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

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.

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A complex structure of arrestin-2 bound to a G protein-coupled receptor

10.1101/822957 ◽

2019 ◽

Author(s):

Wanchao Yin ◽

Zhihai Li ◽

Mingliang Jin ◽

Yu-Ling Yin ◽

Parker W. de Waal ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

G Protein ◽

Complex Structure ◽

Transmembrane Helix ◽

G Protein Coupled Receptors ◽

Dynamics Simulation ◽

Intracellular Loop ◽

Neurotensin Receptor 1 ◽

G Protein Coupled

AbstractArrestins comprise a family of signal regulators of G-protein-coupled receptors (GPCRs), which include arrestins 1 to 4. While arrestins 1 and 4 are visual arrestins dedicated to rhodopsin, arrestins 2 and 3 (Arr2 and Arr3) are β-arrestins known to regulate many nonvisual GPCRs. The dynamic and promiscuous coupling of Arr2 to nonvisual GPCRs has posed technical challenges to tackle the basis of arrestin binding to GPCRs. Here we report the structure of Arr2 in complex with neurotensin receptor 1 (NTSR1), which reveals an overall assembly that is strikingly different from the visual arrestin-rhodopsin complex by a 90° rotation of Arr2 relative to the receptor. In this new configuration, intracellular loop 3 (ICL3) and transmembrane helix 6 (TM6) of the receptor are oriented toward the N-terminal domain of the arrestin, making it possible for GPCRs that lack the C-terminal tail to couple Arr2 through their ICL3. Molecular dynamics simulation and crosslinking data further support the assembly of the Arr2–NTSR1 complex. Sequence analysis and homology modeling suggest that the Arr2–NTSR1 complex structure may provide an alternative template for modeling arrestin-GPCR interactions.

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Faculty Opinions recommendation of Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1091302.545729 ◽

2007 ◽

Author(s):

Arthur Christopoulos

Keyword(s):

Small Molecules ◽

G Protein ◽

Transmembrane Helix ◽

Receptor Activation ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Allosteric Small Molecules Unveil a Role of an Extracellular E2/Transmembrane Helix 7 Junction for G Protein-coupled Receptor Activation

Journal of Biological Chemistry ◽

10.1074/jbc.m705563200 ◽

2007 ◽

Vol 282 (48) ◽

pp. 34968-34976 ◽

Cited By ~ 35

Author(s):

Dorothea Jäger ◽

Caroline Schmalenbach ◽

Stefanie Prilla ◽

Jasmin Schrobang ◽

Anna Kebig ◽

...

Keyword(s):

Small Molecules ◽

G Protein ◽

Transmembrane Helix ◽

Receptor Activation ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

International Journal of Molecular Sciences ◽

10.3390/ijms21113932 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3932 ◽

Cited By ~ 2

Author(s):

Preeti Kumari Chaudhary ◽

Sanggu Kim ◽

Youngheun Jee ◽

Seung-Hun Lee ◽

Kyung-Mee Park ◽

...

Keyword(s):

Platelet Aggregation ◽

G Protein ◽

Platelet Activation ◽

Functional Role ◽

Thrombus Formation ◽

Receptor Activation ◽

G Protein Coupled ◽

Gpcr Desensitization ◽

Stimulation Of

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

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Molecular dynamics of a biophysical model for β2-adrenergic and G protein-coupled receptor activation

Journal of Molecular Graphics and Modelling ◽

10.1016/j.jmgm.2006.02.008 ◽

2006 ◽

Vol 25 (4) ◽

pp. 396-409 ◽

Cited By ~ 22

Author(s):

Lester A. Rubenstein ◽

Randy J. Zauhar ◽

Richard G. Lanzara

Keyword(s):

Molecular Dynamics ◽

G Protein ◽

Receptor Activation ◽

Biophysical Model ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Molecular mechanisms of GABAB receptor activation: new insights from the mechanism of action of CGP7930, a positive allosteric modulator

Biochemical Society Transactions ◽

10.1042/bst0320871 ◽

2004 ◽

Vol 32 (5) ◽

pp. 871-872 ◽

Cited By ~ 15

Author(s):

V. Binet ◽

C. Goudet ◽

C. Brajon ◽

L. Le Corre ◽

F. Acher ◽

...

