Watanabe T, Inui M, Chen B Y, Iga M, Sobue K
Department of Neurochemistry and Neuropharmacology, Osaka University Medical School, Japan.
J Biol Chem. 1994 Jul 1;269(26):17656-62.
Identification of annexin VI-binding proteins is essential to elucidate the physiological functions of annexin VI. Here, we developed the methods to identify an annexin VI-binding protein and characterized the binding. Annexin VI bound to about 14 species of proteins in the whole homogenate of rat forebrain, when examined with 125I-annexin VI using blots of SDS-polyacrylamide gels. The binding was Ca(2+)-dependent and specific for phosphatidylserine (PS) and phosphatidic acid. A line of evidence indicates that the binding of annexin VI to its target proteins is a protein-protein interaction. One of annexin VI-binding proteins with M(r) 240,000 was enriched in the cytoskeletal fraction and was identified as calspectin (brain spectrin or fodrin). When the binding was examined with purified calspectin in the native state, the Ca2+ affinity (KCa) was 7.6 microM, and the affinity for annexin VI (Kd) was 68 nM. Annexin VI bound to beta subunit of calspectin, but not to alpha subunit. The binding site was localized to the NH2-terminal domain of beta subunit, which contains an actin-binding site and exhibits striking homology with the NH2-terminal regions of dystrophin and alpha-actinin. When the effect of annexin VI on the interaction between F-actin and calspectin was examined by low shear viscometry, annexin VI inhibited the F-actin cross-linking activity of calspectin in a Ca2+/PS-dependent manner. Cosedimentation assay showed that annexin VI dissociates calspectin from F-actin in the presence of Ca2+ and PS. These results suggest that annexin VI can dissociate and redistribute calspectin in a Ca2+/phospholipid-dependent manner under the plasma membrane and that annexin VI may be involved in the regulation of the membrane skeleton of neuronal cells in response to Ca2+.
鉴定膜联蛋白VI结合蛋白对于阐明膜联蛋白VI的生理功能至关重要。在此，我们开发了鉴定膜联蛋白VI结合蛋白的方法并对这种结合进行了表征。当使用125I标记的膜联蛋白VI通过SDS-聚丙烯酰胺凝胶印迹进行检测时，膜联蛋白VI与大鼠前脑全匀浆中的约14种蛋白质结合。这种结合是Ca(2+)依赖性的，并且对磷脂酰丝氨酸（PS）和磷脂酸具有特异性。一系列证据表明膜联蛋白VI与其靶蛋白的结合是一种蛋白质-蛋白质相互作用。一种分子量为240,000的膜联蛋白VI结合蛋白在细胞骨架组分中富集，并被鉴定为钙视蛋白（脑血影蛋白或肌纤蛋白）。当用天然状态的纯化钙视蛋白检测这种结合时，Ca2+亲和力（KCa）为7.6 microM，对膜联蛋白VI的亲和力（Kd）为68 nM。膜联蛋白VI与钙视蛋白的β亚基结合，但不与α亚基结合。结合位点定位于β亚基的NH2末端结构域，该结构域包含一个肌动蛋白结合位点，并且与肌营养不良蛋白和α-辅肌动蛋白的NH2末端区域具有显著的同源性。当通过低剪切粘度测定法检测膜联蛋白VI对F-肌动蛋白和钙视蛋白之间相互作用的影响时，膜联蛋白VI以Ca2+/PS依赖性方式抑制钙视蛋白的F-肌动蛋白交联活性。共沉降分析表明，在Ca2+和PS存在的情况下，膜联蛋白VI使钙视蛋白与F-肌动蛋白解离。这些结果表明，膜联蛋白VI可以在质膜下以Ca(2+)/磷脂依赖性方式使钙视蛋白解离并重新分布，并且膜联蛋白VI可能参与神经元细胞的膜骨架响应Ca2+的调节。
