Agonist-induced Receptor Activation Research Articles

Molecular insights into atypical modes of β-arrestin interaction with seven transmembrane receptors.

β-arrestins (βarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of βarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the βarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of βarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of βarr2 from a β strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-βarr complexes with direct implications for exploring novel therapeutic avenues.

The Leu/Val6.51 Side Chain of Cannabinoid Receptors Regulates the Binding Mode of the Alkyl Chain of Δ9-Tetrahydrocannabinol.

(-)-Δ9-trans-tetrahydrocannabinol (THC), which is the principal psychoactive constituent of Cannabis, mediates its action by binding to two members of the G-protein-coupled receptor (GPCR) family: the cannabinoid CB1 (CB1R) and CB2 (CB2R) receptors. Molecular dynamics simulations showed that the pentyl chain of THC could adopts an I-shape conformation, filling an intracellular cavity between Phe3.36 and Trp6.48 for initial agonist-induced receptor activation, in CB1R but not in CB2R. This cavity opens to the five-carbon chain of THC by the conformational change of the γ-branched, flexible, Leu6.51 side chain of CB1R, which is not feasible by the β-branched, mode rigid, Val6.51 side chain of CB2R. In agreement with our computational results, THC could not decrease the forskolin-induced cAMP levels in cells expressing mutant CB1RL6.51V receptor but could activate the mutant CB2RV6.51L receptor as efficiently as wild-type CB1R. Additionally, JWH-133, a full CB2R agonist, contains a branched dimethyl moiety in the ligand chain that bridges Phe3.36 and Val6.51 for receptor activation. In this case, the substitution of Val6.51 to Leu in CB2R makes JWH-133 unable to activate CB2RV6.51L. In conclusion, our combined computational and experimental results have shown that the amino acid at position 6.51 is a key additional player in the initial mechanism of activation of GPCRs that recognize signaling molecules derived from lipid species.

Structural basis of amine odorant perception by a mammal olfactory receptor.

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with β-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.

Nanoluciferase-based complementation assay for systematic profiling of GPCR-GRK interactions.

G protein-coupled receptor kinases (GRKs) are a family of seven soluble receptor-modifying enzymes which are essential regulators of GPCR activity. Following agonist-induced receptor activation and G protein dissociation, GRKs prime the receptor for desensitization through phosphorylation of its C terminus, which subsequently allows arrestins to bind and initiate the receptor internalization process. While GRKs constitute key GPCR-interacting proteins, to date, no method has been put forward to readily and systematically determine the preference of a specific GPCR towards the seven different GRKs (GRK1-7). This chapter describes a simple and standardized approach for systematic profiling of GRK1-7-GPCR interactions relying on the complementation of the split Nanoluciferase (NanoBiT). When applied to a set of GPCRs (MOR, 5-HT1A, B2AR, CXCR3, AVPR2, CGRPR), including two intrinsically β-arrestin-biased receptors (ACKR2 and ACKR3), this methodology yields highly reproducible results highlighting different GRK recruitment profiles. Using this assay, further characterization of MOR, a crucial target in the development of analgesics, reveals not only its GRK fingerprint but also related kinetics and activity of various ligands for a single GRK.

Refrigeration of apheresis platelets in platelet additive solution (PAS-E) supports in vitro platelet quality to maximize the shelf-life.

Refrigeration, or cold-storage, of platelets may be beneficial to extend the limited shelf-life of conventionally stored platelets and support transfusion protocols in rural and military areas. The aim of this study was to compare the morphologic, metabolic, and functional aspects of apheresis platelets stored at room-temperature (RT) or cold conditions, in either plasma or supplemented with platelet additive solution (PAS). Double-dose apheresis platelets were collected in either 100% plasma or 40% plasma/60% PAS-E using the Trima apheresis platform. One component from each group was either stored at RT (20-24°C) or refrigerated (2-6°C). Platelets were tested over a 21-day period. The platelet concentration decreased by approximately 30% in all groups during 21 days of storage (p> .05). Cold-storage reduced glycolytic metabolism, and the pH was maintained above the minimum specification (>6.4) for 21 days only when platelets were stored in PAS. The surface phenotype and the composition of the supernatant were differentially affected by temperature and storage solution. Functional responses (aggregation, agonist-induced receptor activation, clotting time) were improved during cold-storage, and the influence of residual plasma was assay dependent. In vitro platelet quality is differentially affected by storage time, temperature, and solution. Cold-storage, particularly in PAS, better maintains key metabolic, phenotypic, and functional parameters during prolonged storage.

