Identification of secretin, vasoactive intestinal peptide and glucagon binding sites: from chimaeric receptors to point mutations

2002 ◽  
Vol 30 (4) ◽  
pp. 437-441 ◽  
Author(s):  
M. Waelbroeck ◽  
J. Perret ◽  
P. Vertongen ◽  
M. Van Craenenbroeck ◽  
P. Robberecht
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Receptor Activation ◽  
Side Chain ◽  
Extracellular Loop ◽  
Glucagon Receptor ◽  
In The Wild ◽  
Type Receptor ◽  
Receptor Conformation

We have identified two basic residues that are important for the recognition of secretin and vasoactive intestinal peptide (VIP) by their respective receptors. These two peptides containing an Asp residue at position 3 interacted with an arginine residue in transmembrane helix 2 (TM2) of the receptor, and the lysine residue in extracellular loop 1 (ECL1) stabilized the active receptor conformation induced by the ligand. The glucagon receptor possesses a Lys instead of an Arg in TM2, and an Ile instead of Lys in ECL1; it markedly prefers a Gln side chain in position 3 of the ligand. Our results suggested that, in the wild-type receptor, the Ile side chain prevented access to the TM2 Lys side chain, but oriented the glucagon Gln3 side chain to its proper binding site. In the double mutant, the ECL1 Lys allowed an interaction between negatively charged residues in position 3 of glucagon and the TM2 Arg, resulting in efficient receptor activation by [Asp3]glucagon as well as by glucagon.

Mutational analysis of the glucagon receptor: similarities with the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP)/secretin receptors for recognition of the ligand's third residue

2002 ◽  
Vol 362 (2) ◽  
pp. 389-394 ◽  
Author(s):  
Jason PERRET ◽  
Mélanie VAN CRAENENBROECK ◽  
Ingrid LANGER ◽  
Pascale VERTONGEN ◽  
Françoise GREGOIRE ◽  
...  
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
Functional Properties ◽  
Mutational Analysis ◽  
Transmembrane Helix ◽  
Binding Pocket ◽  
Side Chain ◽  
Wild Type ◽  
Glucagon Receptor ◽  
Pituitary Adenylate Cyclase ◽  
In The Wild

Receptor recognition by the Asp3 residues of vasoactive intestinal peptide and secretin requires the presence of a lysine residue close to the second transmembrane helix (TM2)/first extracellular loop junction and an ionic bond with an arginine residue in TM2. We tested whether the glucagon Gln3 residue recognizes the equivalent positions in its receptor. Our data revealed that the binding and functional properties of the wild-type glucagon receptor and the K188R mutant were not significantly different, whereas all agonists had markedly lower potencies and affinities at the I195K mutated receptor. In contrast, glucagon was less potent and the Asp3-, Asn3- and Glu3-glucagon mutants were more potent and efficient at the double-mutated K188R/I195K receptor. Furthermore, these alterations were selective for position 3 of glucagon, as shown by the functional properties of the mutant Glu9- and Lys15-glucagon. Our results suggest that although the Gln3 residue of glucagon did not interact with the equivalent binding pocket as the Asp3 residue of vasoactive intestinal peptide or secretin, the Asp3-glucagon analogue was able to interact with position 188 of the K188R/I195K glucagon receptor. Nevertheless, the Gln3 side chain of glucagon probably binds very close to this region in the wild-type receptor.

Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain

2019 ◽  
Author(s):  
Zichen Wang ◽  
Huaxun Fan ◽  
Xiao Hu ◽  
John Khamo ◽  
Jiajie Diao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Hydrophobic Interactions ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Salt Bridge ◽  
Receptor Activation ◽  
Membrane Dynamics ◽  
Juxtamembrane Domain ◽  
Joint Work

<p>The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP<sub>2 </sub>lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.</p>

scholarly journals Structural insights into ligand efficacy and activation of the glucagon receptor

2019 ◽  
Author(s):  
Daniel Hilger ◽  
Kaavya Krishna Kumar ◽  
Hongli Hu ◽  
Mie Fabricius Pedersen ◽  
Lise Giehm ◽  
...  
Keyword(s):  
G Protein ◽  
Partial Agonist ◽  
Transmembrane Helix ◽  
Peptide Hormone ◽  
Receptor Activation ◽  
Glucagon Receptor ◽  
Structural Framework ◽  
Class B ◽  
Treatment Of Diabetes ◽  
Sugar Levels

