Signaling by receptor tyrosine kinases (RTK) controls major vital processes in developing and adult eukaryotes and is involved in a variety of human pathologies. Epidermal growth factor receptor (EGFR) is the classic RTK, whose signaling and trafficking have been extensively studied, but the molecular mechanisms of endocytosis and spatiotemporal regulation of signaling processes by endocytosis remain poorly understood. Fundamental questions of whether signaling is triggered from endosomes by internalized EGFR and how such signaling is regulated by the endosomal sorting machinery are not addressed. Reciprocally, whether EGFR signaling regulates endocytic trafficking machinery, remains sparsely studied and controversial. Further, the degree and rates of the incorporation of EGFR signaling complexes into intralumenal vesicles of multivesicular endosomes, a process that terminates signaling, are also unknown. In the NIGMS funded research, we have developed single-cell and high-throughput methods to quantitatively monitor endocytic trafficking of endogenous gene- edited EGFR using a pH-sensitive ratiometric fluorescence excitation model. We used these methods to elucidate the mechanism of EGFR endocytosis caused by activation of stress-induced p38-MAP kinase and to determine mechanisms of the crosstalk of this pathway with ligand-induced endocytic pathways. We have developed a pipeline for generation of gene-edited cells expressing endogenous fluorescently tagged EGFR and its downstream signaling effectors, and new approaches to examine the dynamics of these components at high spatial and temporal resolution in living cells. We performed comprehensive phosphoproteomic and proximity proteome mass-spectrometry screenings to identify signaling effectors and regulators of endosomal EGFR. Finally, our preliminary experiments using a novel live-cell assay prompted us to hypothesize that EGFR:adaptor complexes are rapidly incorporated into intralumenal vesicles of multivesicular endosomes which diminishes the capacity of those complexes to signal. These advances have placed us in a unique position to fully define molecular mechanisms of clathrin-mediated internalization of EGFR, an endocytic pathway of EGFR in vivo, and address key fundamental questions of whether EGFR signals from endosomes and how this signaling is terminated. We will exploit the discoveries of the endosomal localization of an actin regulator VAV2 and a protein of uncertain function, TFG (Trk-gene fusion protein), to define their functions in EGFR signaling from endosomes. Using our new assay measuring distribution of signaling complexes within multivesicular endosomes in living cells, we will determine the dynamics of the termination of signaling from endosomal EGFR complexed with various endogenous fluorescently labeled adaptors and enzymes. Together, proposed studies will lead to a comprehensive understanding of the mechanisms of endocytosis and spatiotemporal regulation of signaling processes by endocytosis, and will serve as a paradigm to a similar analysis of the whole RTK family.

The epidermal growth factor receptor (EGFR) plays important roles in mammalian development, wound healing, tissue regeneration and homeostasis, and is involved in various pathologies, most notably, carcinogenesis and metastatic processes. The mechanisms regulating EGFR signaling are incompletely understood. The proposed research will address the fundamental questions of whether specific signaling pathways can be activated by EGFR inside the cells from endosomes and examines the mechanisms regulating this endosomal signaling.