The Cytoplasmic Domain of L-Selectin Interacts with Cytoskeletal Proteins Via ct-Actinin: Receptor Positioning in Microvilli Does Not Require Interaction with et-Actinin

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L-selectin interacts directly with the cytoplasmic actin-binding protein ot-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified ot-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of ot-actinin to the L-selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of ot-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including ct-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that ot-actinin binds directly to L-selectin and that vinculin associates by binding to ot-actinin in vivo to link actin filaments to the L-selectin cytoplasmic domain. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with ot-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of ct-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L-selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L-selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton. YMPHOCYTE migration through lymphoid organs and leukocyte traffic into sites of inflammation are related processes that are both regulated principally at the level of leukocyte interactions with the lumenal surface of postcapillary venules. A number of leukocyte and endothelial cell surface molecules that participate in this interaction have been identified, including members of at least three Please address all correspondence to E M. Pavalko; Department of Physiology and Biophysics, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202-5120. Tel.: (317) 274-3140. Fax: (317) 274-3318. gene families: the integrins, the immunoglobulin (Ig) superfamily, and the selectins (for reviews see Springer, 1994; Butcher, 1991; Arnaout, 1993). Selectins mediate the initial phase of leukocyte recognition of endothelium, which usually takes the form of rolling along the vessel wall at velocities much slower than freely flowing blood elements (Lawrence and Springer, 1991; Bevilacqua and Nelson, 1993). In contrast, integrins mediate the subsequent firm arrest of the leukocytes to the endotheliurn, spreading on the endothelial surface, and transendothelial migration into the tissues (Smith, 1992). While the integrin and Ig families consists of many members and are broadly expressed by numerous cell types, there are only three known selectins, each of which © The Rockefeller University Press, 0021-9525/95/05/1155/10 $2.130 The Journal of Cell Biology, Volume 129, Number 4, May 1995 1155-I 164 1155 on Jne 7, 2017 D ow nladed fom Published May 15, 1995