G Protein-coupled Receptors Dimers Research Articles

The EBI2 receptor is coexpressed with CCR5 in CD4 + T cells and boosts HIV-1 R5 replication.

CCR5, a G protein-coupled receptor (GPCR), is used by most HIV strains as a coreceptor. In this study, we looked for other GPCR able to modify HIV-1 infection. We analyzed the effects of one GPCR coexpressed with CCR5, EBI2, on HIV-1 replicative cycle. We identified GPCR expressed in primary CD4 + CCR5 + T cells by multi-RT-qPCR. We studied GPCR dimerization by FRET technology. Cell lines expressing EBI2 were established by transduction with HIV vectors. HIV-1 entry was quantified with virions harboring β-lactamase fused to the viral protein vpr, early and late HIV-1 transcriptions by qPCR, NFkB nuclear activation by immunofluorescence and transfection, and viral production by measuring p24 concentration in culture supernatant by ELISA. We showed that EBI2 is naturally expressed in primary CD4 + CCR5 + T cells, and that CCR5 and EBI2 heterodimerize. We observed that this coexpression reduced viral entry by 50%. The amount of HIV reverse transcripts was similar in cells expressing or not EBI2. Finally, the presence of EBI2 induced the translocation of NFkB and activated HIV-1 genome expression. Globally, the result was a drastic HIV-1 R5, but not X4, overproduction in EBI2 -transduced cells. EBI2 expression in CD4 + CCR5 + cells boosts HIV-1 R5 productive infection. As the natural ligand for EBI2 is present in blood and lymphoid tissues, the constant EBI2 activation might increase HIV replication in CD4 + T cells. It might be of interest to test the effect of EBI2 antagonists on the residual viral production persisting in patients aviremic under treatment.

G Protein-Coupled Receptor Dimerization-What Next?

Numerous studies highlight the therapeutic potential of G protein-coupled receptor (GPCR) heterodimers, emphasizing their significance in various pathological contexts. Despite extensive basic research and promising outcomes in animal models, the translation of GPCR heterodimer-targeting drugs into clinical use remains limited. The complexities of in vivo conditions, particularly within thecomplex central nervous system, pose challenges in fully replicating physiological environments, hindering clinical success. This review discusses examples of the most studied heterodimers, their involvement in nervous system pathology, and the available data on their potential ligands. In addition, this review highlights the intricate interplay between lipids and GPCRs as a potential key factor in understanding the complexity of cell signaling. The multifaceted role of lipids in modulating the dynamics of GPCR dimerization is explored, shedding light on the elaborate molecular mechanisms governing these interactions.

Inhibitory effect of truncated isoforms on GPCR dimerization predicted by combinatorial computational strategy

G protein-coupled receptors (GPCRs) play a pivotal role in fundamental biological processes and disease development. GPCR isoforms, derived from alternative splicing, can exhibit distinct signaling patterns. Some highly-truncated isoforms can impact functional performance of full-length receptors, suggesting their intriguing regulatory roles. However, how these truncated isoforms interact with full-length counterparts remains largely unexplored. Here, we computationally investigated the interaction patterns of three human GPCRs from three different classes, ADORA1 (Class A), mGlu2 (Class C) and SMO (Class F) with their respective truncated isoforms because their homodimer structures have been experimentally determined, and they have truncated isoforms deposited and identified at protein level in Uniprot database. Combining the neural network-based AlphaFold2 and two physics-based protein-protein docking tools, we generated multiple complex structures and assessed the binding affinity in the context of atomistic molecular dynamics simulations. Our computational results suggested all the four studied truncated isoforms showed potent binding to their counterparts and overlapping interfaces with homodimers, indicating their strong potential to block homodimerization of their counterparts. Our study offers insights into functional significance of GPCR truncated isoforms and supports the ubiquity of their regulatory roles.

