ANNEXIN-II CONTAINS 2 TYPES OF CA2-BINDING SITES()

The annexins are a multigene family of Ca2+-dependent phospholipid-bin ding proteins which contain novel types of Ca2+ sites. Using site-dire cted mutagenesis, we generated mutant proteins that show defects in th e Ca2+-binding sites in a particular member of this family, the src ty rosine kinase substrate annexin II. Analysis of the relative Ca2+-bind ing affinities of annexin II mutants in a combined Ca2+/phospholipid-b inding assay revealed two distinct types of Ca2+-binding sites. Three so-called type II sites are found in annexin repeats 2, 3 and 4 respec tively. Two so-called type III sites are located in the first repeat a nd involve the glutamic acid residues at positions 52 and 95. Both typ es of sites were recently identified by X-ray crystallography in annex ins V and I [Huber, Schneider, Mayr, Romisch and Paques (1990) FEBS Le tt. 275,15-21; Weng, Luecke, Song, Kang, Kim and Huber (1993) Protein Sci. 2, 448-458], indicating that similar principles govern Ca2+ bindi ng to annexins in crystals and in solution. The two types of Ca2+-bind ing sites differ not only in their architecture but also in their affi nity for the bivalent cation. The Ca2+ concentration needed for half-m aximal phosphatidylserine binding is 5-10 muM for an annexin II deriva tive with intact type II but defective type III sites (TM annexin II) whereas a mutant protein containing defective type II but unaltered ty pe III sites (CM annexin II) requires 200-300 muM Ca2+ for the same ac tivity. Annexin II mutants with defects in the type II and/or type III sites also show different subcellular distributions. When expressed t ransiently in HeLa cells, TM annexin II acquires the typical location in the cortical cytoskeleton observed for the wild-type molecule. In c ontrast, 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 associat ion of annexin II with the submembraneous cytoskeleton depends only on the occupation of type II Ca2+-binding sites.