Ligand binding and activation of the CGRP receptor

2007 ◽  
Vol 35 (4) ◽  
pp. 729-732 ◽  
Author(s):  
A.C. Conner ◽  
J. Simms ◽  
J. Barwell ◽  
M. Wheatley ◽  
D.R. Poyner
Keyword(s):  
G Protein ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Extracellular Loop ◽  
C Terminus ◽  
Calcitonin Gene ◽  
The Family ◽  
N Terminus ◽  
G Protein Coupled

The receptor for CGRP (calcitonin gene-related peptide) is a heterodimer between a GPCR (G-protein-coupled receptor), CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). Models have been produced of RAMP1 and CLR. It is likely that the C-terminus of CGRP interacts with the extracellular N-termini of CLR and RAMP1; the extreme N-terminus of CLR is particularly important and may interact directly with CGRP and also with RAMP1. The N-terminus of CGRP interacts with the TM (transmembrane) portion of the receptor; the second ECL (extracellular loop) is especially important. Receptor activation is likely to involve the relative movements of TMs 3 and 6 to create a G-protein-binding pocket, as in Family A GPCRs. Pro321 in TM6 appears to act as a pivot. At the base of TMs 2 and 3, Arg151, His155 and Glu211 may form a loose equivalent of the Family A DRY (Asp-Arg-Tyr) motif. Although the details of this proposed activation mechanism clearly do not apply to all Family B GPCRs, the broad outlines may be conserved.

CSIG-07. ACTIVATION OF THE ADHESION G PROTEIN-COUPLED RECEPTOR GPR133 BY ANTIBODIES AGAINST THE N-TERMINUS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi34-vi34
Author(s):  
Gabriele Stephan ◽  
Joshua Frenster ◽  
Niklas Ravn-Boess ◽  
Devin Bready ◽  
Jordan Wilcox ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Receptor Activation ◽  
Cell Culture Supernatant ◽  
Dependent Manner ◽  
G Protein Coupled Receptor ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus ◽  
G Protein Coupled

Abstract We recently demonstrated that GPR133 (ADGRD1), a member of the adhesion G protein-coupled receptor (aGPCR) family, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We therefore postulate that GPR133 represents a novel target in GBM, which merits development of therapeutics. Like most aGPCRs, GPR133 is characterized by an intracellular C-terminus, 7 plasma membrane-spanning α-helices and a large extracellular N-terminus. The N-terminus possesses a conserved GPCR autoproteolysis-inducing (GAIN) domain that catalyzes cleavage at a GPCR proteolysis site (GPS), resulting in a C-terminal fragment (CTF) and an N-terminal fragment (NTF). We showed that dissociation of the cleaved NTF and CTF at the plasma membrane increases canonical signaling of GPR133, which is mediated by coupling to Gs and increase in cytosolic cAMP. Toward characterizing the effect of biologics on GPR133 function, we overexpressed wild-type or mutant forms of GPR133 in HEK293T cells and patient-derived GBM cells lines. Treatment of these cells with antibodies specifically targeting the NTF of GPR133 increased receptor activation in a dose-dependent manner. No effects were elicited with an antibody against the receptor’s intracellular C-terminus. Interestingly, cells overexpressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. Following antibody treatment, co-purification of the GPR133 NTF and the N-terminal antibody from the cell culture supernatant indicated the formation of antibody-NTF complexes. Analysis of these complexes suggested that antibody binding stimulated the dissociation of the NTF from the CTF. However, the increased flexibility of the GAIN domain and NTF after cleavage, independently of dissociation, may also endow the receptor with responsiveness to the effects of the antibodies. These data constitute a proof-of-concept paradigm of modulation of GPR133 function with antibodies. This work provides rationale for pursuing development of biologics targeting GPR133 in GBM.

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 Universal activation mechanism of class A GPCRs

2019 ◽  
Author(s):  
Qingtong Zhou ◽  
Dehua Yang ◽  
Meng Wu ◽  
Yu Guo ◽  
Wangjing Guo ◽  
...  
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Transmembrane Helix ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Coupling Region ◽  
Activation Pathway ◽  
Class A ◽  
Gpcr Activation

AbstractClass A G protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology. GPCR activation is an allosteric process that links agonist binding to G protein recruitment, with the hallmark outward movement of transmembrane helix 6 (TM6). However, what leads to TM6 movement and the key residue-level changes of this trigger remain less well understood. Here, by analyzing over 230 high-resolution structures of class A GPCRs, we discovered a modular, universal GPCR activation pathway that unites previous findings into a common activation mechanism, directly linking the bottom of ligand-binding pocket with G protein-coupling region. We suggest that the modular nature of the universal GPCR activation pathway allowed for the decoupling of the evolution of the ligand binding site, G protein binding region and the residues important for receptor activation. Such an architecture might have facilitated GPCRs to emerge as a highly successful family of proteins for signal transduction in nature.

scholarly journals Dissociation of the intramolecularly cleaved N- and C-terminal fragments of the adhesion G protein-coupled receptor GPR133 (ADGRD1) increases canonical signaling