AbstractThe glucagon receptor family comprises Class B G protein-coupled receptors (GPCRs) that play a crucial role in regulating blood sugar levels. Receptors of this family represent important therapeutic targets for the treatment of diabetes and obesity. Despite intensive structural studies, we only have a poor understanding of the mechanism of peptide hormone-induced Class B receptor activation. This process involves the formation of a sharp kink in transmembrane helix 6 that moves out to allow formation of the nucleotide-free G protein complex. Here, we present the cryo-EM structure of the glucagon receptor (GCGR), a prototypical Class B GPCR, in complex with an engineered soluble glucagon derivative and the heterotrimeric G-protein, Gs. Comparison with the previously determined crystal structures of GCGR bound to a partial agonist reveals a structural framework to explain the molecular basis of ligand efficacy that is further supported by mutagenesis data.

scholarly journals The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activlation

2022 ◽  
Vol 12 ◽  
Author(s):  
Ian Winfield ◽  
Kerry Barkan ◽  
Sarah Routledge ◽  
Nathan J. Robertson ◽  
Matthew Harris ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
G Protein ◽  
Conformational Changes ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Cgrp Receptor ◽  
Intracellular Loop ◽  
Glucagon Receptor ◽  
G Protein Coupled

The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein β subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gβ subunit.

scholarly journals TSH receptor mutation V509A causes familial hyperthyroidism by release of interhelical constraints between transmembrane helices TMH3 and TMH5

2005 ◽  
Vol 186 (2) ◽  
pp. 377-385 ◽  
Author(s):  
Beate Karges ◽  
Gerd Krause ◽  
Janos Homoki ◽  
Klaus-Michael Debatin ◽  
Nicolas de Roux ◽  
...  
Keyword(s):  
Transmembrane Helix ◽  
Fold Increase ◽  
Receptor Activation ◽  
Side Chain ◽  
Transmembrane Helices ◽  
Camp Production ◽  
Follicular Thyroid Cancer ◽  
Thyroid Adenomas ◽  
Tsh Receptor Mutation

Mutations of the human thyrotrophin receptor (TSH-R) are a cause of thyroid adenomas and hyperthyroidism. Here we study mechanisms of receptor activation in a genomic TSH-R variant V509A located in transmembrane helix (TMH) 3, which we identify in a family with congenital hyperthyroidism, multiple adenomas and follicular thyroid cancer. Using molecular modelling and dynamic simulation, we predicted the release of amino acid residue A593 (located opposite in domain TMH5) from a tight ‘knob-and-hole’ interaction with TMH3, physiologically constrained in the native receptor state by the bulky side chain of V509. To experimentally validate this concept, we generated mutant TSH-R expression constructs for functional in vitro studies. TSH-R mutant V509A showed a 2.8-fold increase in basal cAMP production, confirming constitutive TSH-R activation. The addition of a second site suppressor mutant A593V to TSH-R V509A resulted in the normalization of basal cAMP release, and the dose-responsiveness to TSH ligand was maintained. These data thus demonstrate that TSH-R V509A activation is caused by the release of TMH3–TMH5 interhelical constraints, while the native TSH-R conformation is re-stabilized by the introduction of a spacious valine residue at position 593. In conclusion, we delineate a novel mechanism of constitutive TSH-R activation, leading to thyroid hyperfunction and neoplasia.

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.

Evidence to support a spectrum of active states for the glucagon receptor

2004 ◽  
Vol 32 (6) ◽  
pp. 1037-1039 ◽  
Author(s):  
N. Strudwick ◽  
N. Bhogal ◽  
N.A. Evans ◽  
F.E. Blaney ◽  
J.B.C. Findlay
Keyword(s):  
Partial Agonist ◽  
Transmembrane Helix ◽  
Complex Model ◽  
Wild Type ◽  
Glucagon Receptor ◽  
Activating Mutations ◽  
Ternary Complex Model ◽  
Receptor Mutant ◽  
G Protein Coupled ◽  
Type Receptor

The ternary complex model suggests that G-protein-coupled receptors resonate between inactive (R) and active (R*) forms. Physiologically, R sites ordinarily predominate with a few R* sites giving rise to basal activity. Agonists recognize, stabilize and increase the R* population, thus altering intracellular activity. There is evidence to suggest the possibility of a spectrum of conformations between R and R*. Our aim is to study the consequences of putative GR (glucagon receptor)-activating mutations using glucagon and partial agonist des-His1-[Glu9]glucagon amide (glucagon-NH2). Alanine substitution in TM (transmembrane) helix 2 of Arg173 or of His177 detrimentally affected glucagon and glucagon-NH2 response maxima. TM2 receptor mutant, Phe181-Ala, displayed reduced maximum cAMP accumulation in response to glucagon-NH2. Thr353-Cys (TM6) and Glu406-Ala (TM7) receptors demonstrated constitutive activity and enhanced EC50 values for glucagon-NH2; Arg346-Ala (TM6) and Asn404-Ala (TM7) receptors were activated by sub-fmol glucagon concentrations, yet were not constitutively active and demonstrated wild-type receptor-like EC50 values for glucagon-NH2. Unlike Arg346-Ala receptors, Thr353-Cys, Asn404-Ala and Glu406-Ala receptors demonstrated improved EC50 values for glucagon, whereas their maximal responses to and their affinity for glucagon were comparable with the wild-type receptor. In contrast, despite slightly reduced glucagon-NH2 affinity, Arg346-Ala, Thr353-Cys, Asn404-Ala and Glu406-Ala receptors displayed glucagon-NH2 response maxima that exceeded those seen for wild-type receptors. Interestingly, we observed biphasic glucagon-mediated signalling responses. Our results are consistent with the concept of different agonists promoting the formation of distinct active states from partially active R*low to fully active R*high forms.

