title: Structural domains determining signalling characteristics of the CRH-receptor type 1 variant R1beta and response to PKC phosphorylation.
abstract: 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-R1alpha. Transcription of all 14 exons results in a CRH-R1 variant (CRH-R1beta) 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-R1beta properties, site directed mutagenesis was used to introduce a number of specific mutations into IC1 of CRH-R1beta as well as replace specific phospho-acceptor residues within the aminoacid sequence of CRH-R1alpha and CRH-R1beta. 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-R1beta, 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-R1alpha resistance, but not CRH-R1beta 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.
authors.affiliation: Endocrinology and Metabolism, Division of Clinical Sciences, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.
authors.forename: Thalia
authors.initials: T
authors.lastname: Teli
authors.forename: Danijela
authors.initials: D
authors.lastname: Markovic
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Hewitt
authors.forename: Michael A
authors.initials: MA
authors.lastname: Levine
authors.forename: Edward W
authors.initials: EW
authors.lastname: Hillhouse
authors.forename: Dimitris K
authors.initials: DK
authors.lastname: Grammatopoulos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000502:physiology; D012694:Serine / Q000378:metabolism; D012756:Sheep; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship; D000099052:CRF Receptor, Type 1
chemical_list: D020033:Protein Isoforms; D018019:Receptors, Corticotropin-Releasing Hormone; D043562:Receptors, G-Protein-Coupled; D012694:Serine; D000099052:CRF Receptor, Type 1; D011493:Protein Kinase C