The goal of this research is to develop a novel approach to study the spatial and temporal regulation of SH2-RTK interactions inside living cells and correlate them to specific cellular responses. The key aspects of this new technique is to chemically synthesize a fluorescently labeled PLCgamma SH2 domain to probe SH2-RTK interactions in the intracellular environment. This labeled protein will report binding of its cognitive phosphotyrosine peptide by a shift in fluorescence that will enable quantitation of kinetics and localization. Chemical synthesis of the SH2 domain permits unprecedented control over dye placement within the protein structure for maximal spectral response and minimal binding perturbation. We will microinject the synthetic, labeled PLCgamma SH2 domain into serum starved cells, stimulate with growth factor (ie. EGF) to initiate SH2-RTK interactions, then determine localization and kinetics for these interactions. This research will significantly enhance our understanding of the importance of spatial and temporal regulation in cellular signaling and generate a new technology capable of de-convoluting complicated biochemical reactions inside living cells.
