Structural basis of G protein-coupled receptor function (original) (raw)
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Structural basis of G protein–coupled receptor–G protein interactions
Nature Chemical Biology, 2010
The interaction of G protein-coupled receptors (GPCRs) with heterotrimeric G proteins represents one of the most fundamental biological processes. However, the molecular architecture of the GPCR/G protein complex remains poorly defined. In the present study, we applied a comprehensive GPCR/Gα chemical cross-linking strategy to map a receptor/Gα interface, both prior to and after agonist-induced receptor activation. By employing the M 3 muscarinic acetylcholine receptor (M3R)/Gα q system as a model system, we examined the ability of ~250 combinations of Cys-substituted M3R and Gα q proteins to undergo cross-link formation. We identified many specific M3R/Gα q contact sites, both in the inactive and the active receptor conformation, allowing us to draw conclusions regarding the basic architecture of the M3R/Gα q interface and the nature of the conformational changes following receptor activation. Since heterotrimeric G proteins as well as most GPCRs share a high degree of structural homology, our findings should be of broad general relevance.
Conformational changes involved in G-protein-coupled-receptor activation
Trends in Pharmacological Sciences, 2008
Little is known about the nature of the conformational changes that convert G-protein-coupled receptors (GPCRs), which bind diffusible ligands, from their resting into their active states. To gain structural insight into this process, various laboratories have used disulfide cross-linking strategies involving cysteine-substituted mutant GPCRs. Several recent disulfide cross-linking studies using the M 3 muscarinic acetylcholine receptor as a model system have led to novel insights into the conformational changes associated with the activation of this prototypical class I GPCR. These structural changes are predicted to involve multiple receptor regions, primarily distinct segments of transmembrane helices III, VI and VII and helix 8. Given the high degree of structural homology found among most GPCRs, it is likely that these findings will be of considerable general relevance. A better understanding of the molecular mechanisms underlying GPCR activation might lead to novel strategies aimed at modulating GPCR function for therapeutic purposes.
Homology Modeling and Validation of the Human M1 Muscarinic Acetylcholine Receptor
Molecular Informatics, 2012
In practice 14 % (earlier 30 % ) of the available pharmaceutical compounds develops its effect on this site of action. Although these receptors themselves are important targets, drugs developed with other therapeutic aim can also act on these receptors and cause adverse side effects. Therefore these targets are antitargets as well and the screening for the possible side effects is very important in the early phase of drug development. From the point of view of the GPCRs, the main antitargets are as follows (in paranthesis the action of the compounds and their main adverse effect is enumerated): adrenergic a 1a (antagonist, orthostatic hypotension, dizziness and fainting spells), dopaminergic D 2 (antagonist, extrapyramidal syndrome, tardive dyskinesia), serotonin 5-HT 2C (antagonist, weight gain, obesity), serotonin 5-HT 2B (agonist, valvular heart disease) and muscarinic M1 (antagonist, attention deficits, hallucinations and memory deficits). Nowadays the accurate structure-based description of the GPCRÀligand interaction is more feasible due to the increased number of available crystal structures. Experimental structures of the aforementioned GPCRs are not available, therefore the initial structure has to be built up using a homology modeling (HM) approach and the structure obtained should be submitted for validation (structural and ligand recognition). In the past several years a few 3D structures of the human M1 muscarinic acetylcholine receptor (hM1acr) have been developed using various methods. Recently Haga et al. solved the crystal structure of the human M2 muscarinic acetylcholine receptor (hM2acr), which can be used as the most appropriate template for the 3D structure generation of other muscarinic receptors. The aim of our study was to build a relevant 3D structure (Model#1) of the hM1acr using the most appropriate reference structure, the crystallographic structure of hM2acr as a template. This model was compared to the model developed by McRobb et al. [6b] (a b 2 adrenergic receptor based structure, Model#2) from structural and enrichment factor point of views (using the recently developed GPCR ligand library (GLL) and GPCR decoy database (GDD) set ). These sets of active and decoy compounds were prepared in order to obtain chemically diverse molecules with similar physical properties (molecular weight, formal charge, number of rotatable bonds, number of hy-drogen bond acceptors and donors, octanol-water partition coefficient, and topological polar surface area) which resulted in unbiased enrichment compared to random selection. The structure obtained from HM was analyzed in order to verify its 3D structure from structural points of view. On the Ramachandran surface we did not find any residue in the disallowed region (see Supporting Information, Figure S1) and outliers were not found in the angle and torsional angle space either.
