Protein kinase A-induced negative regulation of the corticotropin- releasing hormone R1α receptor-extracellularly regulated kinase signal transduction pathway: The critical role of Ser301 for signaling switch and selectivity

Activation of CRH receptors type 1 (CRH-R1) by CRH or urocortin (UCN) leads to stimulation of multiple G proteins with consequent effects on diverse signaling cascades in a tissue-specific manner. In human myometrium and human embryonic kidney (HEK)293 cells, binding of UCN to CRH-R1α receptors activates both the Gs and Gq, leading to activation of the adenylyl cyclase/protein kinase A (PKA) and the phospholipase C/protein kinase C and ERK1/2 signaling pathways, respectively. The overall result of these signals is often unpredictable, as these two signaling pathways can interact in many cellular systems, with either potentiation or inhibition of ERK1/2 activity. In the present studies we investigated potential signaling interactions after stimulation of CRH-R1α receptors in human cultured pregnant myometrial cells or HEK293 cells overexpressing recombinant CRH-R1α receptors. We found that the adenylyl cyclase/PKA pathway has the capacity to markedly decrease UCN-induced ERK1/2 activation, and that these effects were due in part to the ability of PKA to phosphorylate the CRH-R1α at position Ser ³⁰¹ in the third intracellular loop. Mutant CRH-R1α receptors with substitutions at position Ser³⁰¹, which is the only potential PKA phosphorylation site, were resistant to PKA-dependent phosphorylation and showed altered signaling characteristics, which were dependent upon the amino acid substitution at this position. We conclude that Ser³⁰¹, which is located in the third intracellular loop of CRK-R1α, is critical for efficient coupling of the receptor to G proteins and to second messenger generation. Phosphorylation by PKA prevents maximal coupling of the CRH-R1α to Gq-protein, and thereby reduces activation of ERK 1/2. (Molecular Endocrinology 18: 624-639, 2004).