Lysosome Biogenesis in Normal and Tumor Cells

描述（由申请人提供）：信号转导和受体介导的膜运输耦合的机制代表了细胞和发育生物学的一个新的和未探索的前沿领域。这种联系的性质将有助于分析贩运在信号质量方面的作用。此外，信号转导和贩运之间的关系的表征可能对我们理解信号转导，内吞途径以及如何利用信号转导/贩运接口进行治疗创新产生深远的影响。该项目将重点关注Rinl，一种新发现的Rab 5鸟嘌呤核苷酸交换因子，及其同源GTdR，Rab 5a，其激活是EGF受体信号转导和EGF受体内化所必需的。Rin 1和Rab 5a调节EGF受体信号传导和运输的机制的阐明可以作为一般酪氨酸激酶受体和可能的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实施。目的1涉及表征的细胞溶质Rin 1-Rab 5a-14-3-3高分子量（HMW）复合物，似乎被招募到膜响应EGF。该复合物将通过鉴定组分蛋白和使用重组蛋白进行重构研究来表征。一个目标是确定在复合物中包含Rab 5a而不是Rab 5 b或Rab 5c的结构要求。目的2关注响应于EGF受体活化的Rinl的磷酸化。我们希望建立磷酸化在调节Rinl：SH 2结构域、Rinl：Vps 9（Rab 5a）鸟嘌呤核苷酸交换结构域中的作用，以及Ras结合结构域与Vps 9结构域在调节GEF活性中的功能关系。长期目标是鉴定形成Rinl/Rab 5a调节的基础的激酶和磷酸酶。该提案的目的3是描绘Rin 1-Rab 5a复合物介导EGF受体内化后内体融合的机制。将进行定位实验，并将使用体外内体融合测定来重建Rinl-Rab 5a依赖性融合。通过募集和激活Rinl-Rab 5a的异型内体融合可以作为内体转运的新范例。Rinl是包括Vps 9-Rab 5 GEF结构域的蛋白质家族的第一个成员。本研究的目的是通过阐明Rin 1在胞吞转运和信号转导中的作用，为今后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.