Regulation of calcyclin (S100A6) binding by alternative splicing in the N-terminal regulatory domain of annexin XI isoforms.
Annexin XI is a Ca2+/phospholipid-binding protein that interacts with a member of S100 protein family, calcyclin (S100A6), in a Ca2+-dependent manner. There are two isoforms of annexin XI, annexin XI-A and -B, generated by alternative splicing in the N-terminal regulatory domain. To determine the role of the alternative splicing region in the calcyclin-binding, we identified and characterized its calcyclin binding site. Experiments with glutathione S-transferase fusion proteins with N-terminal sites of annexin XI-A showed the calcyclin binding site to be in residues Gln49-Thr62 of rabbit annexin XI-A, which contains part of the splicing region. A synthesized peptide corresponding to Tyr43-Thr62 of annexin XI-A inhibited the interaction of annexin XI with calcyclin in liposome co-pelleting assay. The calcyclin binding site possesses a hydrophobic residue cluster conserved among S100 binding sites of annexin I and II. Recombinant annexin XI isoforms were expressed in Sf9 cells using a baculovirus expression system. In contrast to annexin XI-A, it was found that annexin XI-B protein could not bind to calcyclin by the liposome co-pelleting assay. In Sf9 cells coexpressing calcyclin with annexin XI isoforms, the calcyclin binding was observed only for annexin XI-A isoform. These results indicate that the calcyclin binding ability of annexin XI is an annexin XI-A isoform-specific character, suggesting that annexin XI isoforms might play distinct roles in cells through each alternative splicing regions.
Sudo
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Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Nagoya, 466, Japan.
Hidaka
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D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D017302:Annexins / Q000235:genetics* / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D009418:S100 Proteins; D018411:Spodoptera / Q000166:cytology
D017302:Annexins; D002135:Calcium-Binding Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D009418:S100 Proteins; D002118:Calcium