A High-Throughput\nMethod for Screening Peptide Activators\nof G‑Protein-Coupled Receptors

Here, we describe an innovative and\nefficient method\nfor screening\npeptide activators of G-protein-coupled receptors (GPCRs) utilizing\na protein–protein interaction (PPI) approach. We designed a\nlibrary of 92,918 peptides fused with transmembrane domains of glycosylphosphatidylinositol-anchored\nproteins (GPI-APs). We employed a pooled lentiviral system to promote\nthe expression of these proteins at the cellular membrane and evaluate\ntheir ability to activate GPCRs. We then used fluorescence-activated\ncell sorting (FACS) to screen the GPI-AP-peptide library and identify\nnovel peptide activators of the glucagon-like peptide-1 receptor (GLP-1R).\nWe discovered one peptide PepA3 derived from the Frizzled-like (FZ)\ndomain of human Carboxypeptidase Z (CPZ), a regulated secreted metallocarboxypeptidase.\nNotably, PepA3 and its two related variants, PepA and PepA2, activated\nthe GLP-1R receptor with less potency but comparable efficacy to that\nof GLP-1. We then hypothesized that all of these peptides will bind\ndifferently to the GLP-1R than the normal ligand. Our technology could\nidentify novel GPCR-activating peptides for structure–function\nor drug discovery research.