Raffaniello Robert, Fedorova Daria, Ip Dawn, Rafiq Sarwish
Hunter College, New York, NY 10010, USA.
Cell Physiol Biochem. 2009;24(5-6):369-78. doi: 10.1159/000257429. Epub 2009 Nov 4.
Rab proteins are small GTPases required for vesicle trafficking through the secretory and endocytic pathways. Rab GDP-dissociation inhibitor (rab-GDI) regulates Rab protein function and localization by maintaining Rab proteins in the GDP-bound conformation. Two isoforms of rab-GDI are present in most mammalian cells: GDI-1 and GDI-2. It has recently been demonstrated that a Heat shock protein 90 (Hsp90) chaperone complex regulates the interactions between Rab proteins and Rab-GDI-1. The AR42J cell line is derived from rat pancreatic exocrine tumor cells and develops an acinar-like phenotype when treated with dexamethasone (Dex). The aim of the present study was to examine the expression of rab-GDI isoforms and Hsp90 in AR42J cells in the presence or absence of Dex. Rab-GDI:Hsp90 interactions were also examined. Both rab-GDI isoforms were detected in AR42J cells by immunoblotting. In Dex-treated cells, quantitative immunoblotting revealed that rab-GDI-1 expression increased by 28%, although this change was not statistically significant. Rab-GDI-2 levels were unaltered by Dex treatment. Approximately 21% rab-GDI-1 was membrane associated, whereas rab-GDI-2 was exclusively cytosolic. Dex treatment did not affect the subcellular distribution of rab-GDI isoforms. Hsp90 was present in the cytosolic and membrane fractions of AR42J cells and co-immunoprecipitated with cytosolic rab-GDI-1. Moreover, density gradient centrifugation of AR42J cell membranes revealed that Hsp90 and rab-GDI-1 co-localize on low- and high-density membrane fractions, including amylase-containing secretory granules. The Hsp90 inhibitor, geldanamycin, inhibited CCK-8-induced amylase release from these cells in a dose-dependent manner. Our results indicate that as AR42J cells differentiate into acinar-like cells, rab-GDI isoform expression and localization is not significantly altered. Moreover, our findings suggest that Hsp90 regulates agonist-induced secretion in exocrine cells by interacting with rab-GDI-1.
Rab蛋白是小GTP酶，是囊泡通过分泌和内吞途径运输所必需的。Rab GDP解离抑制剂（rab-GDI）通过将Rab蛋白维持在GDP结合构象来调节Rab蛋白的功能和定位。大多数哺乳动物细胞中存在两种rab-GDI亚型：GDI-1和GDI-2。最近有研究表明，热休克蛋白90（Hsp90）伴侣复合物调节Rab蛋白与Rab-GDI-1之间的相互作用。AR42J细胞系源自大鼠胰腺外分泌肿瘤细胞，用地塞米松（Dex）处理后会形成腺泡样表型。本研究的目的是检测在有无Dex的情况下AR42J细胞中rab-GDI亚型和Hsp90的表达。还检测了Rab-GDI:Hsp90的相互作用。通过免疫印迹在AR42J细胞中检测到了两种rab-GDI亚型。在Dex处理的细胞中，定量免疫印迹显示rab-GDI-1表达增加了28%，尽管这一变化无统计学意义。Dex处理未改变Rab-GDI-2的水平。约21%的rab-GDI-1与膜相关，而Rab-GDI-2仅存在于胞质中。Dex处理不影响rab-GDI亚型的亚细胞分布。Hsp90存在于AR42J细胞的胞质和膜部分，并与胞质中的rab-GDI-1共免疫沉淀。此外，对AR42J细胞膜进行密度梯度离心显示，Hsp90和rab-GDI-1共定位于低密度和高密度膜部分，包括含淀粉酶的分泌颗粒。Hsp90抑制剂格尔德霉素以剂量依赖性方式抑制CCK-8诱导的这些细胞中淀粉酶的释放。我们的结果表明，随着AR42J细胞分化为腺泡样细胞，rab-GDI亚型的表达和定位没有明显改变。此外，我们的研究结果表明，Hsp90通过与rab-GDI-1相互作用来调节外分泌细胞中激动剂诱导的分泌。
