Clathrin-independent internalization of the human histamine H1-receptor in CHO-K1 cells

Br J Pharmacol. 2005 Oct;146(4):612-24. doi: 10.1038/sj.bjp.0706337.


The aim of the present study was to investigate the cellular pathway involved in histamine-stimulated internalization of the human H1-receptor in CHO-K1 cells expressing N-terminal myc-tagged H1-receptor (Myc-H1) or N-terminal myc-C-terminal green fluorescent protein (Myc-GFP H1) versions of the receptor. Studies of 3H-mepyramine binding and histamine-stimulated 3H-inositol phosphate accumulation in these cells showed that the Myc-H1 and Myc-GFP H1-receptors had identical pharmacology to the wild-type H1-receptor. The Myc-H1-receptor was rapidly internalized in CHO-K1 cells following stimulation with histamine (0.1 mM). This response occurred within 15 min, and could be prevented by the quaternary H1-receptor antagonist alpha-pirdonium. Similar data were obtained with the Myc-GFP H1-receptors. Internalization of the Myc-GFP H1-receptor was maintained in the absence of extracellular calcium and was not inhibited by the CAM kinase II inhibitor KN-62 (10 microM). Phorbol dibutyrate, an activator of protein kinase C, was also able to stimulate internalization of the H1-receptor. However, inhibition or downregulation of protein kinase C (which significantly modified histamine-stimulated inositol phosphate responses) was without effect on the internalization of the H1-receptor stimulated by histamine. Hypertonic sucrose did not prevent histamine-induced internalization of the Myc-GFP H1-receptor, but was able to attenuate internalization of transferrin via clathrin-mediated endocytosis in the same cells. In contrast, preincubation of cells with filipin or nystatin, which disrupts caveolae and lipid rafts, completely inhibited the histamine-induced internalization of the Myc-GFP H1-receptor, but was without effect on the sequestration of transferrin. The H1-receptor and cholera toxin subunit B were colocalized under resting conditions at the cell surface. Immunohistochemical studies with an antibody to caveolin-1 confirmed that this protein was also localized predominantly to the plasma membrane. However, following stimulation of CHO-Myc-GFP H1 cells with histamine, there was no evidence for internalization of caveolin-1 in parallel with the H1-receptor. These data provide strong evidence that the H1-receptor is internalized via a clathrin-independent mechanism and most likely involves lipid rafts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Clathrin / physiology
  • Coated Pits, Cell-Membrane / metabolism
  • Cricetinae
  • Cricetulus
  • Dose-Response Relationship, Drug
  • Filipin / pharmacology
  • Green Fluorescent Proteins
  • Histamine / pharmacology
  • Histamine H1 Antagonists
  • Humans
  • Membrane Microdomains / drug effects
  • Membrane Microdomains / metabolism*
  • Nystatin / pharmacology
  • Phorbol 12,13-Dibutyrate / pharmacology
  • Protein Transport / drug effects
  • Protein Transport / physiology*
  • Receptors, Histamine H1 / drug effects
  • Receptors, Histamine H1 / genetics
  • Receptors, Histamine H1 / metabolism*
  • Time Factors
  • Transfection


  • Clathrin
  • Histamine H1 Antagonists
  • Receptors, Histamine H1
  • Nystatin
  • Green Fluorescent Proteins
  • Phorbol 12,13-Dibutyrate
  • Histamine
  • Filipin