Identification of Amino Acid Residues Responsible for Agonist-Induced Down-Regulation of Histamine H1 Receptors

  • Horio Shuhei
    Department of Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokushima
  • Ogawa Maki
    Department of Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokushima
  • Kawakami Nozomi
    Department of Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokushima
  • Fujimoto Katsumi
    Department of Dental and Medical Biochemistry, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University
  • Fukui Hiroyuki
    Department of Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokushima

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The histamine H1 receptor (H1R) level is dynamically regulated in vivo under various physiological and pathological conditions. The H1R regulation may consist of various processes, and this study focused on the process of receptor trafficking, that is, receptor internalization to endosomes and the following receptor degradation. First, we identified five possible phosphorylation residues of human H1R, Thr140, Thr142, Ser396, Ser398, and Thr478, based on in vitro phosphorylation studies. Then to determine the role of these residues, we constructed a mutant H1R in which all of these five residues were substituted with alanine. Both wild-type and the mutant receptors expressed in Chinese hamster ovary (CHO) cells had similar values of Kd for [3H]mepyramine binding and Ki for histamine, and these cells showed similar levels of histamine-stimulated inositol phosphate formation. Both types of H1Rs were internalized essentially in the same way upon stimulation with histamine (100 μM) for 30 min. However, down-regulation of the mutant H1R was completely impaired, whereas that of wild-type H1R occurred by approximately 60% by the treatment with 100 μM histamine for 24 h. These results suggest that these residues are responsible for receptor down-regulation but not for receptor internalization. Possibly, phosphorylation of the residues is required for receptor transport from endosomes to lysosomes.<br>

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