G Protein-coupled Receptors Oligomerization Research Articles

Inverse Regulation of C-C Chemokine Receptor 3 Oligomerization by Downstream Proteins Indicates Biased Signal Transduction Pathways.

Oligomerization is one of the important mechanisms for G protein-coupled receptors (GPCRs) to modulate their activity in signal transduction. However, details of how and why the oligomerization of GPCRs regulates their functions under physiological conditions remain largely unknown. Here, using single-molecule photobleaching technology, we show that chemokine ligand 5 (CCL5) and chemokine ligand 8 (CCL8) are similar to the previously reported chemokine ligand 11 (CCL11) and chemokine ligand 24 (CCL24), which can regulate the oligomerization of chemokine receptor 3 (CCR3). Our results further demonstrate that downstream proteins, β-arrestin 2 and Gi protein complex, on the CCR3 signal transduction pathway, can inversely regulate the oligomeric states of CCR3 induced by its binding ligands. This unexpected discovery suggests complex relationships between the oligomeric behaviors of CCR3 and the components of ligands-CCR3-downstream proteins, reflecting the potentially functional impact of the oligomerization on the multiple activation pathways of GPCR, such as biased activation.

Protein-Protein Docking Approach to GPCR Oligomerization.

G-protein-coupled receptors (GPCRs), the largest family of human membrane proteins, play a crucial role in cellular control and are the target of approximately one-third of all drugs on the market. Targeting these complexes with selectivity or formulating small molecules capable of modulating receptor-receptor interactions could potentially offer novel avenues for drug discovery, fostering the development of more refined and safer pharmacotherapies. Due to the lack of experimentally derived X-ray crystallography spectra of GPCR oligomers, there is growing evidence supporting the development of new in silico approaches for predicting GPCR self-assembling structures. The significance of GPCR oligomerization, the challenges in modeling these structures, and the potential of protein-protein docking algorithms to address these challenges are discussed. The study also underscores the use of various software solutions for modeling GPCR oligomeric structures and presents practical cases where these techniques have been successfully applied.

Protein-Protein Docking Approach to GPCR Oligomerization.

G-protein-coupled receptors (GPCRs), the largest family of human membrane proteins, play a crucial role in cellular control and are the target of approximately one-third of all drugs on the market. Targeting these complexes with selectivity or formulating small molecules capable of modulating receptor-receptor interactions could potentially offer novel avenues for drug discovery, fostering the development of more refined and safer pharmacotherapies. Due to the lack of experimentally derived X-ray crystallography spectra of GPCR oligomers, there is growing evidence supporting the development of new in silico approaches for predicting GPCR self-assembling structures. The significance of GPCR oligomerization, the challenges in modeling these structures, and the potential of protein-protein docking algorithms to address these challenges are discussed. The study also underscores the use of various software solutions for modeling GPCR oligomeric structures and presents practical cases where these techniques have been successfully applied.

Visualizing G Protein-Coupled Receptor-Receptor Interactions in Brain Using Proximity Ligation In Situ Assay.

G protein-coupled receptors (GPCRs) constitute the largest family of plasma membrane receptors and the main drug targets in therapeutics. GPCRs can establish direct receptor-receptor interactions (oligomerization), which can also be considered as targets for drug development (GPCR oligomer-based drugs). However, prior to designing any novel GPCR oligomer-based drug development program, demonstrating the existence of a named GPCR oligomer in native tissues is needed as part of its target engagement definition. Here, we discuss the proximity ligation in situ assay (P-LISA), an experimental approach that reveals GPCR oligomerization in native tissues. We provide a detailed step-by-step protocol to perform P-LISA experiments and visualize GPCR oligomers in brain slices. We also provide instructions for slide observation, data acquisition, and quantification. Finally, we discuss the critical aspects determining the success of the technique, namely the fixation process and the validation of the primary antibodies used. Overall, this protocol may be used to straightforwardly visualize GPCR oligomers in the brain. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Visualization of GPCR oligomers by proximity ligation in situ assay (P-LISA) Support Protocol: Slide observation, image acquisition, and quantification.

