ISOPRENYLATED GTP-BINDING PROTEINS AND CELL GROWTH

法呢基或香叶基香叶基类异戊二烯与羧基的共价连接 末端半胱氨酸残基现在被广泛认为是重要的后半胱氨酸残基， 真核细胞中的翻译蛋白质修饰。大多数 异戊二烯化蛋白是Ras相关GTP的“超家族”的成员， 结合蛋白属于Rab亚组的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物之间的相互作用， 一系列异戊烯化依赖的相互作用。总体目标是 该项目的继续是确定结构和 Rab 1B与两个关键调节蛋白的功能相互作用 护送蛋白（REP）和鸟嘌呤核苷酸解离抑制剂（GDI）。 已知REP在体外结合Rab蛋白，形成可溶性复合物 异戊烯化所需B）香叶基香叶基-转移酶II。 由于REP 在异戊二烯化后仍然与Rab蛋白结合， 假定在体内起靶向或“护送”作用。GDI被认为是 从细胞内膜中提取异戊二烯化的Rab蛋白， 在其中核苷酸交换被抑制的可溶池中。拟议 这些研究将在三个方面扩展有关这些蛋白质的知识基础。 的方式(1)Rab 1B中不同的结构域 参与REP和GDI的结合将被测试。为此将 通过突变研究，改变Rab 1B蛋白的能力， 将在无细胞系统和无细胞系统中评估与REP和GDI相关的 共表达Rab 1B和REP或Rab 1B和GDI的转染细胞。(2)的 假设REP在Rab 1B转移到 完整细胞中的内质网或胞质GDI复合物将 通过代谢标记、免疫化学和 采用表位标记蛋白和Rab 1B突变体的分子方法 REP或GDI结合缺陷。(3)假设细胞 Rab 1B的活化含量与细胞增殖有关 将测试正常和Ras转化的成纤维细胞中。潜在 通过改变细胞蛋白质含量来调节Rab 1B， 考虑到，沿着另一种可能性， 主要通过Rab 1D区室化和GDI相互作用的变化。 拟议的研究将有助于从机理上理解 Rab 1D在内质网和高尔基体之间蛋白质运输中的作用。 由于增殖细胞依赖于该途径来递送受体 糖蛋白、生长因子和细胞外基质成分 细胞表面，拟议的研究可能会揭示关键的差异之间 正常细胞和转化细胞可能作为潜在的靶点 治疗干预。

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.