Miyazaki M, Kaibuchi K, Shirataki H, Kohno H, Ueyama T, Nishikawa J, Takai Y
Department of Biochemistry, Kobe University School of Medicine, Japan.
Brain Res Mol Brain Res. 1995 Jan;28(1):29-36. doi: 10.1016/0169-328x(94)00180-m.
Rabphilin-3A is a putative target protein for Rab3A/Smg 25A, which is a member of the Ras-related small GTP-binding protein and implicated in neurotransmitter release from the synapse. Rabphilin-3A is composed of two functionally different domains: the N-terminal Rab3A-binding and the C-terminal phosphatidylserine- and Ca(2+)-binding domains. The C-terminal domain has two copies of an internal repeat that are homologous to the C2 domains of protein kinase C, synaptotagmin, and phospholipase A2 and C-gamma 1, which are known to bind phosphatidylserine and Ca2+. In this study, we attempted to identify the Rabphilin-3A-interacting molecule in bovine brain by use of an overlay assay technique. The 32P-labeled C-terminal fragment of Rabphilin-3A (281-704 amino acids) bound to a protein molecule with a M(r) of about 115 kDa which was immobilized on a nitrocellulose sheet. This protein was highly purified and characterized. The binding of the 32P-labeled C-terminal fragment to this protein was dependent on both phosphatidylserine and Ca2+, and inhibited by an excess amount of the C-terminal fragment and the C2 domain fragment (396-704 amino acids) but not by the N-terminal fragment (1-280 amino acids). These results indicate that Rabphilin-3A binds to a protein molecule with a M(r) of 115 kDa through the C2 domain in the presence of phosphatidylserine and Ca2+.
Rabphilin-3A是Rab3A/Smg 25A的一种假定靶蛋白，Rab3A/Smg 25A是Ras相关小GTP结合蛋白家族的成员，与突触处神经递质的释放有关。Rabphilin-3A由两个功能不同的结构域组成：N端的Rab3A结合结构域和C端的磷脂酰丝氨酸及Ca(2+)结合结构域。C端结构域有两个内部重复序列拷贝，它们与蛋白激酶C、突触结合蛋白以及磷脂酶A2和C-γ1的C2结构域同源，已知这些结构域可结合磷脂酰丝氨酸和Ca2+。在本研究中，我们试图通过使用覆盖分析技术来鉴定牛脑中与Rabphilin-3A相互作用的分子。Rabphilin-3A的32P标记C端片段（281 - 704个氨基酸）与一个固定在硝酸纤维素膜上、分子量约为115 kDa的蛋白质分子结合。该蛋白质被高度纯化并进行了表征。32P标记的C端片段与该蛋白质的结合依赖于磷脂酰丝氨酸和Ca2+，并被过量的C端片段和C2结构域片段（396 - 704个氨基酸）抑制，但不被N端片段（1 - 280个氨基酸）抑制。这些结果表明，在磷脂酰丝氨酸和Ca2+存在的情况下，Rabphilin-3A通过C2结构域与一个分子量为115 kDa的蛋白质分子结合。
