Structural and functional aspects of G protein-coupled receptor oligomerization

1998 ◽  
Vol 76 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Terence E Hébert ◽  
Michel Bouvier
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
Receptor Activation ◽  
Drug Binding ◽  
Receptor Protein ◽  
Surface Receptors ◽  
Receptor Oligomerization ◽  
Gpcr Activation ◽  
Molecular Events ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) represent the single largest family of cell surface receptors involved in signal transduction. It is estimated that several hundred distinct members of this receptor family in humans direct responses to a wide variety of chemical transmitters, including biogenic amines, amino acids, peptides, lipids, nucleosides, and large polypeptides. These transmembrane receptors are key controllers of such diverse physiological processes as neurotransmission, cellular metabolism, secretion, cellular differentiation, and growth as well as inflammatory and immune responses. GPCRs therefore represent major targets for the development of new drug candidates with potential application in all clinical fields. Many currently used therapeutics act by either activating (agonists) or blocking (antagonists) GPCRs. Studies over the past two decades have provided a wealth of information on the biochemical events underlying cellular signalling by GPCRs. However, our understanding of the molecular interactions between ligands and the receptor protein and, particularly, of the structural correlates of receptor activation or inhibition by agonists and inverse agonists, respectively, is still rudimentary. Most of the work in this area has focused on mapping regions of the receptor responsible for drug binding affinity. Although binding of ligand molecules to specific receptors represents the first event in the action of drugs, the efficacy with which this binding is translated into a physiological response remains the only determinant of therapeutic utility. In the last few years, increasing evidence suggested that receptor oligomerization and in particular dimerization may play an important role in the molecular events leading to GPCR activation. In this paper, we review the biochemical and functional evidence supporting this notion.Key words: G proteins, receptors, dimerization, signal transduction, adrenergic.

scholarly journals An Efficient Strategy to Estimate Thermodynamics and Kinetics of G Protein-Coupled Receptor Activation Using Metadynamics and Maximum Caliber

2018 ◽  
Author(s):  
Derya Meral ◽  
Davide Provasi ◽  
Marta Filizola
Keyword(s):  
G Protein ◽  
Adaptive Sampling ◽  
Receptor Activation ◽  
Conformational Space ◽  
Kinetic Properties ◽  
G Protein Coupled Receptor ◽  
Efficient Strategy ◽  
Gpcr Activation ◽  
Kinetic Rates ◽  
G Protein Coupled

ABSTRACTComputational strategies aimed at unveiling the thermodynamic and kinetic properties of G Protein-Coupled Receptor (GPCR) activation require extensive molecular dynamics simulations of the receptor embedded in an explicit lipid-water environment. A possible method for efficiently sampling the conformational space of such a complex system is metadynamics (MetaD) with path collective variables (CV). Here, we applied well-tempered MetaD with path CVs to one of the few GPCRs for which both inactive and fully active experimental structures are available, the μ-opioid receptor (MOR), and assessed the ability of this enhanced sampling method to estimate thermodynamic properties of receptor activation in line with those obtained by more computationally expensive adaptive sampling protocols. While n-body information theory (nBIT) analysis of these simulations confirmed that MetaD can efficiently characterize ligand-induced allosteric communication across the receptor, standard MetaD cannot be used directly to derive kinetic rates because transitions are accelerated by a bias potential. Applying the principle of Maximum Caliber (MaxCal) to the free-energy landscape of morphine-bound MOR reconstructed from MetaD, we obtained Markov State Models (MSMs) that yield kinetic rates of MOR activation in agreement with those obtained by adaptive sampling. Taken together, these results suggest that the MetaD-MaxCal combination creates an efficient strategy for estimating thermodynamic and kinetic properties of GPCR activation at an affordable computational cost.

scholarly journals Shear stress induces Gαq/11 activation independently of G protein-coupled receptor activation in endothelial cells

2017 ◽  
Vol 312 (4) ◽  
pp. C428-C437 ◽  
Author(s):  
Nathaniel G. dela Paz ◽  
Benoît Melchior ◽  
John A. Frangos
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
G Protein ◽  
Molecular Mechanisms ◽  
Fluid Shear Stress ◽  
Receptor Activation ◽  
Proximity Ligation Assay ◽  
Human Coronary Artery ◽  
Gpcr Activation ◽  
G Protein Coupled

