ISOPRENYLATED GTP-BINDING PROTEINS AND CELL GROWTH

法呢基或香叶基香叶基异戊二烯与羧基的共价结合 末端半胱氨酸残基现在被广泛认为是一种重要的后 真核细胞中的翻译蛋白修饰。大多数人 Prenylated Proteins是RAS相关的GTP超家族的成员。 结合蛋白。属于Rab亚群的25种蛋白质发挥作用 在细胞内蛋白质运输中的重要作用。RAB蛋白循环 细胞内膜和可溶性蛋白质复合体之间通过 一系列依赖于预烯基化的相互作用。的总体目标是 本项目的续篇是定义结构和 Rab1B与两种关键调控蛋白Rab的功能相互作用 护航蛋白(REP)和鸟核苷酸解离抑制物(GDI)。 已知Rep在体外能与Rab蛋白结合，形成可溶的复合体 芳基化b)香叶基香叶基转移酶II所需。 在预烯基化后仍然与Rab蛋白相关，它是 被认为在体内起到靶向或“护送”作用。众所周知，GDI可以 从细胞内膜中提取戊烯基化的Rab蛋白并保持 在一个核苷酸交换被抑制的可溶池中。建议数 研究将在三个方面扩大关于这些蛋白质的知识基础 方式。(1)假设Rab1B中的不同结构域 参与绑定到销售代表和GDI将进行测试。这件事会做到的 通过突变研究，改变的Rab1B蛋白的能力 与代表和GDI的关联将在无手机系统和 共表达Rab1B和rep或Rab1B和GDI的细胞。(2) 假设Rep在Rab1B向TRAB的转移中起关键作用 完整细胞的内质网或胞浆GDI复合体将 通过代谢标记、免疫化学和 利用表位标记蛋白和Rab1B突变体的分子方法 REP或GDI绑定有缺陷。(3)假设细胞 激活的Rab1B含量与细胞增殖相关的调节 在正常和RAS转化的成纤维细胞中将进行测试。潜力 将通过改变细胞蛋白质含量来调节Rab1B 考虑到，以及监管发生的替代可能性 主要通过Rab1D区划和GDI相互作用的变化。 拟议的研究将有助于从机械上理解 Rab1D在内质网和高尔基体之间蛋白质转运中的作用。 由于增殖细胞依赖于这一途径来传递受体 糖蛋白、生长因子和细胞外基质成分 细胞表面，拟议的研究可能揭示关键的差异 正常和转化的细胞可能作为潜在的靶细胞 治疗性干预。

Covalent attachment of farnesyl or geranylgeranyl isoprenoids to carboxyl- terminal cysteine residues is now widely recognized as an important post- translational protein modification in eucaryotic cells. The majority of prenylated proteins are members of a "superfamily" of Ras-related GTP- binding proteins. The 25 proteins belonging to the Rab subgroup play important roles in intracellular protein trafficking. Rab proteins cycle between intracellular membranes and soluble protein complexes through a series of prenylation-dependent interactions. The general objective for the continuation of this project is to define the structural and functional interactions of Rab1B with two key regulator proteins; Rab Escort Protein (REP) and Guanine Nucleotide Dissociation Inhibitor (GDI). REP is known to bind Rab proteins in vitro, forming a soluble complex required for prenylation b) geranylgeranyl-transferase II. Since REP remains associated with the Rab protein after prenylation, it is postulated to play a targeting or "escort" role in vivo. GDI is known to extract prenylated Rab proteins from intracellular membranes and hold them in a soluble pool where nucleotide exchange is inhibited. The proposed studies will expand the base of knowledge about these proteins in three ways. (1) The hypothesis that distinct structural domains in Rab1B participate in binding to REP and GDI will be tested. This will be done through mutagenesis studies in which the ability of altered Rab1B proteins to associate with REP and GDI will be assessed in cell-free systems and in transfected cells co-expressing Rab1B & REP or Rab1B & GDI. (2) The hypothesis that REP plays a key role in the transfer of Rab1B to the endoplasmic reticulum or to cytosolic GDI complexes in intact cells will be tested through a combination of metabolic labeling, immunochemical and molecular approaches, employing epitope-tagged proteins and Rab1B mutants defective in REP or GDI binding. (3) The hypothesis that the cellular content of activated Rab1B is regulated in relation to cell proliferation in normal and Ras-transformed fibroblasts will be tested. Potential regulation of Rab1B through changes in cellular protein content will be considered, along with the alternative possibility that regulation occurs mainly through changes in Rab1D compartmentalization and GDI interactions. The proposed studies will contribute to a mechanistic understanding of the role of Rab1D in protein trafficking between the ER and Golgi apparatus. Since proliferating cells depend on this pathway for delivery of receptor glycoproteins, growth factors and extracellular matrix components to the cell surface, the proposed studies may reveal critical differences between normal and transformed cells that may serve as potential targets for therapeutic interventions.