Visualization of EGFR Assembly and Activation Induced by a Protein Nanocage Using Cryo-Electron Tomography.

The epidermal growth factor receptor (EGFR) mediates signal transduction by triggering downstream phosphorylation to regulate cell proliferation. However, the complexity of the cellular environment has limited in situ structural investigations of membrane proteins within their native context. Here, we present a proof-of-concept study integrating protein cage labeling with cryo-electron tomography (cryo-ET) to directly visualize receptor assemblies on the native membrane. Using EGFR as a model system, we demonstrate that the protein cage can associate with multiple EGFR molecules, thereby inducing their oligomerization. The distance between neighboring EGFRs within these assemblies was measured to be 7.1 ± 1.2 nm. Furthermore, we validated the functional relevance of this system by showing that protein cage-induced EGFR assemblies were accompanied by enhanced ligand-independent phosphorylation. In summary, our results establish the feasibility of using protein cage-labeling for the induction and in situ structural analysis of membrane protein oligomerization.