Lysosome Biogenesis in Normal and Tumor Cells

正常细胞和肿瘤细胞中的溶酶体生物发生

• 批准号: 6787209
• 负责人: PHILIP D STAHL
• 金额: $ 42.3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6787209
• 关键词: biological signal transduction; cell line; electron microscopy; endocytosis; enzyme activity; epidermal growth factor; growth factor receptors; guanine nucleotide binding protein; guanine nucleotide exchange factors; guanosinetriphosphatases; intracellular transport; laboratory mouse; light microscopy; lysosomes; membrane fusion; neoplastic cell; phosphorylation; protein localization; protein reconstitution; protein structure function; receptor binding; receptor expression; recombinant proteins

DESCRIPTION (provided by applicant): The mechanism(s) by which signal transduction and receptor-mediated membrane trafficking are coupled represents a new and unexplored frontier for cell and developmental biology. The nature of this linkage will permit an analysis of the role of trafficking in signal quality. Moreover, characterization of the relationship between signaling and trafficking could have profound effects on our understanding of signal transduction, the endocytic pathway and how the signaling/trafficking interface might be exploited for therapeutic innovation. The project will focus on Rinl, a newly discovered Rab5 guanine nucleotide exchange factor, and its cognate GTPase, Rab5a, whose activation is required for EGF receptor signal transduction and for EGF receptor internalization. Elucidation of the mechanisms by which Rin 1 and Rab5a regulate EGF receptor signaling and trafficking may serve as a model for tyrosine kinase receptors in general and possibly other receptor signal transducing proteins. The project will be carried out via three specific aims. Aim 1 deals with the characterization of a cytosolic Rin 1-Rab5a-14-3-3 high molecular weight (HMW) complex that appears to be recruited to membranes in response to EGF. The complex will be characterized by identifying component proteins and by reconstitution studies using recombinant proteins. One goal is to determine the structural requirements for the inclusion of Rab5a--but not Rab5b or Rab5c--in the complex. Aim 2 focuses on phosphorylation of Rinl in response to EGF receptor activation. We hope to establish the role of phosphorylation in regulating the Rinl :SH2 domain, the Rin 1:Vps9 (Rab5a) guanine nucleotide exchange domain, and the functional relationship of the Ras-binding domain to the Vps9 domain in regulating GEF activity. A long-term goal is to identify kinases and phosphatases that form the basis for Rinl/Rab5a regulation. Aim 3 of the proposal is to delineate the mechanism by which the Rin 1-Rab5a complex mediates endosome fusion following EGF receptor internalization. Localization experiments will be carried out and the in vitro endosome fusion assay will be used to reconstitute Rinl-Rab5a-dependent fusion. Heterotypic endosome fusion via recruitment and activation of Rinl-Rab5a may serve as a new paradigm for endosomal transport. Rinl is the first member of a family of proteins that include the Vps9-Rab5 GEF domain. The goal of the present proposal is to set a solid foundation for future study by clarifying the role of Rin 1 in endocytic trafficking and signaling.

描述（由申请人提供）：耦合信号转导和受体介导的膜运输的机制代表了细胞和发育生物学的新的尚未开发的边界。这种联系的性质将允许分析贩运在信号质量中的作用。此外，信号传导与运输之间关系的表征可能会对我们对信号转导，内吞通路以及信号传导/运输界面的理解产生深远的影响，以进行治疗创新。该项目将集中在RINL上，RINL是一种新发现的Rab5鸟嘌呤核苷酸交换因子及其同源GTPase Rab5a，其激活是EGF受体信号转导和EGF受体内在化所必需的。阐明RIN 1和RAB5A调节EGF受体信号传导和运输的机制，可以作为酪氨酸激酶受体的模型，通常是酪氨酸激酶受体的模型，甚至可能是其他受体信号转导蛋白。该项目将通过三个特定目标进行。 AIM 1涉及胞质RIN 1-RAB5A-14-3-3高分子量（HMW）复合物的表征，该复合物似乎是响应于EGF而募集到膜的。该复合物的特征是鉴定成分蛋白质和使用重组蛋白的重组研究。一个目标是确定包含rab5a的结构要求 - 但不是rab5b或rab5c，在综合体中。 AIM 2侧重于响应EGF受体激活的RINL的磷酸化。我们希望确定磷酸化在调节RINL：SH2结构域，RIN 1：VPS9（RAB5A）鸟嘌呤核苷酸交换结构域以及RAS结合域与VPS9结构域在调节GEF活性中与VPS9结构域的功能关系。一个长期目标是鉴定构成RINL/RAB5A调节基础的激酶和磷酸酶。该提案的目标3是描述RIN 1-RAB5A复合物在EGF受体内在化后介导内体融合的机制。将进行定位实验，并将使用内部内体融合测定法来重建RINL-RAB5A依赖性融合。通过募集和激活RINL-RAB5A的异型内体融合可以作为内体运输的新范式。 RINL是包括VPS9-RAB5 GEF域的蛋白质家族的第一个成员。本提案的目的是通过阐明RIN 1在内吞贩运和信号传导中的作用来为将来的研究奠定坚实的基础。