scholarly journals A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor

Cell Research ◽  
2020 ◽  
Vol 30 (12) ◽  
pp. 1098-1108 ◽  
Author(s):  
Wen Sun ◽  
Li-Nan Chen ◽  
Qingtong Zhou ◽  
Li-Hua Zhao ◽  
Dehua Yang ◽  
...  
Keyword(s):  
Drug Targets ◽  
Receptor Activation ◽  
Ligand Specificity ◽  
Middle Region ◽  
Transmembrane Helices ◽  
Intestinal Hormones ◽  
Functional Studies ◽  
C Terminus ◽  
Class B ◽  
G Protein Coupled

AbstractGlucagon-like peptides (GLP-1 and GLP-2) are two proglucagon-derived intestinal hormones that mediate distinct physiological functions through two related receptors (GLP-1R and GLP-2R) which are important drug targets for metabolic disorders and Crohn’s disease, respectively. Despite great progress in GLP-1R structure determination, our understanding on the differences of peptide binding and signal transduction between these two receptors remains elusive. Here we report the electron microscopy structure of the human GLP-2R in complex with GLP-2 and a Gs heterotrimer. To accommodate GLP-2 rather than GLP-1, GLP-2R fine-tunes the conformations of the extracellular parts of transmembrane helices (TMs) 1, 5, 7 and extracellular loop 1 (ECL1). In contrast to GLP-1, the N-terminal histidine of GLP-2 penetrates into the receptor core with a unique orientation. The middle region of GLP-2 engages with TM1 and TM7 more extensively than with ECL2, and the GLP-2 C-terminus closely attaches to ECL1, which is the most protruded among 9 class B G protein-coupled receptors (GPCRs). Functional studies revealed that the above three segments of GLP-2 are essential for GLP-2 recognition and receptor activation, especially the middle region. These results provide new insights into the molecular basis of ligand specificity in class B GPCRs and may facilitate the development of more specific therapeutics.

The role of the extracellular loops of the CGRP receptor, a family B GPCR

2012 ◽  
Vol 40 (2) ◽  
pp. 433-437 ◽  
Author(s):  
James Barwell ◽  
Michael J. Woolley ◽  
Mark Wheatley ◽  
Alex C. Conner ◽  
David R. Poyner
Keyword(s):  
Surface Expression ◽  
Receptor Activation ◽  
Receptor Expression ◽  
Cell Surface Expression ◽  
Calcitonin Receptor ◽  
Transmembrane Helices ◽  
Extracellular Loops ◽  
Calcitonin Gene ◽  
G Protein Coupled

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.

scholarly journals Mechanisms of adhesion G protein–coupled receptor activation

2020 ◽  
Vol 295 (41) ◽  
pp. 14065-14083 ◽  
Author(s):  
Alexander Vizurraga ◽  
Rashmi Adhikari ◽  
Jennifer Yeung ◽  
Maiya Yu ◽  
Gregory G. Tall
Keyword(s):  
G Protein ◽  
Receptor Activation ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Physiological Processes ◽  
Class B ◽  
Peptide Agonist ◽  
G Protein Coupled ◽  
Existing Structure

Adhesion G protein–coupled receptors (AGPCRs) are a thirty-three-member subfamily of Class B GPCRs that control a wide array of physiological processes and are implicated in disease. AGPCRs uniquely contain large, self-proteolyzing extracellular regions that range from hundreds to thousands of residues in length. AGPCR autoproteolysis occurs within the extracellular GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the N terminus of the G protein–coupling seven-transmembrane–spanning bundle. GAIN domain–mediated self-cleavage is constitutive and produces two-fragment holoreceptors that remain bound at the cell surface. It has been of recent interest to understand how AGPCRs are activated in relation to their two-fragment topologies. Dissociation of the AGPCR fragments stimulates G protein signaling through the action of the tethered-peptide agonist stalk that is occluded within the GAIN domain in the holoreceptor form. AGPCRs can also signal independently of fragment dissociation, and a few receptors possess GAIN domains incapable of self-proteolysis. This has resulted in complex theories as to how these receptors are activated in vivo, complicating pharmacological advances. Currently, there is no existing structure of an activated AGPCR to support any of the theories. Further confounding AGPCR research is that many of the receptors remain orphans and lack identified activating ligands. In this review, we provide a detailed layout of the current theorized modes of AGPCR activation with discussion of potential parallels to mechanisms used by other GPCR classes. We provide a classification means for the ligands that have been identified and discuss how these ligands may activate AGPCRs in physiological contexts.