Keyword(s):

G Protein ◽

Mechanism Of Action ◽

Molecular Mechanisms ◽

Gabab Receptor ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Class Iii ◽

G Protein Coupled ◽

New Strategies

The GABAB (γ-aminobutyric acid-B) receptor is composed of two subunits, GABAB1 and GABAB2. Both subunits share structural homology with other class-III G-protein-coupled receptors. They contain two main domains, a heptahelical domain typical of all G-protein-coupled receptors and a large ECD (extracellular domain). It has not been demonstrated whether the association of these two subunits is always required for function. However, GABAB2 plays a major role in coupling with G-proteins, and GABAB1 has been shown to bind GABA. To date, only ligands interacting with GABAB1-ECD have been identified. In the present study, we explored the mechanism of action of CGP7930, a compound described as a positive allosteric regulator of the GABAB receptor. We have shown that it can weakly activate the wild-type GABAB receptor, but also the GABAB2 expressed alone, thus being the first described agonist of GABAB2. CGP7930 retains its weak agonist activity on a GABAB2 subunit deleted of its ECD. Thus the heptahelical domain of GABAB2 behaves similar to a rhodopsin-like receptor. These results open new strategies for studying the mechanism of activation of GABAB receptor and examine any possible role of GABAB2.

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Structural insights into ligand efficacy and activation of the glucagon receptor

10.1101/660837 ◽

2019 ◽

Cited By ~ 5

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.

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Dissecting the allosteric networks governing agonist efficacy and potency in G protein-coupled receptors

10.1101/2021.09.14.460253 ◽

2021 ◽

Author(s):

Franziska Marie Heydenreich ◽

Maria Marti-Solano ◽

Manbir Sandhu ◽

Brian K Kobilka ◽

Michel Bouvier ◽

...

Keyword(s):

G Protein ◽

Conformational Changes ◽

Structural Changes ◽

Receptor Activation ◽

Residue Level ◽

G Protein Coupled Receptors ◽

Maximal Response ◽

Pharmacological Properties ◽

Receptor Ligand ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular responses through conformational changes. Ligand properties are described by the maximum response (efficacy) and the agonist concentration at half-maximal response (potency). Integrating structural changes with pharmacological properties remains challenging and has not yet been performed at the resolution of individual amino acids. We use epinephrine and β2-adrenergic receptor as a model to integrate residue-level pharmacology data with intramolecular residue contact data describing receptor activation. This unveils the allosteric networks driving ligand efficacy and potency. We provide detailed insights into how structural rearrangements are linked to fundamental pharmacological properties at single-residue level in a receptor-ligand system. Our approach can be used to determine such pharmacological networks for any receptor-ligand complex.

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Role of the ERC motif in the proximal part of the second intracellular loop and the C-terminal domain of the human prostaglandin F2α receptor (hFP-R) in G-protein coupling control

Biochemical Journal ◽

10.1042/bj20041321 ◽

2005 ◽

Vol 388 (1) ◽

pp. 317-324 ◽

Cited By ~ 7

Author(s):

Andrea PATHE-NEUSCHÄFER-RUBE ◽

Frank NEUSCHÄFER-RUBE ◽

Gerhard P. PÜSCHEL

Keyword(s):

Focal Adhesion Kinase ◽

G Protein ◽

Focal Adhesion ◽

Inositol Trisphosphate ◽

Receptor Activation ◽

Intracellular Loop ◽

Prostanoid Receptors ◽

Constitutive Activation ◽

Terminal Domain

The human FP-R (F2α prostaglandin receptor) is a Gq-coupled heptahelical ectoreceptor, which is of significant medical interest, since it is a potential target for the treatment of glaucoma and preterm labour. On agonist exposure, it mediates an increase in intracellular inositol phosphate formation. Little is known about the structures that govern the agonist-dependent receptor activation. In other prostanoid receptors, the C-terminal domain has been inferred in the control of agonist-dependent receptor activation. A DRY motif at the beginning of the second intracellular loop is highly conserved throughout the G-protein-coupled receptor family and appears to be crucial for controlling agonist-dependent receptor activation. It is replaced by an ERC motif in the FP-R and no evidence for the relevance of this motif in ligand-dependent activation of prostanoid receptors has been provided so far. The aim of the present study was to elucidate the potential role of the C-terminal domain and the ERC motif in agonist-controlled intracellular signalling in FP-R mutants generated by site-directed mutagenesis. It was found that substitution of the acidic Glu132 in the ERC motif by a threonine residue led to full constitutive activation, whereas truncation of the receptor's C-terminal domain led to partial constitutive activation of all three intracellular signal pathways that had previously been shown to be activated by the FP-R, i.e. inositol trisphosphate formation, focal adhesion kinase activation and T-cell factor signalling. Inositol trisphosphate formation and focal adhesion kinase phosphorylation were further enhanced by ligand binding in cells expressing the truncation mutant but not the E132T (Glu132→Thr) mutant. Thus C-terminal truncation appeared to result in a receptor with partial constitutive activation, whereas substitution of Glu132 by threonine apparently resulted in a receptor with full constitutive activity.

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