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Modeling and docking the endothelin G-protein-coupled receptor.

ABSTRACT: A model of the endothelin G-protein-coupled receptor (ET(A)) has been constructed using a segmented approach. The model was produced using a bovine rhodopsin model as a template for the seven transmembrane alpha-helices. The three cytoplasmic loop regions and the C-terminal region were modeled on NMR structures of corresponding segments from bovine rhodopsin. The three extracellular loops were modeled on homologous loop regions in other proteins of known structure. The N-terminal region was modeled as a three-helix domain based on its homology with a hydrolase protein. To test the model, the FTDOCK algorithm was used to predict the ligand-binding site for the crystal structure of human endothelin. The site of docking is consistent with mutational and biochemical data. The principal sites of interaction in the endothelin ligand all lie on one face of a helix that has been implicated by structure-activity relationship studies as being essential for binding. As further support for the model, attempts to dock bigET, an inactive precursor to endothelin that does not bind to the receptor, found no sites for tight binding. The model of the receptor-ligand complex produced forms a basis for rational drug design of agonists and antagonists for this G-protein-coupled receptor.

SUBMITTER: Orry AJ

PROVIDER: S-EPMC1301185 | biostudies-other | 2000 Dec

REPOSITORIES: biostudies-other

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Modeling and docking the endothelin G-protein-coupled receptor.

Orry A J AJ Wallace B A BA

Biophysical journal 20001201 6

A model of the endothelin G-protein-coupled receptor (ET(A)) has been constructed using a segmented approach. The model was produced using a bovine rhodopsin model as a template for the seven transmembrane alpha-helices. The three cytoplasmic loop regions and the C-terminal region were modeled on NMR structures of corresponding segments from bovine rhodopsin. The three extracellular loops were modeled on homologous loop regions in other proteins of known structure. The N-terminal region was mode ...[more]

PMID: 11106614

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Project description:Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCdelta1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca2+]i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ET(A)R) antagonist, BQ123. To characterize ET(A)R desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ET(A)R responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ET(A)R desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive (D110A,K220R)GRK2, (D110A,K220R)GRK3, (K215R)GRK5, or (K215R)GRK6 constructs. (D110A,K220R)GRK2 expression significantly attenuated ET(A)R desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ET(A)R desensitization. Finally, immunocyotchemical data showed that ET(A)R activation recruited endogenous GRK2 from cytoplasm to membrane.These studies identify GRK2 as a key regulator of ET(A)R responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

Dataset Information

Modeling and docking the endothelin G-protein-coupled receptor.

Publications

Modeling and docking the endothelin G-protein-coupled receptor.

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