Rab8b and its interacting partner TRIP8b are involved in regulated secretion in AtT20 cells
Rab8b and its interacting partner TRIP8b are involved in regulated secretion in AtT20 cells
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DOI：
10.1074/jbc.m010798200
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发表时间：
2001-04-20
影响因子：
4.8
通讯作者：
Ting, AE
中科院分区：
文献类型：
--
作者：
Chen, S;Liang, MC;Ting, AE
Rab蛋白是调节细胞内囊泡运输的小GTP酶家族。Rab 8b，由于其与Rab 8的同源性，已被认为在囊泡运输到质膜中起作用。利用酵母双杂交系统，我们从大鼠脑cDNA文库中鉴定了一个Rab 8b相互作用的克隆，命名为TRIP 8b。该基因编码与过氧化物酶体靶向信号1受体具有同源性的66-kDa蛋白。Rab 8b和TRIP 8b之间的相互作用通过体外结合测定和免疫共沉淀研究进一步验证。Rab 8b突变体的其他实验证明Rab 8b需要鸟嘌呤核苷酸而不是异戊烯化来与TRIP 8b相互作用。Western免疫印迹分析显示TRIP 8b主要在脑中表达。AtT 20细胞的亚细胞分级显示TRIP 8b存在于胞质和膜组分中。为了研究Rab 8b和TRIP 8b在分泌中的功能，我们检测了AtT 20细胞中ACTH的释放。来自稳定表达Rab 8b或TRIP 8b的细胞系的结果表明，这两种蛋白对cAMP诱导的ACTH分泌具有刺激作用。总之，这些数据表明Rab 8b和TRIP 8b相互作用，并参与AtT 20细胞中的调节分泌途径。
Rab proteins are a family of small GTPases that regulate intracellular vesicle traffic. Rab8b, because of its homology with Rab8, has been suggested to function in vesicle transport to the plasma membrane. Using the yeast two-hybrid system, we identified a Rab8b interacting clone, termed TRIP8b, from a rat brain cDNA library. The gene encodes a 66-kDa protein with homology to the peroxisomal targeting signal 1 receptor. The interaction between Rab8b and TRIP8b was further verified by in vitro binding assays and co-immunoprecipitation studies. Additional experiments with Rab8b mutants demonstrated that Rab8b requires a guanine nucleotide but not prenylation for its interaction with TRIP8b. Western immunoblot analysis showed that TRIP8b was primarily expressed in brain. Subcellular fractionation of AtT20 cells revealed that TRIP8b was present in both cytosolic and membrane fractions. To investigate the function of Rab8b and TRIP8b in secretion, we examined the release of ACTH from AtT20 cells, Results from stable cell lines expressing Rab8b or TRIP8b indicated that both proteins had a stimulatory effect on cAMP-induced secretion of ACTH. In summary, these data suggest that Rab8b and TRIP8b interact with each other and are involved in the regulated secretory pathway in AtT20 cells.