G Protein-coupled Receptors Dimers Research Articles

Conformational Dynamics of GPCR Dimers is Dependent on Membrane Cholesterol

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins involved in signal transduction across cell membranes. Increasing evidence points towards a large role of membrane constituents, especially cholesterol, in GPCR structure and function. Here, we analyze the molecular basis of cholesterol modulated GPCR association by coarse-grain molecular dynamics simulations. Several cholesterol interaction sites have been identified on the serotonin1A receptor that are highly dynamic and have a microsecond time scale of exchange with the bulk lipids. We believe that these cholesterol hot-spots are indicative of ‘high occupancy sites’ rather than ‘binding sites’. These specific interactions, along with general membrane effects, have been observed to modulate GPCR organization in a receptor-dependent manner. The plasticity and flexibility of the serotonin1A receptor dimers increase with increased cholesterol concentration. Interestingly, a dimer interface formed by transmembrane helices I was found to be sensitive to cholesterol. The modulation of dimer interface appears to arise from a combination of direct cholesterol occupancy and indirect membrane effects. Importantly, the presence of cholesterol at the dimer interface is correlated with increased dimer plasticity and flexibility. The increased plasticity of the dimer interfaces with increased cholesterol suggests that the formation of higher-order clusters could be facilitated with increased cholesterol. These results represent an important step in characterizing the molecular interactions in GPCR organization with potential relevance to therapeutic interventions.

Binding Interactions of Dopamine and Apomorphine in D2High and D2Low States of Human Dopamine D2 Receptor Using Computational and Experimental Techniques.

We have recently reported G-protein coupled receptor (GPCR) model structures for the active and inactive states of the human dopamine D2 receptor (D2R) using adrenergic crystal structures as templates. Since the therapeutic concentrations of dopamine agonists that suppress the release of prolactin are the same as those that act at the high-affinity state of the D2 receptor (D2High), D2High in the anterior pituitary gland is considered to be the functional state of the receptor. In addition, the therapeutic concentrations of anti-Parkinson drugs are also related to the dissociation constants in the D2High form of the receptor. The discrimination between the high- and low-affinity (D2Low) components of the D2R is not obvious and requires advanced computer-assisted structural biology investigations. Therefore, in this work, the derived D2High and D2Low receptor models (GPCR monomer and dimer three-dimensional structures) are used as drug-binding targets to investigate binding interactions of dopamine and apomorphine. The study reveals a match between the experimental dissociation constants of dopamine and apomorphine at their high- and low-affinity sites of the D2 receptor in monomer and dimer and their calculated dissociation constants. The allosteric receptor-receptor interaction for dopamine D2R dimer is associated with the accessibility of adjacent residues of transmembrane region 4. The measured negative cooperativity between agonist ligand at dopamine D2 receptor is also correctly predicted using the D2R homodimerization model.

Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States

The extent to which Rhodopsin family G-protein-coupled receptors (GPCRs) form invariant oligomers is contentious. Recent single-molecule fluorescence imaging studies mostly argue against the existence of constitutive receptor dimers and instead suggest that GPCRs only dimerize transiently, if at all. However, whether or not even transient dimers exist is not always clear due to difficulties in unambiguously distinguishing genuine interactions from chance colocalizations, particularly with respect to short-lived events. Previous single-molecule studies have depended critically on calculations of chance colocalization rates and/or comparison with unfixed control proteins whose diffusional behavior may or may not differ from that of the test receptor. Here, we describe a single-molecule imaging assay that 1) utilizes comparisons with well-characterized control proteins, i.e., the monomer CD86 and the homodimer CD28, and 2) relies on cell fixation to limit artifacts arising from differences in the distribution and diffusion of test proteins versus these controls. The improved assay reliably reports the stoichiometry of the Glutamate-family GPCR dimer, γ-amino butyric acid receptor b2, whereas two Rhodopsin-family GPCRs, β2-adrenergic receptor and mCannR2, exhibit colocalization levels comparable to those of CD86 monomers, strengthening the case against invariant GPCR oligomerization.

Structural Studies of G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) constitute the largest and the most physiologically important membrane protein family that recognizes a variety of environmental stimuli, and are drug targets in the treatment of numerous diseases. Recent progress on GPCR structural studies shed light on molecular mechanisms of GPCR ligand recognition, activation and allosteric modulation, as well as structural basis of GPCR dimerization. In this review, we will discuss the structural features of GPCRs and structural insights of different aspects of GPCR biological functions.

G Protein-Coupled Receptor Multimers: A Question Still Open Despite the Use of Novel Approaches.

