Calcium-dependent binding of S100C to the N-terminal domain of annexin I

The annexin family of proteins is characterized by a conserved core domain that binds to phospholipids in a Ca2+-dependent manner. Each annexin also has a structurally distinct N-terminal domain that may impart functional specificity. To search for cellular proteins that interact with the N-terminal domain of annexin I, we constructed a fusion protein consisting of glutathione S-transferase fused to amino acids 2-47 of human annexin I(GST AINT; AINT = annexin I N-terminal), Extracts from metabolically labeled A431 cells contained a single protein (M(r) similar to 10,000) that bound to GST-AINT in a Ca2+-dependent manner. A synthetic peptide corresponding to amino acids 2-18 of annexin I inhibited the binding of the 10-kDa protein to GST-AINT with half-maximal inhibition occurring at similar to 15 mu M peptide. In cellular extracts, endogenous annexin I and the 10-kDa protein associated in a reversible Ca2+ dependent manner. Experiments with other annexins and with N-terminal truncated forms of annexin I indicated that the 10-kDa protein bound specifically to a site within the first 12 amino acids of annexin I, The 10-kDa protein was purified from human placenta by hydrophobic and affinity chromatography. Amino acid sequence analysis indicated that the 10-kDa protein is the human homologue of S100C, a recently identified member of the S100 subfamily of EF-hand Ca2+-binding proteins.