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

In recent years, targeted protein degradation (TPD) of plasma membrane proteins by hijacking the ubiquitin proteasome system (UPS) or the lysosomal pathway has emerged as a novel therapeutic avenue in drug development to address and inhibit canonically difficult targets. While TPD strategies have been successful in targeting cell surface receptors, these approaches are limited by the availability of suitable binders to generate heterobifunctional molecules. Here, we present the development of a nanobody (VHH)-based degradation toolbox termed REULR (Receptor Elimination by E3 Ubiquitin Ligase Recruitment). We generated human and mouse cross-reactive nanobodies against five transmembrane PA-TM-RING-type E3 ubiquitin ligases (RNF128, RNF130, RNF167, RNF43, and ZNRF3), covering a broad range and selectivity of tissue expression, with which we characterized the expression in human and mouse cell lines and immune cells (PBMCs). We demonstrate that heterobifunctional REULR molecules can enforce transmembrane E3 ligase interactions with a variety of disease-relevant target receptors (EGFR, EPOR, and PD-1) by induced proximity, resulting in effective membrane clearance of the target receptor at varying levels. In addition, we designed E3 ligase self-degrading molecules, "fratricide" REULRs (RNF128, RNF130, RENF167, RNF43, and ZNRF3), that allow downregulation of one or several E3 ligases from the cell surface and consequently modulate receptor signaling strength. REULR molecules represent a VHH-based modular and versatile "mix and match" targeting strategy for the facile modulation of cell surface proteins by induced proximity to transmembrane PA-TM-RING E3 ligases.

Membrane phosphoinositides regulate GPCR-ß-arrestin complex assembly and dynamics.

Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with ß-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in ß-arrestin recruitment and GPCR-ß-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for ß-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of ß-arrestin and stabilize GPCR-ß-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-ß-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for ß-arrestin recruitment, this provides a mechanism for ß-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.

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

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') or 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 are not optimized for extracellular interactions. Cell-based screening is a promising technology for the deorphanization of 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 transmembrane (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 assays. The newly deorphanized ligands include three receptor protein tyrosine phosphatase (RPTP) ligands and a chemokine-like protein that binds to killer immunoglobulin-like receptors (KIRs). These new interactions provide a resource for future investigations of interactions between the human-secreted and membrane proteomes.

A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions.

Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed ∼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.