The crosslinking of FcepsilonR1 on the surface of mast cells activates intracellular signaling pathways that lead to the lease of bioactive mediators, many of which contribute to the pathogenesis of allergic diseases including bronchial asthma. The gp49 family is a subset of the immunoglobulin (Ig) superfamily whose members have significantly homologous Ig-like domains, some molecules also have multiple immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains. The coligation of mouse gp49 family ITIM receptors, such as gp49Bl and PIR-B to crosslinked FcepsilonR1 on mast cells inhibits the exocytosis of secretory granule mediators in a manner that is dependent on the association of the receptors with the tyrosine phosphatase SHP-1. The broad objective of the proposed research is to address the expression, function, and signal transducing mechanisms of ITIM receptors in mouse mast cells.
Specific Aim 1 will define the diversity of gp49 family ITIM receptor expression in three phenotypically distinct mouse mast cell populations: a committed progenitor derived by culture of bone marrow in stem cell factor (SCF), IL-6, and IL-10 immature mast cells obtained from bone marrow cultured in IL-3: and ex vivo-derived serosal mast cells, which typify a mature mast cell. Novel gp49 family ITIM receptors will be sought by molecular cloning approaches and chromosome """"""""walking,"""""""" analogous to an approach used in Project 1 to expand the family of mouse mast cell proteases. Differences in the expression of existing and any novel gp49 family ITIM receptors will be monitored in the mast cell populations at the mRNA and protein levels.
Specific Aim 2 will compare the capacity of gp49 family ITIM receptors to inhibit the release of secretory granule arachidonate-derived, and cytokine mediators from the distinct mast cell populations In addition, the innate inhibitory capacities of the molecules will be determined by expressing the cytoplasmic domain of each gene family ITIM receptor as a chimera with a common extracellular and transmembrane domain in a rat mast cell line.
Specific Aim 3 will elucidate the mechanisms by which gp49 family ITIM receptors inhibitor mediator release by focusing on structural determinants in the cytoplasmic domains of the molecules and how they interact with proximal inhibitory enzymes, whose substrates will also be identified. Because the proposed studies are designed to increase understanding of how mast cell activation is counter-regulated by endogenous cell surface receptors, this project is closely related to the central theme of this application, namely, that the regulation of mediator release from mast cells is a fundamental determinant in the course of allergic inflammation, including that of the airways.
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