Molecular Pharmacology, 2011
We have used alanine-scanning mutagenesis followed by functional expression and molecular modeling to analyze the roles of the 14 residues, Asn422 to Cys435, C-terminal to transmembrane (TM) helix 7 of the M 1 muscarinic acetylcholine receptor. The results suggest that they form an eighth (H8) helix, associated with the cytoplasmic surface of the cell membrane in the active state of the receptor. We suggest that the amide side chain of Asn422 may act as a cap to the C terminus of TM7, stabilizing its junction with H8, whereas the side chain of Phe429 may restrict the relative movements of H8 and the C terminus of TM7 in the inactive ground state of the receptor. We have identified four residues, Phe425, Arg426, Thr428, and Leu432, which are important for G protein binding and signaling. These may form a docking site for the C-terminal helix of the G protein ␣ subunit, and collaborate with G protein recognition residues elsewhere in the cytoplasmic domain of the receptor to form a coherent surface for G protein binding in the activated state of the receptor.
International Journal of Molecular Sciences
The neurotransmitter molecule acetylcholine is capable of activating five muscarinic acetylcholine receptors, M1 through M5, which belong to the superfamily of G-protein-coupled receptors (GPCRs). These five receptors share high sequence and structure homology; however, the M1, M3, and M5 receptor subtypes signal preferentially through the Gαq/11 subset of G proteins, whereas the M2 and M4 receptor subtypes signal through the Gαi/o subset of G proteins, resulting in very different intracellular signaling cascades and physiological effects. The structural basis for this innate ability of the M1/M3/M5 set of receptors and the highly homologous M2/M4 set of receptors to couple to different G proteins is poorly understood. In this study, we used molecular dynamics (MD) simulations coupled with thermodynamic analyses of M1 and M2 receptors coupled to both Gαi and Gαq to understand the structural basis of the M1 receptor’s preference for the Gαq protein and the M2 receptor’s preference fo...
Proceedings of the …, 1995
Each G protein-coupled receptor recognizes only a distinct subset of the many structurally closely related G proteins expressed within a cell. How this selectivity is achieved at a molecular level is not well understood, particularly since no specific point-to-point contact sites between a receptor and its cognate G protein(s) have been identified. In this study, we demonstrate that a 4-aa epitope on the m2 muscarinic acetylcholine receptor, a prototypical G1/0-coupled receptor, can specifically recognize the C-terminal 5 aa of a subunits of the G1/o protein family. The m2 receptor residues
Biochemistry, 2008
G protein-coupled receptor (GPCR) function can be modulated by different classes of ligands including full and inverse agonists. At present, little is known about the conformational changes that agonist ligands induce in their target GPCRs. In this study, we employed an in situ disulfide cross-linking strategy to monitor ligand-induced structural changes in a series of cysteine (Cys)-substituted mutant M 3 muscarinic acetylcholine receptors. One of our goals was to study whether the cytoplasmic end of transmembrane domain V (TM V), a region known to be critically involved in receptor/G protein coupling, undergoes a major conformational change, similar to the adjacent region of TM VI. Another goal was to determine and compare the disulfide cross-linking patterns observed after treatment of the different mutant receptors with full versus inverse muscarinic agonists. Specifically, we generated 20 double Cys mutant M 3 receptors harboring one Cys substitution within the cytoplasmic end of TM V (L249-I253) and a second one within the cytoplasmic end of TM VI (A489-L492). These receptors were transiently expressed in COS-7 cells and subsequently characterized in pharmacological and disulfide cross-linking studies. Our cross-linking data, in conjunction with a three-dimensional model of the M 3 muscarinic receptor, indicate that M 3 receptor activation does not trigger major structural disturbances within the cytoplasmic segment of TM V, in contrast to the pronounced structural changes predicted to occur at the cytoplasmic end of TM VI. We also demonstrated that full and inverse muscarinic agonists had distinct effects on the efficiency of disulfide bond formation in specific double Cys mutant M 3 receptors. The present study provides novel information about the dynamic changes that accompany M 3 receptor activation and how the receptor conformations induced (or stabilized) by full versus inverse muscarinic agonists differ from each other at the molecular level. Because all class I GPCRs are predicted to share a similar transmembrane topology, the conclusions drawn from the present study should be of broad general relevance.