Biased signaling due to oligomerization of the G protein-coupled platelet-activating factor receptor

G protein-coupled receptors (GPCRs) are important drug targets that mediate various signaling pathways by activating G proteins and engaging β-arrestin proteins. Despite its importance for the development of therapeutics with fewer side effects, the underlying mechanism that controls the balance between these signaling modes of GPCRs remains largely unclear. Here, we show that assembly into dimers and oligomers can largely influence the signaling mode of the platelet-activating factor receptor (PAFR). Single-particle analysis results show that PAFR can form oligomers at low densities through two possible dimer interfaces. Stabilization of PAFR oligomers through cross-linking increases G protein activity, and decreases β-arrestin recruitment and agonist-induced internalization significantly. Reciprocally, β-arrestin prevents PAFR oligomerization. Our results highlight a mechanism involved in the control of receptor signaling, and thereby provide important insights into the relationship between GPCR oligomerization and downstream signaling.

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.

Overcoming the Challenges of Detecting GPCR Oligomerization in the Brain.

G protein-coupled receptors (GPCRs) constitute the largest group of membrane receptor proteins controlling brain activity. Accordingly, GPCRs are the main target of commercial drugs for most neurological and neuropsychiatric disorders. One of the mechanisms by which GPCRs regulate neuronal function is by homo- and heteromerization, with the establishment of direct protein-protein interactions between the same and different GPCRs. The occurrence of GPCR homo- and heteromers in artificial systems is generally well accepted, but more specific methods are necessary to address GPCR oligomerization in the brain. Here, we revise some of the techniques that have mostly contributed to reveal GPCR oligomers in native tissue, which include immunogold electron microscopy, proximity ligation assay (PLA), resonance energy transfer (RET) between fluorescent ligands and the Amplified Luminescent Proximity Homogeneous Assay (ALPHA). Of note, we use the archetypical GPCR oligomer, the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer as an example to illustrate the implementation of these techniques, which can allow visualizing GPCR oligomers in the human brain under normal and pathological conditions. Indeed, GPCR oligomerization may be involved in the pathophysiology of neurological and neuropsychiatric disorders.

Disrupting GPCR Complexes with Smart Drug-like Peptides.

G protein-coupled receptors (GPCRs) are a superfamily of proteins classically described as monomeric transmembrane (TM) receptors. However, increasing evidence indicates that many GPCRs form higher-order assemblies made up of monomers pertaining to identical (homo) or to various (hetero) receptors. The formation and structure of these oligomers, their physiological role and possible therapeutic applications raise a variety of issues that are currently being actively explored. In this context, synthetic peptides derived from TM domains stand out as powerful tools that can be predictably targeted to disrupt GPCR oligomers, especially at the interface level, eventually impairing their action. However, despite such potential, TM-derived, GPCR-disrupting peptides often suffer from inadequate pharmacokinetic properties, such as low bioavailability, a short half-life or rapid clearance, which put into question their therapeutic relevance and promise. In this review, we provide a comprehensive overview of GPCR complexes, with an emphasis on current studies using GPCR-disrupting peptides mimicking TM domains involved in multimerization, and we also highlight recent strategies used to achieve drug-like versions of such TM peptide candidates for therapeutic application.

Mechanistic insight into the impact of a bivalent ligand on the structure and dynamics of a GPCR oligomer

Development of effective bivalent ligands has become the focus of intensive research toward modulation of G protein-coupled receptor (GPCR) oligomers, particularly in the field of GPCR pharmacology. Experimental studies have shown that they increased binding affinity and signaling potency compared to their monovalent counterparts, yet underlying molecular mechanism remains elusive. To address this, we performed accelerated molecular dynamics simulations on bivalent-ligand bound Adenosine 2A receptor (A2AR) dimer in the context of a modeled tetramer, which consists of A2AR and dopamine 2 receptor (D2R) homodimers and their cognate G proteins. Our results demonstrate that bivalent ligand impacted interactions between pharmacophore groups and ligand binding residues, thus modulating allosteric communication network and water channel formed within the receptor. Moreover, it also strengthens contacts between receptor and G protein, by modulating the volume of ligand binding pocket and intracellular domain of the receptor. Importantly, we showed that impact evoked by the bivalent ligand on A2AR dimer was also transmitted to apo D2R, which is part of the neighboring D2R dimer. To the best of our knowledge, this is the first study that provides a mechanistic insight into the impact of a bivalent ligand on dynamics of a GPCR oligomer. Consequently, this will pave the way for development of effective ligands for modulation of GPCR oligomers and hence treatment of crucial diseases such as Parkinson’s disease and cancer.

