Lysosome Biogenesis in Normal and Tumor Cells

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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.