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© The Rockefeller University Press, 0021-9525/1998//859 $5.00
The Journal of Cell Biology, Volume 142, Number 3, , 1998 859-871

Regular Articles

Interactions of the Cytoplasmic Domain of P-Selectin with Clathrin-coated Pits Enhance Leukocyte Adhesion under Flow



Hendra Setiadi*, Gerald Sedgewick{ddagger}, Stanley L. Erlandsen{ddagger}, and Rodger P. McEver*

* Department of Medicine and Department of Biochemistry and Molecular Biology, and W.K. Warren Medical Research Institute, University of Oklahoma Health Sciences Center, and Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104; and {ddagger} Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, Minnesota 55455

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.

Key Words: endocytosis • selectins • adhesion • clathrin • leukocytes

Abbreviations used in this paper: HUVEC, human umbilical vein endothelial cells; PSGL-1, P-selectin glycoprotein ligand-1.

Address all correspondence to Rodger P. McEver, M.D., W.K. Warren Medical Research Institute, University of Oklahoma Health Sciences Center, 825 N.E. 13th Street, Oklahoma City, OK 73104. Tel.: 405-271-6480; Fax: 405-271-3137; E-mail: rodger-mcever{at}ouhsc.edu


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