# R35 - ADMIN SUPPLEMENT - EGF Receptor Endocytosis: Mechanisms and Role in Signaling

> **NIH NIH R35** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2024 · $186,663

## Abstract

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
pr...

## Key facts

- **NIH application ID:** 11032462
- **Project number:** 3R35GM148363-02S1
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** ALEXANDER D SORKIN
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $186,663
- **Award type:** 3
- **Project period:** 2023-06-01 → 2028-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11032462

## Citation

> US National Institutes of Health, RePORTER application 11032462, R35 - ADMIN SUPPLEMENT - EGF Receptor Endocytosis: Mechanisms and Role in Signaling (3R35GM148363-02S1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/11032462. Licensed CC0.

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