No abstract

Site-directed mutagenesis was employed to map and characterize Ca2 + -binding sites in annexin 11, a member of the annexin family of Ca2+-and phospholipid-binding proteins which serves as a major cellular substrate for the tyrosine kinase encoded by the src oncogene. Several single amino acid substitutions were introduced in the human annexin I1 and the various mutant proteins were scored for their affinity towards Ca2+ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J . Biol. Chem. 266, 14732-147391 that a Ca2+-binding site is present in the third of the four repeat segments whch comprise the 33-kDa protein core of annexin 11. In addition to Gly206 and Thr207, which are localized in the highly conserved endonexin fold of the third repeat, Glu246 is involved in the formation of this site. Thus the architecture of this Ca2+-binding site in solution is very similar, if not identical, to that of Ca2+ sites identified recently in annexin V crystals [Huber, R., Schneider, M., Mayr, I., Romisch, J. and Paques, E.-P. (1990) FEBS Lett. 275,[15][16][17][18][19][20][21]. In addition to the site in repeat 3, we have mapped sites of presumably similar architecture in repeats 2 and 4 of annexin 11. Again, an acidic amino acid which is located 40 residues C-terminal to the conserved glycine at position 4 of the endonexin fold is indispensable for highaffinity Ca2+ binding: Asp161 in the second and Asp321 in the fourth repeat. In contrast, repeat 1 does not contain an acidic amino acid at a corresponding position and also shows deviations from the other repeats in the sequence surrounding the conserved glycine. These results on annexin I1 together with the crystallographic information on annexin V reveal that annexins can differ in the position of the Ca2+ sites. Ca2+-binding sites of similar structure are present in repeats 2, 3, and 4 of annexin I1 while in annexin V they occur in repeats 1, 2, and 4. We also synthesized an annexin I1 derivative with mutations in all three Ca2+ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca2+, indicating the presence of (an) additional Ca2 + site(s) of presumably different architecture.The annexins form a multigene family of Ca2+-dependent membrane-binding and phospholipid-binding proteins. Although the precise biological function of the annexins is not known, their biochemical properties imply that the proteins are involved in membrane phenomena, e.g. membrane fusion events during exocytosis, membrane-cytoskeleton linkage, membrane channel formation (for review see Klee, 1988;Moss et al., 1991). In addition to common biochemical properties the annexins share a characteristic structural element, the annexin repeat. This segment of 70-80 amino acids is repeated four (32 -39-kDa annexins) or eight times (68-kDa annexin) along the polypeptide chain of the individual annexin. The annexin repeats show intramolecular as well as intermolecular sequence similarities which are par...

Recombinant annexin II mutants were generated to identify amino acids involved in the formation of the discontinuous cpitope of the monoclonal antibody H28. Analysis of the various mutant proteins by immunoblotting and enzyme-linked immunosorbent assay revealed that residues Lysz7, Arg2, GW, and Arg7 are indispensable for H28 reactivity. Residues in equivalent positions are also in close proximity in the recently determined X-ray structure of annexin V, a different member of the same family of Ca'+/lipid-binding proteins. Thus annexins II and V show a similar threedimensional folding in this region of the molecule. Consequently, !he Ca** binding sites and the residues phosphorylated by pp601~ (Tyti') and protein kinase C (Setis) most likely reside on opposite sides of the annexin II molecule.

Annexin II, a member of the annexin family of Ca(2+)- and lipid-binding proteins, is a major substrate of the pp60src kinase. It is unique within the annexin protein family, since it can form a tight heterotetrameric complex with the cellular protein ligand p11, a member of the S100 protein family. Within the cell, the annexin II2p11(2) complex is localized at the cytoplasmic surface of the plasma membrane in the submembranous cytoskeleton. This intracellular localization is thought to be the consequence of a typical annexin II property observed in vitro, its Ca(2+)-dependent binding to phospholipids and cytoskeletal elements (F-actin, non-erythroid spectrin). We employed site-directed mutagenesis to create mutant annexin II molecules with defects either in the p11-binding site or in the Ca(2+)-binding sites present in repeats 2, 3 and 4. The mutated annexin II derivatives were expressed in HeLa and RMCD cells by transfection of the appropriate DNA constructs in order to analyze the importance of p11- and Ca(2+)-binding for the intracellular localization of annexin II. Immunofluorescence microscopy with a monoclonal antibody that specifically detected the transfected annexin II derivatives indicated that the Ca(2+)-dependent incorporation of annexin II into the submembranous network depended on its ability to form the annexin II/p11 complex and on the presence of intact Ca(2+)-binding sites. Neither monomeric annexin II lacking an intact p11-binding site, nor the annexin II mutant with defects in the Ca(2+)-binding sites in repeats 2, 3 and 4 were associated with the Triton X-100-resistant network of the submembranous cytoskeleton.