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

• Published in: The Journal of cell biology 1998-08, Vol.142 (3), p.859-871
• Main Authors: Setiadi, Hendra, Sedgewick, Gerald, Erlandsen, Stanley L., McEver, Rodger P.
• Format: Article
• Language: English
• Subjects: Adaptor Protein Complex alpha Subunits; Adaptor Proteins, Vesicular Transport; Adhesives; Animals; Cell adhesion; Cell Adhesion - physiology; Cells, Cultured; Cellular biology; CHO Cells; Clathrin - physiology; Coated Pits, Cell-Membrane - physiology; Cricetinae; Endocytosis; Endothelial cells; Endothelium, Vascular - physiology; Humans; Internalization; Leukocytes; Ligands; Membrane Proteins - physiology; Neutrophils; Neutrophils - physiology; P-Selectin - genetics; P-Selectin - physiology; Regular; Rheology; Selectins; Shear stress; Transfection
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.142.3.859

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 α-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.