The goal of this project is to determine the mechanisms that regulate the activities of small, ras-like GTP binding proteins, and to investigate possible functions. A family exists of >20 such proteins, of unknown function, with 20-60% homology to the Ki-ras protein, that probably operate as """"""""molecular switches'. Their medical importance is underlined by the discovery that one member, rap1 (or Krev), suppresses transformation by ras, and another, rho is a target for botulinum toxin. Multiple factors control the switch-states of these proteins, catalyzing GDP/GTP exchange or GTPase activity, or inhibiting GDP release. The ras proteins are also extensively modified, covalently. It is important to understand the functions and control of these GTP-binding proteins. The main focus will be on the rab subclass. The project is divided into 3 main sections: 1. Characterization. Specific antibodies are required against rab peptides and recombinant rab proteins expressed i E. coli. These reagents will be used to determine tissue distribution, subcellular localization and covalent modifications of the rab proteins. The hypothesis will be tested that different C-terminal consensus sequences control modification and localization. 2. Regulation. A ras-specific guanine nucleotide releasing factor has been discovered, that could function as an """"""""on switch"""""""". The existence of similar factors specific to the rab proteins will be investigated. Other factors, that control rab GTPase activity (GAPS) will also be identified and purified, to determine control mechanisms. 3. Function. Three approaches will be used. (a) Affinity columns will be prepared from recombinant rab proteins and used to purify factors that bind specifically to the GTP-states of the proteins. Such factors are potential targets (b) The transforming potential, and transformation-suppressing activity of the rab proteins will be measured, to define the specificity of the properties of ras Krev. (c) Finally, the hypothesis will be tested, in collaboration with other laboratories, that the rab proteins normally function to control exocytosis and/or interorganelle transport, using in vitro assays.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA056300-02
Application #
3200702
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1991-08-15
Project End
1996-01-31
Budget Start
1992-03-01
Budget End
1993-01-31
Support Year
2
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Joberty, G; Stabila, P F; Coppola, T et al. (1999) High affinity Rab3 binding is dispensable for Rabphilin-dependent potentiation of stimulated secretion. J Cell Sci 112 ( Pt 20):3579-87
Chung, S H; Joberty, G; Gelino, E A et al. (1999) Comparison of the effects on secretion in chromaffin and PC12 cells of Rab3 family members and mutants. Evidence that inhibitory effects are independent of direct interaction with Rabphilin3. J Biol Chem 274:18113-20
Joberty, G; Perlungher, R R; Macara, I G (1999) The Borgs, a new family of Cdc42 and TC10 GTPase-interacting proteins. Mol Cell Biol 19:6585-97
Neudauer, C L; Joberty, G; Tatsis, N et al. (1998) Distinct cellular effects and interactions of the Rho-family GTPase TC10. Curr Biol 8:1151-60
McKiernan, C J; Stabila, P F; Macara, I G (1996) Role of the Rab3A-binding domain in targeting of rabphilin-3A to vesicle membranes of PC12 cells. Mol Cell Biol 16:4985-95
Darchen, F; Senyshyn, J; Brondyk, W H et al. (1995) The GTPase Rab3a is associated with large dense core vesicles in bovine chromaffin cells and rat PC12 cells. J Cell Sci 108 ( Pt 4):1639-49
Macara, I G; Brondyk, W H (1995) Oligonucleotide mutagenesis of Rab GTPases. Methods Enzymol 257:107-18
Holz, R W; Brondyk, W H; Senter, R A et al. (1994) Evidence for the involvement of Rab3A in Ca(2+)-dependent exocytosis from adrenal chromaffin cells. J Biol Chem 269:10229-34
Brondyk, W H; McKiernan, C J; Burstein, E S et al. (1993) Mutants of Rab3A analogous to oncogenic Ras mutants. Sensitivity to Rab3A-GTPase activating protein and Rab3A-guanine nucleotide releasing factor. J Biol Chem 268:9410-5
Burstein, E S; Brondyk, W H; Macara, I G et al. (1993) Regulation of the GTPase cycle of the neuronally expressed Ras-like GTP-binding protein Rab3A. J Biol Chem 268:22247-50

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