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.

scholarly journals New Pathogenic Thyrotropin Receptor Mutations Decipher Differentiated Activity Switching at a Conserved Helix 6 Motif of Family A GPCR

2012 ◽  
Vol 97 (2) ◽  
pp. E228-E232 ◽  
Author(s):  
Heike Biebermann ◽  
Franziska Winkler ◽  
Daniela Handke ◽  
Anke Teichmann ◽  
Burkhard Gerling ◽  
...  
Keyword(s):  
Transmembrane Helix ◽  
Helical Conformation ◽  
Side Chain ◽  
Wild Type ◽  
Pathogenic Mechanisms ◽  
Activating Mutations ◽  
Naturally Occurring ◽  
High Conservation ◽  
Inactivating Mutation ◽  
G Protein Coupled

Context: In this paper we report two new TSH receptor (TSHR) mutations. One mutation (Pro6396.50Leu) was identified in two siblings with congenital hypothyroidism, and a second mutation (Cys6366.47Arg) was found in a patient suffering from nonautoimmune hyperthyroidism. Both mutations are located in transmembrane helix (TMH) 6 at the conserved Cys6.47-Trp(Met)6.48-Leu(Ala)6.49-Pro6.50 motif of family A G protein-coupled receptors (GPCR). Objective: To study the pathogenic mechanisms, we tested patients' mutations and further side chain variations regarding their effects on TSHR signaling. Results: Substitution Pro639Leu fully inactivates the promiscuous TSHR for cAMP (Gs) and IP (Gq) signaling. In contrast, Cys636Arg leads to constitutive activation of Gs. Organization of TSHR in oligomers was not modified by mutations at position 636. Interestingly, it is known from crystal structures of GPCR that Pro6.50 is located at a TMH6 kink-distortion, which is a pivot during activation-related helical movements. However, the cell surface expressions of all mutants at position 639 were comparable to wild type, indicating a helical conformation like wild type. Conclusion: Until now, only naturally occurring constitutively activating mutations in TSHR TMH6 have been reported, but here we present the first pathogenic inactivating mutation (Pro639Leu). Our data are indicative of differentiated regulation of Gs and Gq signaling at particular TMH6 positions, but without any effects on TSHR oligomer constellation. Details of signaling modulation by each mutant at positions 6366.47 and 6396.50 help us to understand high conservation of these amino acids in family A GPCR. Described molecular (pathogenic) mechanisms are likely not unique for TSHR.

scholarly journals Selective Modulation of Wild Type Receptor Functions by Mutants of G-Protein-coupled Receptors

1999 ◽  
Vol 274 (18) ◽  
pp. 12548-12554 ◽  
Author(s):  
Christian Le Gouill ◽  
Jean-Luc Parent ◽  
Carolyn-Ann Caron ◽  
Rémi Gaudreau ◽  
Léonid Volkov ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Wild Type ◽  
Selective Modulation ◽  
G Protein Coupled ◽  
Type Receptor

scholarly journals Isolation and characterisation of the marmoset gonadotrophin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe

1999 ◽  
Vol 163 (3) ◽  
pp. 447-456 ◽  
Author(s):  
B Byrne ◽  
A McGregor ◽  
PL Taylor ◽  
R Sellar ◽  
FE Rodger ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Maximal Response ◽  
Wild Type ◽  
Releasing Hormone ◽  
Agonist And Antagonist ◽  
Gonadotrophin Releasing Hormone ◽  
Antagonist Action ◽  
G Protein Coupled ◽  
Type Receptor ◽  
D Value

In order to facilitate the understanding of gonadotrophin-releasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly(10), [d-Trp(6)]-GnRH binding, with a K(d) value similar to those of both the rat and human forms, but with a greatly reduced B(max) value. The ED(50) for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but the maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser(140)Phe rat GnRH-R mutant was generated. The mutant had a K(d) value similar to that of the wild-type rat receptor, although the B(max) value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED(50) value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser(140)Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events.

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.

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.

scholarly journals Functional rescue of a constitutively desensitized β2AR through receptor dimerization

1998 ◽  
Vol 330 (1) ◽  
pp. 287-293 ◽  
Author(s):  
E. Terence HEBERT ◽  
P. Thomas LOISEL ◽  
Lynda ADAM ◽  
Nathalie ETHIER ◽  
ST. Stephane ONGE ◽  
...  
Keyword(s):  
Adrenergic Receptors ◽  
Prolonged Exposure ◽  
Wild Type ◽  
Receptor Dimerization ◽  
Metabolic Labelling ◽  
Functional Consequences ◽  
Receptor Mutant ◽  
Physical Interactions ◽  
Type Receptor ◽  
Positive Effect

We have recently demonstrated that wild-type β2-adrenergic receptors (β2AR) form homodimers and that disruption of receptor dimerization inhibits signalling via Gs [Hebert, Moffett, Morello, Loisel, Bichet, Barret and Bouvier (1996) J. Biol. Chem. 271, 16384-16392]. Here taking advantage of the altered functional properties of a non-palmitoylated, constitutively desensitized mutant β2AR (C341Gβ2AR), we sought to study whether physical interactions between mutant and wild-type β2AR expressed in Sf9 cells could occur and have functional consequences. Using metabolic labelling with [3H]palmitate and co-immunoprecipitation we demonstrated the existence of heterodimerization between wild-type and C341Gβ2AR. Furthermore, we show that, in co-expression experiments, wild-type receptors have a dominant positive effect resulting in the functional complementation of C341Gβ2AR. Indeed, when expressed alone, the mutant C341G receptor displays altered functional characteristics in that (1) the response of the receptor to agonist is reduced as compared to the wild-type receptor and (2) the desensitization of the receptor in response to prolonged exposure to agonist is minimal. In contrast, when C341G and the wild-type β2AR were expressed together, both the response to agonist and subsequent desensitization (at a constant level of total receptor) were equivalent to the wild-type β2AR expressed alone. This dominant positive effect was also seen when C341G was co-expressed with a second receptor mutant in which the two protein kinase A phosphorylation sites (S261, 262, 345, 346A β2AR) were mutated. Taken together these data suggest that intermolecular interactions between receptors may have both functional and structural implications for G-protein-mediated signalling.

scholarly journals Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1

2007 ◽  
Vol 177 (5) ◽  
pp. 905-916 ◽  
Author(s):  
Breann L. Wolfe ◽  
Adriano Marchese ◽  
JoAnn Trejo
Keyword(s):  
G Protein ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Receptor Internalization ◽  
Wild Type ◽  
Endocytic Machinery ◽  
Protease Activated Receptor 1 ◽  
Clathrin Adaptor ◽  
G Protein Coupled ◽  
Type Receptor

Protease-activated receptor-1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization.

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