GPCR Dimerization Research Articles

Determining the oligomer number of native GPCR using florescence correlation spectroscopy and drug-induced inactivation-reactivation.

GPCRs are a major family of homologous proteins and are key mediators of the effects of numerous endogenous neurotransmitters, hormones, cytokines, therapeutic drugs, and drugs-of-abuse. Despite the enormous amount of research on the pharmacological and biochemical properties of GPCRs, there is surprisingly little information on GPCR dimer structure and function in primary cell culture or in vivo. We have used two novel approaches to develop methods to detect and study GPCR dimer function: FCS/PCH and "inactivation-reactivation". This review will focus on the data we have developed and our interpretations of those data. Using FCS/PCH 5-HT2C receptors have been detected directly and appear to exist as dimers, consistent with the inactivation-reactivation data on 5-HT7 and 5-HT2A receptors. Studies of the 5-HT7 and 5-HT2A serotonin receptors have revealed that binding of a pseudo-irreversible antagonist ("inactivator") to one of the orthosteric sites of a homodimer abolishes all receptor activity, and subsequent binding of a competitive antagonist to the orthosteric site of the second protomer releases the inactivator, allowing the receptor to return to an active state. This approach demonstrates allosteric crosstalk between protomers of native GPCR homodimers, indicating that GPCRs do exist and function as homodimers in both recombinant cells and rat primary astrocytes. This technique can be applied universally using intact recombinant or primary cells in culture, membrane homogenate preparations and, potentially, in vivo. This approach can be applied to heterodimers as well as homodimers and may aid in the development of novel drugs with heterodimer selectivity.

CrossTalk opposing view: Weighing the evidence for class A GPCR dimers, the jury is still out.

CrossTalk opposing view: Weighing the evidence for class A GPCR dimers, the jury is still out.

CrossTalk proposal: Weighing the evidence for Class A GPCR dimers, the evidence favours dimers.

CrossTalk proposal: Weighing the evidence for Class A GPCR dimers, the evidence favours dimers.

Platform for examining GPCR dimerization and signaling

Platform for examining GPCR dimerization and signaling

Molecular modeling studies give hint for the existence of a symmetric hβ2R-Gαβγ-homodimer

Several experimental studies suggest that GPCR dimers or oligomers may play an important role in signal transduction. In 2011 the crystal structure of a hβ2R-Gαβγ-complex was published and crystal structures of GPCR dimers are known. But until now, no crystal structure of a GPCR dimer including the Gαβγ-complex is available. In order to obtain detailed insights into interactions within hβ2R dimers including the Gαβγ-complex we performed a potential-energy-surface scan in order to identify favored asymmetric and symmetric hβ2R-Gαβγ-homodimers. This potential energy surface scan suggests, besides the existence of asymmetric dimers, the existence of a symmetric hβ2R-Gαβγ-homodimer with a TM I/VII-contact. A subsequent 20 ns MD simulation of the symmetric homodimer revealed large asymmetric conformational changes of both hβ2Rs, especially regarding TM VII and the interaction network between Asp(2.50), Val(7.44), Ser(7.46) and Tyr(7.43). Since similar conformational changes were not observed during the molecular dynamic simulation of the monomeric hβ2R-Gαβγ-complex, it may be suggested that the conformational changes in the symmetric homodimer are related to the presence of the second hβ2R-Gαβγ-complex. Due to the limitations of simulation time, conformational changes within a time scale of μs or ms may of course not be observed. However, the detected conformational changes, especially in TM VII, correspond to minima on the potential energy surface and thus, this study gives new insights into GPCR dimers on molecular level and furthermore, gives suggestions for site-directed mutagenesis studies.

Synthesis, functional and binding profile of (R)-apomorphine based homobivalent ligands targeting the dopamine D2 receptor

Bivalent ligands represent useful tools to investigate the phenomenon of GPCR dimerization. We synthesized bivalent ligands based on (R)-apomorphine with variations in spacer length, and assessed these compounds in functional and binding assays at the dopamine D2 receptor. The results present novel SAR for bivalent ligands targeting the D2R, and identify a relationship for spacer length with ligand potency, efficacy and affinity.

3P107 GPCRダイマーがシグナルトリガーとしてはたらく : インバースアゴニスト効果の1分子イメジング解析に基づく発見(03.膜蛋白質,ポスター,日本生物物理学会年会第51回(2013年度))

3P107 GPCRダイマーがシグナルトリガーとしてはたらく : インバースアゴニスト効果の1分子イメジング解析に基づく発見(03.膜蛋白質,ポスター,日本生物物理学会年会第51回(2013年度))

Structural aspects of M 3 muscarinic acetylcholine receptor dimer formation and activation

To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M(3) muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. [(35)S]GTPγS binding/Gα(q/11) immunoprecipitation assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (∼50% reduction in receptor-G-protein coupling, as compared to control M3R). These data support the novel concept that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Given the high degree of structural homology among family A GPCRs, these findings should be of broad significance.

Bivalent Dopamine D2 Receptor Ligands: Synthesis and Binding Properties

Dopamine D(2) receptor homodimers might be of particular importance in the pathophysiology of schizophrenia and, thus, serve as promising target proteins for the discovery of atypical antipsychotics. A highly attractive approach to investigate and control GPCR dimerization may be provided by the exploration and characterization of bivalent ligands, which can act as molecular probes simultaneously binding two adjacent binding sites of a dimer. The synthesis of bivalent dopamine D(2) receptor ligands of type 1 is presented, incorporating the privileged structure of 1,4-disubstituted aromatic piperidines/piperazines (1,4-DAPs) and triazolyl-linked spacer elements. Radioligand binding studies provided diagnostic insights when Hill slopes close to two for bivalent ligands with particular spacer lengths and a comparative analysis with respective monovalent control ligands and unsymmetrically substituted analogues indicated a bivalent binding mode with a simultaneous occupancy of two neighboring binding sites.

GPCR dimers moving closer

Time-resolved fluorescence resonance energy transfer (TR-FRET) measurements with selective, fluorescently labeled ligands support the existence of GPCR dimers in native tissues and suggest that activated GPCR dimers are asymmetric.

Role of antibodies in developing drugs that target G-protein-coupled receptor dimers.

G-protein-coupled receptors are important molecular targets in drug discovery. These receptors play a pivotal role in physiological signaling pathways and are targeted by nearly 50% of currently available drugs. Mounting evidence suggests that G-protein-coupled receptors form dimers, and various studies have shown that dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological implications of dimerization in vivo have not been well explored because detection of GPCR dimers in endogenous systems has been a challenging task. One exciting new approach to this challenge is the generation of antibodies against specific G-protein-coupled receptor dimers. Such antibodies could be used as tools for characterization of heteromer-specific function; as reagents for their purification, tissue localization, and regulation in vivo; and as probes for mapping their functional domains. In addition, such antibodies could serve as alternative ligands for G-protein-coupled receptor heteromers. Thus, heteromer-specific antibodies represent novel tools for the exploration and manipulation of G-protein-coupled receptor-dimer pharmacology.

Leukotriene BLT2 Receptor Monomers Activate the Gi2 GTP-binding Protein More Efficiently than Dimers

Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B(4) receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G(i2) protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human alpha(5) integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB(4) binding in the presence of the purified G protein G alpha(i2). The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5'-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified G alpha(i2) beta(1) gamma(2) protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.

G Protein Activation by the Leukotriene B4 Receptor Dimer: EVIDENCE FOR AN ABSENCE OF TRANS-ACTIVATION

There is compelling evidence that G protein-coupled receptors exist as homo- and heterodimers, but the way these assemblies function at the molecular level remains unclear. We used here the purified leukotriene B(4) receptor BLT1 stabilized in its dimeric state to analyze how a receptor dimer activates G proteins. For this, we produced heterodimers between the wild-type BLT1 and a BLT1/ALXR chimera. The latter is no longer activated by leukotriene B(4) but is still activated by ALXR agonists. In this heterodimer, agonist binding to either one of the two protomers induced asymmetric conformational changes within the receptor dimer. Of importance, no G protein activation was observed when using a dimer where the ligand-loaded protomer was not able to trigger GDP/GTP exchange due to specific mutations in its third intracellular loop, establishing that the conformation of the agonist-free protomer is not competent for G protein activation. Taken together, these data indicate that although ligand binding to one protomer in the heterodimer is associated with cross-conformational changes, a trans-activation mechanism where the ligand-free subunit would trigger GDP/GTP exchange cannot be considered in this case for G protein activation. This observation sheds light into the way GPCR dimers, in particular heterodimers, could activate their cognate G proteins.

