Protein-coupled Receptor Oligomerization Research Articles

ADGRG1, an adhesion G protein-coupled receptor, forms oligomers.

G protein-coupled receptor (GPCR) oligomerization is a highly debated topic in the field. While initially believed to function as monomers, current literature increasingly suggests that these cell surface receptors, spanning almost all GPCR families, function as homo- or hetero-oligomers. Yet, the functional consequences of these oligomeric complexes remain largely unknown. Adhesion GPCRs (aGPCRs) present an intriguing family of receptors characterized by their large and multi-domain N-terminal fragments (NTFs), intricate activation mechanisms, and the prevalence of numerous splice variants in almost all family members. In the present study, bioluminescence energy transfer (BRET) and Förster resonance energy transfer (FRET) were used to study the homo-oligomerization of adhesion G protein-coupled receptor G1 (ADGRG1; also known as GPR56) and to assess the involvement of NTFs in these receptor complexes. Based on the results presented herein, we propose that ADGRG1 forms 7-transmembrane-driven homo-oligomers on the plasma membrane. Additionally, Stachel motif interactions appear to influence the conformation of these receptor complexes.

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).

G Protein-Coupled Receptor Heteromers as Putative Pharmacotherapeutic Targets in Autism.

A major challenge in the development of pharmacotherapies for autism is the failure to identify pathophysiological mechanisms that could be targetable. The majority of developing strategies mainly aim at restoring the brain excitatory/inhibitory imbalance described in autism, by targeting glutamate or GABA receptors. Other neurotransmitter systems are critical for the fine-tuning of the brain excitation/inhibition balance. Among these, the dopaminergic, oxytocinergic, serotonergic, and cannabinoid systems have also been implicated in autism and thus represent putative therapeutic targets. One of the latest breakthroughs in pharmacology has been the discovery of G protein-coupled receptor (GPCR) oligomerization. GPCR heteromers are macromolecular complexes composed of at least two different receptors, with biochemical properties that differ from those of their individual components, leading to the activation of different cellular signaling pathways. Interestingly, heteromers of the above-mentioned neurotransmitter receptors have been described (e.g., mGlu2–5HT2A, mGlu5–D2–A2A, D2–OXT, CB1–D2, D2–5HT2A, D1–D2, D2–D3, and OXT–5HT2A). We hypothesize that differences in the GPCR interactome may underlie the etiology/pathophysiology of autism and could drive different treatment responses, as has already been suggested for other brain disorders such as schizophrenia. Targeting GPCR complexes instead of monomers represents a new order of biased agonism/antagonism that may potentially enhance the efficacy of future pharmacotherapies. Here, we present an overview of the crosstalk of the different GPCRs involved in autism and discuss current advances in pharmacological approaches targeting them.

Time-Resolved FRET-Based Assays to Characterize G Protein-Coupled Receptor Hetero-oligomer Pharmacology.

Although G protein-coupled receptor (GPCR) oligomerization is a matter of debate, it has been shown that the nature of the GPCR partners within the oligomers can influence the pharmacological properties of the receptors. Therefore, finding specific ligands for homo- or hetero-oligomers opens new perspectives for drug discovery. However, no efficient experimental strategy to screen for such ligands existed yet. Indeed, conventional binding strategies do not discriminate ligand binding on GPCR monomers, homo- or hetero-oligomers. To address this issue, we recently developed a new assay based on a time-resolved FRET method that is easy to implement and that can focus on ligand binding specifically on the hetero-oligomer.

The Dynamics of GPCR Oligomerization and Their Functional Consequences.

The functional importance of G protein-coupled receptor (GPCR) oligomerization remains controversial. Although obligate dimers of class C GPCRs are well accepted, the generalizability of this phenomenon is still strongly debated with respect to other classes of GPCRs. In this review, we focus on understanding the organization and dynamics between receptor equivalents and their signaling partners in oligomeric receptor complexes, with a view toward integrating disparate viewpoints into a unified understanding. We discuss the nature of functional oligomeric entities, and how asymmetries in receptor structure and function created by oligomers might have implications for receptor function as allosteric machines and for future drug discovery.