The World of GPCR dimers – Mapping dopamine receptor D2 homodimers in different activation states and configuration arrangements

G protein-coupled receptors (GPCRs) are known to dimerize, but the molecular and structural basis of GPCR dimers is not well understood. In this study, we developed a computational framework to generate models of symmetric and asymmetric GPCR dimers using different monomer activation states and identified their most likely interfaces with molecular details. We chose the dopamine receptor D2 (D2R) homodimer as a case study because of its biological relevance and the availability of structural information. Our results showed that transmembrane domains 4 and 5 (TM4 and TM5) are mostly found at the dimer interface of the D2R dimer and that these interfaces have a subset of key residues that are mostly nonpolar from TM4 and TM5, which was in line with experimental studies. In addition, TM2 and TM3 appear to be relevant for D2R dimers. In some cases, the inactive configuration is unaffected by the partnered protomer, whereas in others, the active protomer adopts the properties of an inactive receptor. Additionally, the β-arrestin configuration displayed the properties of an active receptor in the absence of an agonist, suggesting that a switch to another meta-state during dimerization occurred. Our findings are consistent with the experimental data, and this method can be adapted to study heterodimers and potentially extended to include additional proteins such as G proteins or β-arrestins. In summary, this approach provides insight into the impact of the conformational status of partnered protomers on the overall quaternary GPCR macromolecular structure and dynamics.

The transmembrane domains of GPCR dimers as targets for drug development

G-protein-coupled receptors (GPCRs) can form homodimers or heterodimers that modulate specific signal transduction pathways to regulate a wide range of physiological and pathological functions. As such, GPCR dimers are novel drug targets for disorders including depression, hypertension, diabetes, and vascular dementia. The interaction between two receptors in a GPCR dimer involves a conformational change in the transmembrane domain (TMD). It has been demonstrated that the TMD has an important role in GPCR dimer formation and stability in vitro and in vivo. Moreover, increasing evidence shows that the TMD of GPCRs affects the function of dimers. Therefore, the TMD of GPCRs is an emerging target for the development of drugs to treat diseases that involve GPCR dimerization.

Probing GPCR Dimerization Using Peptides.

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins and the most common and extensively studied pharmacological target. Numerous studies over the last decade have confirmed that GPCRs do not only exist and function in their monomeric form but in fact, have the ability to form dimers or higher order oligomers with other GPCRs, as well as other classes of receptors. GPCR oligomers have become increasingly attractive to investigate as they have the ability to modulate the pharmacological responses of the receptors which in turn, could have important functional roles in diseases, such as cancer and several neurological & neuropsychiatric disorders. Despite the growing evidence in the field of GPCR oligomerisation, the lack of structural information, as well as targeting the ‘undruggable’ protein-protein interactions (PPIs) involved in these complexes, has presented difficulties. Outside the field of GPCRs, targeting PPIs has been widely studied, with a variety of techniques being investigated; from small-molecule inhibitors to disrupting peptides. In this review, we will demonstrate several physiologically relevant GPCR dimers and discuss an array of strategies and techniques that can be employed when targeting these complexes, as well as provide ideas for future development.

A model for how Gβγ couples Gα to GPCR.

Representing ∼5% of the human genome, G-protein-coupled receptors (GPCRs) are a primary target for drug discovery; however, the molecular details of how they couple to heterotrimeric G protein subunits are incompletely understood. Here, I propose a hypothetical initial docking model for the encounter between GPCR and Gβγ that is defined by transient interactions between the cytosolic surface of the GPCR and the prenyl moiety and the tripeptide motif, asparagine-proline-phenylalanine (NPF), in the C-terminus of the Gγ subunit. Analysis of class A GPCRs reveals a conserved NPF binding site formed by the interaction of the TM1 and H8. Functional studies using differentially prenylated proteins and peptides further suggest that the intracellular hydrophobic core of the GPCR is a prenyl binding site. Upon binding TM1 and H8 of GPCRs, the propensity of the C-terminal region of Gγ to convert into an α helix allows it to extend into the hydrophobic core of the GPCR, facilitating the GPCR active state. Conservation of the NPF motif in Gγ isoforms and interacting residues in TM1 and H8 suggest that this is a general mechanism of GPCR-G protein signaling. Analysis of the rhodopsin dimer also suggests that Gγ-rhodopsin interactions may facilitate GPCR dimer transactivation.

