Clathrin-independent internalization of the human histamine H 1-receptor in CHO-K1 cells

Abstract

1. The aim of the present study was to investigate the cellular pathway involved in histamine-stimulated internalization of the human H 1-receptor in CHO-K1 cells expressing N-terminal myc-tagged 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 3H-mepyramine binding and histamine-stimulated 3H-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 α-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 μM). 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. © 2005 Nature Publishing Group All rights reserved.