Impairment of agonist-induced M2 muscarinic receptor activation by autoantibodies from chagasic patients with cardiovascular dysautonomia

Previous studies showed that circulating autoantibodies against M2 muscarinic receptors (anti-M2R Ab) are associated with decreased cardiac parasympathetic modulation in patients with chronic Chagas disease (CD). Here we investigated whether the exposure of M2R to such antibodies could impair agonist-induced receptor activation, leading to the inhibition of associated signaling pathways. Preincubation of M2R-expressing HEK 293T cells with serum IgG fractions from chagasic patients with cardiovascular dysautonomia, followed by the addition of carbachol, resulted in the attenuation of agonist-induced Gi protein activation and arrestin-2 recruitment. These effects were not mimicked by the corresponding Fab fractions, suggesting that they occur through receptor crosslinking. IgG autoantibodies did not enhance M2R/arrestin interaction or promote M2R internalization, suggesting that their inhibitory effects are not likely a result of short-term receptor regulation. Rather, these immunoglobulins could function as negative allosteric modulators of acetylcholine-mediated responses, thereby contributing to the development of parasympathetic dysfunction in patients with CD.

Assessments of cellular melatonin receptor signaling pathways: β-arrestin recruitment, receptor internalization, and impedance variations

Melatonin receptors belong to the family of G-protein coupled receptors. Agonist-induced receptor activation is terminated with the recruitment of β-arrestin, which leads to receptor internalization. Furthermore, agonist binding induces a shift in cellular shape that translates into a change in the electric impedance of the cell. In the present study, we employed engineered cells to study these internalization-related processes in the context of the two melatonin receptors, MT1 and MT2. To assess these three receptor internalization-related functions and validate the results, we employed four classical ligands of melatonin receptors: the natural agonist melatonin; the super-agonist 2-iodo-melatonin and the two antagonists luzindole and 4-phenyl-2-propionamidotetralin. The assessments confirmed the nature of the agonistic ligands but showed that 4-phenyl-2-propionamidotetralin, a described antagonist, is a biased partial agonist at MT2 with poorer affinity for MT1. The methods are now available to be applied to any receptor system for which multiple signaling pathways must be evaluated for new molecules.

Helix-constrained nociceptin peptides are potent agonists and antagonists of ORL-1 and nociception.

Nociceptin (orphanin FQ) is a 17-residue neuropeptide hormone with roles in both nociception and analgesia. It is an opioid-like peptide that binds to and activates the G-protein-coupled receptor opioid receptor-like-1 (ORL-1, NOP, orphanin FQ receptor, kappa-type 3 opioid receptor) on central and peripheral nervous tissue, without activating classic delta-, kappa-, or mu-opioid receptors or being inhibited by the classic opioid antagonist naloxone. The three-dimensional structure of ORL-1 was recently published, and the activation mechanism is believed to involve capture by ORL-1 of the high-affinity binding, prohelical C-terminus. This likely anchors the receptor-activating N-terminus of nociception nearby for insertion in the membrane-spanning helices of ORL-1. In search of higher agonist potency, two lysine and two aspartate residues were strategically incorporated into the receptor-binding C-terminus of the nociceptin sequence and two Lys(i)→Asp(i+4) side chain-side chain condensations were used to generate lactam cross-links that constrained nociceptin into a highly stable α-helix in water. A cell-based assay was developed using natively expressed ORL-1 receptors on mouse neuroblastoma cells to measure phosphorylated ERK as a reporter of agonist-induced receptor activation and intracellular signaling. Agonist activity was increased up to 20-fold over native nociceptin using a combination of this helix-inducing strategy and other amino acid modifications. An NMR-derived three-dimensional solution structure is described for a potent ORL-1 agonist derived from nociceptin, along with structure-activity relationships leading to the most potent known α-helical ORL-1 agonist (EC₅₀ 40 pM, pERK, Neuro-2a cells) and antagonist (IC₅₀ 7 nM, pERK, Neuro-2a cells). These α-helix-constrained mimetics of nociceptin(1-17) had enhanced serum stability relative to unconstrained peptide analogues and nociceptin itself, were not cytotoxic, and displayed potent thermal analgesic and antianalgesic properties in rats (ED₅₀ 70 pmol, IC₅₀ 10 nmol, s.c.), suggesting promising uses in vivo for the treatment of pain and other ORL-1-mediated responses.