Quantitative Super-Resolution Imaging for the Analysis of GPCR Oligomerization.

G-protein coupled receptors (GPCRs) are known to form homo- and hetero- oligomers which are considered critical to modulate their function. However, studying the existence and functional implication of these complexes is not straightforward as controversial results are obtained depending on the method of analysis employed. Here, we use a quantitative single molecule super-resolution imaging technique named qPAINT to quantify complex formation within an example GPCR. qPAINT, based upon DNA-PAINT, takes advantage of the binding kinetics between fluorescently labelled DNA imager strands to complementary DNA docking strands coupled to protein targeting antibodies to quantify the protein copy number in nanoscale dimensions. We demonstrate qPAINT analysis via a novel pipeline to study the oligomerization of the purinergic receptor Y2 (P2Y2), a rhodopsin-like GPCR, highly expressed in the pancreatic cancer cell line AsPC-1, under control, agonistic and antagonistic conditions. Results reveal that whilst the density of P2Y2 receptors remained unchanged, antagonistic conditions displayed reduced percentage of oligomers, and smaller numbers of receptors in complexes. Yet, the oligomeric state of the receptors was not affected by agonist treatment, in line with previous reports. Understanding P2Y2 oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y2 mechanistic action and therapeutic targeting.

Homo-oligomerization of the human adenosine A2A receptor is driven by the intrinsically disordered C-terminus.

G protein-coupled receptors (GPCRs) have long been shown to exist as oligomers with functional properties distinct from those of the monomeric counterparts, but the driving factors of oligomerization remain relatively unexplored. Herein, we focus on the human adenosine A2A receptor (A2AR), a model GPCR that forms oligomers both in vitro and in vivo. Combining experimental and computational approaches, we discover that the intrinsically disordered C-terminus of A2AR drives receptor homo-oligomerization. The formation of A2AR oligomers declines progressively with the shortening of the C-terminus. Multiple interaction types are responsible for A2AR oligomerization, including disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. These interactions are enhanced by depletion interactions, giving rise to a tunable network of bonds that allow A2AR oligomers to adopt multiple interfaces. This study uncovers the disordered C-terminus as a prominent driving factor for the oligomerization of a GPCR, offering important insight into the effect of C-terminus modification on receptor oligomerization of A2AR and other GPCRs reconstituted in vitro for biophysical studies.

Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells.

Oligomerization of membrane proteins has received intense research interest because of their importance in cellular signaling and the large pharmacological and clinical potential this offers. Fluorescence imaging methods are emerging as a valid tool to quantify membrane protein oligomerization at high spatial and temporal resolution. Here, we provide a detailed protocol for an image-based method to determine the number and oligomerization state of fluorescently labeled prototypical G-protein-coupled receptors (GPCRs) on the basis of small out-of-equilibrium fluctuations in fluorescence (i.e., molecular brightness) in single cells. The protocol provides a step-by-step procedure that includes instructions for (i) a flexible labeling strategy for the protein of interest (using fluorescent proteins, small self-labeling tags or bio-orthogonal labeling) and the appropriate controls, (ii) performing temporal and spatial brightness image acquisition on a confocal microscope and (iii) analyzing and interpreting the data, excluding clusters and intensity hot-spots commonly observed in receptor distributions. Although specifically tailored for GPCRs, this protocol can be applied to diverse classes of membrane proteins of interest. The complete protocol can be implemented in 1 month.

Interface Prediction for GPCR Oligomerization Between Transmembrane Helices.