2020 ◽  
Author(s):  
Joshua D. Frenster ◽  
Gabriele Stephan ◽  
Niklas Ravn-Boess ◽  
Devin Bready ◽  
Jordan Wilcox ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Receptor Activation ◽  
List Type ◽  
G Protein Coupled Receptor ◽  
Major Mechanism ◽  
N Terminus ◽  
Adhesion Gpcr ◽  
G Protein Coupled

SUMMARYGPR133 (ADGRD1), an adhesion G protein-coupled receptor (GPCR), is necessary for growth of glioblastoma (GBM), a brain malignancy. The extracellular N-terminus of GPR133 is thought to be autoproteolytically cleaved into an N-terminal and a C-terminal fragment (NTF and CTF). Nevertheless, the role of this cleavage in receptor activation remains unclear. Here, we show that the wild-type (WT) receptor is cleaved after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant (H543R) in patient-derived GBM cultures and HEK293T cells. However, the resulting NTF and CTF remain non-covalently bound until the receptor is trafficked to the plasma membrane, where we find NTF-CTF dissociation. Using a fusion of the hPAR1 receptor N-terminus and the CTF of GPR133, we demonstrate that thrombin-induced cleavage and shedding of the hPAR1 NTF increases receptor signaling. This study supports a model where dissociation of the NTF at the plasma membrane promotes GPR133 activation.Highlights-GPR133 is intramolecularly cleaved in patient-derived GBM cultures-Cleaved GPR133 signals at higher efficacy than the uncleavable GPR133 H543R mutant-The N- and C-terminal fragments (NTF and CTF) of GPR133 dissociate at the plasma membrane-Acute thrombin-induced cleavage of the human PAR1 NTF from the GPR133 CTF increases signalingeTOC BlurbFrenster et al. demonstrate intramolecular cleavage of the adhesion GPCR GPR133 in glioblastoma and HEK293T cells. The resulting N- and C-terminal fragments dissociate at the plasma membrane to increase canonical signaling. The findings suggest dissociation of GPR133’s N-terminus at the plasma membrane represents a major mechanism of receptor activation.

scholarly journals The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

2013 ◽  
Vol 10 (88) ◽  
pp. 20130589 ◽  
Author(s):  
Michael. J. Woolley ◽  
Harriet A. Watkins ◽  
Bruck Taddese ◽  
Z. Gamze Karakullukcu ◽  
James Barwell ◽  
...  
Keyword(s):  
Experimental Approach ◽  
Point Mutations ◽  
Receptor Activation ◽  
Calcitonin Receptor ◽  
Extracellular Loop ◽  
Cgrp Receptor ◽  
Alanine Scan ◽  
Calcitonin Gene ◽  
G Protein Coupled

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.

scholarly journals Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases

2015 ◽  
Vol 290 (34) ◽  
pp. 20629-20647 ◽  
Author(s):  
Konstantin E. Komolov ◽  
Anshul Bhardwaj ◽  
Jeffrey L. Benovic
Keyword(s):  
G Protein ◽  
High Mobility ◽  
Binding Pocket ◽  
Structural Features ◽  
Kinase Domain ◽  
G Protein Coupled Receptor ◽  
The Family ◽  
Receptor Kinases ◽  
Novel Interactions ◽  
G Protein Coupled

G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5′-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs.

Pharmacological Characterization of Receptor Redistribution and β-Arrestin Recruitment Assays for the Cannabinoid Receptor 1

2009 ◽  
Vol 14 (7) ◽  
pp. 811-823 ◽  
Author(s):  
Miranda M.C. van der Lee ◽  
Marion Blomenröhr ◽  
Antoon A. van der Doelen ◽  
Jesse W.Y. Wat ◽  
Niels Smits ◽  
...  
Keyword(s):  
G Protein ◽  
Cannabinoid Receptor ◽  
Receptor Activation ◽  
Binding Affinities ◽  
Cannabinoid Receptor 1 ◽  
Pharmacological Characterization ◽  
Cellular Effects ◽  
Partial Agonists ◽  
C Terminus ◽  
G Protein Coupled

Receptor redistribution and β-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular β-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution® assay and 2 nonimaging-based β-arrestin recruitment assays, Tango™ and PathHunter ™, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of Gαi coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the 3 assays, and are comparable with the measured ligand binding affinities. The inverse agonist SR141716 decreased basal signal in all 3 assays, but only in the Tango bla assay a reliable EC50 could be determined for this compound, suggesting that Tango is the most suitable assay for the identification of new inverse agonists. Both the Redistribution and the PathHunter assay could discriminate partial agonists from full agonists, whereas in the Tango assay partial agonists behaved as full agonists. Only the PathHunter cells allowed detection of cannabinoid receptor activation via β-arrestin recruitment and Gαi-protein-mediated inhibition of cAMP, thus enabling the identification of biased ligands that differ in these cellular effects. The characteristics and limitations of the different assays are discussed. ( Journal of Biomolecular Screening 2009:811-823)

scholarly journals β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

2021 ◽  
Vol 118 (11) ◽  
pp. e2017201118
Author(s):  
Keehun Kim ◽  
Shayla Paulekas ◽  
Fredrik Sadler ◽  
Tejas M. Gupte ◽  
Michael Ritt ◽  
...  
Keyword(s):  
G Protein ◽  
Dynamic Range ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Two Stage ◽  
Protein Activation ◽  
Intrinsic Efficacy ◽  
C Terminus ◽  
G Protein Activation ◽  
Structural Determinant