GPCR oligomerization analysis by means of BRET and dFRAP.

The spatiotemporal characterization of protein-protein interactions is essential to understand nearly all cellular events. Several methodological strategies derived from noninvasive luminescence- and fluorescence-based approaches allow the detection of specific protein-protein interactions in living cells. Here, we describe the application of bioluminescence resonance energy transfer (BRET) and donor fluorescent recovery after photobleaching (dFRAP) approaches to the study of G protein-coupled receptor (GPCR) oligomerization. These technologies alone - or in combination with complementary methods - can become extremely powerful approaches for visualizing these cellular protein-protein interactions, even between more than two proteins. Overall, these methods might become extremely important tools in GPCR drug discovery.

G Protein–Coupled Receptor Oligomerization Revisited: Functional and Pharmacological Perspectives

Most evidence indicates that, as for family C G protein-coupled receptors (GPCRs), family A GPCRs form homo- and heteromers. Homodimers seem to be a predominant species, with potential dynamic formation of higher-order oligomers, particularly tetramers. Although monomeric GPCRs can activate G proteins, the pentameric structure constituted by one GPCR homodimer and one heterotrimeric G protein may provide a main functional unit, and oligomeric entities can be viewed as multiples of dimers. It still needs to be resolved if GPCR heteromers are preferentially heterodimers or if they are mostly constituted by heteromers of homodimers. Allosteric mechanisms determine a multiplicity of possible unique pharmacological properties of GPCR homomers and heteromers. Some general mechanisms seem to apply, particularly at the level of ligand-binding properties. In the frame of the dimer-cooperativity model, the two-state dimer model provides the most practical method to analyze ligand-GPCR interactions when considering receptor homomers. In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to different intrinsic efficacy of ligands for different signaling pathways (functional selectivity). This gives a rationale for the use of GPCR oligomers, and particularly heteromers, as novel targets for drug development. Herein, we review the functional and pharmacological properties of GPCR oligomers and provide some guidelines for the application of discrete direct screening and high-throughput screening approaches to the discovery of receptor-heteromer selective compounds.

The Prevalence, Maintenance, and Relevance of G Protein–Coupled Receptor Oligomerization

Over the past decade, ideas and experimental support for the hypothesis that G protein-coupled receptors may exist as dimeric or oligomeric complexes moved initially from heresy to orthodoxy, to the current situation in which the capacity of such receptors to interact is generally accepted but the prevalence, maintenance, and relevance of such interactions to both pharmacology and function remain unclear. A vast body of data obtained following transfection of cultured cells is still to be translated to native systems and, even where this has been attempted, results often remain controversial and contradictory. This review will consider approaches that are currently being applied and why these might be challenging to interpret, and will suggest means to overcome these limitations.

Transcriptional profiling of striatal neurons in response to single or concurrent activation of dopamine D2, adenosine A2A and metabotropic glutamate type 5 receptors: Focus on beta-synuclein expression

G protein-coupled receptor oligomerization is a concept which is changing the understanding of classical pharmacology. Both, oligomerization and functional interaction between adenosine A2A, dopamine D2 and metabotropic glutamate type 5 receptors have been demonstrated in the striatum. However, the transcriptional consequences of receptors co-activation are still unexplored. We aim here to determine the changes in gene expression of striatal primary cultured neurons upon isolated or simultaneous receptor activation. Interestingly, we found that 95 genes of the total analyzed (15,866 transcripts and variants) changed their expression in response to simultaneous stimulation of all three receptors. Among these genes, we focused on the β-synuclein (β-Syn) gene (SCNB). Quantitative PCR verified the magnitude and direction of change in expression of SCNB. Since β-Syn belongs to the homologous synuclein family and may be considered a natural regulator of α-synuclein (α-Syn), it has been proposed that β-Syn might act protectively against α-Syn neuropathology.

