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PreprintCited by 17Extracellular vesicles in diseaseAdvanced Electron Microscopy Techniques and ApplicationsCellular transport and secretion

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Structure and organization of full-length Epidermal Growth Factor Receptor in extracellular vesicles by cryo-electron tomography

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Gonzalez-Magaldi, Monica, Gullapalli, Anuradha, Papoulas, Ophelia, Liu, Chang, Leung, Adelaide Y.-H., Guo, Luqiang, Brilot, Axel, Marcotte, Edward M., Ke, Zunlong, Leahy, Daniel J.·2024·Proceedings of the National Academy of Sciences
DOI 10.1101/2024.11.25.625301PMC PMC12167996

Preprint — published as 10.1073/pnas.2424678122 · Paper page

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Abstract

We report here transport of the Epidermal Growth Factor Receptor (EGFR), Insulin Receptor, 7-pass transmembrane receptor Smoothened, and 13-pass Sodium-iodide symporter to extracellular vesicles (EVs) for structural and functional studies. Mass spectrometry confirmed the transported proteins as the most abundant in EV membranes, and the presence of many receptor-interacting proteins demonstrates the utility of EVs for characterizing membrane protein interactomes. Cryo-electron tomography of EGFR-containing EVs reveals that EGFR forms clusters in the presence of EGF with a ∼3 nm gap between the inner membrane and cytoplasmic density. EGFR extracellular regions do not form regular arrays, suggesting that clustering is mediated by the intracellular region. Subtomogram averaging of the EGFR extracellular region (ECR) yielded a 15 Å map into which the crystal structure of the ligand-bound EGFR ECR dimer fits well. These findings refine our understanding of EGFR activation, clustering, and signaling, and they establish EVs as a versatile platform for structural and functional characterization of human membrane proteins in a native-like environment. Significance Statement: Atomic or near-atomic resolution structural studies of proteins embedded in cell membranes have proven challenging. We show that transporting integral membrane proteins to cell-derived extracellular vesicles enables structural and functional studies of human membrane proteins in a native membrane environment. We have used this approach to visualize an active form of full-length Epidermal Growth Factor Receptor (EGFR) and show that it forms clusters in the membrane and projects its cytoplasmic signaling domains ∼3 nm away from the membrane surface. EGFR is essential for normal development, but abnormal EGFR activity is associated with several human cancers and is the target of many anticancer therapies. Our studies refine current models of how ligand binding to EGFR transmits signals across cell membranes.

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    “ET and mass spectrometric analyses of EGFR - containing EVs indicate that EGFR represents at least 80% and likely more of the ECR particles visible in electron micrographs ( 49 ), which simplifies particle identification in low - resolution cryo - electron tomograms ( 57 ).”

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Metadata

Authors
Gonzalez-Magaldi, M., Gullapalli, A., Papoulas, O., Liu, C., Leung, A. Y.-H., Guo, L., Brilot, A., Marcotte, E. M., Ke, Z., & Leahy, D. J.
Affiliations
The University of Texas at Austin (US)
Corresponding author
  • Ke, Zunlong
  • Leahy, Daniel J.
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
openRxiv
Published
2024-11-28
Volume
122
Issue
23
Pages
e2424678122-e2424678122
ISSN
0027-8424 (ISSN-L)
DOI
10.1101/2024.11.25.625301
PMC
PMC12167996
OpenAlex
W4410948342
Cited by
17
References
1
Citations by year
2026: 11 · 2025: 6
FWCI
5.11
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Funders
  • Welch Foundation (F-1515)
  • Cancer Prevention and Research Institute of Texas (RR160023, RR230050)
  • National Institute of General Medical Sciences (R35GM122480)
  • Life Sciences Division, Army Research Office (@911NF-12-1-0390)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
epidermal growth factor receptor, extracellular vesicles, receptor clustering, EGF binding, membrane protein structure, EGFR activation, subtomogram averaging, insulin receptor, Smoothened, sodium iodide symporter

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