Plasma membrane proteins, including receptors and nutrient transporters, undergo internalization into the endosomal compartment, from where these proteins are recycled back to the plasma membrane. This endosomal recycling process is essential to cellular homeostasis. Our laboratories have discovered two interrelated complexes that play key roles in endosomal recycling known as CCC and retriever. Both of these assemblies work in concert with WASH, an endosomally-localized actin nucleating pentameric complex. Significantly, perturbations of these systems have far-reaching consequences, including developmental anomalies, altered copper and lipid handling, and defective glucose tolerance. These physiologic alterations can be traced to faulty trafficking of key receptors and transporters including Notch, ATP7A/ATP7B, LDLR and GLUT2. At its core, the CCC complex contains COMMD proteins, in association with two coiled-coil proteins, CCDC22 and CCDC93. We recently demonstrated that CCC regulates the WASH complex by limiting the amount of endosomal PI(3)P through an interaction with the PI(3)P phosphatase MTMR2. We also uncovered that the CCC complex recruits a cargo recognition complex, termed retriever, whose function is to identify proteins that need to be recycled. While we made great progress in dissecting the mechanistic underpinnings of these systems, our understanding is still rudimentary. The overall goal of this project is to provide a deep mechanistic picture of CCC-mediated regulation of WASH and retriever, focusing our attention on endosomal recycling of nutrient regulators. Based on new emerging data, this proposal will test the hypothesis that the CCC complex coordinates both PI(3)P levels and Rab21 activation to promote retriever-mediated recycling of surface proteins. This project will focus on the following specific aims: (1) Determine the mechanism by which CCC regulates MTMR actions on endosomes. In this aim we will examine how the CCC complex coordinates MTMR actions, particularly by MTMR5, together with activation of Rab21 and retriever recruitment. (2) Define the mechanism by which PI(3)P regulates the recruitment and activity of the WASH complex. In this aim, we will define the mechanisms by which PI(3)P-binding domains in FAM21 and WASH regulate the endosomal recruitment and activity of the WASH complex, and how these activities ultimately impact endosomal trafficking. (3) Examine retriever-dependent regulation of cargo proteins in vivo. In this aim we will utilize newly generated mouse models of retriever deficiency (conditional Vps35l, Vps26c alleles) to examine the contribution of this system to ATP7B and LDLR trafficking in the liver. Furthermore, we will utilize proteomics to define the breadth of plasma membrane proteins regulated by retriever, CCC, and WASH in the mammalian liver. Altogether, this project will elucidate important principles that govern the trafficking of a myriad of cargoes that traverse the endosomal system. Deregulation of these pathways have a broad impact on normal organismal/cellular physiology, thus its scientific impact will have far reaching implications for several biomedical fields.
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