Wang Y, Sugita S, Sudhof T C
Center for Basic Neuroscience, Department of Molecular Genetics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9111, USA.
J Biol Chem. 2000 Jun 30;275(26):20033-44. doi: 10.1074/jbc.M909008199.
RIM1 is a putative effector protein for Rab3s, synaptic GTP-binding proteins. RIM1 is localized close to the active zone at the synapse, where it interacts in a GTP-dependent manner with Rab3 located on synaptic vesicles. We now describe a second RIM protein, called RIM2, that is highly homologous to RIM1 and also expressed primarily in brain. Like RIM1, RIM2 contains an N-terminal zinc finger domain that binds to Rab3 as a function of GTP, a central PDZ domain, and two C-terminal C(2) domains that are separated by long alternatively spliced sequences. Unexpectedly, the 3'-end of the RIM2 gene produces an independent mRNA that encodes a smaller protein referred as NIM2. NIM2 is composed of a unique N-terminal sequence followed by the C-terminal part of RIM2. Data bank searches identified a third RIM/NIM-related gene, which encodes a NIM isoform referred to as NIM3; no RIM transcript from this gene was detected. To test if NIMs, like RIMs, may function in secretion, we investigated the effect of NIM3 on calcium-triggered exocytosis in PC12 cells. NIM3 induced a dramatic increase in calcium-evoked exocytosis (50%), with no significant effect on base-line release, suggesting that NIMs, like RIMs, regulate exocytosis The combination of conserved and variable sequences in RIMs and NIMs indicates that the individual domains of these proteins provide binding sites for interacting molecules during exocytosis, as shown for the zinc finger domain of RIM, which binds to GTP-bound Rab3s. To search for additional interacting proteins for RIMs, we employed yeast two-hybrid screens with the C-terminal half of RIM1. Two members of a new family of homologous brain proteins, referred to as RIM-binding proteins (RIM-BPs), were identified. RIM-BPs bind to RIM in yeast two-hybrid and GST pull-down assays, suggesting a specific interaction. In RIMs, the binding site for RIM-BPs consists of a conserved proline-rich sequence between the two C(2) domains, N-terminal to the beginning of NIMs. RIM-BPs are composed of multiple domains, including three fibronectin type III-domains and three Src homology 3 domains, of which the second Src homology 3 domain binds to RIMs. With the RIM-BPs, we have identified a partner for RIMs that may bind to RIMs at the synapse in addition to Rab3.
RIM1是一种假定的Rab3s效应蛋白，Rab3s是突触GTP结合蛋白。RIM1定位于突触处的活性区附近，在那里它以GTP依赖的方式与位于突触小泡上的Rab3相互作用。我们现在描述第二种RIM蛋白，称为RIM2，它与RIM1高度同源，也主要在脑中表达。与RIM1一样，RIM2包含一个N端锌指结构域，该结构域作为GTP的函数与Rab3结合，一个中央PDZ结构域，以及两个C端C(2)结构域，这两个结构域由长的可变剪接序列分隔。出乎意料的是，RIM2基因的3'端产生一种独立的mRNA，其编码一种较小的蛋白，称为NIM2。NIM2由一个独特的N端序列和RIM2的C端部分组成。数据库搜索鉴定出第三个与RIM/NIM相关的基因，其编码一种称为NIM3的NIM异构体；未检测到来自该基因的RIM转录本。为了测试NIMs是否像RIMs一样可能在分泌中起作用，我们研究了NIM3对PC12细胞中钙触发的胞吐作用的影响。NIM3诱导钙诱发的胞吐作用显著增加（50%），对基线释放无显著影响，表明NIMs像RIMs一样调节胞吐作用。RIMs和NIMs中保守序列和可变序列的组合表明，这些蛋白质的各个结构域在胞吐过程中为相互作用分子提供结合位点，如RIM的锌指结构域与结合GTP的Rab3s结合所示。为了寻找RIMs的其他相互作用蛋白，我们用RIM1的C端一半进行酵母双杂交筛选。鉴定出一个新的同源脑蛋白家族的两个成员，称为RIM结合蛋白（RIM-BPs）。在酵母双杂交和GST下拉实验中，RIM-BPs与RIM结合，表明存在特异性相互作用。在RIMs中，RIM-BPs的结合位点由两个C(2)结构域之间、NIMs起始位置N端的一个保守的富含脯氨酸序列组成。RIM-BPs由多个结构域组成，包括三个纤连蛋白III型结构域和三个Src同源3结构域，其中第二个Src同源3结构域与RIMs结合。通过RIM-BPs，我们鉴定出RIMs的一个伴侣，除了Rab3之外，它可能在突触处与RIMs结合。
