Pavalko F M, Walker D M, Graham L, Goheen M, Doerschuk C M, Kansas G S
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202-5120, USA.
J Cell Biol. 1995 May;129(4):1155-64. doi: 10.1083/jcb.129.4.1155.
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.
白细胞黏附分子L-选择素介导与淋巴结高内皮微静脉（HEV）的结合，并有助于白细胞在炎症部位的内皮上滚动。先前的研究表明，L-选择素细胞质尾部截短11个氨基酸会消除体内与淋巴结HEV的结合以及白细胞滚动，但该观察结果的分子基础尚未确定。本研究检测了L-选择素与细胞骨架蛋白之间的潜在相互作用。我们发现L-选择素的细胞质结构域直接与细胞质肌动蛋白结合蛋白α-辅肌动蛋白相互作用，并与纽蛋白以及可能与踝蛋白形成复合物。使用与微量滴定板结合的全长L-选择素细胞质结构域进行的固相结合试验表明，纯化的α-辅肌动蛋白与L-选择素存在直接、特异性和可饱和的结合（解离常数Kd = 550 nM），但纯化的踝蛋白或纽蛋白无直接结合。有趣的是，尽管无法检测到踝蛋白与L-选择素的直接结合，但踝蛋白增强了α-辅肌动蛋白与L-选择素细胞质结构域肽的结合。只有在存在α-辅肌动蛋白的情况下，才能检测到纽蛋白与L-选择素细胞质结构域肽的结合。L-选择素与包括α-辅肌动蛋白和纽蛋白在内的细胞骨架蛋白复合物一起从转染了L-选择素的细胞中共沉淀，这与α-辅肌动蛋白直接结合L-选择素且纽蛋白在体内通过与α-辅肌动蛋白结合将肌动蛋白丝连接到L-选择素细胞质结构域的可能性一致。相反，缺乏细胞质结构域COOH末端11个氨基酸的L-选择素缺失突变体未能与α-辅肌动蛋白或纽蛋白共沉淀。令人惊讶的是，这种突变的L-选择素正常定位于细胞表面的微绒毛突起上。这些数据表明，L-选择素细胞质结构域的COOH末端11个氨基酸是通过α-辅肌动蛋白和纽蛋白复合物介导与肌动蛋白细胞骨架相互作用所必需的，但细胞质结构域的这一部分对于L-选择素在细胞表面的正确定位并非必需。因此，正确的L-选择素受体定位不足以介导L-选择素介导的白细胞黏附，这表明这种黏附可能还需要与细胞骨架的直接相互作用。
