Mechanistic Studies of EGFR/ErbB Receptor Tyrosine Kinases

EGFR/ErbB 受体酪氨酸激酶的机制研究

• 批准号: 8436210
• 负责人: PHILIP A COLE
• 金额: $ 29.7万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8436210
• 关键词: Address; Amino Acids; Antibodies; Antineoplastic Agents; Behavior; Binding; Biological Assay; C-terminal; Cancer Etiology; Cell Differentiation process; Cell Proliferation; Cells; Cetuximab; Chinese Hamster Ovary Cell; Clinical; Colon; Communication; Development; Dimerization; Disease; Docking; Drug Targeting; Drug resistance; ERBB2 gene; ERBB3 gene; Embryonic Development; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; ErbB Receptor Family Protein; ErbB4 gene; Erbitux; Erlotinib; FDA approved; Family; Family member; Head and Neck Cancer; Homologous Gene; Human; In Vitro; Individual; Knowledge; Laboratories; Learning; Length; Ligand Binding; Ligands; Lung; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Monoclonal Antibodies; Mutate; Mutation; Oncogenic; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Phosphotyrosine; Predisposition; Protein Microchips; Protein Tyrosine Kinase; Receptor Protein-Tyrosine Kinases; Regulation; Relative (related person); Rest; Roche brand of trastuzumab; Roentgen Rays; Severities; Signal Transduction; Site; Specificity; Substrate Specificity; Tail; Testing; Time; Trastuzumab; Western Blotting; Work; base; cancer therapy; cancer type; cell growth; dimer; experience; extracellular; glycosylation; inhibitor/antagonist; insight; interest; kinase inhibitor; lapatinib; malignant breast neoplasm; member; milligram; mutant; nanodisk; overexpression; programs; receptor; resistance mutation; response; small molecule

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.

描述（由申请人提供）：表皮生长因子受体（EGFR/ErbB）受体酪氨酸激酶家族在人类中包括四个家族成员：EGFR (HER1, ErbB1), HER2 (ErbB2, Neu), HER3 （ErbB3）和HER4 （ErbB4）。每个家族成员由一个细胞外配体结合区、一个单膜跨越区、一个细胞质酪氨酸激酶和一个约230个氨基酸的c端尾部组成。所有EGFR/ErbB家族成员都是正常胚胎发育所必需的，每个ErbB的异常活动与人类癌症有关。特别是，过度表达或活跃的EGFR和HER2与肺癌、结肠癌和头颈癌（EGFR）或乳腺癌（HER2）的严重程度增加有关，并且一些靶向EGFR和HER2的药物是fda批准的抗癌疗法。EGFR活性的典型模型是，与细胞外区域结合的配体稳定了受体的特定二聚体构象，其中其激酶活性受到刺激，导致受体c尾和其他底物的磷酸化，下游效应物的定位和/或活性发生变化，并启动改变细胞生长和分化的信号级联反应。HER2和HER3是不典型erbb，因为HER2没有已知的配体，HER3由于其激酶结构域的关键突变而很少或没有激酶活性。HER2和HER3似乎都是其他ErbB家族成员的异源二聚体伴侣，然而，HER2/HER3异源二聚体是许多标准中最有效的信号传导和致癌ErbB配对。通过对ErbB片段的结构和功能研究，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一直受到研究完整ErbB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对配体激活和抑制状态下的EGFR酶活性进行了严格的表征，并开始对肺癌中发现的EGFR激活突变的影响进行了表征。我们建议通过扩展纯化EGFR的酶学和功能研究，提供正常和疾病状态下ErbB活性及其调控的全面、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，并通过将这些突变与针对EGFR特定区域的其他突变相结合，确定这些突变发挥作用的机制；（ii） EGFR抑制剂和抑制剂组合针对特定形式的活化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，以及（iv）体外底物