Understanding EGF receptor activation by growth factors and oncogenic mutations

 描述（由申请人提供）：表皮生长因子受体（EGFR）通常被描述为“原型”受体酪氨酸激酶（RTK），其活化通常被认为是通过直接生长因子诱导的受体二聚化发生的。然而，用这样一个简单的模型，很难解释不同的活化配体，如TGF α，β细胞素，HB-EGF，epiregulin，epigen和双调蛋白如何通过相同的受体引发不同的细胞信号-可能是通过不同的自磷酸化模式或通过运输差异。此外，一个简单的二聚化模型不能解释如何突变激活形式的EGFR在肺癌（激酶突变）和胶质母细胞瘤（细胞外错义突变或缺失的EGFRvIII）将有不同的抑制剂敏感性，如已报道的。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性结合配体。因此，我们专注于详细了解这种受体的变构调节，以期了解EGFR信号传导特异性，并确定哪些致癌突变促进对哪类EGFR抑制剂的敏感性。这些研究也可能为靶向治疗提供新的策略。 为了深入了解整个膜的变构变化的通信，我们提出了EGFR激活其不同的配体-或致癌突变-使用各种技术的结构和生化研究。重要的是，我们还将使用氢/氘交换质谱法（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细胞外区域以及由配体或突变激活的完整EGFR进行HX-MS研究，以深入了解细胞外结构域、激酶结构域和连接区域的构象波动。我们将确定配体结合或致癌突变如何改变完整EGFR的构象、动力学和激活程度，以及这些变化如何跨膜传播。我们的初步数据还表明，HX-MS分析将提供有价值的洞察混乱的羧基末端尾巴的作用，以及它如何可能有助于信号特异性。 我们的总体目标是弥合目前EGFR家族成员结构信息之间的鸿沟，我们对细胞中跨膜信号传导特性的理解，以及癌症中的激活（和抑制）机制。我们的具体目标解决两个主要问题：1如何激活复合物的EGFR诱导其七种不同的配体在结构，活性和动力学不同？2胶质母细胞瘤中发现的EGFR致癌形式在结构、活性和动力学方面与配体激活受体相比如何-以及差异如何导致激酶抑制剂选择性改变？

 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?