MCB-9812248 Hahn, Klaus Project Summary The spatial organization of signaling components is critically important in the regulation of signaling pathways within intact cells. Signals are often produced only when signaling molecules are brought together in supramolecular complexes, or when a protein is activated in a specific subcellular location. Although there is clear evidence for the importance of spatial organization in signal transduction, it has been very difficult to gather quantitative information about protein-protein interactions in the intact cell. The purpose of this project is to examine the location and kinetics of receptor-dependent tyrosine kinase activity in intact cells. Receptor tyrosine kinases can trigger multifarious cellular responses produced by different extracellular stimuli even though signaling is mediated through interactions of the phosphorylated receptors with a common set of binding domains (SH2 domains) of downstream molecules. Evidence suggests that the different responses are produced by variations in the intracellular locations and kinetics of the interactions between the SH2 domain with its partner receptors. A main part of the project will be to synthesize novel fluorescent reporters for the SH2 domain of phospholipase C gamma. The goal is to attach the fluorescent dye to this SH2 domain at a position that makes its fluorescence highly sensitive to its molecular environment. If the method is successful, this dye will report where and when the SH2 domain binds to specific activated receptors, in this case epidermal growth factor receptor or nerve growth factor receptor. Thus, the formation, breakdown and trafficking of these complexes can be monitored in real time. Development of the SH2-based indicator will employ peptide synthesis of the labeled SH2 domain and a novel combinatorial screening assay that will enable the selection of that labeled peptide with the optimal fluorescent properties. While the focus of this project will be on SH2 interactions, this approach should be generally applicable for the design of reporter molecules to study a wide range of protein-protein interactions in living cells.