Oligomers of G protein-coupled receptors (GPCRs) are closely related to their biochemical and biological functions and have been conserved during the course of molecular evolution. The mechanisms of GPCR interactions and the reason why GPCRs interact between themselves have remained elusive. Accurate interface prediction is useful to generate guidelines for mutation and inhibition experiments and would accelerate investigations of the molecular mechanisms of GPCR oligomerization and signaling. We have developed a method to predict the interfaces for GPCR oligomerization. Our method detects clusters of conserved residues along the surfaces of transmembrane helices, using a multiple sequence alignment and a target GPCR or closely related structure. This chapter outlines our method and introduces some problems that occur with it, along with our future direction to extend the method for interface predictions of general membrane proteins.

In-Cell Detection of Conformational Substates of a G Protein-Coupled Receptor Quaternary Structure: Modulation of Substate Probability by Cognate Ligand Binding.

While the notion that G protein-coupled receptors (GPCRs) associate into homo- and hetero-oligomers has gained more recognition in recent years, a lack of consensus remains among researchers regarding the functional relevance of GPCR oligomerization. A technique, Förster resonance energy transfer (FRET) spectrometry, allows for the determination of the oligomeric (or quaternary) structure of proteins in living cells via analysis of efficiency distributions of energy transferred from optically excited fluorescent tags acting as donors of energy to fluorescent tags acting as acceptors of energy and residing within the same oligomer. In this study, we significantly improved the resolution of FRET spectrometry to detect subtle differences in quaternary structures of GPCR oligomers within living cells. We then used this approach to study the conformational substates of oligomers of the sterile 2 α-factor receptor (Ste2), a class D GPCR found in the yeast Saccharomyces cerevisiae of mating type a. Ste2 has previously been shown to form tetramers at relatively low expression levels (11 to 140 molecules/μm2) in the absence of its cognate ligand, the α-factor pheromone. The significantly improved FRET spectrometry technique allowed us to detect multiple distinct quaternary conformational substates of Ste2 oligomers, and to assess how the α-factor ligand altered the proportion of such substates. The ability to determine quaternary structure substates of GPCRs provides exquisite means to elucidate functional relevance of GPCR oligomerization.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

SummaryLanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

Cholesterol-mediated oligomerization pathways of serotonin G-coupled receptor 5-HT2C

Serotonin (5-HT) receptors have been shown to homodimerize and heterodimerize with other G protein-coupled receptors (GPCRs), although the details of this process have not yet been elucidated. Here we use coarse-grained molecular dynamics on monomeric 5-HT2C receptors to predict the transmembrane (TM) helices involved in such associations. All these simulations were carried out both in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers and in mixed composition POPC-Cholesterol ones, to show whether the presence of cholesterol could directly influence and drive the dimeric association. The goal is to get insights on the self-assembly pathway leading to GPCRs 5-HT2C oligomerization, which is supposed to be the basis of its constitutional activity. From the analysis of the molecular dynamics trajectories, we observed the formation of 5-HT2C oligomers through self-assembly and we identified the main domains involved in the receptor dimerization. In particular, dimers and oligomers from the two different environments show TM4-TM5 and TM1-TM7-H8 as the preferential dimerization interfaces. Nevertheless, substantial differences arise for oligomers in POPC and in POPC-Chol membranes: in POPC-Chol the variability of dimers interfaces is strictly limited to the TM1-TM7-H8 and TM4-TM5 interfaces and the dimorphism depends on cholesterol that directly participates in its formation. These results are in agreement with both experimental evidences and other computational studies conducted on other GPCRs oligomerization.