Structural mechanism of ligand activation in human GABAB receptor

Human GABAB receptor is a G-protein coupled receptor central to inhibitory neurotransmission in the brain. It functions as an obligatory heterodimer of GBR1 and GBR2 subunits. Here we present the first crystal structures of a heterodimeric complex between the extracellular domains of GBR1 and GBR2 in the apo, agonist-bound, and antagonist-bound forms. The apo and antagonist-bound structures represent the resting state of the receptor; the agonist-bound complex corresponds to the active state. Both subunits adopt an open conformation at rest, and only GBR1 closes upon agonist-induced receptor activation. The agonists and antagonists are anchored in the interdomain crevice of GBR1 by an overlapping set of residues. An antagonist confines GBR1 to the open conformation of the inactive state, while an agonist induces its domain closure for activation. Our data reveals a unique activation mechanism for GABAB receptor that involves the formation of a novel heterodimer interface between subunits.

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB1 cannabinoid receptors (CB1Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB1R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB1Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB1Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.

A Selective Antagonist Reveals a Potential Role of G Protein–Coupled Receptor 55 in Platelet and Endothelial Cell Function

The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²⁺ release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB₁ or CB₂). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.

Multiparametric Homogeneous Method for Identification of Ligand Binding to G Protein-Coupled Receptors: Receptor–Ligand Binding and β-Arrestin Assay

Two homogeneous assay systems have been combined to provide a new cell-based functional assay. The assay can be used to identify ligand binding to β(2)-adrenergic receptors, but also the downstream response can be determined in the same assay. Both the quenching resonance energy transfer (QRET) and the DiscoveRx PathHunter assay formats allow the use of intact cells. The homogeneous QRET technique is a single-label approach based on nonspecific quenching of the time-resolved luminescence, enabling agonist and antagonist receptor binding measurements. The commercial PathHunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the PathHunter technology the enzyme complementation is recorded immediately downstream of agonist-induced receptor activation. The new multiparametric detection technology combines these two assay methods enabling the identification of agonist, and antagonist binding to the receptor, and the agonist-induced response. Using the QRET and the PathHunter methods a panel of β(2)-adrenergic receptor ligands (epinephrine, terbutaline, metaproterenol, salmeterol, propranolol, alprenolol, bisoprolol, ICI 118,551, and bucindolol) was tested to prove the assay performance. The signal-to-background ratio for tested ligands ranged from 5 to 11 and from 6 to 18 with QRET and PathHunter, respectively. Combined homogeneous assay technique can provide an informative method for screening purposes and an efficient way to monitor receptor-ligand interaction, thus separating agonist from antagonist.

Histamine Modulates Isoproterenol Efficacy at the β2 Adrenoceptor: Inferences Regarding Allosteric Modulation by Imidazole-Containing Compounds

In the last few years, evidence has been mounting of the possible allosteric modulation of second messenger formation on β2-adrenoceptors (β2ARs) induced by non-arylethylamine compounds. We herein addressed this issue by carrying out functional and molecular modeling studies to explore the possibility that imidazole-containing compounds (ICCs) affect the formation and accumulation of cAMP by mediating β2AR activation. Results show that histamine (an ICC) had no effect on basal cAMP accumulation in COS-7 cells transfected with the human β2AR (10.4 ± 1.1 pmol/mg protein), but significantly augmented the receptor response to the agonist isoproterenol (137 ± 7% of controls, EC50 10.5 μM, pEC50 5.87 ± 0.06). Moreover, histamine (10 μM) did not affect the isoproterenol inhibition of 3H]-dihydroalprenolol binding to hβ2ARs in membranes of transfected COS-7 cells. Q-site Finder program and molecular docking studies identified possible sites of interaction for ICCs on the β2AR, but the estimated affinities were lower than those reported for well-known β2AR ligands on the orthosteric site. On the basis of the present experimental and theoretical data, we postulate that direct ICC-hβ2AR interactions can allosterically modulate agonist-induced receptor activation. Implications of these findings for drug design are discussed.

