ISOPRENYLATED GTP-BINDING PROTEINS AND CELL GROWTH

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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.