Update of the GRIP web service

G protein-coupled receptors (GPCRs) can form homodimers, heterodimers, or higher-order molecular complexes (oligomers). The reports on the change of functions through the oligomerization have been accumulated. Inhibition of GPCR oligomerization without affecting the protomer’s overall structure would clarify the oligomer-specific functions although inhibition experiments are costly and require accurate information about the interface location. Unfortunately, the number of experimentally determined interfaces is limited. The precise prediction of the oligomerization interfaces is, therefore, useful for inhibition experiments to examine the oligomer-specific functions, which would accelerate investigations of the GPCR signaling. However, interface prediction for GPCR oligomerization is difficult because different GPCR subtypes belonging to the same subfamily often use different structural regions as their interfaces. We previously developed a high-performance method to predict the interfaces for GPCR oligomerization, by identifying the conserved surfaces with the sequence and structure information. Then, the structural characteristic of a GPCR structure is regarded to be a thick-tube like conformation that is approximately perpendicular to the membrane plane. Our method had successfully predicted all of the interfaces available on that day. We had launched a web server for our interface prediction of GPCRs (GRIP). We have improved the previous version of GRIP server and enhanced its usability. First, we discarded the approximation of the GPCR structure as the thick-tube-like conformation. This improvement increased the number of structures for the prediction. Second, the FUGUE-based template recommendation service was introduced to facilitate the choice of an appropriate structure for the prediction. The new prediction server is available at http://grip.b.dendai.ac.jp/∼grip/.

Oligomerization of G protein-coupled receptors: Still doubted?

Experimental data accumulated during the last 10 years strongly support the existence and functional significance of oligomerization of G protein-coupled receptors (GPCRs). In this essay, we discuss the unique biochemical properties of GPCR oligomers in the frame of "allosterism" and how these properties can be used to identify GPCR oligomers in artificial systems and in native tissues. We also address how the significant advances in biophysical, computational and crystallization techniques have provided significant structural insights about the mechanisms behind allosterism in GPCRs, giving distinct clues about the mechanisms of allosteric interactions in GPCR homomers and heteromers. Finally, we elaborate on the emerging picture of the role of GPCR oligomers as components of pre-coupled macromolecular complexes that include different G proteins and effectors, such as adenylyl cyclase. Allosteric properties of GPCR oligomers can therefore extend to ligand interactions through the different components of the macromolecular complexes.

Exploring functional consequences of GPCR oligomerization requires a different lens.

As the largest family of cell surface receptors, G protein-coupled receptors (GPCRs) represent an important strategic class of therapeutic targets. Attaining a clearer perspective of how such signaling complexes set molecular events in motion could have significant impact on our understanding and treatment of human diseases. As such, many experimental approaches have set out to better understand signaling networks associated with individual receptors to understand signaling architectures and their relationship to signaling outcomes. However, designing in vitro assays aimed at addressing signaling events downstream of single GPCRs must also take into account their propensity to form homo- and heterooligomeric complexes. In the context of GPCR oligomers, physical interactions with a partner protein can have a number of potential consequences, which we will explore in this review. We will also discuss methods used to identify putative dimer partners as well as the various techniques used to study the functional consequences of such complex formation. Since the full functional significance and physiological relevance of GPCR oligomers remains incompletely understood, owing in part to technical limitations, new tools to elucidate molecular mechanisms underlying allosteric co-regulation occurring between two GPCRs are required. Accordingly, using the example of the FP/AT1R heterodimer, we discuss the potential of the FlAsH-BRET approach as a simple tool to reveal how allosteric information is transmitted via conformational rearrangements within putative GPCR complexes and as a means to deorphanize receptors.

Integrated structural modeling and super-resolution imaging resolve GPCR oligomers.

Formation of G protein-coupled receptors (GPCRs) dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. For the Class A/rhodopsin subfamily of glycoprotein hormone receptors (GpHRs), di/oligomerization has been demonstrated to play a significant role in regulating its signaling activity at a cellular and physiological level and even pathophysiologically. Here we will describe and discuss the developments in our understanding of GPCR oligomerization, in both health and disease, from the study of this unique and complex subfamily of GPCRs with light on the luteinizing hormone receptor (LHR). Focus will be put on the results of an approach relying on the combination of atomistic modeling by protein-protein docking with super-resolution imaging. The latter could resolve single LHR molecules to ~8nm resolution in functional asymmetric dimers and oligomers, using dual-color photoactivatable dyes and localization microscopy (PD-PALM). Structural modeling of functionally asymmetric LHR trimers and tetramers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.