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) internalization in Chinese hamster ovary cells using two assays; flow cytometric quantification of cell surface myc-ETA and in situ localization of Cy5-labeled ET-1. In both assays, agonist-dependent internalization of myc-ETA was inhibited by nystatin and filipin, both of which disrupt internalization via caveolae, whereas it was barely affected by chlorpromazine and hypertonic sucrose, 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 chlorpromazine. These results strongly argue that ETA is internalized via caveolae but not clathrin-coated pits. Treatment of the cells with cholesterol oxidase reduced cellular cholesterol and caused intracellular translocation of caveolin-1 but did not affect cell surface localization of myc-ETA. In cholesterol oxidase-treated cells, however, both chlorpromazine and hypertonic sucrose effectively blocked ET-1-induced myc-ETA internalization and nystatin was less effective than in untreated cells. Accordingly, expression of a dominant negative form of beta-arrestin blocked myc-ETA internalization in cholesterol oxidase-treated cells but not in untreated cells. These results suggest that, in Chinese hamster ovary cells, 1) agonist-occupied ETA can be internalized 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.
Okamoto
Y
Y
Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.
Ninomiya
H
H
Miwa
S
S
Masaki
T
T
D000818:Animals; D016466:CHO Cells; D002232:Carbocyanines / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins; D002746:Chlorpromazine / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism*; D002789:Cholesterol Oxidase / Q000494:pharmacology; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D006224:Cricetinae; D004705:Endocytosis; D019332:Endothelin-1 / Q000494:pharmacology; D005372:Filipin / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009761:Nystatin / Q000494:pharmacology; D010084:Oxidation-Reduction; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000378:metabolism*
D002232:Carbocyanines; D051242:Caveolin 1; D022461:Caveolins; D019332:Endothelin-1; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin; C085321:cyanine dye 5; D009761:Nystatin; D005372:Filipin; D002784:Cholesterol; D002789:Cholesterol Oxidase; D002746:Chlorpromazine