Agonist-induced phosphorylation of the human corticotropin-releasing factor type 1 receptor (hCRF(1)-R) was investigated using an influenza hemagglutinin (HA) epitope-tagged receptor transiently expressed in COS-7 cells. The HA-hCRF(1)-R migrated as a broad band (M-r 60,000-70,000) in SDS-PAGE and showed increased mobility (M-r similar to 48,000) after enzymatic deglycosylation with peptide-N-glycosidase F, consistent with the predicted size (47 kDa) of the nonglycosylated HA-hCRF(1)-R protein. A marked increase in HA-hCRF(1)-R phosphorylation was observed in HA-hCRF,R-expressing COS-7 cells exposed to 1 mu M ovine CRF for 5 min, whereas activation of protein kinase A (PRA) by 50 mu M forskolin, or of Ca2+/calmodulin (CaM)dependent kinases by 10 mu M ionomycin, had little effect. These findings are consistent with preliminary data suggesting that CRF1-R phosphorylation mediated by G protein receptor kinase 3 (GRK3), but not by PKA or CaM-dependent kinases, has an important role in the homologous desensitization of brain CRF1-Rs,