ACTIVATION OF RTK KINASE ACTIVITY

本提案的总体目标是调查 配体结合激活受体酪氨酸激酶（RTK）的激酶。 我们将使用血小板衍生生长因子（PDGF）α和β 受体作为模型系统。 突变两个酪氨酸残基， PDGFR β受体（β PDGFR）的质膜（JM）结构域严重 削弱受体的配体刺激的激酶活性。 以来 突变的受体保留了基础激酶活性，并且可以在 在体外，这些突变似乎主要干扰配体- 刺激受体的激活，而不是引起结构性的 破坏酶活性的改变。 而且 高度相关的PDGFR α受体（alphaPDGFR）不受 类似的突变 这些发现提出了一个中心假设， β PDGFR的JM结构域在以下方面起着关键作用： 激活β PDGFR的激酶活性。 我们将测试这一假设，并探讨参与的机制， β PDGFR激酶激活在以下一组特定目的中。 1. β-PDGFR突变体的PDGF刺激激酶活性是否可以被 通过与α PDGFR异源二聚化来拯救它 2. 构建并表征嵌合受体，其中JM结构域 alpha和betaPDGFR是互换的。 3. 测试Src是否有助于配体依赖性激活 β PDGFR的激酶活性。 4. 确定β PDGFR是否可以被Src磷酸化， 磷酸化位点，以及对受体催化活性的影响。 这项建议的产物将是关于激酶如何 RTK的活动正在进行。 由于RTK规范了许多重要的 生物过程中，这些信息将有广泛的应用。 为 例如，能够防止驱动RTK的激活 血管生成将显著改善糖尿病和 黄斑变性视网膜病变

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.