Annexin VI-binding proteins in brain. Interaction of annexin VI with a membrane skeletal protein, calspectin (brain spectrin or fodrin).
Identification of annexin VI-binding proteins is essential to elucidate the physiological functions of annexin VI. Here, we developed the methods 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 125I-annexin VI using blots of SDS-polyacrylamide gels. The binding was Ca(2+)-dependent and specific for phosphatidylserine (PS) and phosphatidic acid. A line of evidence indicates that the binding of annexin VI to its target proteins is a protein-protein interaction. One of annexin VI-binding proteins 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+ affinity (KCa) was 7.6 microM, and the affinity for annexin VI (Kd) was 68 nM. Annexin VI bound to beta subunit of calspectin, but not to alpha subunit. The binding site was localized to the NH2-terminal domain of beta subunit, which contains an actin-binding site and exhibits striking homology with the NH2-terminal regions of dystrophin and alpha-actinin. When the effect of annexin VI on the interaction between F-actin and calspectin was examined by low shear viscometry, annexin VI inhibited the F-actin cross-linking activity of calspectin in a Ca2+/PS-dependent manner. Cosedimentation assay showed that annexin VI dissociates calspectin from F-actin in the presence of Ca2+ and PS. These results 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 skeleton of neuronal cells in response to Ca2+.
Department of Neurochemistry and Neuropharmacology, Osaka University Medical School, Japan.
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Watanabe
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Inui
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Chen
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Iga
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Sobue
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017317:Annexin A6 / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D013049:Spectrin / Q000378:metabolism*
D000199:Actins; D017317:Annexin A6; D002118:Calcium; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D008840:Microfilament Proteins; D010743:Phospholipids; D013049:Spectrin