and Specific Aims.) Stimulated exocytosis is a key regulatory process by which cells control the release of signalling and effector molecules into their environment, and also control the composition of their surface membranes. It has recently become apparent that small Ras-related GTPases of the Rab family play a ubiquitous and critical role in intracellular membrane transfers, including exocytosis. Like other family members of the extended GTPase superfamily, Rab proteins have been found to possess predictable biochemical activities, such as regulated nucleotide exchange and regulated GTPase activities. These biochemical activities provide a powerful rationale for the identification of associated proteins. It is the central hypothesis of this application that the putative exocytic Rab in rat basophilic leukemia (RBL) cells, Rab3""""""""e"""""""", can be used as a biochemical and molecular genetic tool to discover other components of the regulated exocytic machine in RBL cells.
The Specific Aims are to: 1) clone Rab3""""""""e"""""""" cDNA from an RBL cell library; 2) establish the cell-specific localization of Rab3""""""""e"""""""" from alveolar T2 cells and from RBL cells by in situ hybridization and Northern blot hybridization; 3) establish the subcellular localization of Rab3""""""""e"""""""" from T2 cells and from RBL cells by expression of epitope-tagged constructs of both in RBL cells, and by immunocytochemistry in T2 cells and RBL cells using anti-peptide antisera and Rab3""""""""e""""""""-specific anti-holoprotein sera; 4) establish the molecular function of Rab3""""""""e"""""""" from T2 cells and from RBL cells in regulated exocytosis by loss of exocytic function through the expression of dominant suppressor mutants and the use of antisense oligonucleotides in RBL cells; 5) analyze structure/function relationships for Rab3""""""""e"""""""" in RBL cells using transient and stable transfections of mutant and chimeric Rab3""""""""e"""""""", and through the use of peptides from the """"""""effector"""""""" region and other domains; 6) employ the RBL cell as a system for discovery of Rab3""""""""e""""""""-associated components of the regulated exocytic machine using protein biochemical assays based on nucleotide exchange and GTPase activities; 7) identify other proteins, whose interactions with Rab3""""""""e"""""""" cannot be biochemically predicted, by physical and genetic methods; and 8) identify alveolar T2 cell homologues of the RBL exocytic machine by homology cloning using probes derived from RBL cells. Elucidation of the molecular components of the regulated exocytic machine may be an important step in understanding and in attempting to pharmacologically influence secretory function in alveolar T2 cells and tissue mast cells.
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