Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform

Abstract

Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ('orphans') and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIR's). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.

AlphaFold2 enables accurate deorphanization of ligands to single-pass receptors

“…To test if we could predict receptors for orphan secreted ligands using the single-pass transmembrane library, we tested the screen using 15 ligands based on a previously curated library of potentially high-value orphan secreted proteins 15 ( Extended Data Table S3 ). Due to limited graphics processing unit (GPU) resources, we limited our test to binding partners that could be predicted using central processing units (CPUs).…”

“…Similarly, binding screens using an individual ligand applied to a receptor in solution are time-consuming, not applicable for all receptor types, and may lack the cellular environment necessary for posttranslational modifications or co-receptor binding 13,14 . Lastly, cell-based CRISPR screens have recently been utilized for the deorphanization of ligands, but are limited by the ability to gain sufficient receptor expression and the lack of expression of essential coreceptors 13,15 .…”

Receptor Elimination by E3 Ubiquitin Ligase Recruitment (REULR): A Targeted Protein Degradation Toolbox

“…A raw list of reported ubiquitin sites was obtained from PhosphoSitePlus (PSP; https://www.phosphosite.org) and matched to a curated list of the human membrane proteome (Siepe et al, 2022) to generate a master list of cell surface receptors with reported ubiquitination sites.…”

Receptor Elimination by E3 Ubiquitin Ligase Recruitment (REULR): A Targeted Protein Degradation Toolbox

“…A raw list of reported ubiquitin sites was obtained from PhosphoSitePlus (PSP; https://www. phosphosite.org) and matched to a curated list of the human membrane proteome 65 to generate a master list of cell surface receptors with reported ubiquitination sites.…”