Mechanochemical signal transduction occurs when mechanical forces, such as fluid shear stress, are converted into biochemical responses within the cell. The molecular mechanisms by which endothelial cells (ECs) sense/transduce shear stress into biological signals, including the nature of the mechanosensor, are still unclear. G proteins and G protein-coupled receptors (GPCRs) have been postulated independently to mediate mechanotransduction. In this study, we used in situ proximity ligation assay (PLA) to investigate the role of a specific GPCR/Gαq/11 pair in EC shear stress-induced mechanotransduction. We demonstrated that sphingosine 1-phosphate (S1P) stimulation causes a rapid dissociation at 0.5 min of Gαq/11 from its receptor S1P3, followed by an increased association within 2 min of GPCR kinase-2 (GRK2) and β-arrestin-1/2 with S1P3 in human coronary artery ECs, which are consistent with GPCR/Gαq/11 activation and receptor desensitization/internalization. The G protein activator AlF4 resulted in increased dissociation of Gαq/11 from S1P3, but no increase in association between S1P3 and either GRK2 or β-arrestin-1/2. The G protein inhibitor guanosine 5′-(β-thio) diphosphate (GDP-β-S) and the S1P3 antagonist VPC23019 both prevented S1P-induced activation. Shear stress also caused the rapid activation within 7 s of S1P3/Gαq/11. There were no increased associations between S1P3 and GRK2 or S1P3 and β-arrestin-1/2 until 5 min. GDP-β-S, but not VPC23019, prevented dissociation of Gαq/11 from S1P3 in response to shear stress. Shear stress did not induce rapid dephosphorylation of β-arrestin-1 or rapid internalization of S1P3, indicating no GPCR activation. These findings suggest that Gαq/11 participates in the sensing/transducing of shear stress independently of GPCR activation in ECs.

scholarly journals Correlated motions of conserved polar motifs lay out a plausible mechanism of G protein-coupled receptor activation.

2020 ◽  
Author(s):  
Argha Mitra ◽  
Arijit Sarkar ◽  
Marton Richard Szabo ◽  
Attila Borics
Keyword(s):  
G Protein ◽  
Intracellular Signaling ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Current Theory ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Binding Interface ◽  
Gpcr Activation ◽  
G Protein Coupled

Recent advancements in the field of experimental structural biology have provided high-resolution structures of active and inactive state G protein-coupled receptors (GPCRs), a highly important pharmaceutical target family, but the process of transition between these states is poorly understood. According to the current theory, GPCRs exist in structurally distinct, dynamically interconverting functional states of which populations are shifted upon binding of ligands and intracellular signaling proteins. However, explanation of the activation mechanism on an entirely structural basis gets complicated when multiple activation pathways and active receptor states are considered. Our unbiased, atomistic molecular dynamics simulations of the mu-opioid receptor in a physiological environment revealed that external stimulus is propagated to the intracellular surface of the receptor through subtle, concerted movements of highly conserved polar amino acid side chains along the 7th transmembrane helix. To amend the widely accepted theory we suggest that the initiation event of GPCR activation is the shift of macroscopic polarization between the ortho- and allosteric binding pockets and the intracellular G protein-binding interface.

scholarly journals Activation pathway of a G protein-coupled receptor uncovers conformational intermediates as targets for allosteric drug design

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shaoyong Lu ◽  
Xinheng He ◽  
Zhao Yang ◽  
Zongtao Chai ◽  
Shuhua Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
Large Scale ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Therapeutic Drug ◽  
Activation Pathway ◽  
Transition Mechanism ◽  
Gpcr Activation ◽  
State Models ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) are the most common proteins targeted by approved drugs. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. Here, we apply a combined computational and experimental framework integrating extensive molecular dynamics simulations, Markov state models, site-directed mutagenesis, and conformational biosensors to investigate the conformational landscape of the angiotensin II (AngII) type 1 receptor (AT1 receptor) — a prototypical class A GPCR—activation. Our findings suggest a synergistic transition mechanism for AT1 receptor activation. A key intermediate state is identified in the activation pathway, which possesses a cryptic binding site within the intracellular region of the receptor. Mutation of this cryptic site prevents activation of the downstream G protein signaling and β-arrestin-mediated pathways by the endogenous AngII octapeptide agonist, suggesting an allosteric regulatory mechanism. Together, these findings provide a deeper understanding of AT1 receptor activation at an atomic level and suggest avenues for the design of allosteric AT1 receptor modulators with a broad range of applications in GPCR biology, biophysics, and medicinal chemistry.

