Cell surface receptors are critical for cell signaling and constitute a quarter of all human genes. Despite their importance and abundance, receptor interaction networks remain understudied because of difficulties associated with maintaining membrane proteins in their native conformation and their typically weak interactions. To overcome these challenges, Genentech researchers developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor–ligand interactions in membranes. The researchers demonstrate that this platform is broadly applicable to a variety of membrane proteins, enabling enhanced detection of extracellular interactions over a wide range of binding affinities. They were able to recapitulate and expand the interactome for prominent members of the B7 family of immunoregulatory proteins such as PD-L1/CD274 and B7-H3/CD276. Moreover, when applied to the orphan cancer-associated fibroblast protein, LRRC15, they identified a membrane-dependent interaction with the tumor stroma marker TEM1/CD248. Furthermore, this platform enabled profiling of cellular receptors for target-expressing as well as endogenous extracellular vesicles. Overall, this study presents a sensitive and easy to use screening platform that bypasses membrane protein purification and enables characterization of interactomes for any cell surface–expressed target of interest in its native state.
EVs enable STM protein display for receptor–ligand discovery
(A) EVs provide an endogenous membrane-like environment for receptors. (B) Screen-compatible rEVs can be generated by overexpressing HIV gag protein and a protein-of-interest either as a full-length protein or as a gD-GPI–tagged ectodomain. (C) Western blots of whole-cell lysates or EVs expressing different full-length, untagged receptors. (D) Full-length PVR expressing rEVs or (E) PVR gD-GPI EVs expressing gag-NeonGreen are incubated with cells transfected with known full-length PVR ligands, CD226, CD96, KIR2DL5A, PVRL1, and TIGIT. Green represents the direct fluorescence from EVs. (Scale bar, 20 µm.) (F) BLI experiment with either CD226-Fc or a control human IgG on the sensors dipped into 2.5 nM of rEVs expressing full-length PVR or gD-GPI ectodomains. The experimental conditions have been labeled in the figure to facilitate interpretation.