title: Regulation of gonadotropin-releasing hormone receptors by protein kinase C: inside out signalling and evidence for multiple active conformations.
abstract: Desensitization and internalization of G protein-coupled receptors can be mediated by phosphorylation within the C-terminal tail, facilitating beta-arrestin binding and targeting the receptor for internalization. Type II GnRH receptors (GnRH-Rs) show such regulation, but type I GnRH-Rs lack C-tails and are not rapidly desensitized or internalized. Here we show contrasting susceptibility of type I (human and sheep) and II (Xenopus) GnRH-Rs to regulation by protein kinase C (PKC). When human (h) or Xenopus (X) GnRH-Rs were expressed using recombinant adenovirus, PKC activation increased radioligand binding to XGnRH-Rs but not to hGnRH-Rs. A dominant-negative dynamin mutant (K44A) inhibited internalization of XGnRH-Rs (but not hGnRH-Rs) without influencing PKC regulation of XGnRH-R binding. PKC activation increased the affinity of XGnRH-Rs for the type II GnRH ligand and increased effects of low concentrations of GnRH-II on the [Ca(2+)](i) but had no effect on type I ligand binding to hGnRH-Rs, sGnRH-Rs or XGnRH-Rs, or to chimeric receptors with the XGnRH-R C-tail added to a type I receptor. Binding of type II ligand to human or sheep receptors was also unaffected but was increased in the chimeras. Mutation of both PKC-phosphorylation consensus sites in the XGnRH-R tail did not prevent the PKC-mediated increases in binding or alter agonist-induced translocation of beta-arrestin2/green fluorescent protein or inhibition of inositol phosphate accumulation by beta-arrestin2/green fluorescent protein. Thus, it appears that there are two distinct active conformations of XGnRH-Rs (differing in affinity for type I and II ligands) and that these cells exhibit a novel form of inside-out signaling in which PKC feeds back to influence receptor affinity.
authors.affiliation: Laboratory for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol BS1 3NY, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Caunt
authors.forename: James N
authors.initials: JN
authors.lastname: Hislop
authors.forename: Eamonn
authors.initials: E
authors.lastname: Kelly
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Matharu
authors.forename: Lisa D
authors.initials: LD
authors.lastname: Green
authors.forename: Kathleen R
authors.initials: KR
authors.lastname: Sedgley
authors.forename: Ann R
authors.initials: AR
authors.lastname: Finch
authors.forename: Craig A
authors.initials: CA
authors.lastname: McArdle
mesh_terms: D001665:Binding Sites; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000502:physiology*; D011966:Receptors, LHRH / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate