Shirataki H, Kaibuchi K, Sakoda T, Kishida S, Yamaguchi T, Wada K, Miyazaki M, Takai Y
Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Japan.
Mol Cell Biol. 1993 Apr;13(4):2061-8. doi: 10.1128/mcb.13.4.2061-2068.1993.
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 Ca(2+)-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 gamma S-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.
在之前的一项研究中（H. 白滝、海渕启、山口彻、和田健、堀内浩、高井洋，《生物化学杂志》267:10946 - 10949，1992年），我们从牛脑粗膜中高度纯化了推测的smg p25A/rab3A p25的靶蛋白，smg p25A/rab3A p25是一种与ras p21相关的小GTP结合蛋白，参与神经递质释放。在本研究中，我们从牛脑cDNA文库中分离并测序了该蛋白的cDNA。该cDNA有一个开放阅读框，编码一个704个氨基酸的蛋白，计算的分子量为77,976。我们暂时将此蛋白称为rabphilin - 3A。rabphilin - 3A的结构分析显示存在两个内部重复序列拷贝，它们与蛋白激酶C的C2结构域同源，就像已知定位于突触小泡膜并以钙依赖方式结合膜磷脂的突触结合蛋白一样。分离的cDNA在COS7细胞中表达，编码的蛋白能被抗rabphilin - 3A多克隆抗体识别，并且其大小与通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳从牛脑中纯化的rabphilin - 3A相同。此外，从牛脑中纯化的rabphilin - 3A和重组rabphilin - 3A都能与GTPγS结合形式的rab3A p25形成复合物，但不能与GDP结合形式的rab3A p25形成复合物。免疫印迹和Northern（RNA）印迹分析表明rabphilin - 3A在牛脑和大鼠脑中高度表达。这些结果表明rabphilin - 3A是一种具有C2结构域并选择性地与GTP结合形式的rab3A p25相互作用的新型蛋白。
