Studies on the Structure of the G-Protein-Coupled Receptor Rhodopsin Including the Putative G-Protein Binding Site in Unactivated and Activated Forms†

Congenital nephrogenic diabetes insipidus (CNDI) is a genetic disorder caused by mutations in arginine vasopressin receptor 2 (AVPR2) or aquaporin 2 genes, rendering collecting duct cells insensitive to the peptide hormone arginine vasopressin stimulation for water reabsorption. This study reports a first identified AVPR2 mutation in Taiwan and demonstrates our effort to understand the pathogenesis caused by applying computational structural analysis tools. The CNDI condition of an 8-month-old male patient was confirmed according to symptoms, family history, and DNA sequence analysis. The patient was identified to have a valine 279 deletion–mutation in the AVPR2 gene. Cellular experiments using mutant protein transfected cells revealed that mutated AVPR2 is expressed successfully in cells and localized on cell surfaces. We further analyzed the pathogenesis of the mutation at sub-molecular levels via long-term molecular dynamics (MD) simulations and structural analysis. The MD simulations showed while the structure of the extracellular ligand-binding domain remains unchanged, the mutation alters the direction of dynamic motion of AVPR2 transmembrane helix 6 toward the center of the G-protein binding site, obstructing the binding of G-protein, thus likely disabling downstream signaling. This study demonstrated that the computational approaches can be powerful tools for obtaining valuable information on the pathogenesis induced by mutations in G-protein-coupled receptors. These methods can also be helpful in providing clues on potential therapeutic strategies for CNDI.

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Mapping the Ligand-Binding Site on a G Protein-Coupled Receptor (GPCR) Using Genetically Encoded Photocrosslinkers

Biochemistry ◽

10.1021/bi200214r ◽

2011 ◽

Vol 50 (17) ◽

pp. 3411-3413 ◽

Cited By ~ 68

Author(s):

Amy Grunbeck ◽

Thomas Huber ◽

Pallavi Sachdev ◽

Thomas P. Sakmar

Keyword(s):

Ligand Binding ◽

Binding Site ◽

G Protein ◽

Ligand Binding Site ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Room 302, 10/16/2000 9: 00 AM - 10: 30 AM (PD) Direct Interaction of Halothane with a Ligand Binding Site in a G Protein Coupled Receptor

Anesthesiology ◽

10.1097/00000542-200009001-00122 ◽

2000 ◽

Vol 93 (3A) ◽

pp. A-122

Author(s):

Yumiko Ishizawa ◽

Paul A. Liebman ◽

Roderic G. Eckenhoff

Keyword(s):

Ligand Binding ◽

Binding Site ◽

G Protein ◽

Direct Interaction ◽

Ligand Binding Site ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Accelerated molecular dynamics simulations of ligand binding to a muscarinic G-protein-coupled receptor

Quarterly Reviews of Biophysics ◽

10.1017/s0033583515000153 ◽

2015 ◽

Vol 48 (4) ◽

pp. 479-487 ◽

Cited By ~ 83

Author(s):

Kalli Kappel ◽

Yinglong Miao ◽

J. Andrew McCammon

Keyword(s):

Molecular Dynamics ◽

Ligand Binding ◽

Binding Site ◽

G Protein ◽

Partial Agonist ◽

Md Simulations ◽

G Protein Coupled Receptor ◽

Accelerated Molecular Dynamics ◽

Dynamics Simulations ◽

G Protein Coupled

AbstractElucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein–ligand binding, especially the drug recognition of GPCRs.

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On the spatial disposition of the fifth transmembrane helix and the structural integrity of the transmembrane binding site in the opioid and ORL1 G protein-coupled receptor family

Protein Engineering Design and Selection ◽

10.1093/protein/13.7.477 ◽

2000 ◽

Vol 13 (7) ◽

pp. 477-490 ◽

Cited By ~ 2

Author(s):

Christopher M. Topham ◽

Lionel Moulédous ◽

Jean-Claude Meunier

Keyword(s):

Binding Site ◽

G Protein ◽

Structural Integrity ◽

Transmembrane Helix ◽

G Protein Coupled Receptor ◽

G Protein Coupled ◽

Receptor Family

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Characterization of the G protein-coupled receptor kinase 6 promoter reveals a functional CREB binding site

PLoS ONE ◽

10.1371/journal.pone.0247087 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0247087

Author(s):

Maike Stegen ◽

Andrea Engler ◽

Crista Ochsenfarth ◽

Iris Manthey ◽

Jürgen Peters ◽

...

Keyword(s):

Protein Expression ◽

Binding Site ◽

G Protein ◽

Promoter Activity ◽

Luciferase Reporter ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

Western Blots ◽

G Protein Coupled ◽

Creb Binding Site

Background G protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. GRK6 seems to play a role in regulation of inflammatory processes, but mechanisms of transcriptional regulation of GRK6 expression in inflammatory cell lines have not been characterized. Protein kinase C (PKC) signalling is also involved in inflammatory regulation and an impact of PKC activation on GRK6 protein expression was described previously. Thus, the aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression. Methods Five deletion constructs of the GRK6 promoter were cloned. After transient transfection into a human T cell line, promoter activity was assessed using luciferase reporter gene assays. Putative transcription factor binding sites were identified, mutated, and binding was investigated using electrophoretic mobility shift assays (EMSA). Following stimulation with a PKC activator, GRK6 expression on mRNA and protein levels was assessed by reverse transcriptase qPCR and Western blots. Results Investigation of the GRK6 promoter revealed a putative cAMP responsive element (CRE), whose mutation led to decreased promoter activity (p = 0.0006). Functionality of the CRE binding protein (CREB) binding site was verified in EMSA blots. Stimulation with a PKC activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression. Conclusion We characterized the human GRK6 promoter and identified promoter activity to be influenced by a CREB binding site. PKC might be one determinant contributing to altered GRK6 expression.

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