title: C-terminal of human histamine H1receptors regulates their agonist-induced clathrin-mediated internalization and G-protein signaling.
abstract: It has been suggested that the agonist-induced internalization of G-protein-coupled receptors from the cell surface into intracellular compartments regulates cellular responsiveness. We previously reported that Gq/11-protein-coupled human histamine H1receptors internalized via clathrin-dependent mechanisms upon stimulation with histamine. However, the molecular determinants of H1receptors responsible for agonist-induced internalization remain unclear. In this study, we evaluated the roles of the intracellular C-terminal of human histamine H1receptors tagged with hemagglutinin (HA) at the N-terminal in histamine-induced internalization in Chinese hamster ovary cells. The histamine-induced internalization was evaluated by the receptor binding assay with [3H]mepyramine and confocal immunofluorescence microscopy with an anti-HA antibody. We found that histamine-induced internalization was inhibited under hypertonic conditions or by pitstop, a clathrin terminal domain inhibitor, but not by filipin or nystatin, disruptors of the caveolar structure and function. The histamine-induced internalization was also inhibited by truncation of a single amino acid, Ser487, located at the end of the intracellular C-terminal of H1receptors, but not by its mutation to alanine. In contrast, the receptor-G-protein coupling, which was evaluated by histamine-induced accumulation of [3H]inositol phosphates, was potentiated by truncation of Ser487, but was lost by its mutation to alanine. These results suggest that the intracellular C-terminal of human H1receptors, which only comprises 17 amino acids (Cys471-Ser487), plays crucial roles in both clathrin-dependent internalization of H1receptors and G-protein signaling, in which truncation of Ser487 and its mutation to alanine are revealed to result in biased signaling toward activation of G-proteins and clathrin-mediated internalization, respectively.
authors: Department of Pharmacodynamics, Meiji Pharmaceutical University, Tokyo, Japan.
authors: Shigeru
authors: S
authors: Hishinuma
authors: Department of Pharmacodynamics, Meiji Pharmaceutical University, Tokyo, Japan.
authors: Hiroki
authors: H
authors: Nozawa
authors: Department of Pharmacodynamics, Meiji Pharmaceutical University, Tokyo, Japan.
authors: Chizuru
authors: C
authors: Akatsu
authors: Department of Pharmacodynamics, Meiji Pharmaceutical University, Tokyo, Japan.
authors: Masaru
authors: M
authors: Shoji
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002966:Clathrin / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology; D017442:Histamine Agonists / Q000494:pharmacology*; D006634:Histamine H1 Antagonists / Q000494:pharmacology; D006801:Humans; D011969:Receptors, Histamine H1 / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D002966:Clathrin; D017442:Histamine Agonists; D006634:Histamine H1 Antagonists; D011969:Receptors, Histamine H1; D006632:Histamine; D043802:GTP-Binding Protein alpha Subunits, Gq-G11
keywords: C-terminal; biased signaling; clathrin; histamine H1 receptor; internalization; intracellular trafficking