Design and Synthesis of Specific Probes for Human 5-HT4 Receptor Dimerization Studies

Recently, human 5-HT4 receptors have been demonstrated to form constitutive dimers in living cells. To evaluate the role of dimerization on the 5-HT4 receptor function, we investigated the conception and the synthesis of bivalent molecules able to influence the dimerization process. Their conception is based on a model of the 5-HT4 receptor dimer derived from protein/protein docking experiments. These bivalent ligands are constituted by two ML10302 units, a specific 5-HT4 ligand, linked through a spacer of different sizes and natures. These synthesized bivalent ligands were evaluated in binding assays and cyclic AMP production on the 5-HT4(e/g) receptor isoform stably transfected in C6 glial cells. Our data showed that bivalent ligands conserved a similar affinity compared to the basal ML10302 unit. Nevertheless, according to the nature and the size of the spacer, the pharmacological profile of ML10302 is more or less conserved. In view of the interest of bivalent ligands for investigating the GPCR dimerization process, these 5-HT4 specific bivalent ligands constitute valuable pharmacological tools for the study of 5-HT4 receptor dimerization.

Bioluminescence Resonance Energy Transfer Reveals Ligand-induced Conformational Changes in CXCR4 Homo- and Heterodimers

Homo- and heterodimerization have emerged as prominent features of G-protein-coupled receptors with possible impact on the regulation of their activity. Using a sensitive bioluminescence resonance energy transfer system, we investigated the formation of CXCR4 and CCR2 chemokine receptor dimers. We found that both receptors exist as constitutive homo- and heterodimers and that ligands induce conformational changes within the pre-formed dimers without promoting receptor dimer formation or disassembly. Ligands with different intrinsic efficacies yielded distinct bioluminescence resonance energy transfer modulations, indicating the stabilization of distinct receptor conformations. We also found that peptides derived from the transmembrane domains of CXCR4 inhibited activation of this receptor by blocking the ligand-induced conformational transitions of the dimer. Taken together, our data support a model in which chemokine receptor homo- and heterodimers form spontaneously and respond to ligand binding as units that undergo conformational changes involving both protomers even when only one of the two ligand binding sites is occupied.

Glucose sensing via the protein kinase A pathway in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe primarily detects glucose via a cAMP-signalling pathway. Components of this pathway include the Git3 G-protein-coupled receptor and a heterotrimeric G-protein, from which the Gpa2 Galpha subunit activates adenylate cyclase (Git2/Cyr1). Three additional proteins, Git1, Git7 and Git10 are required to generate a cAMP response even in a strain expressing an activated form of Gpa2, which is capable of bypassing the loss of the GPCR and Gbetagamma dimer. Therefore, Git1, Git7 and Git10 either act in a G-protein-independent manner or are required to stabilize or assemble a functional signalling complex. Although prior data suggested that the Cgs2 cAMP phosphodiesterase (PDE) does not regulate the cAMP response, we now have evidence that along with adenylate cyclase regulation, PDE activation is important for limiting the response to glucose. Finally, regulation of protein kinase A activation appears to involve both traditional post-translational regulation of the function of the components of the cAMP pathway and glucose-dependent transcriptional regulation of some of these cAMP pathway genes.

