CONTROL OF SECRETION BY SMALL GTP BINDING PROTEINS

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Mast cells play a central role in allergic diseases, including asthma, and also have an essential role in defense against overwhelming bacterial infection. The principal effector mechanism is secretion of the contents of performed granules which contain a variety of inflammatory mediators. Our long term goals are to know the molecular composition of the mast cell exocytic machine, understand its function, and determine its role in mammalian physiology. Rab3 proteins are small GTPases that modulate exocytic and endocytic function in diverse cell types, but whose molecular function remains poorly understood. We have found that Rab3D is localized on mast cell granules and translocates to the plasma membrane upon exocytosis. We hypothesize that its absence due to gene ablation will result in alterations in the structural organization of the secretory compartment of cells normally expressing Rab3D; changes in the cellular physiology of secretion and endocytosis leading to alterations in the allergenic and microbial defence functions of mast cells; changes in biochemical interactions among other components of the exocytic machine, particularly the fusion protein Syntaxin and its regulatory Sec1; and changes in whole animal physiology under conditions of allergic or infectious challenge. We will test these hypotheses in Rab3D deletant mice that we are generating, in existing Rab3A deletant mice since Rab3A is found in the cytoplasm of mast cells, and in double deletant mice. Our biochemical and cell physiological analyses will focus on mast cells harvested from deletant mice, and the morphologic analysis will focus both on mast cells and on the pancreas which is also rich in Rab3D. Accomplishment of our goals will provide insight into the molecular function of Rab3 proteins and advance understanding of the role of mast cells in protective and pathophysiologic immune function.