Signal transduction through the multi-subunit antigen receptors of immune system cells (the TCR, the BCR, the Fc epsilon receptor and others) depends on conserved tyrosines within the immunoreceptor tyrosine-based activation motifs (ITAMs) of the receptor subunit cytoplasmic domains. A consensus model of this signalling pathway has developed in which Src-family tyrosine kinases are inherently associated with the antigen receptor cytoplasmic tails and are activated by receptor crosslinking. This initial response permits the recruitment and activation of additional Src-family kinases, the phosphorylation of ITAMs, and the binding of Syk tyrosine kinase family members to phospho-ITAMs, resulting in Syk activation and signal transduction. The data obtained provided important building blocks for this model, including evidence that the ITAM-containing gamma subunit is the principal signal transducing subunit of the heterotrimeric (alpha beta gamma 2) Fc epsilon receptor, and that Syk is the critical kinase coupling this receptor to downstream responses. Nevertheless, many challenges remain. First, the model assumes that the presence of an ITAM predicts the ability to activate tyrosine kinases. However, tyrosine kinase activation could not be demonstrated for the ITAM-containing Fc epsilon beta subunit C-terminal cytoplasmic domain, and cocrosslinking studies led to the hypothesis that beta-c subunit crosslinking instead activates a tyrosine phosphatase that antagonizes Fc epsilon receptor mediated responses. In the renewal period, the applicant proposes to localize the negative signalling activity of TTbeta-c to specific beta-c cytoplasmic tail domains and will determine if this domain sequesters Lyn or activates a phosphatase, possibly PTP-1C. Novel beta-c-binding proteins will be isolated by GST:beta-c affinity chromatography for functional and biochemical characterization. Second, the consensus model provides no function for additional features of antigen receptors, including the Fc epsilon R1 beta subunit N-terminal cytoplasmic domain. This will be addressed by determining if crosslinking chimeric receptors whose cytoplasmic tail consists of Fc epsilon R1 beta-N alters Fc epsilon R1-mediated signalling responses. Finally, the consensus model attributes no signalling activity to the antigen binding subunits. The applicant has identified two antibodies to the Fc epsilon R1 alpha subunit with similar binding activity but divergent tyrosine phosphorylation and signalling responses. The applicant, therefore, hypothesizes that ligand binding subunits can additionally direct crosslinked receptors to signalling pathways that favor either kinase or phosphatase activation. This hypothesis will be tested, and also the contribution of dimer topography selection will be determined. The results of these studies will reveal specific mechanisms involved in Fc epsilon R1-mediated signalling, identify new targets for the control and treatment of allergic diseases, and contribute to the next generation of models of antigen receptor signalling.
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