Hetero-oligomerization between adenosine A1 and thromboxane A2 receptors and cellular signal transduction on stimulation with high and low concentrations of agonists for both receptors

Growing evidence indicates that G protein-coupled receptors can form homo- and hetero-oligomers to diversify signal transduction. However, the molecular mechanisms and physiological significance of G protein-coupled receptor-oligomers are not fully understood. Both ADOR1 (adenosine A1 receptor) and TBXA2R (thromboxane A2 receptor α; TPα receptor), members of the G protein-coupled receptor family, act on astrocytes and renal mesangial cells, suggesting certain functional correlations. In this study, we explored the possibility that adenosine A1 and TPα receptors form hetero-oligomers with novel pharmacological profiles. We showed that these receptors hetero-oligomerize by conducting coimmunoprecipitation and bioluminescence resonance energy transfer (BRET2) assays in adenosine A1 receptor and TPα receptor-cotransfected HEK293T cells. Furthermore, coexpression of the receptors affected signal transduction including the accumulation of cyclic AMP and phosphorylation of extracellular signal-regulated kinase-1 and -2 was significantly increased by high and low concentrations of adenosine A1 receptor agonist and TPα agonists, respectively. Our study provides evidence of hetero-oligomerization between adenosine A1 and TPα receptors for the first time, and suggests that this oligomerization affects signal transduction responding to different concentrations of receptor agonists.

Chaperones contribute to G protein coupled receptor oligomerization, but do not participate in assembly of the G protein with the receptor signaling complex

BackgroundPrevious studies have demonstrated that seven transmembrane receptors (7TM-Rs) can associate with various chaperones to control their maturation and export. It has been shown for a few years now that 7TM-Rs can form homo or heterooligomeric complexes. Due to the difficulty to study heterooligomers in a context devoid of homooligomers signaling, very little is known on heterooligomerization. β2AR-AT1R receptor complexes have been found on cells and ligand activation of one receptor affects signaling of the partner. Yet, very little is known about the mechanisms linking those receptors together. We propose to examine the role of chaperones in the maturation of homo- and heterodimers of the β2AR and AT1R. It would not be surprising that strict cellular mechanisms exist to ensure that only properly folded receptors are inserted into the plasma membrane.ResultsOur goal is to understand the process whereby the adrenergic and angiotensin receptors attain their proper mature conformation. We determined whether any of the common chaperones are physically associated with the fully and/or immature β2AR and AT1R receptors forms and if they play any role in the selective recruitment of G proteins subunits to receptor complexes. Our results suggest that when a pair of receptors is expressed in such way that one is retained in the endoplasmic reticulum (ER), this immature receptor will dictate the chaperones interacting with the receptor complex. We showed that ERp57 is important for receptor dimerization of AT1R homo and β2AR/AT1R receptor dimers, but plays no role in the β2AR homodimerization. Then, we verified if some of those chaperones could play a role in the assembly of the heterotrimeric G protein subunits with the receptor complex, but none appeared to be essential.ConclusionsOverall, our results suggest that variations among receptor oligomers occur early in the synthesis/maturation processes, and that chaperones will interact more specifically with some receptor pairs than others to allow the formation of certain receptor pairs, while others will contribute to the folding and maturation of receptors without any effect on receptor assembly within a signaling complex.

G protein-coupled receptor oligomerization for what?

Although the G protein-coupled receptor (GPCR) oligomerization has been questioned during the last decade, under some premises the existence of a supramolecular organization of these receptors begins now to be widely accepted by the scientific community. Indeed, GPCR oligomers may enhance the diversity and performance by which extracellular signals are transferred to the G proteins in the process of receptor transduction, although the mechanism that underlie this phenomenon remains still unexplained. Recently, a trans-conformational switching model has been proposed as a mechanism allowing direct inhibition of receptor activation. Thus, heterotropic receptor–receptor allosteric regulations are behind the GPCR oligomeric function. Accordingly, we revise here how GPCR oligomerization impinge in several important receptor functions like biosynthesis, plasma membrane diffusion or velocity, pharmacology and signaling. Overall, the rationale of receptor oligomerization might lie in the cellular need of sensing complex extracellular signals and to translate into a simple computational mode.

