RABPHILIN-3A, A PUTATIVE TARGET PROTEIN FOR SMG P25A RAB3A P25 SMALL GTP-BINDING PROTEIN RELATED TO SYNAPTOTAGMIN
RABPHILIN-3A, A PUTATIVE TARGET PROTEIN FOR SMG P25A RAB3A P25 SMALL GTP-BINDING PROTEIN RELATED TO SYNAPTOTAGMIN
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DOI：
10.1128/mcb.13.4.2061
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发表时间：
1993-04-01
影响因子：
5.3
通讯作者：
TAKAI, Y
中科院分区：
文献类型：
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作者：
SHIRATAKI, H;KAIBUCHI, K;TAKAI, Y
在以前的研究中（H。白泷湾Kaibuchi，T.山口K.韦达，H. Horiuchi和Y. Takai，J.Biol.Chem.267：10946-10949，1992），我们从牛脑粗膜中高度纯化了smg p25 A/rab 3A p25的推定靶蛋白，该蛋白是一种与神经递质释放有关的ras p21相关的小GTP结合蛋白。在这项研究中，我们已经分离和测序该蛋白的cDNA从牛脑cDNA文库。该cDNA具有开放阅读框，编码704个氨基酸的蛋白质，计算的M（r）为77，976。我们暂时将这种蛋白质称为rabphilin-3A。rabphilin-3A的结构分析揭示了存在两个拷贝的内部重复序列，其与突触结合蛋白所述的蛋白激酶C的C2结构域同源，已知其位于突触囊泡的膜中并以Ca 2+依赖性方式结合膜磷脂。分离的cDNA在COS 7细胞中表达，编码的蛋白质被识别与抗rabphilin-3A的多克隆抗体，并在大小上与rabphilin-3A纯化的牛脑十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。此外，从牛脑中纯化的rabphilin-3A和重组rabphilin-3A都与rab 3A p25的GTP-γ S结合形式形成复合物，但不与rab 3A p25的GDP结合形式形成复合物。免疫印迹和北方（RNA）印迹分析表明，rabphilin-3A在牛和大鼠脑中高度表达。这些结果表明，rabphilin-3A是一种新的蛋白质，具有C2结构域，并选择性地与GTP结合形式的rab 3A p25相互作用。
In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca2+-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP-gammaS-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.