ANNEXIN-VI-BINDING PROTEINS IN BRAIN - INTERACTION OF ANNEXIN VI WITHA MEMBRANE SKELETAL PROTEIN, CALSPECTIN (BRAIN SPECTRIN OR FODRIN)

Identification of annexin VI-binding proteins is essential to elucidat e the physiological functions of annexin VI. Here, we developed the me thods to identify an annexin VI-binding protein and characterized the binding. Annexin VI bound to about 14 species of proteins in the whole homogenate of rat forebrain, when examined with I-125-annexin VI usin g blots of SDS-polyacrylamide gels. The binding was Ca2+-dependent and specific for phosphatidylserine (PS) and phosphatidic acid. A line of evidence indicates that the binding of annexin VI to its target prote ins is a protein-protein interaction. One of annexin VI-binding protei ns with M(r) 240,000 was enriched in the cytoskeletal fraction and was identified as calspectin (brain spectrin or fodrin). When the binding was examined with purified calspectin in the native state, the Ca2+ a ffinity (K-Ca) was 7.6 mu M, and the affinity for annexin VI (K-d) was 68 nM. Annexin VI bound to beta subunit of calspectin, but not to alp ha subunit. The binding site was localized to the NH2-terminal domain of beta subunit, which contains an actin-binding site and exhibits str iking homology with the NH2-terminal regions of dystrophin and alpha-a ctinin. When the effect of annexin VI on the interaction between F-act in and calspectin was examined by low shear viscometry, annexin VI inh ibited the F-actin cross-linking activity of calspectin in a Ca2+/PS d ependent manner. Cosedimentation assay showed that annexin VI dissocia tes calspectin from F-actin in the presence of Ca2+ and PS. These resu lts suggest that annexin VI can dissociate and redistribute calspectin in a Ca2+/phospholipid-dependent manner under the plasma membrane and that annexin VI may be involved in the regulation of the membrane ske leton of neuronal cells in response to Ca2+.