title: Dimerization and phosphorylation of thyrotropin-releasing hormone receptors are modulated by agonist stimulation.
abstract: Dimerization and phosphorylation of thyrotropin-releasing hormone (TRH) receptors was characterized using HEK293 and pituitary GHFT cells expressing epitope-tagged receptors. TRH receptors tagged with FLAG and hemagglutinin epitopes were co-precipitated only if they were co-expressed, and 10-30% of receptors were isolated as hemagglutinin/FLAG-receptor dimers under basal conditions. The abundance of receptor dimers was increased when cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a dose-dependent manner. TRH increased phosphorylation of both receptor monomers and dimers, documented by incorporation of (32)P and an upshift in receptor mobility reversed by phosphatase treatment. The ability of TRH to increase receptor phosphorylation and dimerization did not depend on signal transduction, because it was not inhibited by the phospholipase C inhibitor. Receptor phosphorylation required an agonist but was not blocked by the casein kinase II inhibitor apigenin, the protein kinase C inhibitor GF109203X, or expression of a dominant negative form of G protein-coupled receptor kinase 2. TRH receptors lacking most of the cytoplasmic carboxyl terminus formed dimers constitutively but failed to undergo agonist-induced dimerization and phosphorylation. TRH also increased phosphorylation and dimerization of TRH receptors expressed in GHFT pre-lactotroph cells.
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: Chang-Cheng
authors.initials: CC
authors.lastname: Zhu
authors.forename: Laurie B
authors.initials: LB
authors.lastname: Cook
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Hinkle
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D047310:Apigenin; D002462:Cell Membrane / Q000502:physiology; D019281:Dimerization; D004963:Estrenes / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D008954:Models, Biological; D009842:Oligopeptides; D010455:Peptides; D010766:Phosphorylation; D010902:Pituitary Gland / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000819:agonists* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
chemical_list: D000970:Antineoplastic Agents; D004963:Estrenes; D005419:Flavonoids; D009842:Oligopeptides; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D011760:Pyrrolidinones; D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D047310:Apigenin; C085706:FLAG peptide; D019204:GTP-Binding Proteins