Heteromerization of G protein-coupled receptors (GPCRs) can significantly change the functional properties of involved receptors. Various biochemical and biophysical methodologies have been developed in the last two decades to identify and functionally evaluate GPCR heteromers in heterologous cells, with recent approaches focusing on GPCR complex stoichiometry and stability. Yet validation of these observations in native tissues is still lagging behind for the majority of GPCR heteromers. Remarkably, recent studies, particularly some involving advanced fluorescence microscopy techniques, are contributing to our current knowledge of aspects that were not well known until now, such as GPCR complex stoichiometry and stability. In parallel, a growing effort is being applied to move the field forward into native systems. This short review will highlight recent developments to study the stoichiometry and stability of GPCR complexes and methodologies to detect native GPCR dimers.

A G protein-coupled receptor dimer imaging assay reveals selectively modified pharmacology of neuropeptide Y Y1/Y5 receptor heterodimers.

The ability of G protein-coupled receptors (GPCRs) to form dimers, and particularly heterodimers, offers potential for targeted therapeutics with improved selectivity. However, studying dimer pharmacology is challenging, because of signaling cross-talk or because dimerization may often be transient in nature. Here we develop a system to isolate the pharmacology of precisely defined GPCR dimers, trapped by bimolecular fluorescence complementation (BiFC). Specific effects of agonist activation on such dimers are quantified using automated imaging and analysis of their internalization, controlled for by simultaneous assessment of endocytosis of one coexpressed protomer population. We applied this BiFC system to study example neuropeptide Y (NPY) Y1 receptor dimers. Incorporation of binding-site or phosphorylation-site mutations into just one protomer of a Y1/Y1 BiFC homodimer had no impact on efficient NPY-stimulated endocytosis, demonstrating that single-site agonist occupancy, and one phosphorylated monomer within this dimer, was sufficient. For two Y1 receptor heterodimer combinations (with the Y4 receptor or β2-adrenoceptor), agonist and antagonist pharmacology was explained by independent actions on the respective orthosteric binding sites. However, Y1/Y5 receptor BiFC dimers, compared with the constituent subtypes, were characterized by reduced potency and efficacy of Y5-selective peptide agonists, the inactivity of Y1-selective antagonists, and a change from surmountable to nonsurmountable antagonism for three unrelated Y5 antagonists. Thus, allosteric interactions between Y1 and Y5 receptors modify the pharmacology of the heterodimer, with implications for potential antiobesity agents that target centrally coexpressed Y1 and Y5 receptors to suppress appetite.

Major ligand-induced rearrangement of the heptahelical domain interface in a GPCR dimer.

G protein-coupled receptors (GPCRs) are major players in cell communication. Although they form functional monomers, increasing evidence indicates that GPCR dimerization has a critical role in cooperative phenomena that are important for cell signal integration. However, the structural bases of these phenomena remain elusive. Here, using well-characterized receptor dimers, the metabotropic glutamate receptors (mGluRs), we show that structural changes at the dimer interface are linked to receptor activation. We demonstrate that the main dimer interface is formed by transmembrane α helix 4 (TM4) and TM5 in the inactive state and by TM6 in the active state. This major change in the dimer interface is required for receptor activity because locking the TM4-TM5 interface prevents activation by agonist, whereas locking the TM6 interface leads to a constitutively active receptor. These data provide important information on the activation mechanism of mGluRs and improve our understanding of the structural basis of the negative cooperativity observed in these GPCR dimers.

Computational approaches for modeling GPCR dimerization.

Growing experimental evidences suggest that dimerization and oligomerization are important for G Protein- Coupled Receptors (GPCRs) function. The detailed structural information of dimeric/oligomeric GPCRs would be very important to understand their function. Although it is encouraging that recently several experimental GPCR structures in oligomeric forms have appeared, experimental determination of GPCR structures in oligomeric forms is still a big challenge, especially in mimicking the membrane environment. Therefore, development of computational approaches to predict dimerization of GPCRs will be highly valuable. In this review, we summarize computational approaches that have been developed and used for modeling of GPCR dimerization. In addition, we introduce a novel two-dimensional Brownian Dynamics based protein docking approach, which we have recently adapted, for GPCR dimer prediction.

Improved Methodical Approach for Quantitative BRET Analysis of G Protein Coupled Receptor Dimerization

G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, β2 adrenergic and CB1 cannabinoid receptors.

Application of BRET for studying G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute one of the largest classes of cell surface receptors. GPCR biology has been a subject of widespread interest owing to the functional relevance of these receptors and their potential importance in the development of new drugs. At present, over 30% of all launched drugs target these receptors. GPCRs have been considered for a long time to function as monomeric entities and the idea of GPCR dimerization and oligomerization was initially accepted with disbelief. However, a significant amount of experimental and molecular modeling evidence accumulated during the last several years suggests that the process of GPCRs dimer or oligomer formation is a general phenomenon, in some cases even essential for receptor function. Among the many methods to study GPCR dimerization and oligomerization, modern biophysical techniques such as those based on resonance energy transfer (RET) and particularly bioluminescence resonance energy transfer (BRET) have played a leading role. RET methods are commonly applied as non-destructive indicators of specific protein-protein interactions (PPIs) in living cells. Data from numerous BRET experiments support the idea that the process of GPCR oligomerization may be relevant in many physiological and pathological conditions. The application of BRET to the study of GPCRs is not only limited to the assessment of receptor oligomerization but also expands to the investigation of the interactions of GPCRs with other proteins, including G proteins, G protein-coupled receptor kinases, β-arrestins or receptor tyrosine kinases, as well as to the characterization of GPCR activation and signaling. In this review, we briefly summarize the fundaments of BRET, discuss new trends in this technology and describe the wide range of applications of BRET to study GPCRs.