Cooperative Conformational Changes in a G-protein-coupled Receptor Dimer, the Leukotriene B4 Receptor BLT1

We have used an isolated receptor, the leukotriene B(4) receptor BLT1, to analyze the mechanism of receptor activation in a G-protein-coupled receptor dimer. The isolated receptor is essentially a dimer whether the agonist is present or not, provided the detergent used stabilizes the inactive dimeric assembly. We have produced a receptor mutant where Cys(97) in the third transmembrane domain has been replaced by a serine. This mutation leads to an approximately 100-fold decrease in the affinity for the agonist. 5-Hydroxytryptophan has then been introduced at position 234 in the C97A mutant sixth transmembrane domain. Agonist binding to the labeled receptor is associated with variations in the fluorescence properties of 5-hydroxytryptophan due to specific agonist-induced conformational changes. The C97A mutant labeled with 5-hydroxytryptophan has then been associated with a wild-type receptor in a dimeric complex that has been subsequently purified. The purified complex activates its G-protein partner in a similar manner as the wild-type homodimer. Due to the difference in the affinity for the agonist between the wild-type and mutant protomers in this dimer, we have been able to reach a state where one of the protomers, the mutant, is in its unliganded state, whereas the other, the wild type, is loaded with the agonist. We show that agonist binding to the wild-type receptor induces specific changes in the conformation of the unliganded protomer, as evidenced by the variations in the emission of the 5-hydroxytryptophan residue in the mutant receptor. These data provide a direct demonstration for agonist-induced cooperative conformational changes in a GPCR dimer.

Signal Transduction by GABAB Receptor Heterodimers

GABAB receptors are G-protein-coupled receptors that mediate inhibition throughout the central and peripheral nervous systems. A single cloned receptor, GABABR1, which has at least three alternatively spliced forms, appears to account for the vast majority of binding sites in the brain for high-affinity antagonists. In heterologous expression systems GABABR1 is poorly expressed on the plasma membrane and largely fails to couple to ion channels. A second gene, GABABR2, which exhibits moderately low homology to GABABR1, permits surface expression of GABABR1 and the appearance of baclofen-sensitive K+ and Ca+1 currents. We review the data that supports a model of the native GABAB receptor as a heterodimer composed of GABABR1 and GABABR2 proteins. New data from mutagenesis experiments are presented that point to amino acid residues on GABABR1 critical for ligand activation of the heterodimer. The possible role of GABABR2 in signal transduction is also discussed. The interdependent nature of the two subunits for receptor function makes the GABAB receptor a useful model to explore the larger significance of GPCR dimerization for G-protein activation.

G-protein-coupled receptor dimers in the lime light

It will be important to apply these optical resonance energy transfer techniques to examine the dimerization of other GPCRs in intact cells. GPCRs can be classified into three major subfamilies (rhodopsin, calcitonin and metabotropic receptor subfamilies) and members of all three subfamilies have been shown to exist as homodimers12xSee all References12. Several recent studies have also revealed heterodimerization of GPCRs (between closely related as well as distantly related members), which alters the functional properties of these receptors13xGABAB receptors – the first 7TM heterodimers. Marshall, F.H. et al. Trends Pharmacol. Sci. 1999; 20: 396–399Abstract | Full Text | Full Text PDF | PubMed | Scopus (245)See all References, 14xG-protein-coupled receptor hetero-dimerization modulates receptor function. Jordan, B.A. and Devi, L.A. Nature. 1999; 399: 697–700Crossref | PubMed | Scopus (833)See all References, 15xReceptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Rocheville, M. et al. Science. 2000; 288: 154–157Crossref | PubMed | Scopus (638)See all References. Biochemical analyses have shown that in some GPCRs, agonist treatment leads to stabilization and/or an increase in the level of dimers, whereas in others agonist treatment has no effect or leads to a decrease in the level of dimers12xSee all References12. These optical resonance energy transfer techniques provide a novel and clear protocol to examine both homodimerization and heterodimerization of GPCRs in living cells. These methods are free from the complications that arise from more traditional biochemical methods of identifying dimers, and we should expect to see more studies using BRET and FRET given their simplicity and straightforwardness.Dimerization of GPCRs is a novel mechanism for receptor activation. It remains to be established whether GPCR dimerization occurs in vivo (i.e. in a living organism). Recent studies using energy transfer techniques to demonstrate the existence of dimers in living cells is a major step in this direction.