scholarly journals Mutagenesis facilitated crystallization of GLP-1R

IUCrJ ◽  
2019 ◽  
Vol 6 (6) ◽  
pp. 996-1006 ◽  
Author(s):  
Yueming Xu ◽  
Yuxia Wang ◽  
Yang Wang ◽  
Kaiwen Liu ◽  
Yao Peng ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Crystal Packing ◽  
Peptide Hormone ◽  
Current Data ◽  
Transmembrane Helices ◽  
Dynamic Simulations ◽  
Class B ◽  
Glucagon Like Peptide 1 ◽  
Diabetes Drug ◽  
G Protein Coupled

The class B family of G-protein-coupled receptors (GPCRs) has long been a paradigm for peptide hormone recognition and signal transduction. One class B GPCR, the glucagon-like peptide-1 receptor (GLP-1R), has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease. The previously determined structures of inactive GLP-1R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of 98 designed mutations. Here we systematically summarize all 98 mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical hydrophobic interactions shows the highest success rate. We further investigate four back mutations by thermal-shift assay, crystallization and molecular dynamic simulations, and conclude that mutation I1962.66bF increases thermal stability intrinsically and that mutation S2714.47bA decreases crystal packing entropy extrinsically, while mutations S1932.63bC and M2333.36bC may be dispensable since these two cysteines are not disulfide-linked. Our results indicate intrinsic connections between different regions of GPCR transmembrane helices and the current data suggest a general mutagenesis principle for structural determination of GPCRs and other membrane proteins.

scholarly journals SNAP-tagged nanobodies enable reversible optical control of a G protein-coupled receptor via a remotely tethered photoswitchable ligand

2018 ◽  
Author(s):  
Helen Farrants ◽  
Amanda Acosta Ruiz ◽  
Vanessa A. Gutzeit ◽  
Dirk Trauner ◽  
Kai Johnsson ◽  
...  
Keyword(s):  
G Protein ◽  
Drug Targets ◽  
Metabotropic Glutamate Receptor ◽  
Receptor Activation ◽  
Metabotropic Glutamate ◽  
Extracellular Signals ◽  
Physiological Processes ◽  
Wide Range ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) mediate the transduction of extracellular signals into complex intracellular responses. Despite their ubiquitous roles in physiological processes and as drug targets for a wide range of disorders, the precise mechanisms of GPCR function at the molecular, cellular, and systems levels remain partially understood. In order to dissect the function of individual receptors subtypes with high spatiotemporal precision, various optogenetic and photopharmacological approaches have been reported that use the power of light for receptor activation and deactivation. Here, we introduce a novel and, to date, most remote way of applying photoswitchable orthogonally remotely-tethered ligands (PORTLs) by using a SNAP-tag fused nanobody. Our nanobody-photoswitch conjugates (NPCs) can be used to target a GFP-fused metabotropic glutamate receptor by either gene-free application of purified complexes or co-expression of genetically encoded nanobodies to yield robust, reversible control of agonist binding and subsequent downstream activation. By harboring and combining the selectivity and flexibility of both nanobodies and self-labelling enzymes, we set the stage for targeting endogenous receptors in vivo.

The third extracellular loop of G-protein-coupled receptors: more than just a linker between two important transmembrane helices

2004 ◽  
Vol 32 (6) ◽  
pp. 1048-1050 ◽  
Author(s):  
Z. Lawson ◽  
M. Wheatley
Keyword(s):  
G Protein ◽  
Tertiary Structure ◽  
Large Family ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Transmembrane Helices ◽  
Ligand Selectivity ◽  
Interaction Sites ◽  
Amine Neurotransmitters ◽  
G Protein Coupled

GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins, which mediate their effects by coupling with G-proteins. Despite responding to a range of very diverse stimuli, these receptors exhibit a conserved tertiary structure comprising a bundle of seven TM (transmembrane) helices linked by alternating ECLs (extracellular loops) and ICLs (intracellular loops). The hydrophobic environment formed by the cluster of TM helices is functionally important. For example, the 11-cis retinal chromophore of rhodopsin forms a protonated Schiff base linkage to a lysine in TM7, deep within the helical bundle, and small ligands, such as amine neurotransmitters and non-peptide analogues of peptide hormones, also bind within the corresponding region of their cognate receptors. In addition, activation of GPCRs involves relative movement of TM helices to present G-protein interaction sites across the intracellular face of the receptor. Consequently, it might be assumed that the ECLs of the GPCR are inert peptide linkers that merely connect important TM helices. Focusing on ECL3 (third ECL), it is becoming increasingly apparent that this extracellular domain can fulfil a range of important roles with respect to GPCR signalling, including agonist binding, ligand selectivity and receptor activation.

Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors

2001 ◽  
Vol 359 (3) ◽  
pp. 465-484 ◽  
Author(s):  
Emmanuel HERMANS ◽  
R. A. John CHALLISS
Keyword(s):  
G Protein ◽  
Drug Targets ◽  
Metabotropic Glutamate Receptors ◽  
Gabab Receptor ◽  
Receptor Activation ◽  
Metabotropic Glutamate ◽  
Calcium Sensing ◽  
Group I ◽  
G Protein Coupled ◽  
The Brain

In 1991 a new type of G-protein-coupled receptor (GPCR) was cloned, the type 1a metabotropic glutamate (mGlu) receptor, which, despite possessing the defining seven-transmembrane topology of the GPCR superfamily, bore little resemblance to the growing number of other cloned GPCRs. Subsequent studies have shown that there are eight mammalian mGlu receptors that, together with the calcium-sensing receptor, the GABAB receptor (where GABA is γ-aminobutyric acid) and a subset of pheromone, olfactory and taste receptors, make up GPCR family C. Currently available data suggest that family C GPCRs share a number of structural, biochemical and regulatory characteristics, which differ markedly from those of the other GPCR families, most notably the rhodopsin/family A GPCRs that have been most widely studied to date. This review will focus on the group I mGlu receptors (mGlu1 and mGlu5). This subgroup of receptors is widely and differentially expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in the control of an array of key signalling events, including roles in the adaptative changes needed for long-term depression or potentiation of neuronal synaptic connectivity. In addition to playing critical physiological roles within the brain, the mGlu receptors are also currently the focus of considerable attention because of their potential as drug targets for the treatment of a variety of neurological and psychiatric disorders.

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 Ligand-Induced and -Independent Internalization of the Chemerin Receptor GPR1

2021 ◽  
Author(s):  
Tobias F. Fischer ◽  
Anne S. Czerniak ◽  
Tina Weiß ◽  
Clara T. Schoeder ◽  
Philipp Wolf ◽  
...  
Keyword(s):  
Binding Mode ◽  
Loop Structure ◽  
Ligand Specificity ◽  
Extracellular Loop ◽  
Extracellular Loops ◽  
C Terminus ◽  
Loop 2 ◽  
G Protein Coupled ◽  
Migration Of Macrophages

Abstract 1. Tight regulation of cytokines is essential for the initiation and resolution of inflammation. Chemerin, a mediator of innate immunity, mainly acts on chemokine-like receptor 1 (CMKLR1) to induce the migration of macrophages and dendritic cells. The role of the second chemerin receptor, G protein-coupled receptor 1 (GPR1), is still unclear. Here we demonstrate that GPR1 shows ligand-induced arrestin3 recruitment and internalization. The chemerin C-terminus triggers this activation by folding into a loop structure, binding to aromatic residues in the extracellular loops of GPR1. While this overall binding mode is shared between GPR1 and CMKLR1, differences in their respective extracellular loop 2 allowed for the design of the first GPR1-selective peptide. However, our results suggest that ligand-induced arrestin recruitment is not the only mode of action of GPR1. This receptor also displays constitutive internalization and recycling, which allows GPR1 to internalize inactive peptides efficiently by an activation-independent pathway. Our results demonstrate that GPR1 takes a dual role in regulating chemerin activity: As a signaling receptor for arrestin-based signaling on one hand, and as a scavenging receptor with broader ligand specificity on the other.

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 Regulating G protein-coupled receptors by topological inversion

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Bray Denard ◽  
Sungwon Han ◽  
JungYeon Kim ◽  
Elliott M Ross ◽  
Jin Ye
Keyword(s):  
G Protein ◽  
Transmembrane Helix ◽  
Peritoneal Macrophages ◽  
G Protein Coupled Receptors ◽  
Editorial Note ◽  
Transmembrane Helices ◽  
Ccr5 Chemokine Receptor ◽  
C Terminus ◽  
G Protein Coupled ◽  
Mouse Peritoneal Macrophages

G protein-coupled receptors (GPCRs) are a family of proteins containing seven transmembrane helices, with the N- and C-terminus of the protein located at the extracellular space and cytosol, respectively. Here, we report that ceramide or related sphingolipids might invert the topology of many GPCRs that contain a GXXXN motif in their first transmembrane helix. The functional significance of this topological regulation is illustrated by the CCR5 chemokine receptor. In the absence of lipopolysaccharide (LPS), CCR5 adopts a topology consistent with that of GPCR, allowing mouse peritoneal macrophages to migrate toward its ligand CCL5. LPS stimulation results in increased production of dihydroceramide, which inverts the topology of CCR5, preventing macrophages from migrating toward CCL5. These results suggest that GPCRs may not always adopt the same topology and can be regulated through topological inversion.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that major issues remain unresolved (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

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