A unique peptide recognition mechanism by the human relaxin family peptide receptor 4 (RXFP4)

Members of the insulin superfamily regulate a variety of biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs), which are class A G protein-coupled receptors (GPCRs), to exert pleiotropic actions. Here, we report three cryo-electron microscopy structures of RXFP4–Gi protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053, both were discovered through medicinal chemistry efforts. The B chain of INSL5 adopts a single α-helix that penetrates into the orthostatic pocket, while the A chain sits above the orthosteric pocket to interact with the extracellular surface of RXFP4, revealing a unique peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. DC591053 selectively mimicked the action of INSL5 at RXFP4 whereas compound 4 activated both RXFP3 and RXFP4. Comparison of peptide binding modes within the insulin superfamily displayed diverse interaction mechanisms distinct to each type of the peptides. Our findings not only provide valuable insights into ligand recognition and subtype selectivity among class A GPCRs, but also expand the knowledge of signaling mechanisms in the insulin superfamily. ### Competing Interest Statement The authors have declared no competing interest.