Voltage-Dependent Modulation of α2A Adrenergic Receptor Conformations

G protein-coupled receptors (GPCRs) are proteins that span the cell membrane seven times. They are stimulated by extracellular agonists and activate heterotrimeric G proteins to elicit intracellular responses. Recent evidence suggests that GPCRs are voltage-sensitive: They exhibit gating currents similar to ion channels and respond to depolarization of the plasma membrane with changes in agonist affinity. We used a FRET-based biosensor of the α2A adrenergic receptor to analyze voltage dependence of receptor activation in HEK 293 cells by means of voltage-clamp recording. The biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (NE) and receptor activation was measured as decrease in FRET ratio. Receptor stimulation by NE was reduced by membrane depolarization and enhanced by hyperpolarization. This effect was present in wilde-type receptors and transduced to the level of G protein activation, which we determined in a FRET assay that directly detects Gαi protein activation. Depolarization-mediated inhibition of NE activated receptors was strong at low concentrations (500 nM: 60 % inhibition) but almost undetectable at saturating agonist concentrations (100 μM: 9 % inhibition). Both agonist-induced and hyperpolarization-induced receptor activation exhibited a similar monoexponential time course. For both activation modes, speed of activation was primarily dependent on agonist concentration, indicating that depolarization lowers the apparent affinity of the NE receptor interaction and thus causes receptor deactivation by means of NE release. Application of clonidine (1 μM, VM=-90 mV) resulted in a FRET response that was inhibited by 40 % at +60 mV. In contrast to NE, strong receptor-inhibition at +60 mV was present even at super-saturating concentrations of clonidine (100 μM). Therefore we conclude that negative membrane potentials promote active conformations of the α2A adrenergic receptor, increase affinity of full agonists and enhance G protein signaling.

Delta opioid receptor analgesia

Delta opioid receptors represent a promising target for the development of novel analgesics. A number of tools have been developed recently that have significantly improved our knowledge of δ receptor function in pain control. These include several novel δ agonists with potent analgesic properties, and genetic mouse models with targeted mutations in the δ opioid receptor gene. Also, recent findings have further documented the regulation of δ receptor function at cellular level, which impacts on the pain-reducing activity of the receptor. These regulatory mechanisms occur at transcriptional and post-translational levels, along agonist-induced receptor activation, signaling and trafficking, or in interaction with other receptors and neuromodulatory systems. All these tools for in-vivo research, and proposed mechanisms at molecular level, have tremendously increased our understanding of δ receptor physiology, and contribute to designing innovative strategies for the treatment of chronic pain and other diseases such as mood disorders.

Compartmentalization of the GABABReceptor Signaling Complex Is Required for Presynaptic Inhibition at Hippocampal Synapses

Presynaptic inhibition via G-protein-coupled receptors (GPCRs) and voltage-gated Ca(2+) channels constitutes a widespread regulatory mechanism of synaptic strength. Yet, the mechanism of intermolecular coupling underlying GPCR-mediated signaling at central synapses remains unresolved. Using FRET spectroscopy, we provide evidence for formation of spatially restricted (<100 Å) complexes between GABA(B) receptors composed of GB(1a)/GB(2) subunits, Gα(o)β(1)γ(2) G-protein heterotrimer, and Ca(V)2.2 channels in hippocampal boutons. GABA release was not required for the assembly but for structural reorganization of the precoupled complex. Unexpectedly, GB(1a) deletion disrupted intermolecular associations within the complex. The GB(1a) proximal C-terminal domain was essential for association of the receptor, Ca(V)2.2 and Gβγ, but was dispensable for agonist-induced receptor activation and cAMP inhibition. Functionally, boutons lacking this complex-formation domain displayed impaired presynaptic inhibition of Ca(2+) transients and synaptic vesicle release. Thus, compartmentalization of the GABA(B1a) receptor, Gβγ, and Ca(V)2.2 channel in a signaling complex is required for presynaptic inhibition at hippocampal synapses.

Erratum to: Is prenatal myo‐inositol deficiency a mechanism of CNS injury in galactosemia?

Erratum to: J Inherit Metab Dis DOI 10.1007/s10545-010-9260-x Unfortunately, in the original published article, there are some errors. The following paragraphs should read: Heading Evidence for myo-inositol deficiency Lines “In high concentrations, galactose-1-phosphate may … to …. Myo-inositol transporter activity” should read: In high concentrations, galactose-1 phosphate may inhibit myo-inositol recycling in developing neurons following agonist-induced signal transduction events, as it may lead to trapping of myo-inositol as inositol monophosphate. Independently, intracellular galactitol accumulation via osmoregulatory perurbations may reduce myo-inositol transporter activity in fetal neurons. Heading Physiology and biochemistry of myo-inositol Lines “Almost all of the PtdIns-4,5-P2 … to …. (Berridge and Irvine 1989).” should read: Almost all of the PtdIns-4,5-P2 that is involved in membrane signaling events, especially in the CNS, contains arachidonate in the sn1 position and stearate in the sn2 position. Following agonist-induced receptor activation, PtdIns-4,5-P2 is hydrolyzed by a phospholipase C to myo-inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) and diacylglycerol. Subsequently, the water soluble second messenger, Ins-1,4,5-P3, binds to an Ins-1,4,5-P3 receptor on an internal membrane to activate a calcium channel resulting in a transient burst in cytosolic calcium activity (Berridge and Irvine 1989). Heading Physiology and biochemistry of myo-inositol Lines “Also, PtdIns deficiency may lead to impaired … to … Loretscher and Lavery 2002).” should read: Also, PtdIns deficiency may lead to impaired synthesis of ectoproteins that are anchored to the plasma membrane via PtdIns such as alkaline phosphatase, 5′-nucleotidase, acetylcholinesterase, folate receptor and Thy-1 (Sharom and Lehto 2002; Loretscher and Lavery 2002). In the legend to Fig. 4, last two lines, it should read: … sodium/myo-inositol cotransporter1. (not … sodium/myo-inositolcotransporter1.)