Lighting up multiprotein complexes: lessons from GPCR oligomerization

Spatiotemporal characterization of protein-protein interactions (PPIs) is essential in determining the molecular mechanisms of intracellular signaling processes. In this review, we discuss how new methodological strategies derived from non-invasive fluorescence- and luminescence-based approaches (FRET, BRET, BiFC and BiLC), when applied to the study of G protein-coupled receptor (GPCR) oligomerization, can be used to detect specific PPIs in live cells. These technologies alone or in concert with complementary methods (SRET, BRET or BiFC, and SNAP-tag or TR-FRET) can be extremely powerful approaches for PPI visualization, even between more than two proteins. Here we provide a comprehensive update on all the biotechnological aspects, including the strengths and weaknesses, of new fluorescence- and luminescence-based methodologies, with a specific focus on their application for studying PPIs.

Progress in Elucidating the Structural and Dynamic Character of G Protein-Coupled Receptor Oligomers for Use in Drug Discovery

G Protein-Coupled Receptors (GPCRs) are the most targeted group of proteins for the development of therapeutic drugs. Until the last decade, structural information about this family of membrane proteins was relatively scarce, and their mechanisms of ligand binding and signal transduction were modeled on the assumption that GPCRs existed and functioned as monomeric entities. New crystal structures of native and engineered GPCRs, together with important biochemical and biophysical data that reveal structural details of the activation mechanism(s) of this receptor family, provide a valuable framework to improve dynamic molecular models of GPCRs with the ultimate goal of elucidating their allostery and functional selectivity. Since the dynamic movements of single GPCR protomers are likely to be affected by the presence of neighboring interacting subunits, oligomeric arrangements should be taken into account to improve the predictive ability of computer-assisted structural models of GPCRs for effective use in drug design.

Metabotropic glutamate type 5, dopamine D2 and adenosine A2a receptors form higher‐order oligomers in living cells

G protein-coupled receptors are known to form homo- and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu(5) receptors (mGlu(5)R) and dopamine D(2) receptors (D(2)R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu(5)R, D(2)R and adenosine A(2A) receptor (A(2A)R). Interestingly, by using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu(5)R, D(2)R and A(2A)R in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein-coupled receptor oligomers in general and the mGlu(5)R/D(2)R/A(2A)R oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders.

Fluorescent and Bioluminescent Protein-Fragment Complementation Assays in the Study of G Protein-Coupled Receptor Oligomerization and Signaling

Most cellular functions, including signaling by G protein-coupled receptors (GPCRs), are mediated by protein-protein interactions, making the identification and localization of protein complexes key to the understanding of cellular processes. In complement to traditional biochemical techniques, noninvasive resonance energy transfer (RET) and protein-fragment complementation assays (PCAs) now allow protein interactions to be detected in the context of living cells. In this review, fluorescent and bioluminescent PCAs are discussed and their application illustrated with studies on GPCR signaling. Newly developed techniques combining PCA and RET assays for the detection of ternary and quaternary protein complexes are also presented.

Detection of higher-order G protein-coupled receptor oligomers by a combined BRET–BiFC technique

Despite some caveats, G protein-coupled receptor oligomerization is a phenomenon that is becoming largely accepted. Within these oligomers, however, stoichiometry remains to be elucidated. Here, by using bimolecular fluorescence complementation, we visualized adenosine A 2A receptor homodimers in living cells, showing no apparent difference in the subcellular distribution when compared to the YFP-labelled adenosine A 2A receptor protomer. Interestingly, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques allowed us to detect the occurrence of adenosine A 2A receptors oligomers containing more than two protomers. These results provide new insights into the molecular composition of G protein-coupled receptor oligomers. Structured summary MINT- 6700472: A2A (uniprotkb: P29274), A2A (uniprotkb: P29274) and A2A (uniprotkb: P29274) physically interact (MI: 0218) by bioluminescence resonance energy transfer (MI: 0012) MINT- 6699330: A2A (uniprotkb: P29274) and A2A (uniprotkb: P29274) physically interact (MI: 0218) by bimolecular fluorescence complementation (MI: 0809) MINT- 6699346: A2A (uniprotkb: P29274) and A2A (uniprotkb: P29274) physically interact (MI: 0218) by bioluminescence resonance energy transfer (MI: 0012)

