Jost M, Weber K, Gerke V
Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Göttingen, Germany.
Biochem J. 1994 Mar 15;298 Pt 3(Pt 3):553-9. doi: 10.1042/bj2980553.
The annexins are a multigene family of Ca(2+)-dependent phospholipid-binding proteins which contain novel types of Ca2+ sites. Using site-directed mutagenesis, we generated mutant proteins that show defects in the Ca(2+)-binding sites in a particular member of this family, the src tyrosine kinase substrate annexin II. Analysis of the relative Ca(2+)-binding affinities of annexin II mutants in a combined Ca2+/phospholipid-binding assay revealed two distinct types of Ca(2+)-binding sites. Three so-called type II sites are found in annexin repeats 2, 3 and 4 respectively. Two so-called type III sites are located in the first repeat and involve the glutamic acid residues at positions 52 and 95. Both types of sites were recently identified by X-ray crystallography in annexins V and I [Huber, Schneider, Mayr, Römisch and Paques (1990) FEBS Lett. 275, 15-21; Weng, Luecke, Song, Kang, Kim and Huber (1993) Protein Sci. 2, 448-458], indicating that similar principles govern Ca2+ binding to annexins in crystals and in solution. The two types of Ca(2+)-binding sites differ not only in their architecture but also in their affinity for the bivalent cation. The Ca2+ concentration needed for half-maximal phosphatidylserine binding is 5-10 microM for an annexin II derivative with intact type II but defective type III sites (TM annexin II) whereas a mutant protein containing defective type II but unaltered type III sites (CM annexin II) requires 200-300 microM Ca2+ for the same activity. Annexin II mutants with defects in the type II and/or type III sites also show different subcellular distributions. When expressed transiently in HeLa cells, TM annexin II acquires the typical location in the cortical cytoskeleton observed for the wild-type molecule. In contrast, CM annexin II remains essentially cytosolic, as does a mutant protein containing defects in both type II and type III Ca(2+)-binding sites (TCM annexin II). This indicates that the intracellular association of annexin II with the submembraneous cytoskeleton depends only on the occupation of type II Ca(2+)-binding sites.
膜联蛋白是一个多基因家族的依赖Ca(2+)的磷脂结合蛋白，其中包含新型的Ca2+位点。利用定点诱变技术，我们生成了在该家族的一个特定成员——src酪氨酸激酶底物膜联蛋白II的Ca(2+)结合位点存在缺陷的突变蛋白。在Ca2+/磷脂结合联合测定中对膜联蛋白II突变体的相对Ca(2+)结合亲和力进行分析，揭示了两种不同类型的Ca(2+)结合位点。分别在膜联蛋白重复序列2、3和4中发现了三个所谓的II型位点。两个所谓的III型位点位于第一个重复序列中，涉及第52位和第95位的谷氨酸残基。最近通过X射线晶体学在膜联蛋白V和I中鉴定出了这两种类型的位点[胡贝尔、施奈德、迈尔、勒米施和帕克斯（1990年）《欧洲生物化学学会联合会快报》275，15 - 21；翁、吕克、宋、康、金和胡贝尔（1993年）《蛋白质科学》2，448 - 458]，这表明在晶体和溶液中，Ca2+与膜联蛋白结合遵循相似的原理。这两种类型的Ca(2+)结合位点不仅在结构上不同，而且对二价阳离子的亲和力也不同。对于具有完整II型但III型位点有缺陷的膜联蛋白II衍生物（TM膜联蛋白II），半最大磷脂酰丝氨酸结合所需的Ca2+浓度为5 - 微摩尔，而含有缺陷II型但III型位点未改变的突变蛋白（CM膜联蛋白II）进行相同活性则需要200 - 300微摩尔Ca2+。在II型和/或III型位点存在缺陷的膜联蛋白II突变体也表现出不同的亚细胞分布。当在HeLa细胞中瞬时表达时，TM膜联蛋白II获得了野生型分子在皮质细胞骨架中的典型定位。相比之下，CM膜联蛋白II基本上仍位于胞质溶胶中，含有II型和III型Ca(2+)结合位点均有缺陷的突变蛋白（TCM膜联蛋白II）也是如此。这表明膜联蛋白II与膜下细胞骨架的细胞内结合仅取决于II型Ca(2+)结合位点的占据情况。
