited the observation that GCR1, a plant GPCR, has homology with Family A, Family B and Family E GPCRs. The resulting alignment for transmembrane helix 3 is presented.

A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single beta2AR is incorporated per rHDL particle. The monomeric beta2AR efficiently activates Gs and displays GTP-sensitive allosteric ligand-binding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization.

Basic Concepts in G-Protein-Coupled Receptor Homo- and Heterodimerization

Until recently, heptahelical G-protein-coupled receptors (GPCRs) were considered to be expressed as monomers on the cell surface of neuronal and non-neuronal cells. It is now becoming evident that this view must be overtly changed since these receptors can form homodimers, heterodimers, and higher-order oligomers on the plasma membrane. Here we discuss some of the basics and some new concepts of receptor homo- and heteromerization. Dimers-oligomers modify pharmacology, trafficking, and signaling of receptors. First of all, GPCR dimers must be considered as the main molecules that are targeted by neurotransmitters or by drugs. Thus, binding data must be fitted to dimer-based models. In these models, it is considered that the conformational changes transmitted within the dimer molecule lead to cooperativity. Cooperativity must be taken into account in the binding of agonists-antagonists-drugs and also in the binding of the so-called allosteric modulators. Cooperativity results from the intramolecular cross-talk in the homodimer. As an intramolecular cross-talk in the heterodimer, the binding of one neurotransmitter to one receptor often affects the binding of the second neurotransmitter to the partner receptor. Coactivation of the two receptors in a heterodimer can change completely the signaling pathway triggered by the neurotransmitter as well as the trafficking of the receptors. Heterodimer-specific drugs or dual drugs able to activate the two receptors in the heterodimer simultaneously emerge as novel and promising drugs for a variety of central nervous system (CNS) therapeutic applications.

Asymmetric conformational changes in a GPCR dimer controlled by G-proteins

G-protein-coupled receptors (GPCRs) are key players in cell communication. Although long considered as monomeric, it now appears that these heptahelical proteins can form homo- or heterodimers. Here, we analyzed the conformational changes in each subunit of a receptor dimer resulting from agonist binding to either one or both subunits by measuring the fluorescent properties of a leukotriene B(4) receptor dimer with a single 5-hydroxytryptophan-labeled protomer. We show that a receptor dimer with only a single agonist-occupied subunit can trigger G-protein activation. We also show that the two subunits of the receptor dimer in the G-protein-coupled state differ in their conformation, even when both are liganded by the agonist. No such asymmetric conformational changes are observed in the absence of G-protein, indicating that the interaction of the G-protein with the receptor dimer brings specific constraints that prevent a symmetric functioning of this dimer. These data open new options for the differential signaling properties of GPCR dimers.

Regulation of Anterograde Transport of α2-Adrenergic Receptors by the N Termini at Multiple Intracellular Compartments

The studies on the intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly focused on the C termini of the receptors. In this report we determined the role of the extracellular N termini of alpha(2)-adrenergic receptors (alpha(2)-ARs) in the anterograde transport from the endoplasmic reticulum (ER) through the Golgi to the cell surface. The N-terminal-truncated alpha(2B)-AR mutant is completely unable to target to the cell surface. A single Met-6 residue is essential for the export of alpha(2B)-AR from the ER, likely through modulating correct alpha(2B)-AR folding in the ER. The Tyr-Ser motif, highly conserved in the membrane-proximal N termini of all alpha(2)-AR subtypes, is required for the exit of alpha(2A)-AR and alpha(2B)-AR from the Golgi apparatus, thus representing a novel Tyr-based motif modulating GPCR transport at the Golgi level. These data provide the first evidence indicating an essential role of the N termini of GPCRs in the export from distinct intracellular compartments along the secretory pathway.

Serotonin 5-HT2C Receptor Homodimer Biogenesis in the Endoplasmic Reticulum: REAL-TIME VISUALIZATION WITH CONFOCAL FLUORESCENCE RESONANCE ENERGY TRANSFER

Dimerization is a common property of G-protein-coupled receptors (GPCR). While the formation of GPCR dimers/oligomers has been reported to play important roles in regulating receptor expression, ligand binding, and second messenger activation, less is known about how and where GPCR dimerization occurs. The present study was performed to identify the precise cellular compartment in which class A GPCR dimer/oligomer biogenesis occurs. We addressed this issue using confocal microscopy and fluorescence resonance energy transfer (FRET) to monitor GPCR proximity within discrete intracellular compartments of intact living cells. Time-lapse confocal imaging was used to follow CFP- and YFP-tagged serotonin 5-HT2C receptors during biosynthesis in the endoplasmic reticulum (ER), trafficking through the Golgi apparatus and subsequent expression on the plasma membrane. Real-time monitoring of FRET between CFP- and YFP-tagged 5-HT2C receptors was performed by acceptor photobleaching within discrete regions of the ER, Golgi, and plasma membrane. The FRET signal was dependent on the ratio of CFP- to YFP-tagged 5-HT2C receptors expressed in each region and was independent of receptor expression level, as predicted for proteins in a non-random, clustered distribution. FRET efficiencies measured in the ER, Golgi, and plasma membrane were similar. These experiments provide direct evidence for homodimerization/oligomerization of class A GPCR in the ER and Golgi of intact living cells, and suggest that dimer/oligomer formation is a naturally occurring step in 5-HT2C receptor maturation and processing.

