Annexin II contains two types of Ca(2+)-binding sites.
Annexin II contains two types of Ca(2+)-binding sites.
复制标题
膜联蛋白 II 包含两种类型的 Ca(2 ) 结合位点。
作者：
M. Jost;K. Weber;V. Gerke
膜联蛋白是一个多基因的Ca（2+）依赖性磷脂结合蛋白家族，它含有新型的Ca2+位点。通过位点定向诱变，我们产生了突变蛋白，这些突变蛋白在该家族的特定成员src酪氨酸激酶底物膜联蛋白II的Ca（2+）结合位点上显示缺陷。在Ca2+/磷脂结合试验中，对膜联蛋白II突变体的相对Ca（2+）结合亲和力进行分析，发现了两种不同类型的Ca（2+）结合位点。三个所谓的II型位点分别位于膜联蛋白重复序列2、3和4。两个所谓的III型位点位于第一个重复序列中，涉及位置52和95的谷氨酸残基。最近通过x射线晶体学在膜联蛋白V和I中发现了这两种类型的位点[Huber， Schneider, Mayr, Römisch and Paques (1990) FEBS Lett. 275, 15-21；Weng, Luecke, Song, Kang， Kim和Huber (1993) Protein Sci. 2,448 -458]，表明Ca2+在晶体和溶液中与膜联蛋白结合的原理相似。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型但缺陷III型位点的膜联蛋白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5-10微米，而含有缺陷II型但未改变III型位点的突变蛋白（CM膜联蛋白II）需要200-300微米的Ca2+才能达到相同的活性。在II型和/或III型位点有缺陷的膜联蛋白II突变体也表现出不同的亚细胞分布。当在HeLa细胞中短暂表达时，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位置。相比之下，CM膜联蛋白II基本上仍然是细胞质的，在II型和III型Ca（2+）结合位点（TCM膜联蛋白II）中含有缺陷的突变蛋白也是如此。这表明膜联蛋白II与膜下细胞骨架的细胞内结合仅依赖于II型Ca（2+）结合位点的占据。
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.