Why is dimerization essential for class-C GPCR function? New insights from mGluR1 crystal structure analysis.

G-protein coupled receptors (GPCRs) play an essential role in eukaryotic cells signaling. According to phylogenetic analysis, most GPCRs belong to one of four classes, i.e. A, B, C, and Frizzled (Lagerstrom and Schioth, 2008). The class-C GPCR family contains metabotropic glutamate receptors (mGluR), γ-aminobutyric acid B receptors (GABAB receptor or GBR), several taste-sensing receptors (e.g. TAS1), and a Ca2+-sensing receptor (CaS). One characteristic feature of the class-C GPCRs is their dimerization, either into homo- or hetero-dimers, which is obligatory for function (Kniazeff et al., 2011). Each subunit in the dimer usually consists of an amino (N)-terminal extracellular domain (ECD) which binds orthosteric agonists, a linker peptide, a 7-transmembrane (TM) domain, and a cytosolic tail of varying lengths at the carboxyl (C)-terminus. It is well-established that all domains spare the linker peptide participating in the dimerization process (Rondard et al., 2008; Huang et al., 2011; Rondard et al., 2006). Further, it is believed that, after transmission of the ligand-binding signal from the ECD region to the 7-TM domain, the activation of the class-C GPCR dimer is associated with a conformational change of the 7-TM domain on the cytosol side, enabling binding and activation of downstream G-proteins. In contrast, the precise details of the mechanisms for signal transduction from the ligand binding site in the ECD region to the cytosolic side of the 7-TM domain, where downstream G-proteins are activated, remain to be elucidated.

Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units

The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists.

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β2-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β2-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β2-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.

Novel Structural and Functional Insights into M3 Muscarinic Receptor Dimer/Oligomer Formation

Class A G protein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M3 muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M., Heusel, M., Liu, T., Costanzi, S., and Wess, J. (2011) J. Biol. Chem. 286, 28584-28598). To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within two distinct cytoplasmic M3R regions, the C-terminal portion of the second intracellular loop (i2) and helix H8 (H8). The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of two structurally distinct M3R dimer interfaces, one involving i2/i2 contacts (TM4-TM5-i2 interface) and the other one characterized by H8-H8 interactions (TM1-TM2-H8 interface). Specific H8-H8 disulfide cross-links led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation or mobility of H8 are critical for efficient receptor-G protein coupling. Our findings provide novel structural and functional insights into the mechanisms involved in M3R dimerization (oligomerization). Because the M3R shows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest.

Simultaneous method for analyzing dimerization and signaling of G‐protein‐coupled receptor in yeast by dual‐color reporter system

Understanding the role of G-protein-coupled receptor (GPCR) dimerization in cellular function has now become a major research focus. The potentially large functional and physiological diversity of dimerization among GPCRs is expected to provide opportunities for novel drug discovery. However, there is currently a lack of cell-based assays capable of specific profiling for the functional consequences of dimerization linked to ligand-mediated signaling. Here, we present an advanced method to simultaneously analyze the dimerization and ligand response of GPCRs using two yeast-based systems for split-ubiquitin two-hybrid assay and G-protein signaling assay. To permit simultaneous detection, we established a two-color (dualcolor) fluorescence reporter gene assay using enhanced green fluorescent protein (EGFP) and a far-red derivative of the tetrameric fluorescent protein DsRed-Express2 (E2-Crimson). In the present study, we tested our method first by analyzing dimerization and ligand-mediated signaling by the yeast endogenous pheromone receptor (Ste2p). Second, we showed that the system facilitated mutational analysis of domains involved in dimerization and signaling by Ste2p. Third, we successfully demonstrated that the system could simultaneously monitor homo- and hetero-dimerization and somatostatin-induced signaling in the test case of the human SSTR5 somatostatin receptor. Our strategy is expected to provide a useful tool for the elucidation of molecular biological functions of GPCR dimers and for the screening of GPCR dimer-specific agonistic ligands.

