ANNEXIN-II CONTAINS 2 TYPES OF CA2+-BINDING SITES

作者：JOST M; WEBER K; GERKE V

来源：Biochemical Journal, 1994, 298: 553-559.

The annexins are a multigene family of Ca2+-dependent phospholipid-binding proteins which contain novel types of Ca2+ sites. Using site-directed mutagenesis, we generated mutant proteins that show defects in the Ca2+-binding sites in a particular member of this family, the src tyrosine kinase substrate annexin II. Analysis of the relative Ca2+-binding affinities of annexin II mutants in a combined Ca2+/phospholipid-binding assay revealed two distinct types of Ca2+-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, Romisch 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 Ca2+-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 muM 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 muM 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 Ca2+-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 Ca2+-binding sites.

• 出版日期1994-3-15