title: Differential responses of corticotropin-releasing hormone receptor type 1 variants to protein kinase C phosphorylation.
abstract: Corticotropin-releasing hormone (CRH) regulates diverse biological functions in mammals, through activation of two types of specific G protein-coupled receptors that are expressed as multiple mRNA spliced variants. In most cells, the type 1alpha CRH receptor (CRH-R1alpha) preferentially activates the G(s)-adenylyl cyclase signaling cascade. CRH-R1alpha-mediated signaling activity is impaired by insertion of 29 amino acids in the first intracellular loop, a sequence modification that is characteristic of the human-specific CRH-R1beta variant. In various tissues, CRH signaling events are regulated by protein kinase C (PKC). The CRH receptors contain multiple putative PKC phosphorylation sites that represent potential targets. To investigate this, we expressed recombinant CRH-R1alpha or CRH-R1beta in human embryonic kidney 293 cells and analyzed signaling events after PKC activation. Agonist (oxytocin) or phorbol 12-myristate 13-acetate-induced activation of PKC led to phosphorylation of both CRH-R1 variants. However, CRH-R1alpha and CRH-R1beta exhibited different functional responses to PKC-induced phosphorylation, with only the CRH-R1beta susceptible to cAMP signaling desensitization. This was associated with a significant decrease of accessible CRH-R1beta receptors expressed on the cell surface. Both CRH-R1 variants were susceptible to homologous desensitization and internalization following treatment with CRH; however, PKC activation increased internalization of CRH-R1beta but not CRH-R1alpha in a beta-arrestin-independent manner. Our findings indicate that CRH-R1alpha and -R1beta exhibit differential responses to PKC-induced phosphorylation, and this might represent an important mechanism for functional regulation of CRH signaling in target cells.
authors.affiliation: Endocrinology and Metabolism, Division of Clinical Sciences, Warwick Medical School, University of Warwick, Coventry, UK.
authors.forename: Danijela
authors.initials: D
authors.lastname: Markovic
authors.forename: Nikolleta
authors.initials: N
authors.lastname: Papadopoulou
authors.forename: Thalia
authors.initials: T
authors.lastname: Teli
authors.forename: Harpal
authors.initials: H
authors.lastname: Randeva
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: D000818:Animals; D019393:Arrestin / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D000099052:CRF Receptor, Type 1
chemical_list: D019393:Arrestin; D012333:RNA, Messenger; D018019:Receptors, Corticotropin-Releasing Hormone; D005576:Colforsin; D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D011493:Protein Kinase C; D019205:GTP-Binding Protein alpha Subunits, Gs; D013755:Tetradecanoylphorbol Acetate