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

[Image: see text] 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.