ACTIVATION OF RTK KINASE ACTIVITY

这项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。突变了两个酪氨酸残基 严重的PDGFRβ受体膜旁(JM)结构域 损害受体的配体刺激的激酶活性。自.以来 突变的受体保留了基础激酶的活性，并可以在 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 破坏酶活性的变化。此外， 高度相关的PDGFRα受体(αPDGFR)不受 类似的突变。这些发现引出了中心假说 赠款提案；BetaPDGFR的JM域在以下方面发挥关键作用 激活β-PDGFR的激酶活性。 我们将检验这一假说，并探索 在以下一组特定目标中，β-PDGFR激酶被激活。 1.β-PDGFR突变体的PDGF刺激的激酶活性能否 通过与阿尔法PDGFR的异二聚化来拯救它？ 2.构建和鉴定JM结构域的嵌合受体 α和β的PDGFR是互换的。 3.测试Src是否有助于配体依赖的激活 活体环境下的β-PDGFR的激酶活性。 4.确定βPDGFR是否可以被Src磷酸化，确定 磷酸化的位点，以及对受体催化活性的影响。 这一提议的产物将是关于激酶如何 RTK的活动正在进行。因为RTK规范了许多重要的 生物过程中，这种信息将会有广泛的应用。为 实例中，能够阻止激活驱动程序的RTK 血管新生将显著改善糖尿病和糖尿病的治疗 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.