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。特别是，这涉及到结合生长因子heregulin的ErbB-4受体的两步切割。ErbB-4的分裂可被heregulin或TPA刺激，并导致ErbB-4细胞质结构域（包括其酪氨酸激酶）从质膜到细胞核的重新定位。该申请旨在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。第一个目标将描述ErbB-4外结构域的初始和必需分泌酶（金属蛋白酶）依赖的切割。这将包括鉴定裂解位点，受体和蛋白酶突变体的构建，评估ErbB-4结构对蛋白酶识别的贡献，以及切割和信号转导与heregulin诱导的ErbB-4向膜微域易位的关系。第二个目标将集中在随后的γ分泌酶（早老素）介导的分裂，从质膜释放ErbB-4细胞质结构域。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细胞质结构域的核易位。最后的目的是构建解决ErbB-4切割和细胞质区域核重新定位的生理意义。这将包括细胞核细胞质结构域功能的细胞生物学实验，如酪氨酸磷酸化和基因表达的激活。蛋白水解过程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”方法来评估这种新的受体加工途径在动物中的生理意义。