Activation mechanism of the class D fungal GPCR dimer Ste2

The fungal class D1 G-protein-coupled receptor (GPCR) Ste2 has a different arrangement of transmembrane helices compared with mammalian GPCRs and a distinct mode of coupling to the heterotrimeric G protein Gpa1–Ste2–Ste181. In addition, Ste2 lacks conserved sequence motifs such as DRY, PIF and NPXXY, which are associated with the activation of class A GPCRs2. This suggested that the activation mechanism of Ste2 may also differ. Here we determined structures of Saccharomyces cerevisiae Ste2 in the absence of G protein in two different conformations bound to the native agonist α-factor, bound to an antagonist and without ligand. These structures revealed that Ste2 is indeed activated differently from other GPCRs. In the inactive state, the cytoplasmic end of transmembrane helix H7 is unstructured and packs between helices H1–H6, blocking the G protein coupling site. Agonist binding results in the outward movement of the extracellular ends of H6 and H7 by 6 Å. On the intracellular surface, the G protein coupling site is formed by a 20 Å outward movement of the unstructured region in H7 that unblocks the site, and a 12 Å inward movement of H6. This is a distinct mechanism in GPCRs, in which the movement of H6 and H7 upon agonist binding facilitates G protein coupling.

Allosteric ligands control the activation of a class C GPCR heterodimer by acting at the transmembrane interface

G protein-coupled receptors (GPCRs) are among the most promising drug targets. They often form homo- and heterodimers with allosteric cross-talk between receptor entities, which contributes to fine-tuning of transmembrane signaling. Specifically controlling the activity of GPCR dimers with ligands is a good approach to clarify their physiological roles and validate them as drug targets. Here, we examined the mode of action of positive allosteric modulators (PAMs) that bind at the interface of the transmembrane domains of the heterodimeric GABAB receptor. Our site-directed mutagenesis results show that mutations of this interface impact the function of the three PAMs tested. The data support the inference that they act at the active interface between both transmembrane domains, the binding site involving residues of the TM6s of the GABAB1 and the GABAB2 subunit. Importantly, the agonist activity of these PAMs involves a key region in the central core of the GABAB2 transmembrane domain, which also controls the constitutive activity of the GABAB receptor. This region corresponds to the sodium ion binding site in class A GPCRs that controls the basal state of the receptors. Overall, these data reveal the possibility of developing allosteric compounds able to specifically modulate the activity of GPCR homo- and heterodimers by acting at their transmembrane interface.

Exploring the signaling space of a GPCR using bivalent ligands with a rigid oligoproline backbone

G protein-coupled receptors (GPCRs) are one of the most important drug-target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-Å distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-Å distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-Å distance have different potencies toward β-arrestin-1 and β-arrestin-2, and 3) the divalent agonistic ligand with 10-Å distance specifically reduces Gq potency without affecting β-arrestin recruitment, pointing toward an allosteric effect. In summary, we show that rigid oligoproline backbones represent a tool to develop ligands with biased GPCR signaling.

Modeling the Heterodimer Interfaces of Melatonin Receptors.

Melatonin receptors are Class A G protein-coupled receptors (GPCRs) that regulate a plethora of physiological activities in response to the rhythmic secretion of melatonin from the pineal gland. Melatonin is a key regulator in the control of circadian rhythm and has multiple functional roles in retinal physiology, memory, immunomodulation and tumorigenesis. The two subtypes of human melatonin receptors, termed MT1 and MT2, utilize overlapping signaling pathways although biased signaling properties have been reported in some cellular systems. With the emerging concept of GPCR dimerization, melatonin receptor heterodimers have been proposed to participate in system-biased signaling. Here, we used computational approaches to map the dimerization interfaces of known heterodimers of melatonin receptors, including MT1/MT2, MT1/GPR50, MT2/GPR50, and MT2/5-HT2C. By homology modeling and membrane protein docking analyses, we have identified putative preferred interface interactions within the different pairs of melatonin receptor dimers and provided plausible structural explanations for some of the unique pharmacological features of specific heterodimers previously reported. A thorough understanding of the molecular basis of melatonin receptor heterodimers may enable the development of new therapeutic approaches against aliments involving these heterodimeric receptors.

BRET Analysis of GPCR Dimers in Neurons and Non-Neuronal Cells: Evidence for Inactive, Agonist, and Constitutive Conformations.

