In mast cells, basophils and the RBL-2H3 mast cell line, antigens that crosslink the high affinity IgE receptor, Fc-epsilonR1, lead to the release of histamine and other inflammatory agents by degranulation. Although receptor-mediated degranulation is GTP-dependent, the Fc- epsilonR1 is not a classical G-protein linked receptor. Rather it belongs to the family of immune system antigen receptors, including the T cell receptor, the mIg receptor of B cells and the Fc-gamma receptors, that modulate cellular function through the activation of cytoplasmic tyrosine kinases. Growth factor receptors also signal through tyrosine kinase activation; recent studies in the EGF and PDGF receptor systems have shown that the prototypic low molecular weight G-protein, ras, is activated when the autophosphorylated receptor stimulates the SH2/SH3 adaptor protein, Grb2, that complexes with the guanine nucleotide exchange factor, Sos. Rab3 proteins are ras-related proteins that are implicated in synaptic vesicle fusion and regulated exocytosis from endocrine cells. Recently, we obtained a full-length clone of a rab3 isoform, rab3D, from RBL-2H3 cells. Rab3D's hypervariable regions contain two proline-rich motifs, PXXP, that fit the criteria for binding to SH3 (Src homology 3) domains. We hypothesize that rab3D is the principal mediator of GTP-dependent, Ca2+- regulated secretion in mast cells. We hypothesize further that its activation occurs by a signaling cascade in which Fc-epsilonR1-associated tyrosine kinases activate a SH2/SH3-containing adaptor protein/guanine nucleotide exchange factor complex that in turn activates rab3D. Immunolocalization studies with rab3D-specific antibodies will be used to demonstrate the association of rab3D with RBL-2H3 cell secretory granules. The hypothesis that rab3D activity is essential for degranulation will be tested by studies of granule morphology and secretion in stable RBL-2H3 transfectants overexpressing mutant rab3D proteins that are defective in guanine nucleotide binding or GTPase activity. Rab3D mutants which lack the two PXXP motifs will be designed to test the hypothesis that these SH3-binding regions are essential to couple Fc-epsilonR1 crosslinking to secretion PXXP-motif peptides will be used as affinity supports to purify and characterize SH3 domain-containing proteins which may serve as adaptors, guanine nucleotide exchange factors, or both for rab3D. Finally, the conformationally locked rab3D mutants will be used in co-precipitation studies and as affinity substrates in order to identify proteins which interact with rab3D downstream of activation; these proteins are likely to represent components of the mast cell granule's docking or fusion machinery. The proposed studies are expected to provide a complete description of the pathway coupling Fc-epsilonR1 crosslinking to secretion in mast cells. They will serve as a model for understanding mechanisms of regulated secretion in other immune system cells.
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