Structure and organization of full-length epidermal growth factor receptor in extracellular vesicles by cryo-electron tomography

Abstract

We report here transport of full-length 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 are the most abundant in EV membranes, and the presence of many receptor-interacting proteins in EVs demonstrates their utility for characterizing membrane protein interactomes. Cryo-electron tomography of EGFR-containing EVs reveals that EGFR forms clusters in both the presence and absence of EGF with a ~3 nm gap between the inner membrane and cytoplasmic density. EGFR extracellular region (ECR) dimers do not form regular arrays in these clusters. Subtomogram averaging of the 150 kDa EGF-bound EGFR ECR dimer 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 establish EVs as a versatile platform for structural and functional characterization of human membrane proteins in cell-derived membranes.

Keywords: EGFR; cryo-ET; extracellular vesicles; mass spectrometry; subtomogram averaging.

Fig. 1.

Transport of human proteins to EVs. (A) Schematic diagram of “transporter” proteins containing the EABR fused to the N-terminal region of influenza neuraminidase followed by a peptide binding module. (B) Schematic diagram of a protein of interest with the EABR fused to its C-terminus to promote transport to EVs and a Strep-tag II epitope fused to its N terminus to enable purification of EVs using a SA affinity column. (C) Western blot analysis of affinity-purified EVs derived from Expi293F cells transfected with the SA transporter diagrammed in (A) along with Strep-tag II (ST) labeled EGFR. (D) Western blot analysis of affinity-purified EVs derived from Expi293F cells transfected with EABR-tagged EGFR. The probing antibody is indicated below each blot. (E) Cartoon representation of EV formation stimulated by expression of EABR-labeled EGFR.

Fig. 2.

Affinity purification and characterization of EGFR-containing EVs. (A) Anti-EGFR western blot of EGFR-containing EVs eluted from a SA column. (B) Coomassie Blue stained Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis analysis of affinity-purified EGFR-containing EVs. EGFR is indicated by a red arrowhead. (C) Cryo-electron micrograph of affinity-purified EGFR-containing EVs. (D) Mass spectrometric riBAQ analysis of mole fractions of transmembrane proteins in affinity-purified EGFR-containing EVs. (E) Plot of the log(Fold-change) (logFC) in the mass spectrometric counts of all proteins in EVs produced by transfection with EGFR-EABR relative to EVs without EGFR. The position of EGFR is indicated along with the positions of several proteins known to be involved in EGFR signaling. A full list of abbreviations and interacting proteins can be found in SI Appendix.

Fig. 3.

Tomograms of EGFR-containing EVs. (A) Section of a tomogram of EGFR-containing EVs following application of ISONET to compensate for the missing wedge. Individual EGFR ECRs circled in red. (B and C) Enlarged views of two EVs from panel A showing the ECR (pink), membrane (tan), and intracellular region (yellow). Several EGFR ECRs are circled in red. (D) A section of a tomogram of an EGFR-containing EV is shown. (E) An enlarged region of the tomographic section shown in panel D is shown with ribbon diagrams of the EGFR ECR dimer (RCSB 3NJP; EGF is colored red and the EGFR subunits are colored cyan and light gray) manually positioned in the tomogram. (F–H) Images of individual EGFR molecules from tomograms of EGFR-containing EVs are shown alongside the same image with the crystal structures of the EGFR ECR dimer colored as in panel E and the asymmetric EGFR kinase dimer (RCSB 3GOP; one kinase subunit is colored light pink and the other light green).

Fig. 4.

The ECRs of clustered EGFRs do not form regular arrays. (Top) Tomographic sections of three different EVs (A–C) fully embedded in ice, viewed perpendicular to the plane of the EM grid. Yellow lines indicate the approximate positions of the corresponding x-y planes shown below each section in the Middle panels. (Middle) Tomographic sections of the same tomograms in the Top row but viewed in the plane of the EM grid near the edges of the EVs. (Bottom) Top–down views of cross-sections of the EGFR dimer derived from the crystal structure (RCSB: 3NJP), which are bowtie-shaped and ~4.5 × 11 nm, positioned as in the Middle panels.

Fig. 5.

Characterization of intravesicular density. (A) 2D tomographic section (Left) of an EGFR:EGF-containing EV showing regions with (orange) and without (blue) an EGFR cluster. The average radial density of these regions is shown in the Middle and Right panels. (B) 2D sections of a 3D subtomogram average of regions of four comparably sized EVs that contain EGFR:EGF (Left) or are devoid of EGFR:EGF (Right). The ECR, outer membrane (OM), inner membrane (IM), and ICR are indicated. Averaged radial density (C) and isosurface view (D) of the subtomogram averages shown in panel B.

Fig. 6.

Subtomogram averages of the EGFR:EGF ECR in EVs strongly resemble the crystal structure. (A) Orthogonal views of the crystal structure of a dimer of the EGFR ECR complexed with EGF (RCSB 3NJP; the EGFR subunits are colored cyan and light gray and EGF is red) are shown fit to the 15 Å subtomogram average map of the EGFR ECRs on the surface of EVs. Sites of potential N-linked glycosylation are shown in yellow spheres for either N-acetyl glucosamines modeled in the crystal structure or Asparagine residues at consensus glycosylation sites. (B) Orthogonal views of the 15 Å map generated from the crystal structure of EGFR dimers bound to EGF as shown in panel A). (C) Subtomogram average of the intracellular regions of 16 isolated EGFRs with a ribbon the crystal structure of the EGFR asymmetric kinase dimer (RCSB 3GOP) shown with one light pink and one light green kinase subunit.