Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells

We investigated the mechanism of endothelin receptor type A (ETA) internali zation in Chinese hamster ovary cells using two assays; flow cytometric qua ntification of cell surface myc-ETA and in, situ localization of Cy5-labele d ET-1, In both assays, agonist-dependent internalization of myc-ETA was in hibited by nystatin and filipin, both of which disrupt internalization via caveolae, whereas it was barely affected by chlorpromazine and hypertonic s ucrose, both of which disrupt internalization via clathrin coated pits. In addition to myc-ETA, ET-1 caused intracellular translocation of caveolin-1 and this translocation was also blocked by nystatin but not by chlorpromazi ne, These results strongly argue that ETA is internalized via caveolae but not clathrin-coated pits. Treatment of the cells with cholesterol oxidase r educed cellular cholesterol and caused intracellular translocation of caveo lin-1 but did not affect cell surface localization of myc-ETA. In cholester ol oxidase-treated cells, however, both chlorpromazine and hypertonic sucro se effectively blocked ET-1-induced myc-ETA internalization and nystatin wa s less effective than in untreated cells. Accordingly, expression of a domi nant negative form of beta-arrestin blocked myc-ETA internalization in chol esterol oxidase-treated cells but not in untreated cells. These results sug gest that, in Chinese hamster ovary cells, 1) agonist-occupied ETA can be i nternalized either via caveolae or clathrin-coated pits; 2) of the two, the former is the default pathway; and 3) the oxidative state of cell surface cholesterol is one of the factors involved in the pathway selection.