The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-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 alpha-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 alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-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 alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha-actinin binds directly to L-selectin and that vinculin associates by binding to alpha-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 alpha-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 alpha-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.
Pavalko
F M
FM
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202-5120, USA.
Walker
D M
DM
Graham
L
L
Goheen
M
M
Doerschuk
C M
CM
Kansas
G S
GS
D000185:Actinin / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism / Q000648:ultrastructure; D015815:Cell Adhesion Molecules / Q000302:isolation & purification / Q000378:metabolism*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D006801:Humans; D019041:L-Selectin; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D010446:Peptide Fragments; D000185:Actinin; D019041:L-Selectin