Molecular mechanisms involved in vasoactive intestinal peptide receptor activation and regulation: current knowledge, similarities to and differences from the A family of G-protein-coupled receptors

2007 ◽  
Vol 35 (4) ◽  
pp. 724-728 ◽  
Author(s):  
I. Langer ◽  
P. Robberecht
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
G Protein ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Peptide Receptor ◽  
Vasoactive Intestinal Peptide Receptor ◽  
G Protein Coupled

An actual paradigm for activation and regulation of the GPCR (G-protein-coupled receptors)/seven-transmembrane helix family of receptors essentially emerges from extensive studies of the largest family of receptors, the GPCR-A/rhodopsin family. The mechanisms regulating the GPCR-B family signal transduction are less precisely understood due in part to the lack of the conserved signatures of the GPCR-A family (E/DRY, NPXXY) and in part to the absence of a reliable receptor modelling, although some studies suggest that both families share similar features. Here, we try to highlight the current knowledge of the activation and the regulation of the VIP (vasoactive intestinal peptide) receptors, namely VPAC (VIP/pituitary adenylate cyclase-activating peptide receptor) 1 and 2. This includes search for amino acids involved in the stabilization of the receptor active conformation and in coupling to G-proteins, signalling pathways activated in response to VIP, agonist-dependent receptor down-regulation, phosphorylation and internalization as well as pharmacological consequences of receptor hetero-dimerization.

scholarly journals Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain

2019 ◽  
Author(s):  
Zichen Wang ◽  
Huaxun Fan ◽  
Xiao Hu ◽  
John Khamo ◽  
Jiajie Diao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Hydrophobic Interactions ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Salt Bridge ◽  
Receptor Activation ◽  
Membrane Dynamics ◽  
Juxtamembrane Domain ◽  
Joint Work

<p>The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP<sub>2 </sub>lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.</p>

scholarly journals Receptor-operated Ca2+ channels in gastric parietal cells: gastrin and carbachol induce Ca2+ influx in depleting intracellular Ca2+ stores

1993 ◽  
Vol 289 (1) ◽  
pp. 117-124 ◽  
Author(s):  
S Roche ◽  
J P Bali ◽  
R Magous
Keyword(s):  
Steady State ◽  
Receptor Activation ◽  
Parietal Cells ◽  
The Other ◽  
Bivalent Cation ◽  
Gtp Binding ◽  
Gastric Parietal Cells ◽  
Type Receptor ◽  
Ca2 Channels ◽  
Stimulation Of

The mechanism whereby gastrin-type receptor and muscarinic M3-type receptor regulate free intracellular Ca2+ concentration ([Ca2+]i) was studied in rabbit gastric parietal cells stimulated by either gastrin or carbachol. Both agonists induced a biphasic [Ca2+]i response: a transient [Ca2+]i rise, followed by a sustained steady state depending on extracellular Ca2+. Gastrin and carbachol also caused a rapid and transient increase in Mn2+ influx (a tracer for bivalent-cation entry). Pre-stimulation of cells with one agonist drastically decreased both [Ca2+]i increase and Mn2+ influx induced by the other. Neither diltiazem nor pertussistoxin treatment had any effect on agonist-stimulated Mn2+ entry. Thapsigargin, a Ca(2+)-pump inhibitor, induced a biphasic [Ca2+]i increase, and enhanced the rate of Mn2+ entry. Preincubation of cells with thapsigargin inhibits the [Ca2+]i increase as well as Mn2+ entry stimulated by gastrin or by carbachol. Thapsigargin induced a weak but significant increase in Ins(1,4,5)P3 content, but this agent had no effect on the agonist-evoked Ins(1,4,5)P3 response. In permeabilized parietal cells, Ins(1,4,5)P3 and caffeine caused an immediate Ca2+ release from intracellular pools, followed by a reloading of Ca2+ pools which can be prevented in the presence of thapsigargin. We conclude that (i) gastrin and carbachol mobilize common Ca2+ intracellular stores, (ii) Ca2+ permeability secondary to receptor activation involves neither a voltage-sensitive Ca2+ channel nor a GTP-binding protein from the G1 family, and (iii) agonists regulate common Ca2+ channels in depleting intracellular Ca2+ stores.

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