Toward activated homology models of the human M1 muscarinic acetylcholine receptor
Journal of Molecular Graphics and Modelling, 2014
Structure-based virtual screening offers a good opportunity for the discovery of selective M 1 muscarinic acetylcholine receptor (mAChR) agonists for the treatment of Alzheimer's disease. However, no 3-D structure of an M 1 mAChR is yet available and the homology models that have been previously reported are only able to identify antagonists in virtual screening experiments. In this study, we generated a homology model of the human M 1 mAChR, based on the crystal structure of an M 3 mAChR as the template. This initial model was modified, using the agonist-bound crystal structure of a ˇ2-adrenergic receptor as a guide, to give two possible activated structures. The T192 side chain was adjusted in both structures and one of the structures also had the whole of transmembrane (TM) 5 rotated and tilted toward the inner channel of the transmembrane region. The binding sites of all three structures were then refined by induced-fit docking (IFD) with acetylcholine. Virtual screening experiments showed that all three refined models could efficiently differentiate agonists from decoy molecules, with the TM5-modified models also giving good agonist/antagonist selectivity. The whole range of agonists and antagonists was observed to bind within the orthosteric site of the structure obtained by IFD refinement alone, implying that it has inactive state character. In contrast, the two TM5-modified structures were unable to accommodate the antagonists, supporting the proposition that they possess activated state character.
Chemistry & Biodiversity, 2006
Three-dimensional models of the five human muscarinic receptors were obtained from their known sequences. Homology modelling based on the crystallographic structure of bovine rhodopsin yielded models compatible with known results from site-directed mutagenesis studies. The only exceptions were the cytoplasmic loop 3 (CL3) in the five receptors, and the large C-terminal domain in M 1. Here, homology modelling with other closely related proteins allowed to solve these gaps. A detailed comparative discussion of the five models is given. The second part of the work involved docking experiments with the physiological ligand acetylcholine, again yielding results entirely compatible with results from mutagenesis experiments. The study revealed analogies and differences between the five receptors in the residues, and interactions leading to the recognition and binding of acetylcholine.
Journal of Biological Chemistry, 2006
Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cisretinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M 3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys 220 residue, Gln 207 , Gly 211 , Arg 213 , Gly 218 , Ile 222 , Phe 224 , Leu 225 , and Pro 228 were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance. . 3 The abbreviations used are: GPCR, G protein-coupled receptor; EGFP, enhanced green fluorescent protein; HA tag, hemagglutinin tag; [ 3 H]NMS, N-[ 3 H]methylscopolamine; i3 loop, the third intracellular loop of G protein-coupled receptors; M3R, M 3 muscarinic receptor; M3R(⌬i3), M 3 muscarinic receptor lacking amino acids Ala 274 -Lys 469 ; M3R⌬(Nterm), M 3 muscarinic receptor lacking amino acids Thr 2 -His 62 ; N-term, the extracellular N-terminal segment of G protein-coupled receptors; o2 loop, the second extracellular loop of G protein-coupled receptors; SC medium, synthetic complete medium; TM I-VII, the seven transmembrane domains of G protein-coupled receptors; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; FLIPR, fluorometric imaging plate reader.