Structural domains determining signalling characteristics of the CRH-receptor type 1 variant R1beta and response to PKC phosphorylation. Export

@article{citeulike:5776539, 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.}, author = {Teli, Thalia and Markovic, Danijela and Hewitt, Margaret E. and Levine, Michael A. and Hillhouse, Edward W. and Grammatopoulos, Dimitris K.}, citeulike-article-id = {5776539}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/j.cellsig.2007.08.014}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/17913459}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=17913459}, doi = {10.1016/j.cellsig.2007.08.014}, issn = {0898-6568}, journal = {Cellular signalling}, keywords = {iliad}, month = {January}, number = {1}, pages = {40--49}, posted-at = {2009-09-11 18:23:28}, priority = {2}, title = {Structural domains determining signalling characteristics of the CRH-receptor type 1 variant R1beta and response to PKC phosphorylation.}, url = {http://dx.doi.org/10.1016/j.cellsig.2007.08.014}, volume = {20}, year = {2008} }

TY - JOUR ID - citeulike:5776539 L3 - citeulike-article-id:5776539 N2 - 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. IS - 1 JF - Cellular signalling SN - 0898-6568 EP - 49 TI - Structural domains determining signalling characteristics of the CRH-receptor type 1 variant R1beta and response to PKC phosphorylation. VL - 20 SP - 40 KW - iliad AU - Teli, Thalia AU - Markovic, Danijela AU - Hewitt, Margaret AU - Levine, Michael AU - Hillhouse, Edward AU - Grammatopoulos, Dimitris PY - 2008/01// UR - http://dx.doi.org/10.1016/j.cellsig.2007.08.014 ER -