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.
Setiadi
H
H
Department of Medicine, University of Oklahoma Health Sciences Center, and Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
Sedgewick
G
G
Erlandsen
S L
SL
McEver
R P
RP
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