Alternative splicing in the first α-helical region of the Rab-binding domain of Rim regulates Rab3A binding activity:: is Rim a Rab3 effector protein during evolution?
Alternative splicing in the first α-helical region of the Rab-binding domain of Rim regulates Rab3A binding activity:: is Rim a Rab3 effector protein during evolution?
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DOI：
10.1111/j.1365-2443.2004.00767.x
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发表时间：
2004-09-01
期刊：
影响因子：
2.1
通讯作者：
Fukuda, M
中科院分区：
文献类型：
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作者：
Fukuda, M
Rim1 和 Rim2 最初被描述为参与分泌囊泡胞吐作用调节的特定 Rab3A 效应蛋白。 Rim 的假定 Rab3A 结合域 (RBD) 由两个 α 螺旋区域（称为 RBD1 和 RBD2）组成，并由两个锌指基序分隔。尽管已知Rim的RBD1中的选择性剪接会产生长形式和短形式的RBD（分别命名为Rim1和Rim1Delta56-105，以及Rim2（+40A）和Rim2），并且长形式的Rim1和短形式的Rim2在小鼠大脑中占主导地位，但RBD1中的选择性剪接的生理意义从未被阐明。在本研究中，我发现 Rim RBD1 中的选择性剪接改变了 Rab3A 对 Rims 的结合亲和力，并发现在 Rim2 的 RBD1 中插入 40 个氨基酸（即 Rim2(+40A)）会显着降低其 Rab3A 结合活性（亲和力降低超过 50 倍）。类似地，Rim1Delta56-105 表现出比长形式的 Rim1 更高的与 Rab3A 的结合亲和力。 PC12 细胞中 Rim RBD 的短形式表达与内源性 Rab3A 很好地共定位，而 PC12 细胞中 Rim RBD 的长形式表达导致细胞质和核定位。此外，我发现秀丽隐杆线虫Rim/UNC-10（ce-Rim）和果蝇Rim（dm-Rim）分别不与ce-Rab3和dm-Rab3相互作用，表明Rim的Rab3效应子功能在进化过程中并未保留。基于这些发现，我提出分泌囊泡胞吐作用期间 Rim 的 Rab3A 效应器功能仅限于哺乳动物 Rim RBD 的短形式。
Rim1 and Rim2 were originally described as specific Rab3A-effector proteins involved in the regulation of secretory vesicle exocytosis. The putative Rab3A-binding domain (RBD) of Rim consists of two alpha-helical regions (named RBD1 and RBD2) separated by two zinc finger motifs. Although alternative splicing in the RBD1 of Rim is known to produce long and short forms of RBD (named Rim1 and Rim1Delta56-105, and Rim2(+40A) and Rim2, respectively), with the long form of Rim1 and short form of Rim2 being dominant in mouse brain, the physiological significance of the alternative splicing in RBD1 has never been elucidated. In the present study I discovered that alternative splicing in Rim RBD1 alters Rab3A binding affinity to Rims, and found that insertion of 40 amino acids into the RBD1 of Rim2 (i.e. Rim2(+40A)) dramatically reduced its Rab3A binding activity (more than a 50-fold decrease in affinity). Similarly, Rim1Delta56-105 exhibited higher affinity binding to Rab3A than the long form of Rim1. Expression of the short forms of the Rim RBD in PC12 cells co-localized well with endogenous Rab3A, whereas expression of the long forms of the Rim RBD in PC12 cells resulted in cytoplasimc and nuclear localization. Moreover, I found that Caenorhabditis elegans Rim/UNC-10 (ce-Rim) and Drosophila Rim (dm-Rim) do not interact with ce-Rab3 and dm-Rab3, respectively, indicating that the Rab3-effector function of Rim has not been retained during evolution. Based on these findings, I propose that the Rab3A-effector function of Rim during secretory vesicle exocytosis is limited to the short form of the mammalian Rim RBD alone.