Self Timothy J, Oakley Sarah M, Hill Stephen J
Institute of Cell Signalling, Medical School, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH.
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.
本研究的目的是探究在表达N端带有myc标签的H1受体（Myc-H1）或N端myc-C端绿色荧光蛋白（Myc-GFP H1）的CHO-K1细胞中，组胺刺激人H1受体内化所涉及的细胞途径。对这些细胞中3H-美吡拉敏结合以及组胺刺激的3H-肌醇磷酸积累的研究表明，Myc-H1和Myc-GFP H1受体与野生型H1受体具有相同的药理学特性。组胺（0.1 mM）刺激后，Myc-H1受体在CHO-K1细胞中迅速内化。这种反应在15分钟内发生，并且可被季铵类H1受体拮抗剂α-匹多溴铵阻止。Myc-GFP H1受体也获得了类似的数据。在无细胞外钙的情况下，Myc-GFP H1受体的内化得以维持，并且不受钙调蛋白激酶II抑制剂KN-62（10 μM）的抑制。蛋白激酶C的激活剂佛波酯二丁酯也能够刺激H1受体的内化。然而，蛋白激酶C的抑制或下调（这显著改变了组胺刺激的肌醇磷酸反应）对组胺刺激的H1受体内化没有影响。高渗蔗糖不能阻止组胺诱导的Myc-GFP H1受体内化，但能够减弱同一细胞中通过网格蛋白介导的内吞作用对转铁蛋白的内化。相反，用制霉菌素或菲律宾菌素对细胞进行预孵育，它们会破坏小窝和脂筏，完全抑制组胺诱导的Myc-GFP H1受体内化，但对转铁蛋白的摄取没有影响。在静息条件下，H1受体和霍乱毒素B亚基在细胞表面共定位。用抗小窝蛋白-1抗体进行的免疫组织化学研究证实，该蛋白也主要定位于质膜。然而，用组胺刺激CHO-Myc-GFP H1细胞后，没有证据表明小窝蛋白-1与H1受体同时内化。这些数据提供了强有力的证据，表明H1受体通过非网格蛋白依赖机制内化，并且很可能涉及脂筏。
