Secretase Processing of ErbB-4 Receptor Tyrosine Kinase

ErbB-4 受体酪氨酸激酶的分泌酶加工

• 批准号: 6506422
• 负责人: GRAHAM F CARPENTER
• 金额: $ 30.14万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6506422
• 关键词: bioassay; biological signal transduction; cell line; chemical cleavage; enzyme activity; enzyme inhibitors; enzyme mechanism; fluorescence resonance energy transfer; gene mutation; gene targeting; genetically modified animals; growth factor receptors; laboratory mouse; metalloendopeptidases; microarray technology; neoplastic cell; phosphorylation; presenilin; protein localization; protein structure function; protein transport; protein tyrosine kinase; proteolysis; receptor expression; transcription factor; yeast two hybrid system

DESCRIPTION (provided by applicant): The focus of this application is to explore a novel pathway for the proteolytic processing of receptor tyrosine kinases. In particular, this involves the two step cleavage of the ErbB-4 receptor that binds the growth factor heregulin. Cleavage of ErbB-4 can be stimulated by heregulin or TPA and results in the relocalization of the ErbB-4 cytoplasmic domain, including its tyrosine kinase, from the plasma membrane to the nucleus. The application seeks to elucidate the biochemical parameters of the two cleavage events, as well as the biological significance of the processing pathway for ErbB-4 mediated cellular responses. The first aim will characterize the initial and essential secretase (metalloprotease)-dependent cleavage of the ErbB-4 ectodomain. This will include identification of the cleavage site, construction of receptor and protease mutants, evaluation of the contributions of ErbB-4 structure to protease recognition, and the relationship of cleavage and signal transduction to heregulin-induced translocation of ErbB-4 to membrane microdomains. The second aim will concentrate on the subsequent gamma-secretase (presenilin) mediated cleavage that releases the ErbB-4 cytoplasmic domain from the plasma membrane. This will include analysis of the gamma-secretase cleavage site, the role of PDZ domain proteins and presenilins, and nuclear translocation of the ErbB-4 cytoplasmic domain. The final aim is constructed to address the physiological significance of ErbB-4 cleavage and nuclear re-localization of the cytoplasmic domain. This will include cell biological experiments of cytoplasmic domain functions in the nucleus, such as tyrosine phosphorylation and the activation of gene expression. Selective inhibitors of proteolytic processing will be used to assess the role of this pathway in ErbB-4 mediated biological responses. Lastly, a mouse "knockin" approach is proposed to assess the physiological significance of this novel receptor processing pathway in the animal.

描述（由申请人提供）：本申请的重点是探索受体酪氨酸激酶蛋白水解加工的新途径。特别地，这涉及结合生长因子调蛋白的ErbB-4受体的两步切割。ErbB-4的切割可以被调蛋白或TPA刺激，并导致ErbB-4胞质结构域（包括其酪氨酸激酶）从质膜重新定位到细胞核。该申请试图阐明两个裂解事件的生化参数，以及ErbB-4介导的细胞反应的加工途径的生物学意义。 第一个目标将表征ErbB-4胞外域的初始和基本分泌酶（金属蛋白酶）依赖性切割。这将包括裂解位点的鉴定，受体和蛋白酶突变体的构建，ErbB-4结构对蛋白酶识别的贡献的评价，以及裂解和信号转导与调蛋白诱导的ErbB-4向膜微区的易位的关系。第二个目标将集中在随后的γ-分泌酶（早老素）介导的切割，释放ErbB-4细胞质结构域从质膜。这将包括γ-分泌酶切割位点的分析，PDZ结构域蛋白和早老素的作用，以及ErbB-4胞质结构域的核转位。最终的目的是解决ErbB-4切割和细胞质结构域的核重新定位的生理意义。这将包括细胞生物学实验的胞质结构域功能在细胞核中，如酪氨酸磷酸化和基因表达的激活。蛋白水解加工的选择性抑制剂将用于评估该途径在ErbB-4介导的生物反应中的作用。最后，提出了一种小鼠“敲入”的方法来评估这种新的受体处理途径在动物中的生理意义。