Probing the Existence of G Protein-Coupled Receptor Dimers by Positive and Negative Ligand-Dependent Cooperative Binding

An increasing amount of ligand binding data on G protein-coupled receptors (GPCRs) is not compatible with the prediction of the simple mass action law. This may be related to the propensity of most GPCRs, if not all, to oligomerize. Indeed, one of the consequences of receptor oligomerization could be a possible cross-talk between the protomers, which in turn could lead to negative or positive cooperative ligand binding. We prove here that this can be demonstrated experimentally. Saturation, dissociation, and competition binding experiments were performed on vasopressin and oxytocin receptors expressed in Chinese hamster ovary or COS-7 cells. Linear, concave, and convex Scatchard plots were then obtained, depending on the ligand used. Moreover, some competition curves exhibited an increase of the radiotracer binding for low concentrations of competitors, suggesting a cooperative binding process. These data demonstrate that various vasopressin analogs display either positive or negative cooperative binding. Because positive cooperative binding cannot be explained without considering receptor as multivalent, these binding data support the concept of GPCR dimerization process. The results, which are in good accordance with the predictions of previous mathematical models, suggest that binding experiments can be used to probe the existence of receptor dimers.

Dynamic models of G-protein coupled receptor dimers: indications of asymmetry in the rhodopsin dimer from molecular dynamics simulations in a POPC bilayer.

Based on the growing evidence that G-protein coupled receptors (GPCRs) form homo- and hetero-oligomers, models of GPCR signaling are now considering macromolecular assemblies rather than monomers, with the homo-dimer regarded as the minimal oligomeric arrangement required for functional coupling to the G-protein. The dynamic mechanisms of such signaling assemblies are unknown. To gain some insight into properties of GPCR dimers that may be relevant to functional mechanisms, we study their current structural prototype, rhodopsin. We have carried out nanosecond time-scale molecular dynamics (MD) simulations of a rhodopsin dimer and compared the results to the monomer simulated in the same type of bilayer membrane model composed of an equilibrated unit cell of hydrated palmitoyl-oleoyl-phosphatidyl choline (POPC). The dynamic representation of the homo-dimer reveals the location of structural changes in several regions of the monomeric subunits. These changes appear to be more pronounced at the dimerization interface that had been shown to be involved in the activation process [Proc Natl Acad Sci USA 102:17495, 2005]. The results are consistent with a model of GPCR activation that involves allosteric modulation through a single GPCR subunit per dimer.

Computational methods in drug design: Modeling G protein-coupled receptor monomers, dimers, and oligomers

G protein-coupled receptors (GPCRs) are membrane proteins that serve as very important links through which cellular signal transduction mechanisms are activated. Many vital physiological events such as sensory perception, immune defense, cell communication, chemotaxis, and neurotransmission are mediated by GPCRs. Not surprisingly, GPCRs are major targets for drug development today. Most modeling studies in the GPCR field have focused upon the creation of a model of a single GPCR (ie, a GPCR monomer) based upon the crystal structure of the Class A GPCR, rhodopsin. However, the emerging concept of GPCR dimerization has challenged our notions of the monomeric GPCR as functional unit. Recent work has shown not only that many GPCRs exist as homo- and heterodimers but also that GPCR oligomeric assembly may have important functional roles. This review focuses first on methodology for the creation of monomeric GPCR models. Special emphasis is given to the identification of localized regions where the structure of a GPCR may diverge from that of bovine rhodopsin. The review then focuses on GPCR dimers and oligomers and the bioinformatics methods available for identifying homo- and heterodimer interfaces.

Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells

Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the alpha2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits alpha o, beta1, and gamma2 and a number of GPCRs including M4, alpha2A, the adenosine A1 receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and Galpha proteins shows specificity: there is interaction between the alpha2A receptor and Go, but little interaction between the alpha2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heterotrimer are present at the cell membrane in the resting state in a pentameric complex.

Inhibition of Serotonin 5-Hydroxytryptamine2C Receptor Function through Heterodimerization: RECEPTOR DIMERS BIND TWO MOLECULES OF LIGAND AND ONE G-PROTEIN

