ACTIVATION OF RTK KINASE ACTIVITY

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The overall goal of this proposal is to investigate the mechanism by which ligand-binding activates the kinase of receptor tyrosine kinases (RTKs). We will use the platelet-derived growth factor (PDGF) alpha and beta receptors as a model system. Mutating two tyrosine residues in the juxtamembrane (JM) domain of the PDGFR beta receptor (betaPDGFR) severely impairs the ligand-stimulated kinase activity of the receptor. Since the mutated receptor retains basal kinase activity, and can be activated in vitro, it appears that these mutations interfere primarily with the ligand- stimulated activation of the receptor, as opposed to causing a structural alteration that destroys the activity of the enzyme. Furthermore, the highly related PDGFR alpha receptor (alphaPDGFR) is unaffected by the analogous mutations. These findings give rise to the central hypothesis of the grant proposal; The JM domain of the betaPDGFR plays a key role in activation of the betaPDGFR's kinase activity. We will test this hypothesis, and explore the mechanism involved in betaPDGFR kinase activation in the following set of Specific Aims. 1. Can the PDGF-stimulated kinase activity of the betaPDGFR mutant be rescued by heterodimerizing it with the alpha PDGFR? 2. Construct and characterize chimeric receptors in which the JM domains of the alpha and betaPDGFR are interchanged. 3. Test if Src contributes to ligand-dependent activation of the betaPDGFR's kinase activity in an in vivo setting. 4. Determine if the betaPDGFR can be phosphorylated by Src, identify the sites phosphorylated, and the affect on the receptor's catalytic activity. The product of this proposal will be information regarding how the kinase activity of RTKs is engaged. Because RTKs regulate so many important biological processes, this information will have a wide application. For instance, the ability to prevent activation of the RTKs that drive angiogenesis will dramatically improve the treatment of both diabetic and macular degenerative retinopathy.