G-protein-coupled receptors (GPCRs) are dimeric proteins, but the functional consequences of the process are still debated. Active GPCR conformations are promoted either by agonists or constitutive activity. Inverse agonists decrease constitutive activity by promoting inactive conformations. The histamine H3 receptor (H3R) is the target of choice for the study of GPCRs because it displays high constitutive activity. Here, we study the dimerization of recombinant and brain H3R and explore the effects of H3R ligands of different intrinsic efficacy on dimerization. Co-immunoprecipitations and Western blots showed that H3R dimers co-exist with monomers in transfected HEK 293 cells and in rodent brains. Bioluminescence energy transfer (BRET) analysis confirmed the existence of spontaneous H3R dimers, not only in living HEK 293 cells but also in transfected cortical neurons. In both cells, agonists and constitutive activity of the H3R decreased BRET signals, whereas inverse agonists and GTPγS, which promote inactive conformations, increased BRET signals. These findings show the existence of spontaneous H3R dimers not only in heterologous systems but also in native tissues, which are able to adopt a number of allosteric conformations, from more inactive to more active states.

Functional independence of endogenous μ- and δ-opioid receptors co-expressed in cholinergic interneurons.

Class A G-protein-coupled receptors (GPCRs) normally function as monomers, although evidence from heterologous expression systems suggests that they may sometimes form homodimers and/or heterodimers. This study aims to evaluate possible functional interplay of endogenous µ- and δ-opioid receptors (MORs and DORs) in mouse neurons. Detecting GPCR dimers in native tissues, however, has been challenging. Previously, MORs and DORs co-expressed in transfected cells have been reported to form heterodimers, and their possible co-localization in neurons has been studied in knock-in mice expressing genetically engineered receptors fused to fluorescent proteins. Here, we find that single cholinergic neurons in the mouse striatum endogenously express both MORs and DORs. The receptors on neurons from live brain slices were fluorescently labeled with a ligand-directed labeling reagent, NAI-A594. The selective activation of MORs and DORs, with DAMGO (µ-agonist) and deltorphin (δ-agonist) inhibited spontaneous firing in all cells examined. In the continued presence of agonist, the firing rate returned to baseline as the result of receptor desensitization with the application of deltorphin but was less observed with the application of DAMGO. In addition, agonist-induced internalization of DORs but not MORs was detected. When MORs and DORs were activated simultaneously with [Met5]-enkephalin, desensitization of MORs was facilitated but internalization was not increased. Together, these results indicate that while MORs and DORs are expressed in single striatal cholinergic interneurons, the two receptors function independently.

Structural Characterization of Receptor-Receptor Interactions in the Allosteric Modulation of G Protein-Coupled Receptor (GPCR) Dimers.

G protein-coupled receptor (GPCR) oligomerization, while contentious, continues to attract the attention of researchers. Numerous experimental investigations have validated the presence of GPCR dimers, and the relevance of dimerization in the effectuation of physiological functions intensifies the attractiveness of this concept as a potential therapeutic target. GPCRs, as a single entity, have been the main source of scrutiny for drug design objectives for multiple diseases such as cancer, inflammation, cardiac, and respiratory diseases. The existence of dimers broadens the research scope of GPCR functions, revealing new signaling pathways that can be targeted for disease pathogenesis that have not previously been reported when GPCRs were only viewed in their monomeric form. This review will highlight several aspects of GPCR dimerization, which include a summary of the structural elucidation of the allosteric modulation of class C GPCR activation offered through recent solutions to the three-dimensional, full-length structures of metabotropic glutamate receptor and γ-aminobutyric acid B receptor as well as the role of dimerization in the modification of GPCR function and allostery. With the growing influence of computational methods in the study of GPCRs, we will also be reviewing recent computational tools that have been utilized to map protein–protein interactions (PPI).

Single-molecule FRET imaging of GPCR dimers in living cells.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

Homobivalent Dopamine D2 Receptor Ligands Modulate the Dynamic Equilibrium of D2 Monomers and Homo- and Heterodimers.

