Regulation of corticotropin-releasing hormone receptor type 1alpha signaling: structural determinants for G protein-coupled receptor kinase-mediated phosphorylation and agonist-mediated desensitization.
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-R1alpha 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-R1alpha functional response within 2 h of removal of CRH pretreatment. In HEK 293 cells, desensitization of CRH-R1alpha was associated with receptor phosphorylation and subsequent endocytosis. To analyze the mechanism leading to CRH-R1alpha desensitization, we overexpressed a truncated beta-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-R1alpha, and that recruitment of GRK3 requires Gbetagamma-subunits as well as beta-arrestin. Site-directed mutagenesis of Ser and Thr residues in the CRH-R1alpha C terminus, identified Thr399 as important for GRK-induced receptor phosphorylation and desensitization.We conclude that homologous desensitization of CRH-R1alpha involves the coordinated action of multiple GRK isoforms, Gbeta gamma dimers and beta-arrestin. Based on our identification of key amino acid(s) for GRK-dependent phosphorylation, we demonstrate the importance of the CRH-R1alpha carboxyl tail for regulation of receptor activity.
Teli
Thalia
T
Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom.
Markovic
Danijela
D
Levine
Michael A
MA
Hillhouse
Edward W
EW
Grammatopoulos
Dimitris K
DK
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005260:Female; D006493:Heparin / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D032342:Muscle Cells / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D012756:Sheep; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D000071557:beta-Arrestins; D000099052:CRF Receptor, Type 1
D019390:Arrestins; D020033:Protein Isoforms; D018019:Receptors, Corticotropin-Releasing Hormone; D000071557:beta-Arrestins; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D000099052:CRF Receptor, Type 1; D006493:Heparin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases