title: Clustering endothelial E-selectin in clathrin-coated pits and lipid rafts enhances leukocyte adhesion under flow.
abstract: During inflammation, E-selectin expressed on cytokine-activated endothelial cells mediates leukocyte rolling under flow. E-selectin undergoes endocytosis and may associate with lipid rafts. We asked whether distribution of E-selectin in membrane domains affects its functions. E-selectin was internalized in transfected CHO cells or cytokine-activated human umbilical vein endothelial cells (HUVECs). Confocal microscopy demonstrated colocalization of E-selectin with alpha-adaptin, a clathrin-associated protein. Deleting the cytoplasmic domain of E-selectin or disrupting clathrin-coated pits with hypertonic medium blocked internalization of E-selectin, reduced colocalization of E-selectin with alpha-adaptin, and inhibited E-selectin-mediated neutrophil rolling under flow. Unlike CHO cells, HUVECs expressed a small percentage of E-selectin in lipid rafts. Even fewer neutrophils rolled on E-selectin in HUVECs treated with hypertonic medium and with methyl-beta-cyclodextrin, which disrupts lipid rafts. These data demonstrate that E-selectin clusters in both clathrin-coated pits and lipid rafts of endothelial cells but is internalized in clathrin-coated pits. Distribution in both domains markedly enhances E-selectin's ability to mediate leukocyte rolling under flow.
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
authors.forename: Hendra
authors.initials: H
authors.lastname: Setiadi
authors.forename: Rodger P
authors.initials: RP
authors.lastname: McEver
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D002478:Cells, Cultured; D022163:Clathrin-Coated Vesicles / Q000502:physiology*; D003034:Coated Pits, Cell-Membrane / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D019040:E-Selectin / Q000502:physiology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007249:Inflammation / Q000503:physiopathology; D007375:Interleukin-1 / Q000494:pharmacology; D007962:Leukocytes / Q000502:physiology*; D009504:Neutrophils / Q000502:physiology*; D014162:Transfection; D014471:Umbilical Veins
chemical_list: D019040:E-Selectin; D007375:Interleukin-1