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1 The aim of the present study was to investigate the cellular pathway involved in histaminestimulated internalization of the human H 1 -receptor in CHO-K1 cells expressing N-terminal myctagged H 1 -receptor (Myc-H 1 ) or N-terminal myc-C-terminal green fluorescent protein (Myc-GFP H 1 ) versions of the receptor. 2 Studies of 3 H-mepyramine binding and histamine-stimulated 3 H-inositol phosphate accumulation in these cells showed that the Myc-H 1 and Myc-GFP H 1 -receptors had identical pharmacology to the wild-type H 1 -receptor. 3 The Myc-H 1 -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 H 1 -receptor antagonist a-pirdonium. Similar data were obtained with the Myc-GFP H 1 -receptors. 4 Internalization of the Myc-GFP H 1 -receptor was maintained in the absence of extracellular calcium and was not inhibited by the CAM kinase II inhibitor KN-62 (10 mM). 5 Phorbol dibutyrate, an activator of protein kinase C, was also able to stimulate internalization of the H 1 -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 H 1 -receptor stimulated by histamine. 6 Hypertonic sucrose did not prevent histamine-induced internalization of the Myc-GFP H 1 -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 H 1 -receptor, but was without effect on the sequestration of transferrin. 7 The H 1 -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 H 1 cells with histamine, there was no evidence for internalization of caveolin-1 in parallel with the H 1 -receptor. 8 These data provide strong evidence that the H 1 -receptor is internalized via a clathrin-independent mechanism and most likely involves lipid rafts.

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