Growth control represents a balance of positive and negative growth stimuli, and is dependent on the precise relay of intracellular signals. The broad goals of this research proposal are to understand the process of signal transduction, as it relates to growth control. This information will provide the foundation for designing strategies for the effective treatment of pathological conditions such ad cancer, which arise from unmoderated proliferation. The proposed research examines the mechanism of signal transduction of the platelet-derived growth factor receptor (PDGFR). The intracellular domain of the PDGFR is a tyrosine kinase which is activated by the binding of PDGF. This results in the tyrosine phosphorylation of numerous intracellular proteins including the receptor itself. Receptor autophosphorylation permits the stable association of several signal transduction enzymes. Our studies with receptor mutants have shown that those receptors that fail to associate with any of the signal transduction molecules also fail to mediate a biological signal which strongly suggests that the receptor associated proteins are the most likely intracellular mediators of PDGF's mitogenic signal. I propose to define the relative contribution of each of the receptor- associated proteins to PDGF-mediated signal transduction. This will be accomplished by generating random receptor mutants, screening for those that constitutively mediate a biological signal, and then identifying the proteins that associate. By focusing on the constitutively activated receptor mutants that bind a reduced set of signal relay molecules, we will define the minimal number of proteins needed to bind to the PDGFR in order to relay PDGF's biological signal. In addition, by determining the precise location of the activating mutations we will identify regions of the receptor that regulate the binding of these signal transduction molecules. The proteins that associate with the PDGFR have in large part been identified, and this information has provided valuable insight into how the receptor transmits a biological signal. Two of the receptor-associated proteins, a 120 and 65 kd species, have not yet been identified. As the second aim of this application, I propose to isolate the cDNAs corresponding to these two proteins, so that they could be identified and studied further. This will be accomplished by screening a cDNA expression library with the phosphorylated receptor, to which we have demonstrated that the 120 and 65 kd proteins bind in vitro. These two approaches are both the most likely to succeed and the most direct way to answering the question of how PDGF's mitogenic signal is relayed intracellularly. In addition, given that the proteins that associate with the PDGFR also associate with a number of other growth factor receptors and activated tyrosine kinases, what we learn of the mechanism of signal relay by these associated proteins for the PDGFR system will be widely received in the field of signal transduction.