Classical pharmacological models have incorporated an “intrinsic efficacy” parameter to capture system-independent effects of G protein–coupled receptor (GPCR) ligands. However, the nonlinear serial amplification of downstream signaling limits quantitation of ligand intrinsic efficacy. A recent biophysical study has characterized a ligand “molecular efficacy” that quantifies the influence of ligand-dependent receptor conformation on G protein activation. Nonetheless, the structural translation of ligand molecular efficacy into G protein activation remains unclear and forms the focus of this study. We first establish a robust, accessible, and sensitive assay to probe GPCR interaction with G protein and the Gα C terminus (G-peptide), an established structural determinant of G protein selectivity. We circumvent the need for extensive purification protocols by the single-step incorporation of receptor and G protein elements into giant plasma membrane vesicles (GPMVs). We use previously established SPASM FRET sensors to control the stoichiometry and effective concentration of receptor–G protein interactions. We demonstrate that GPMV-incorporated sensors (v-SPASM sensors) provide enhanced dynamic range, expression-insensitive readout, and a reagent level assay that yields single point measurements of ligand molecular efficacy. Leveraging this technology, we establish the receptor–G-peptide interaction as a sufficient structural determinant of this receptor-level parameter. Combining v-SPASM measurements with molecular dynamics (MD) simulations, we elucidate a two-stage receptor activation mechanism, wherein receptor–G-peptide interactions in an intermediate orientation alter the receptor conformational landscape to facilitate engagement of a fully coupled orientation that tunes G protein activation.

scholarly journals Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism

2015 ◽  
Vol 112 (48) ◽  
pp. 14966-14971 ◽  
Author(s):  
Yiqun Yu ◽  
Claire A. de March ◽  
Mengjue J. Ni ◽  
Kaylin A. Adipietro ◽  
Jérôme Golebiowski ◽  
...  
Keyword(s):  
G Protein ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Activation Mechanism ◽  
Odorant Receptors ◽  
Structural Basis ◽  
Single Mutation ◽  
Response Properties ◽  
Basal Activity ◽  
G Protein Coupled

Mammals detect and discriminate numerous odors via a large family of G protein-coupled odorant receptors (ORs). However, little is known about the molecular and structural basis underlying OR response properties. Using site-directed mutagenesis and computational modeling, we studied ORs sharing high sequence homology but with different response properties. When tested in heterologous cells by diverse odorants, MOR256-3 responded broadly to many odorants, whereas MOR256-8 responded weakly to a few odorants. Out of 36 mutant MOR256-3 ORs, the majority altered the responses to different odorants in a similar manner and the overall response of an OR was positively correlated with its basal activity, an indication of ligand-independent receptor activation. Strikingly, a single mutation in MOR256-8 was sufficient to confer both high basal activity and broad responsiveness to this receptor. These results suggest that broad responsiveness of an OR is at least partially attributed to its activation likelihood.

scholarly journals Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

2015 ◽  
Vol 112 (18) ◽  
pp. 5702-5707 ◽  
Author(s):  
Daniel G. Isom ◽  
Henrik G. Dohlman
Keyword(s):  
G Protein ◽  
Sequence Similarity ◽  
Transmembrane Helix ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Transmembrane Receptors ◽  
Domains Of Life ◽  
G Protein Coupled ◽  
Ionizable Groups

Seven-transmembrane receptors (7TMRs) have evolved in prokaryotes and eukaryotes over hundreds of millions of years. Comparative structural analysis suggests that these receptors may share a remote evolutionary origin, despite their lack of sequence similarity. Here we used structure-based computations to compare 221 7TMRs from all domains of life. Unexpectedly, we discovered that these receptors contain spatially conserved networks of buried ionizable groups. In microbial 7TMRs these networks are used to pump ions across the cell membrane in response to light. In animal 7TMRs, which include light- and ligand-activated G protein-coupled receptors (GPCRs), homologous networks were found to be characteristic of activated receptor conformations. These networks are likely relevant to receptor function because they connect the ligand-binding pocket of the receptor to the nucleotide-binding pocket of the G protein. We propose that agonist and G protein binding facilitate the formation of these electrostatic networks and promote important structural rearrangements such as the displacement of transmembrane helix-6. We anticipate that robust classification of activated GPCR structures will aid the identification of ligands that target activated GPCR structural states.

Sign in / Sign up

Export Citation Format

Share Document