A Platform for Extracellular Interactome Discovery Identifies Novel Functional Binding Partners for the Immune Receptors B7-H3/CD276 and PVR/CD155

The Conditioned Media AlphaScreen represents a new platform for high throughput and sensitivity detection of interactions between plasma membrane proteins. The interleukin receptor IL20RA is identified as a binding partner for the orphan checkpoint inhibitor B7-H3. Further, a new functional interaction between the KIR2DL5 and the immune receptor PVR regulates natural killer cell cytolysis of tumor cells. This technology represents a versatile approach for receptor interactome discovery that provides insights into plasma membrane protein biology. [Display omitted] Highlights •New platform for high throughput detection of transient interactions between membrane proteins.•IL20RA is a receptor for the orphan checkpoint inhibitor B7-H3.•The natural killer cell protein KIR2DL5A binds the immune receptor PVR.•KIR2DL5 binding to PVR regulates natural killer cell cytotoxicity and inhibits tumor cell killing.•Elucidation of receptor interactomes to gain insights into extracellular protein biology. Receptors expressed on the plasma membrane and their interacting partners critically regulate cellular communication during homeostasis and disease, and as such represent main therapeutic targets. Despite its importance for drug development, receptor-ligand proteomics has remained a daunting field, in part because of the challenges associated to the study of membrane-expressed proteins. Here, to enable sensitive detection of receptor-ligand interactions in high throughput, we implement a new platform, the Conditioned Media AlphaScreen, for interrogation of a library consisting of most single transmembrane human proteins. Using this method to study key immune receptors, we identify and further validate the interleukin receptor IL20RA as the first binding partner for the checkpoint inhibitor B7-H3. Further, KIR2DL5, a natural killer cell protein that had remained orphan, is uncovered as a functional binding partner for the poliovirus receptor (PVR). This interaction is characterized using orthogonal assays, which demonstrate that PVR specifically engages KIR2DL5 on natural killer cells leading to inhibition of cytotoxicity. Altogether, these results reveal unappreciated links between protein families that may importantly influence receptor-driven functions during disease. Applicable to any target of interest, this technology represents a versatile and powerful approach for elucidation of receptor-ligand interactomes, which is essential to understand basic aspects of the biol