Structural domains determining signalling characteristics of the CRH-receptor type 1 variant R1 beta 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-R1 alpha. Transcription of all 14 exons results in a CRH-R1 variant (CRH-R1 beta) 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-R1 beta properties, site directed mutagenesis was used to introduce a number of specific mutations into IC1 of CRH-R1 beta as well as replace specific phospho-acceptor residues within the aminoacid sequence of CRH-R1 alpha and CRH-R1 beta 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-R1 beta, which contains multiple positive charged aminoacids (F-170-R-174), as an important structural determinant for the impaired cAMP response. Furthermore, serine at position 408 in the carboxy-termimis appears to be important for mediating CRI-R1 alpha resistance, but not CRH-R1 beta 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. (C) 2007 Elsevier Inc. All rights reserved.