Chung S H, Joberty G, Gelino E A, Macara I G, Holz R W
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, USA.
J Biol Chem. 1999 Jun 18;274(25):18113-20. doi: 10.1074/jbc.274.25.18113.
The Rab class of low molecular weight GTPases has been implicated in the regulation of vesicular trafficking between membrane compartments in eukaryotic cells. The Rab3 family consisting of four highly homologous isoforms is associated with secretory granules and synaptic vesicles. Many different types of experiments indicate that Rab3a is a negative regulator of exocytosis and that its GTP-bound form interacts with Rabphilin3, a possible effector. Overexpression of Rabphilin3 in chromaffin cells enhances secretion. We have investigated the expression, localization, and effects on secretion of the various members of the Rab3 family in bovine chromaffin and PC12 cells. We found that Rab3a, Rab3b, Rab3c, and Rab3d are expressed to varying degrees in PC12 cells and in a fraction enriched in chromaffin granule membranes from the adrenal medulla. Immunocytochemistry revealed that all members of the family when overexpressed in PC12 cells localize to secretory granules. Binding constants for the interaction of the GTP-bound forms of Rab3a, Rab3b, Rab3c, and Rab3d with Rabphilin3 were comparable (Kd = 10-20 nM). Overexpression of each of the four members of the Rab3 family inhibited secretion. Mutations in Rab3a were identified that strongly impaired the ability of the GTP-bound form to interact with Rabphilin3. The mutated proteins inhibited secretion similarly to wild type Rab3a. Although Rab3a and Rabphilin3 are located on the same secretory granule or secretory vesicle and interact both in vitro and in situ, it is concluded that the inhibition of secretion by overexpression of Rab3a is unrelated to its ability to interact with Rabphilin3.
低分子量GTP酶的Rab家族与真核细胞内膜隔室之间的囊泡运输调节有关。由四种高度同源的亚型组成的Rab3家族与分泌颗粒和突触小泡相关。许多不同类型的实验表明，Rab3a是胞吐作用的负调节因子，其GTP结合形式与可能的效应分子Rabphilin3相互作用。在嗜铬细胞中过表达Rabphilin3可增强分泌。我们研究了Rab3家族各成员在牛嗜铬细胞和PC12细胞中的表达、定位及其对分泌的影响。我们发现Rab3a、Rab3b、Rab3c和Rab3d在PC12细胞以及富含肾上腺髓质嗜铬颗粒膜的部分中均有不同程度的表达。免疫细胞化学显示，该家族所有成员在PC12细胞中过表达时均定位于分泌颗粒。Rab3a、Rab3b、Rab3c和Rab3d的GTP结合形式与Rabphilin3相互作用的结合常数相当（Kd = 10 - 20 nM）。Rab3家族的四个成员各自过表达均抑制分泌。已鉴定出Rab3a中的突变，这些突变严重损害了GTP结合形式与Rabphilin3相互作用的能力。突变蛋白与野生型Rab3a类似地抑制分泌。尽管Rab3a和Rabphilin3位于同一分泌颗粒或分泌小泡上，且在体外和原位均相互作用，但得出的结论是，Rab3a过表达对分泌的抑制与其与Rabphilin3相互作用的能力无关。