Plasma membrane diffusion of g protein-coupled receptor oligomers

G protein-coupled receptors are known to form homo-and heteromers at the plasma membrane, but the molecular properties of these oligomers are relatively unknown. Here, we show a method that allows the diffusion of G protein-coupled receptors oligomers in the plasma membrane to be monitored in single cells by combining Bimolecular Fluorescence Complementation and Fluorescence Correlation Spectroscopy. With this approach we have measured, for the first time, the membrane diffusional characteristics of adenosine A 1 and A 2A receptor homo-and heterodimers in Chinese Hamster Ovary cells. Interestingly, both homodimers display similar diffusion co-efficients ( D) when expressed in living cells ( D = 5.0 and 4.8 × 10 − 9 cm 2/s, respectively) but the heterodimer formed by these receptors exhibit a significantly faster plasma membrane diffusion co-efficent ( D = 5.6 × 10 − 9 cm 2/s) when compared to the adenosine A 1 receptor tagged with the full-length yellow fluorescent protein ( D = 4.0 × 10 − 9 cm 2/s). Overall, these results demonstrate differences in plasma membrane diffusion between adenosine receptor homo-and heterodimers, providing new insights into the molecular plasticity of G protein-coupled receptor oligomerization.

Light resonance energy transfer‐based methods in the study of G protein‐coupled receptor oligomerization

Since most of the functions in cells are mediated by multimeric protein complexes, the determination of protein-protein interactions is an important step in the study of cellular mechanisms. Traditionally, after screening for possible target interactors by means of a yeast two-hybrid screen, several methods are used to validate the initial result before carrying out functional experiments. Nowadays, non-invasive fluorescence-based methods like Bioluminescence Resonance Energy Transfer (BRET) and Fluorescence Resonance Energy Transfer (FRET) are widely used in the study of protein-protein interactions in living cells. In the present review, we address the individual strengths and weaknesses of both RET approaches, providing information on their possible future use in the study of G protein-coupled receptor oligomerization.

Cross-inhibition of angiotensin AT 1 receptors supports the concept of receptor oligomerization

G protein-coupled receptors are cell surface receptors that mediate the effects of extracellular signals in the endocrine/paracrine and sensory systems. Experimental evidence is accumulating, which suggest that these receptors form dimers or higher order oligomers. The functional relevance of G protein-coupled receptor dimerization or oligomerization has been raised in a number of different processes, including ontogeny, internalization, ligand-induced regulation, pharmacological diversity and signal transduction of these receptors. Agonist-independent homo- and hetero-oligomerization of the angiotensin AT 1 receptor has been reported, and it has been suggested that hetero-oligomerization with β-adrenergic receptors leads to cross-inhibition of these receptors. Much less is known about the functional interactions between AT 1 receptor homo-oligomers. The aim of the present study was to analyze the functional interactions between these homo-oligomers by determining the functions of normal, AT 1 receptor blocker (candesartan) resistant (S109Y) and G protein coupling deficient (DRY/AAY) AT 1 receptors (co-)expressed in COS-7 cells. Although we have found no evidence that stimulation of a G protein coupling deficient receptor could cross-activate co-expressed normal receptors, candesartan binding to a signaling deficient receptor caused cross-inhibition of co-expressed candesartan resistant AT 1 receptors. Since the studied mutations were in the third intracellular helix of the receptor, the observed effects cannot be explained with domain swapping. These data suggest that AT 1 receptor blockers cause cross-inhibition of homo-oligomerized AT 1 receptors, and support the concept that receptor dimers/oligomers serve as the functional unit of G protein-coupled receptors.