Lysosome Biogenesis in Normal and Tumor Cells

描述(申请人提供)：信号转导和受体介导膜转运的耦合机制(S)代表了细胞和发育生物学的一个新的和未被探索的前沿。这种联系的性质将使分析贩运在信号质量方面的作用成为可能。此外，表征信号和贩运之间的关系可能会对我们理解信号转导、细胞内途径以及如何利用信号/贩运接口进行治疗创新产生深远的影响。该项目将专注于新发现的Rab5鸟嘌呤核苷酸交换因子RINL及其同源GTPase Rab5a，其激活是EGF受体信号转导和EGF受体内化所必需的。阐明Rin-1和Rab5a调节EGF受体信号和转运的机制可能成为研究酪氨酸激酶受体和其他受体信号转导蛋白的模型。该项目将通过三个具体目标进行。目的1研究胞内Rin 1-Rab5a-14-3-3高分子量(HMW)复合体的特性，该复合体似乎能被EGF征集到细胞膜上。该复合体将通过鉴定组成蛋白和使用重组蛋白进行重组研究来表征。一个目标是确定在复合体中包含Rab5a--但不包括Rab5b或Rab5c--的结构要求。目的2研究EGF受体激活后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中的作用，以及Ras结合结构域与Vps9结构域在调节全球环境基金活性中的功能关系。一个长期的目标是确定构成RINL/Rab5a调控基础的激酶和磷酸酶。该提案的目的3是描述Rin 1-Rab5a复合体在EGF受体内化后介导内体融合的机制。将进行定位实验，并将使用体外内体融合实验来重建依赖RINL-Rab5a的融合。RINL-Rab5a通过RINL-Rab5a的募集和激活进行异型内体融合，可能成为内体转运的新范式。RINL是包括Vps9-Rab5全球环境基金结构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在细胞内转运和信号转导中的作用，为未来的研究奠定坚实的基础。

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.