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-R1α 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-R1α functional response within 2 h of removal of CRH pretreatment. In HEK 293 cells, desensitization of CRH-R1α was associated with receptor phosphorylation and subsequent endocytosis. To analyze the mechanism leading to CRH-R1α desensitization, we overexpressed a truncated β-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-R1α, and that recruitment of GRK3 requires Gβγ-subunits as well as β-arrestin. Site-directed mutagenesis of Ser and Thr residues in the CRH-R1α C terminus, identified Thr399 as important for GRK-induced receptor phosphorylation and desensitization. We conclude that homologous desensitization of CRH-R1α involves the coordinated action of multiple GRK isoforms, Gβ γ dimers and β-arrestin. Based on our identification of key amino acid(s) for GRK-dependent phosphorylation, we demonstrate the importance of the CRH-R1α carboxyl tail for regulation of receptor activity.
ASJC Scopus subject areas
- Molecular Biology
- Endocrinology, Diabetes and Metabolism