The high affinity IgE receptor, FcepsilonR1, of mast cells belongs to an important family of multi-chain immune system receptors that includes the T cell receptor (TCR), the mIg receptor on B cells an the Fcgamma receptors. Cross-linking this heterotrimeric (alphabetagamma2) receptor on RBL-2H3 rat tumor mast cells activates two receptor-bound protein- tyrosine kinases, Lyn and PTK72, and stimulates a cascade of biochemical, ionic and functional responses, including protein-tyrosine phosphorylation, protein-serine/threonine phosphorylation, phospholipase C activation, Ca2+ stores release, Ca2+ influx, secretion, ruffling, spreading, F-actin polymerization and actin plaque assembly. Receptor cross-linking is followed by receptor localization to coated pits and endocytosis. The structural complexity of the FcepsilonR1 has made it difficult to isolate functional domains responsible for this array of responses. We propose to define the separate and/or cooperative activities of the individual subunits of the FcepsilonR1 by use of chimeric receptors consisting of the extracellular and transmembrane domains of the IL-2 receptor (the Tac antigen) linked to full length or truncated cytoplasmic domains of the FcepsilonR1 alpha, beta and gamma subunits. RBL-2H3 cells carrying the native FcepsilonR1 will be transfected with plasmids carrying cDNAs for these chimeric receptors and analyzed systematically for responses mediated through the transfected receptors and through the co-expressed native FcepsilonR1, that will serve as an internal control for our studies. The results of assays in transfected cells for receptor-mediated increases in reactivity towards anti-phosphotyrosine antibody, 1,4,5-IP3 synthesis, Ca2+ mobilization, secretion and membrane and cytoskeletal responses will provide early and critical information linking particular subunits of the FcepsilonR1 to specific functional responses. Transfectants that show receptor-mediated increases in anti-phosphotyrosine reactivity will be studied further both to define the range of proteins that are tyrosine phosphorylated by specific chimeric receptor-kinase complexes and to identify receptor subunits and subunit domains that specifically associate with the protein-tyrosine kinases, Lyn and PTK72. Studies by ratio imaging microscopy will link particular receptor subunits to the control of Ca2+ stores release and Ca2+ influx. We will identify subunit cytoplasmic domains that activate the 3-phosphate-containing phosphatidylinositol pathway. We will also identify subunit cytoplasmic domains that control the coated pit-mediated endocytosis of cross-linked receptors. The use of chimeric receptors to isolate signal transduction events mediated through individual cytoplasmic domains of this complex receptor is an important step towards constructing a functional map of the oligomeric FcepsilonR1 and, more generally, towards understanding signal transduction mediated through the family of multi-chain receptors of the immune system.
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