RAS-LIKE GTP-BINDING PROTEINS--REGULATION AND FUNCTION

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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.