A membrane protein display platform for receptor interactome discovery

Abstract

Significance Membrane proteins and their interactions are major drug targets because of their central roles in regulating cellular communication and signal transduction. Despite this, membrane receptors remain underrepresented in our knowledge base of protein–protein interactions because of the limitations of most available techniques. Here, we developed a vesicle-based membrane-protein display platform as a fast, reproducible, and broadly applicable method for unbiased receptor–ligand interaction discovery. Using this technology, we elucidated known and new single-pass transmembrane-binding partners for several immune checkpoint molecules as well as an architecturally distinct single-pass transmembrane protein that has resisted deorphanization by existing techniques. This method is well suited for extracellular protein interaction discovery for difficult-to-purify membrane proteins and whole vesicles. 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, we developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor–ligand interactions in membranes. We 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. We 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, we 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.