scholarly journals Receptor-Drug Interaction: Europium Employment for Studying the Biochemical Pathway of G-Protein-Coupled Receptor Activation

2007 ◽  
Vol 2007 ◽  
pp. 1-8 ◽  
Author(s):  
Colabufo Nicola Antonio ◽  
Perrone Maria Grazia ◽  
Contino Marialessandra ◽  
Berardi Francesco ◽  
Perrone Roberto
Keyword(s):  
Drug Interaction ◽  
G Protein ◽  
Receptor Activation ◽  
Intracellular Camp ◽  
Biochemical Pathway ◽  
Antagonist Activity ◽  
Gpcr Activation ◽  
Highly Sensitive ◽  
Radiolabeled Compounds ◽  
G Protein Coupled

In medicinal chemistry field, the biochemical pathways, involved in 7-transmembrane domains G-protein coupled receptors (GPCRs) activation, are commonly studied to establish the activity of ligands towards GPCRs. The most studied steps are the measurement of activated GTP-α subunit and stimulated intracellular cAMP. At the present, many researchers defined agonist or antagonist activity of potential GPCRs drugs employing [35S]GTPγS or [3H]cAMP as probes. Recently, the corresponding lanthanide labels Eu-GTP and Eu-cAMP as alternative to radiochemicals have been developed because they are highly sensitive, easy to automate, easily synthesized, they display a much longer shelf-life and they can be used in multilabel experiments. In the present review, the receptor-drug interaction by europium employment for studying the biochemical pathway of GPCR activation has been focused. Moreover, comparative studies between lanthanide label probes and the corresponding radiolabeled compounds have been carried out.

scholarly journals Cell-Based High-Throughput Screening Assay System for Monitoring G Protein-Coupled Receptor Activation Using β-Galactosidase Enzyme Complementation Technology

2002 ◽  
Vol 7 (5) ◽  
pp. 451-459 ◽  
Author(s):  
Yu-Xin Yan ◽  
Deborah M. Boldt-Houle ◽  
Bonnie P. Tillotson ◽  
Melissa A. Gee ◽  
Brian J. D'Eon ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Receptor Activation ◽  
Assay System ◽  
Functional Assay ◽  
Regulation Mechanism ◽  
G Protein Coupled Receptor ◽  
Gpcr Activation ◽  
G Protein Coupled

A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between β-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX™ system, uses a pair of inactive β-galactosidase (β-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and β-arrestin-β-gal fusion proteins are generated. Following ligand stimulation, β-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the β-gal mutant fragments. GPCR activation is measured directly by quantitating restored β-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the β2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The β2-adrenergic receptor cell line was screened with the LOPAC™ compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.

scholarly journals Correlated Motions of Conserved Polar Motifs Lay out a Plausible Mechanism of G Protein-Coupled Receptor Activation

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 670
Author(s):  
Argha Mitra ◽  
Arijit Sarkar ◽  
Márton Richárd Szabó ◽  
Attila Borics
Keyword(s):  
G Protein ◽  
Intracellular Signaling ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Binding Interface ◽  
Gpcr Activation ◽  
Macroscopic Polarization ◽  
G Protein Coupled

Recent advancements in the field of experimental structural biology have provided high-resolution structures of active and inactive state G protein-coupled receptors (GPCRs), a highly important pharmaceutical target family, but the process of transition between these states is poorly understood. According to the current theory, GPCRs exist in structurally distinct, dynamically interconverting functional states of which populations are shifted upon binding of ligands and intracellular signaling proteins. However, explanation of the activation mechanism, on an entirely structural basis, gets complicated when multiple activation pathways and active receptor states are considered. Our unbiased, atomistic molecular dynamics simulations of the μ opioid receptor (MOP) revealed that transmission of external stimulus to the intracellular surface of the receptor is accompanied by subtle, concerted movements of highly conserved polar amino acid side chains along the 7th transmembrane helix. This may entail the rearrangement of polar species and the shift of macroscopic polarization in the transmembrane domain, triggered by agonist binding. Based on our observations and numerous independent indications, we suggest amending the widely accepted theory that the initiation event of GPCR activation is the shift of macroscopic polarization between the ortho- and allosteric binding pockets and the intracellular G protein-binding interface.

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