A High-Throughput Method for Screening Peptide Activators of G-Protein-Coupled Receptors

Here, we describe an innovative and efficient method for screening peptide activators of G-protein-coupled receptors (GPCRs) utilizing a protein-protein interaction (PPI) approach. We designed a library of 92,918 peptides fused with transmembrane domains of glycosylphosphatidylinositol-anchored proteins (GPI-APs). We employed a pooled lentiviral system to promote the expression of these proteins at the cellular membrane and evaluate their ability to activate GPCRs. We then used fluorescence-activated cell sorting (FACS) to screen the GPI-AP-peptide library and identify novel peptide activators of the glucagon-like peptide-1 receptor (GLP-1R). We discovered one peptide PepA3 derived from the Frizzled-like (FZ) domain of human Carboxypeptidase Z (CPZ), a regulated secreted metallocarboxypeptidase. Notably, PepA3 and its two related variants, PepA and PepA2, activated the GLP-1R receptor with less potency but comparable efficacy to that of GLP-1. We then hypothesized that all of these peptides will bind differently to the GLP-1R than the normal ligand. Our technology could identify novel GPCR-activating peptides for structure-function or drug discovery research.