Interactions of the cytoplasmic domain of P-selectin with clathrin-coated pits enhance leukocyte adhesion under flow.
Flowing leukocytes tether to and roll on P-selectin, a receptor on endothelial cells that is rapidly internalized in clathrin-coated pits. We asked whether the association of P-selectin with clathrin-coated pits contributes to its adhesive function. Under flow, rolling neutrophils accumulated efficiently on CHO cells expressing wild-type P-selectin or a P-selectin construct with a substitution in the cytoplasmic domain that caused even faster internalization than that of the wild-type protein. By contrast, far fewer rolling neutrophils accumulated on CHO cells expressing P-selectin constructs with a deletion or a substitution in the cytoplasmic domain that impaired internalization. Neutrophils rolled on the internalization-competent constructs with greater adhesive strength, slower velocity, and more uniform motion. Flowing neutrophils tethered equivalently to internalization-competent or internalization-defective P-selectin, but after tethering, they rolled further on internalization-competent P-selectin. Confocal microscopy demonstrated colocalization of alpha-adaptin, a component of clathrin-coated pits, with wild-type P-selectin, but not with P-selectin lacking the cytoplasmic domain. Treatment of CHO cells or endothelial cells with hypertonic medium reversibly impaired the clathrin-mediated internalization of P-selectin and its ability to support neutrophil rolling. Interactions of the cytoplasmic domain of P-selectin with clathrin-coated pits provide a novel mechanism to enhance leukocyte adhesion under flow.
Department of Medicine, University of Oklahoma Health Sciences Center, and Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
H
H
Setiadi
G
G
Sedgewick
S L
SL
Erlandsen
R P
RP
McEver
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D002478:Cells, Cultured; D002966:Clathrin / Q000502:physiology*; D003034:Coated Pits, Cell-Membrane / Q000502:physiology*; D006224:Cricetinae; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000502:physiology; D009504:Neutrophils / Q000502:physiology*; D019007:P-Selectin / Q000235:genetics / Q000502:physiology*; D012212:Rheology; D014162:Transfection
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D008565:Membrane Proteins; D019007:P-Selectin