Dopamine D2 receptors (D2Rs) are major targets in the treatment of psychiatric and neurodegenerative diseases. As with many other G protein-coupled receptors (GPCRs), D2Rs interact within the cellular membrane, leading to a transient receptor homo- or heterodimerization. These interactions are known to alter ligand binding, signaling, and receptor trafficking. Bivalent ligands are ideally suited to target GPCR dimers and are composed of two pharmacophores connected by a spacer element. If properly designed, bivalent ligands are able to engange the two orthosteric binding sites of a GPCR dimer simultaneously. Taking advantage of previously developed ligands for heterodimers of D2R and the neurotensin receptor 1 (NTSR1), we synthesized homobivalent ligands targeting D2R. Employing bioluminescence resonance energy transfer, we found that the bivalent ligands 3b and 4b comprising a 92-atom spacer are able to foster D2R-homodimerization while simultaneously reducing interactions of D2R with NTSR1. Both receptors are coexpressed in the central nervous system and involved in important physiological processes. The newly developed bivalent ligands are excellent tools to further understand the pharmacological consequences of D2R homo- and heterodimerization. Not limited to the dopaminergic system, modifying class A GPCRs' dynamic equilibrium between monomers, homomers, and heteromers with bivalent ligands may represent a novel pharmacological concept paving the way toward innovative drugs.

A method for identifying G protein-coupled receptor dimers and their interfaces

The G protein-coupled receptor (GPCR) dimer interface plays an important role in the formation and stabilization of the dimer. Therefore, identifying the potential receptor-receptor interface is an important part of studying GPCRs. Various strategies have been employed to study the GPCR dimer interface and explore its functional significance, but experimental methods lack robustness and calculations are laborious. Herein, we report a combined optimized experimental and calculation approach for identifying and structurally characterizing GPCR dimer interfaces, and constructing atomic resolution models. Using a transmembrane domain (TM) peptide containing a human immunodeficiency virus trans-acting transcriptional activator (HIV-TAT) protein transduction motif, matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDITOF-MS), and bioluminescence resonance energy transfer (BRET), we successfully identified Apelin receptor (APJ)/Nociceptin receptor 1 (ORL1) and APJ/Vasopressin receptor 2 (V2R) heterodimer interfaces, and two key sites mediating dimerization. This method can identify dimer interfaces of GPCR homodimers and heterodimers.

FLIM–FRET-Based Structural Characterization of a Class-A GPCR Dimer in the Cell Membrane

Class-A G protein-coupled receptors (GPCRs) are known to homo-dimerize in the membrane. Yet, methods to characterize the structure of GPCR dimer in the native environment are lacking. Accordingly, the molecular basis and functional relevance of the class-A GPCR dimerization remain unclear. Here, we present the dimeric structural model of GPR17 in the cell membrane. The dimer mainly involves transmembrane helix 5 (TM5) at the interface, with F229 in TM5, a critical residue. An F229A mutation makes GPR17 monomeric regardless of the expression level of the receptor. Monomeric mutants of GPR17 display impaired ERK1/2 activation and cannot be properly internalized upon agonist treatment. Conversely, the F229C mutant is cross-linked as a dimer and behaves like wild-type. Importantly, the GPR17 dimer structure has been modeled using sparse inter-protomer FRET distance restraints obtained from fluorescence lifetime imaging microscopy. The same approach can be applied to characterizing the interactions of other important membrane proteins in the cell.

Comprehensive Characterization of Lipid-Guided G Protein-Coupled Receptor Dimerization.

Dimerization of G protein-coupled receptors (GPCRs) is considered to take part in regulating the highly dynamic nature of receptor function. Intensive research unraveled a large variety of different dimer configurations with potentially distinct activity profiles. Studies are complicated by the critical role of the membrane environment for receptor dimerization, and experimental deficiencies in modulating the same. Here we chose a molecular dynamics strategy to characterize the potential of the large chemical lipid repertoire to steer dimerization fingerprints of the neurotensin 1 receptor. Unfavorable hydrophobic mismatch results in excessive dimerization whereas particular lipid features, e.g., anionic headgroups, induce specific dimer interfaces via direct protein-lipid interactions. Polyunsaturated fatty acids attenuate compact dimer formation by facilitated adhesion to the protein transmembrane surface, and receptor lipidation-induced conformational changes confer modulated protein-lipid and protein-protein interactions. Our results highlight the striking role of the membrane environment on GPCR dimerization with potential functional consequences.

Cryo-EM structure of the native rhodopsin dimer in nanodiscs

Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.