Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix

L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial ven ules (HEV). This study examines the biochemical basis and regulation of int eractions between L-selectin, an integral transmembrane protein, and the ly mphocyte cytoskeleton, Using a detergent-based extraction procedure, consti tutive associations between L-selectin and the insoluble cytoskeletal matri x could not be detected. However, engagement of the L-selectin lectin domai n by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-der ived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytos keleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosi ne kinase activity (staurosporine, calphostin C, or genistein), although L- selectin-mediated adhesion of human PBL was markedly suppressed by these ag ents, Exposure of human PBL or murine pre-B transfectants expressing full-l ength human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enh anced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-sel ectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimul ation and did not support adhesion to HEV. These studies, when taken togeth er with the previously demonstrated interaction between the L-selectin cyto plasmic domain and the cytoskeletal linker protein cu-actinin, strongly imp licate the actin-based cytoskeleton in dynamically controlling L-selectin a dhesion.