Understanding EGF receptor activation by growth factors and oncogenic mutations

 描述(申请人提供)：表皮生长因子受体(EGFR)通常被描述为一种典型的受体酪氨酸激酶(RTK)，其激活通常被认为是通过直接的生长因子诱导的受体二聚体发生的。然而，对于这样一个简单的模型，很难解释不同的激活配体，如TGFa、β-细胞蛋白、HB-EGF、表观调节蛋白、EpiGen和双调节蛋白如何通过相同的受体--可能通过不同的自磷酸化模式或通过不同的转运差异--诱导不同的细胞信号。此外，简单的二聚化模型不能解释肺癌(有激酶突变)和胶质母细胞瘤(有细胞外错义突变或缺失，如EGFRvIII)中突变激活的EGFR形式如何具有明显的抑制敏感性，如已报道的那样。这些观察结果和许多其他结果表明，EGFR是一种复杂的变构调节受体，我们知道它以负协同作用结合配体。因此，我们专注于详细了解该受体的变构调节，以期了解EGFR信号的特异性，并确定哪些癌基因突变促进了对哪些类别的EGFR抑制剂的敏感性。这些研究还可能为靶向治疗提供新的策略。为了深入了解跨膜的变构变化的沟通，我们建议使用各种技术对不同配体激活的EGFR或由致癌突变激活的EGFR进行结构和生化研究。重要的是，我们还将使用氢/氢交换质谱仪(HX-MS)分析受体的构象动力学。将对野生型和突变受体的分离胞外区以及由配体或突变激活的完整EGFR进行HX-MS研究，以深入了解胞外区、激酶区和连接区的构象波动。我们将确定配体结合或致癌突变如何改变完整的EGFR的构象、动力学和激活程度，以及这些变化是如何跨膜传播的。我们的初步数据还表明，HX-MS分析将为无序的羧基末端尾巴的作用提供有价值的见解，以及它可能如何有助于信号特异性。我们的总体目标是弥合目前关于EGFR家族成员的结构信息、我们对它们在细胞中的跨膜信号特性的理解以及在癌症中激活(和抑制)机制之间的鸿沟。我们的具体目标是解决两个主要问题：1由七种不同的配体诱导的EGFR激活的复合体在结构、活性和动力学方面有何不同？2在胶质母细胞瘤中发现的致癌形式的EGFR在结构、活性和动力学方面如何与配体激活的受体进行比较-以及差异如何导致激酶抑制剂选择性的改变？

 DESCRIPTION (provided by applicant): The epidermal growth factor receptor (EGFR) is often described as a `prototypic' receptor tyrosine kinase (RTK), and its activation is typically considered to occur through straightforward growth factor-induced receptor dimerization. With such a simple model, however, it is difficult to explain how different activating ligands such as TGFa, betacellulin, HB-EGF, epiregulin, epigen, and amphiregulin can elicit distinct cellular signals through the same receptor - possibly through different patterns of autophosphorylation or through trafficking differences. Moreover, a simple dimerization model cannot explain how mutationally activated forms of EGFR in lung cancer (with kinase mutations) and glioblastoma (with extracellular missense mutations or deletions as in EGFRvIII) would have distinct inhibitor sensitivities, as has been reported. These observations and many others suggest that EGFR is a complex allosterically regulated receptor, which we know binds ligands with negative cooperativity. We are therefore focused on understanding allosteric regulation of this receptor in detail, with a view to understanding EGFR signaling specificity and identifying which oncogenic mutations promote sensitivity to which classes of EGFR inhibitors. These studies may also suggest new strategies for targeted therapeutics. To gain insight into the communication of allosteric changes across the membrane, we propose structural and biochemical studies of EGFR activated by its different ligands - or by oncogenic mutations - using a variety of techniques. Importantly, we will also analyze the receptor's conformational dynamics using hydrogen/deuterium exchange mass spectrometry (HX-MS). HX-MS studies will be performed for isolated extracellular regions of wild-type and mutated receptors and for intact EGFR activated by ligands or mutations - to provide insight into conformational fluctuations in the extracellular domain, kinase domain, and linking regions. We will determine how ligand binding or oncogenic mutation alters the conformation, dynamics, and extent of activation of the intact EGFR, and how these changes are propagated across the membrane. Our preliminary data also suggest that HX-MS analyses will provide valuable insight into the role of the disordered carboxy-terminal tail, and how it might contribute to signaling specificity. Our overall goal is t bridge the current gulf between the structural information about EGFR family members, our understanding of their transmembrane signaling properties in cells, and mechanisms of activation (and inhibition) in cancer. Our Specific Aims address two main questions: 1 how do activated complexes of EGFR induced by its seven different ligands differ in structure, activity, and dynamics? 2 How do oncogenic forms of EGFR found in glioblastoma compare in structure, activity, and dynamics to the ligand-activated receptor - and how do differences lead to altered kinase inhibitor selectivity?