Clathrin-independent internalization of the human histamine H1-receptor in CHO-K1 cells.
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.
Self
Timothy J
TJ
Institute of Cell Signalling, Medical School, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH.
Oakley
Sarah M
SM
Hill
Stephen J
SJ
D000818:Animals; D016466:CHO Cells; D002966:Clathrin / Q000502:physiology; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D005372:Filipin / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006632:Histamine / Q000494:pharmacology; D006634:Histamine H1 Antagonists; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D009761:Nystatin / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D011969:Receptors, Histamine H1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D002966:Clathrin; D006634:Histamine H1 Antagonists; D011969:Receptors, Histamine H1; D009761:Nystatin; D049452:Green Fluorescent Proteins; D015240:Phorbol 12,13-Dibutyrate; D006632:Histamine; D005372:Filipin