Annexin II contains two types of Ca(2+)-binding sites.
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.
Jost
M
M
Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Göttingen, Germany.
Weber
K
K
Gerke
V
V
D000595:Amino Acid Sequence; D000818:Animals; D017306:Annexin A2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002413:Cations, Divalent; D003460:Crystallization; D018360:Crystallography, X-Ray; D003599:Cytoskeleton / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005971:Glutamates; D018698:Glutamic Acid; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013552:Swine
D017306:Annexin A2; D002413:Cations, Divalent; D005971:Glutamates; D010718:Phosphatidylserines; D010743:Phospholipids; D011994:Recombinant Proteins; D018698:Glutamic Acid; D002118:Calcium