Our long-term goal in this proposal is to identify and understand the cellular components that transport internalized endocytic cargo from endosomes to the Golgi apparatus (retrograde recycling), and to understand how this process regulates, and is integrated within, intercellular signaling pathways. Retrograde recycling has recently been linked to multiple metazoan-specific processes such as the generation of Wnt signaling gradients, AMPA-receptor signaling in the nervous system, clearance of dead apoptotic cells, and regulation of epithelial polarity. Wnt signaling requires endosome-to-Golgi retrieval of the Wnt ligand chaperone MIG-14/Wntless, and the molecular details of MIG-14/Wntless recycling have proven to be highly conserved in C. elegans, Drosophila, and mammalian cells. Without retrograde recycling, transmembrane cargo proteins such as MIG-14/Wntless aberrantly enter the lysosome and are degraded, thus depleting the functional pool of such proteins from the cell. To gain novel insight into how retrograde recycling functions, and how it influences intercellular signaling mechanisms in multicellular animals, we have pioneered analysis of the polarized intestinal epithelium of C. elegans. In novel preliminary work we discovered that TGF?-signaling is impaired in retrograde recycling mutants. We plan to further test the hypothesis that TGF?-receptors are retrograde cargo, and test phylogenetic conservation of this mechanism in Drosophila and mammalian cells. In addition, our team has discovered previously unsuspected requirements for endosome regulatory proteins beyond the well-studied retromer complex. These novel regulators include the J-domain co-chaperone RME-8. We plan to test the hypothesis that RME-8 regulates the disassembly of the endosomal flat clathrin lattice, mediating a feedback loop between retromer and the ESCRT-driven degradation machinery that is present on the same endosomes. In addition we have identified an entire additional protein complex involved in retrograde recycling of MIG-14/Wntless. This protein complex appears to be associated with the recycling endosome, and contains modules for membrane binding, membrane bending, and actin nucleation. We plan to determine how this protein complex influences MIG-14/Wntless recycling, test its ability to influence other retrograde cargo including TGF?-receptors, and test for conservation of function in mammalian cells. We expect that our findings will provide unique insight into how trafficking mechanisms influence signaling pathways, and will improve our ability to treat diverse diseases that arise from misregulation of receptor signaling strength, such as in cancers, and other diseases associated with misregulated retrograde recycling including Alzheimer's and Parkinson's diseases.