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C‐terminal of human histamine H 1 receptors regulates their agonist‐induced clathrin‐mediated internalization and G‐protein signaling
Author(s) -
Hishinuma Shigeru,
Nozawa Hiroki,
Akatsu Chizuru,
Shoji Masaru
Publication year - 2016
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.13834
Subject(s) - internalization , clathrin , microbiology and biotechnology , receptor , histamine , histamine h1 receptor , biology , histamine receptor , endocytosis , g protein coupled receptor , biochemistry , endocrinology , antagonist
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 G q/11 ‐protein‐coupled human histamine H 1 receptors internalized via clathrin‐dependent mechanisms upon stimulation with histamine. However, the molecular determinants of H 1 receptors responsible for agonist‐induced internalization remain unclear. In this study, we evaluated the roles of the intracellular C‐terminal of human histamine H 1 receptors 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 [ 3 H]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 H 1 receptors, but not by its mutation to alanine. In contrast, the receptor‐G‐protein coupling, which was evaluated by histamine‐induced accumulation of [ 3 H]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 H 1 receptors, which only comprises 17 amino acids (Cys471‐Ser487), plays crucial roles in both clathrin‐dependent internalization of H 1 receptors 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.