Functional significance of serotonin receptor dimerization

The original model of G-protein activation by a single G-protein-coupled receptor (GPCR) is giving way to a new model, wherein two protomers of a GPCR dimer interact with a single G-protein. This article will review the evidence suggesting that 5-HT receptors form dimers/oligomers and will compare the findings with the results obtained from the studies with other biogenic amine receptors. Topics to be covered include the origin or biogenesis of dimer formation, potential dimer interface(s), and oligomer size (dimer vs. tetramer or higher order). The functional significance will be discussed in terms of G-protein activation following ligand binding to one or two protomers in a dimeric structure, the formation of heterodimers, and the development of bivalent ligands.

Rapid, Facile Detection of Heterodimer Partners for Target Human G-Protein-Coupled Receptors Using a Modified Split-Ubiquitin Membrane Yeast Two-Hybrid System

Potentially immeasurable heterodimer combinations of human G-protein-coupled receptors (GPCRs) result in a great deal of physiological diversity and provide a new opportunity for drug discovery. However, due to the existence of numerous combinations, the sets of GPCR dimers are almost entirely unknown and thus their dominant roles are still poorly understood. Thus, the identification of GPCR dimer pairs has been a major challenge. Here, we established a specialized method to screen potential heterodimer partners of human GPCRs based on the split-ubiquitin membrane yeast two-hybrid system. We demonstrate that the mitogen-activated protein kinase (MAPK) signal-independent method can detect ligand-induced conformational changes and rapidly identify heterodimer partners for target GPCRs. Our data present the abilities to apply for the intermolecular mapping of interactions among GPCRs and to uncover potential GPCR targets for the development of new therapeutic agents.

Class A G-Protein-Coupled Receptor (GPCR) Dimers and Bivalent Ligands

G-protein-coupled receptors (GPCRs) represent the largest family of membrane proteins involved in cellular signal transduction and are activated by various different ligand types including photons, peptides, proteins, but also small molecules like biogenic amines. Therefore, GPCRs are involved in diverse physiological processes and provide valuable drug targets for numerous diseases. Emerging body of evidence suggests that GPCRs exist as monomers or cross-react forming dimers and higher-ordered oligomers. In this Perspective we will review current biochemical and biophysical techniques to visualize GPCR dimerization, functional consequences of homo- and heterodimers, and approaches of medicinal chemists to target these receptor complexes with homo- and heterobivalent ligands.

Glutamate Acts as a Partial Inverse Agonist to Metabotropic Glutamate Receptor with a Single Amino Acid Mutation in the Transmembrane Domain

Metabotropic glutamate receptor (mGluR), a prototypical family 3 G protein-coupled receptor (GPCR), has served as a model for studying GPCR dimerization, and growing evidence has revealed that a glutamate-induced dimeric rearrangement promotes activation of the receptor. However, structural information of the seven-transmembrane domain is severely limited, in contrast to the well studied family 1 GPCRs including rhodopsins and adrenergic receptors. Homology modeling of mGluR8 transmembrane domain with rhodopsin as a template suggested the presence of a conserved water-mediated hydrogen-bonding network between helices VI and VII, which presumably constrains the receptor in an inactive conformation. We therefore conducted a mutational analysis to assess structural similarities between mGluR and family 1 GPCRs. Mutational experiments confirmed that the disruption of the hydrogen-bonding network by T789Y(6.43) mutation induced high constitutive activity. Unexpectedly, this high constitutive activity was suppressed by glutamate, the natural agonist ligand, indicating that glutamate acts as a partial inverse agonist to this mutant. Fluorescence energy transfer analysis of T789Y(6.43) suggested that the glutamate-induced reduction of the activity originated not from the dimeric rearrangement but from conformational changes within each protomer. Double mutational analysis showed that the specific interaction between Tyr-789(6.43) and Gly-831(7.45) in T789Y(6.43) mutant was important for this phenotype. Therefore, the present study is consistent with the notion that the metabotropic glutamate receptor shares a common activation mechanism with family 1 GPCRs, where rearrangement between helices VI and VII causes the active state formation.

Crystal structure of oligomeric β1-adrenergic G protein–coupled receptors in ligand-free basal state

G protein-coupled receptors (GPCRs) mediate transmembrane signaling. Before ligand binding, GPCRs exist in a basal state. Crystal structures of several GPCRs bound with antagonists or agonists have been solved. However, the crystal structure of the ligand-free basal state of a GPCR, the starting point of GPCR activation and function, has not been determined. Here we report the X-ray crystal structure of the first ligand-free basal state of a GPCR in a lipid membrane-like environment. Oligomeric turkey β1-adrenergic receptors display two alternating dimer interfaces. One interface involves the transmembrane domain (TM) 1, TM2, the C-terminal H8, and the extracellular loop 1. The other interface engages residues from TM4, TM5, the intracellular loop 2 and the extracellular loop 2. Structural comparisons show that this ligand-free state is in an inactive conformation. This provides the structural information regarding GPCR dimerization and oligomerization.