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Regulation of Corticotropin-Releasing Hormone Receptor Type 1 Signaling: Structural Determinants for G Protein-Coupled Receptor Kinase-Mediated Phosphorylation and Agonist-Mediated Desensitization

Sir Quinton Hazell Molecular Medicine Research Centre (T.T., D.M., D.K.G.), Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom; Division of Paediatrics (M.A.L.), The Children’s Hospital of The Cleveland Clinic Foundation, Cleveland, Ohio 44195; and The Leeds Institute of Health (E.W.H.), Genetics and Therapeutics, University of Leeds, Leeds LS2 9NL, United Kingdom

Address all correspondence and requests for reprints to: Dr. D.Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. E-mail: d.grammatopoulos{at}warwick.ac.uk.

Attenuation of CRH receptor type 1 (CRH-R1) signaling activity might involve desensitization and uncoupling of CRH-R1 from intracellular effectors. We investigated the desensitization of native CRH-R in human myometrial cells from pregnant women and recombinant CRH-R1 stably overexpressed in human embryonic kidney (HEK) 293 cells. In both cell types, CRH-R1-mediated adenylyl cyclase activation was susceptible to homologous desensitization induced by pretreatment with high concentrations of CRH. Time course studies showed half-maximal desensitization occurring after approximately 40 min of pretreatment and full recovery of CRH-R1 functional response within 2 h of removal of CRH pretreatment. In HEK 293 cells, desensitization of CRH-R1 was associated with receptor phosphorylation and subsequent endocytosis. To analyze the mechanism leading to CRH-R1 desensitization, we overexpressed a truncated ß-arrestin (319–418) and performed coimmunoprecipitation and G protein-coupled receptor kinase (GRK) translocation studies. We found that GRK3 and GRK6 are the main isoforms that interact with CRH-R1, and that recruitment of GRK3 requires Gß-subunits as well as ß-arrestin. Site-directed mutagenesis of Ser and Thr residues in the CRH-R1 C terminus, identified Thr³⁹⁹ as important for GRK-induced receptor phosphorylation and desensitization.

We conclude that homologous desensitization of CRH-R1 involves the coordinated action of multiple GRK isoforms, Gß dimers and ß-arrestin. Based on our identification of key amino acid(s) for GRK-dependent phosphorylation, we demonstrate the importance of the CRH-R1 carboxyl tail for regulation of receptor activity.

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