Although dimerization appears to be a common property of G-protein-coupled receptors (GPCRs), it remains unclear whether a GPCR dimer binds one or two molecules of ligand and whether ligand binding results in activation of one or two G-proteins when measured using functional assays in intact living cells. Previously, we demonstrated that serotonin 5-hydroxytryptamine2C (5-HT2C) receptors form homodimers (Herrick-Davis, K., Grinde, E., and Mazurkiewicz, J. (2004) Biochemistry 43, 13963-13971). In the present study, an inactive 5-HT(2C) receptor was created and coexpressed with wild-type 5-HT2C receptors to determine whether dimerization regulates receptor function and to determine the ligand/dimer/G-protein stoichiometry in living cells. Mutagenesis of Ser138 to Arg (S138R) produced a 5-HT2C receptor incapable of binding ligand or stimulating inositol phosphate (IP) signaling. Confocal fluorescence imaging revealed plasma membrane expression of yellow fluorescent protein-tagged S138R receptors. Expression of wild-type 5-HT2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT2C receptors was visualized in living cells using confocal fluorescence resonance energy transfer (FRET). FRET was dependent on the donor/acceptor ratio and independent of the receptor expression level. Therefore, inactive 5-HT2C receptors inhibit wild-type 5-HT2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane. These results are consistent with a model in which one GPCR dimer binds two molecules of ligand and one G-protein and indicate that dimerization is essential for 5-HT receptor function.

The quaternary structure of G protein‐coupled receptors

G protein-coupled receptors (GPCRs) are seven-transmembrane-helix polypeptides that are encoded by somewhere in the region of 3% of the genes of mammalian genomes and are the most actively targeted class of polypeptides for small molecule drug design. Despite this, and observations scattered throughout the literature for a number of years suggesting that GPCRs can exist as dimers or higher-order oligomers, it is only in the last five years that analysis of the structural organization of GPCRs has become wide-ranging. Although a number of key issues, including the molecular basis of dimerization ⁄oligomerization, the importance of dimerization ⁄oligomerization for function, and the physiological occurrence and relevance of hetero-dimers containing GPCRs produced by two different genes, remain both contentious and central research themes, there is now little doubt that dimerization ⁄oligomerization does occur. The four minireviews in this issue of FEBS Journal address different aspects of GPCR dimerization ⁄oligomerization. In the first of these Michel Bouvier and I discuss, and address critically, the approaches that have and are being employed to examine GPCR dimerization ⁄ oligomerization. Given the large number of studies that are being performed in this area and the limitations of each individual technique we conclude that concurrent application of a range of approaches that employ distinct techniques is required to provide convincing data. At the current time experimental information on the mechanisms of dimerization and the interfaces involved is both limited and fragmentary. Indeed, models for dimerization include both ‘linear packing’ models in which the external faces of specific transmembrane helices provide the interface and ‘domain swap’ models in which the two elements of the dimer exchange transmembrane segments. Informatics can suggest plausible mechanisms and in the second article Marta Filizola and Harel Weinstein consider the contributions that informatic studies have made to this field and the manner in which iterative cycles of informatic analysis and direct experiments may lead to clearer understanding. The third article by Roberto Maggio and colleagues focuses on the development of distinct pharmacology and function associated with GPCR hetero-dimer formation. As noted above, there is considerable interest in the possibility that at least a proportion of GPCR ‘hetero-dimers’ represent ‘domain swaps’. In such a situation it is almost axiomatic that differences in the details of ligand binding would be produced in comparison with ‘linear packing’ dimers. The extent of existence of GPCR hetero-dimers in physiological settings remains to be established but if they are relatively common they may represent a novel and currently unappreciated set of drug targets for the pharmaceutical industry. Although the first three articles all concentrate on members of the rhodopsin-like (or family A) GPCRs because numerically these are by far the largest group, the first clear example of GPCR hetero-dimerization and its importance for function came from the family C GPCRs. These all contain a long N-terminal extracellular domain in which the natural ligands bind within a ‘venus flytrap’ structure. Crystal structures of such domains in both empty and ligand-bound arrangements have provided detailed insights into conformational reorganization of such GPCR dimers in the presence of ligands. The ability to create chimeric family C GPCRs by combining the extracellular domain of one with the transmembrane and intracellular element of a second has also provided dramatic insights into the importance of dimerization for function, and in the final article in the series Jean-Phillipe Pin and colleagues discuss such studies and the development of allosteric modulators of function of family C GPCRs, another area with major implications for the development of novel classes of small molecule therapeutics.

Evidence for a single heptahelical domain being turned on upon activation of a dimeric GPCR

G-protein-coupled receptors (GPCRs) have been shown to form dimers, but the relevance of this phenomenon in G-protein activation is not known. Among the large GPCR family, metabotropic glutamate (mGlu) receptors are constitutive dimers. Here we examined whether both heptahelical domains (HDs) are turned on upon full receptor activation. To that aim, we measured G-protein coupling efficacy of dimeric mGlu receptors in which one subunit bears specific mutations. We show that a mutation in the third intracellular loop (i3 loop) known to prevent G-protein activation in a single subunit decreases coupling efficacy. However, when a single HD is blocked in its inactive state using an inverse agonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), no decrease in receptor activity is observed. Interestingly, in a receptor dimer in which the subunit that binds MPEP is mutated in its i3 loop, MPEP enhances agonist-induced activity, reflecting a 'better' activation of the adjacent HD. These data are consistent with a model in which a single HD is turned on upon activation of such homodimeric receptors and raise important issues in deciphering the functional role of GPCR dimer formation for G-protein activation.