Functional coupling of Cys-226 and Cys-296 in the glucagon-like peptide-1 (GLP-1) receptor indicates a disulfide bond that is close to the activation pocket

G protein-coupled receptors (GPCRs) are seven transmembrane α-helical (7TM) integral membrane proteins that play a central role in both cell signaling and in the action of many pharmaceuticals. The crystal structures of several Family A GPCRs have shown the presence of a disulfide bond linking transmembrane helix 3 (TM3) to the second extracellular loop (ECL2), enabling ECL2 to stabilize and contribute to the ligand binding pocket. Family B GPCRs share no significant sequence identity with those in Family A but nevertheless share two conserved cysteines in topologically equivalent positions. Since there are no available crystal structures for the 7TM domain of any Family B GPCR, we used mutagenesis alongside pharmacological analysis to investigate the role of ECL2 and the conserved cysteine residues. We mutated Cys-226, at the extracellular end of TM3 of the glucagon-like peptide-1 (GLP-1) receptor, to alanine and observed a 38-fold reduction in GLP-1 potency. Interestingly, this potency loss was restored by the additional substitution of Cys-296 in ECL2 to alanine. Alongside the complete conservation of these cysteine residues in Family B GPCRs, this functional coupling suggested the presence of a disulfide bond. Further mutagenesis demonstrated that the low potency observed at the C226A mutant, compared with the C226A-C296A double mutant, was the result of the bulky nature of the released Cys-296 side chain. Since this suggested that ECL2 was in close proximity to the agonist activation pocket, an alanine scan of ECL2 was carried out which confirmed the important role of this loop in agonist-induced receptor activation.

Structural basis of G protein–coupled receptor–G protein interactions

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-G protein alpha subunit (Galpha) chemical cross-linking strategy to map a receptor-Galpha interface, both before and after agonist-induced receptor activation. Using the M(3) muscarinic acetylcholine receptor (M3R)-Galpha(q) system as a model system, we examined the ability of approximately 250 combinations of cysteine-substituted M3R and Galpha(q) proteins to undergo cross-link formation. We identified many specific M3R-Galpha(q) contact sites, in both the inactive and active receptor conformations, allowing us to draw conclusions regarding the basic architecture of the M3R-Galpha(q) interface and the nature of the conformational changes following receptor activation. As heterotrimeric G proteins as well as most GPCRs share a high degree of structural homology, our findings should be of broad general relevance.

Crosslinking Analysis of the M3 Muscarinic Acetylcholine Receptor/G alpha q Interface in Both the Inactive and Active States

The interaction of G protein‐coupled receptors (GPCRs) with heterotrimeric G proteins represents one of the most fundamental biological processes. Although detailed structural information is now available for several GPCRs and heterotrimeric G proteins, the molecular architecture of the GPCR/G protein complex remains poorly defined, primarily due to the lack of a high‐resolution X‐ray structure. 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 M3 muscarinic acetylcholine receptor (M3R)/Gαq system as a model system, we examined the ability of ~250 combinations of Cys‐substituted M3R and Gαq proteins to undergo cross‐link formation. We identified many specific M3R/Gαq–contact sites, both in the inactive and the active receptor conformation. Co‐immunoprecipitation and mass spectrometry experiments confirmed the molecular identity of the M3R/Gαq complexes. The observed M3R/Gαq cross‐linking patterns yielded a sufficient number of structural constraints allowing us to generate the first evidenced‐based model of a GPCR/Gα complex. Since heterotrimeric G proteins as well as most GPCRs share a high degree of structural homology, our findings should be of great general relevance.