Mechanistic Studies of EGFR/ErbB Receptor Tyrosine Kinases

描述（由申请方提供）：受体酪氨酸激酶的表皮生长因子受体（EGFR/ErbB）家族包括4个人类家族成员：EGFR（HER 1、ErbB 1）、HER 2（ErbB 2、Neu）、HER 3（ErbB 3）和HER 4（ErbB 4）。每个家族成员由细胞外配体结合区、单个跨膜区、细胞质酪氨酸激酶和约230个氨基酸的C末端尾组成。所有EGFR/ErbB家族成员对正常胚胎发育都是必需的，并且每个ErbB的异常活性与人类癌症相关。特别是，过表达或活性EGFR和HER 2与肺癌、结肠癌和头颈癌（EGFR）或乳腺癌（HER 2）的严重程度增加相关，并且靶向EGFR和HER 2的几种药物是FDA批准的抗癌疗法。EGFR活性的典型模型是与胞外区结合的配体稳定受体的特异性二聚体构象，其中其激酶活性被刺激，这导致受体C-尾和其他底物的磷酸化，下游效应物的定位和/或活性的变化，以及改变细胞生长和分化的信号传导级联的启动。HER 2和HER 3是非典型ErbB，因为HER 2没有已知的配体，而HER 3由于其激酶结构域中的关键突变而几乎没有或没有激酶活性。然而，HER 2和HER 3似乎都是其他ErbB家族成员的异二聚体伴侣，并且根据许多标准，HER 2/HER 3异二聚体是最有效的信号传导和致癌ErbB配对。从ErbB片段的结构和功能研究中，我们已经了解了很多关于EGFR/ErbB家族成员的信息，但是我们对EGFR/ErbB活性的分子内和分子间调控的了解却受到了研究完整ErbBs的困难的影响。在过去的五年中，我们的实验室合作表达和纯化了近毫克量的稳定，活性全长和近全长形式的EGFR。我们已经使用这种材料来提供配体激活和抑制状态下EGFR酶活性的严格表征，并已开始提供肺癌中发现的EGFR激活突变的影响的表征。我们建议通过扩展我们对纯化EGFR的酶学和功能研究来提供ErbB活性及其在正常和疾病状态下的调节的全面的机制表征，以确定（i）EGFR的特定癌症相关突变对EGFR活性的影响，以及通过这些突变与靶向EGFR特定区域的额外突变的组合，这些突变发挥其作用的机制，（ii）EGFR抑制剂和抑制剂组合靶向特定形式的活化EGFR的能力，（iii）EGFR糖基化和EGFR C-尾对EGFR活性和底物特异性的影响，和（iv）EGFR的体外底物 EGFR和突变EGFR使用17，000蛋白质微阵列。我们最近已经扩大了我们的表达和纯化的HER 2，HER 3和HER 4，我们建议进行类似的机制和酶学研究的内在和调节这些ErbBs的活动。

DESCRIPTION (provided by applicant): The epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases includes four family members in humans: EGFR (HER1, ErbB1), HER2 (ErbB2, Neu), HER3 (ErbB3), and HER4 (ErbB4). Each family member consists of an extracellular ligand-binding region, a single membrane-spanning region, a cytoplasmic tyrosine kinase, and a C-terminal tail of ~230 amino acids. All EGFR/ErbB family members are essential for normal embryonic development, and abnormal activity of each ErbB has been associated with human cancer. In particular, overexpressed or active EGFR and HER2 are associated increased severity of lung, colon, and head-and-neck cancers (EGFR) or breast cancer (HER2), and several drugs targeting EGFR and HER2 are FDA-approved anticancer therapies. The canonical model of EGFR activity is that a ligand binding to the extracellular region stabilizes a specific dimeric conformation of the receptor in which its kinase activity is stimulated, which results in phosphorylation of the receptor C-tail and other substrates, changes in the localization and/or activity of downstream effectors, and initiation of signaling cascades that alter cell growth and differentiation. HER2 and HER3 are atypical ErbBs in that HER2 has no known ligand and HER3 has little or no kinase activity owing to key mutations in its kinase domain. Both HER2 and HER3 appear to function as heterodimeric partners of other ErbB family members, however, and the HER2/HER3 heterodimer is the most potent signaling and oncogenic ErbB pairing by many criteria. Much has been learned about EGFR/ErbB family members from structural and functional studies of ErbB fragments, but our knowledge of intra- and intermolecular regulation of EGFR/ErbB activity has suffered from difficulties studying intact ErbBs. In the last five years our laboratories have collaborated to express and purify near milligram amounts of stable, active full-length and near full-length forms of EGFR. We have used this material to provide a rigorous characterization of EGFR enzymatic activity in ligand-activated and inhibited states and have begun to provide a characterization of the effects of EGFR-activating mutations found in lung cancer. We propose to provide a comprehensive, mechanistic characterization of ErbB activity and its regulation in normal and diseases states by extending our enzymological and functional studies of purified EGFR to determine (i) the effects of specific cancer-associated mutations of EGFR on EGFR activity and, through combination of these mutations with additional mutations targeting specific regions of EGFR, the mechanism by which these mutations exert their effects, (ii) the ability of EGFR inhibitors and combinations of inhibitors to target specific forms of activated EGFR, (iii) the influence of EGFR glycosylation and the EGFR C-tail on EGFR activity and substrate specificity, and (iv) the in vitro substrates of EGFR and mutant EGFRs using a 17,000 protein microarray. We have recently extended our expression and purification to HER2, HER3, and HER4, and we propose to carry out similar mechanistic and enzymological studies of the intrinsic and regulated activity of these ErbBs.