Structural domains determining signalling characteristics of the CRH-receptor type 1 variant R1β and response to PKC phosphorylation

Mammalian adaptive mechanisms to stressful stimuli involve release of corticotropin-releasing hormone (CRH) and downstream activation of specific G-protein-coupled 7 transmembrane domain receptors. These CRH receptors (CRH-R) are expressed as multiple mRNA spliced variants. In contrast to other mammals, the human type 1 CRH-R gene contains an additional exon (exon 6) that needs to be spliced out in order to generate the fully active CRH-R1α. Transcription of all 14 exons results in a CRH-R1 variant (CRH-R1β) with an extended 1st intracellular loop (IC1); this sequence modification impairs signalling activity and alters receptor responsiveness to PKC-induced phosphorylation that leads to signalling desensitization and receptor endocytosis. To elucidate structure–function relationships and delineate sequences involved in CRH-R1β properties, site directed mutagenesis was used to introduce a number of specific mutations into IC1 of CRH-R1β as well as replace specific phospho-acceptor residues within the aminoacid sequence of CRH-R1α and CRH-R1β. Mutant receptors were transiently expressed in human embryonic kidney (HEK293) cells and tested for their abilities to increase intracellular cAMP and their response to PKC-induced phosphorylation. Results identified a penta-aminoacid cassette within the 29-aminoacid insert of CRH-R1β, which contains multiple positive charged aminoacids (F170-R174), as an important structural determinant for the impaired cAMP response. Furthermore, serine at position 408 in the carboxy-terminus appears to be important for mediating CRH-R1α resistance, but not CRH-R1β susceptibility, to PKC-induced desensitization and internalization. These findings provide new insights about the structural determinants of CRH-R1 coupling to Gs proteins and response to protein kinase phosphorylation.