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Submitted on July 7, 2004
Accepted on October 15, 2004
signalling: structural determinants for G protein-coupled receptor kinase-mediated phosphorylation and agonist-mediated desensitization
Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK, Division of Paediatrics, The Children's Hospital of The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA, The Leeds Institute of Health, Genetics and Therapeutics, University of Leeds, Leeds, LS2 9NL, UK
* To whom correspondence should be addressed. E-mail: d.grammatopoulos{at}warwick.ac.uk.
Attenuation of corticotropin-releasing hormone 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 over-expressed in HEK293 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 
40 min of pretreatment and full recovery of CRH-R1 functional response within 2 h of removal of CRH pretreatment. In HEK293 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 
-arrestin (319-418), and performed co-immunoprecipitation and GRK translocation studies. We found that GRK3 and GRK6 are the main isoforms that interact with CRH-R1, and that recruitment of GRK3 requires G
-subunits as well as -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 co-ordinated action of multiple GRK isoforms, G dimers and -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.
-arrestin •
phosphorylation
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