Ligand recognition mechanism of the human relaxin family peptide receptor 4 (RXFP4)

Abstract

Members of the insulin superfamily regulate pleiotropic 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). 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. The B chain of INSL5 adopts a single α-helix that penetrates into the orthosteric pocket, while the A chain sits above the orthosteric pocket, revealing a peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. Our findings not only provide insights into ligand recognition and subtype selectivity among class A G protein-coupled receptors, but also expand the knowledge of signaling mechanisms in the insulin superfamily.

Structural insights into ligand recognition and subtype selectivity of the human melanocortin-3 and melanocortin-5 receptors

“…7a ). Structural comparison of MC3R–G s and MC5R–G s complexes with other class A GPCRs demonstrates a high degree of similarity and suggests that MC3R and MC5R follow common G protein coupling mechanisms 39 , consistent with that observed in MC1R (PDB code: 7F4D) 27 , MC4R (PDB code: 7F53) 26 and dopamine receptor D1 (DRD1, PDB code: 7CKW) 40 (Fig. 7b ).…”

“…All the three bound peptides in this study adopt a “U-shape” conformation with the middle region (X–X–R–W motif) being the deepest segment within the receptor core 26 – 29 , 38 . Such a peptide binding mode is distinct from those observed in other class A GPCRs including relaxin family peptide receptor 4 (RXPF4) 39 , C-X-C motif chemokine receptor 2 (CXCR2) 41 and cholecystokinin A receptor (CCKAR) 42 and also class B1 GPCRs including glucagon-like peptide-1 receptor (GLP-1R) 43 , glucose-dependent insulinotropic polypeptide receptor (GIPR) 44 and glucagon receptor (GCGR) 45 . Combined with mutagenesis experiments, these structures provide molecular basis of ligand recognition, subtype selectivity and divalent ion modulation of MC3R and MC5R, and will certainly be of value for the design of better therapeutics with desired pharmacological profiles.…”

“…Substitutions of these receptor residues by either alanine or asparagine/glutamine decreased the potency, efficacy and affinity to various extents. In a previously reported SHU9119–MC4R complex structure 39 , Ca 2+ was observed as a co-factor and found to increase the affinity and potency of the endogenous agonist α-MSH at MC4R. The presence of Ca 2+ preferentially increased the γ-MSH-induced cAMP accumulation at MC3R/MC1R and ACTH-induced cAMP accumulation at MC2R, compared to that of α-MSH at MC5R, suggesting that MC1R, MC2R and MC3R were more likely to be stabilized by Ca 2+ .…”

The diverse influences of relaxin-like peptide family on tumor progression: Potential opportunities and emerging challenges

“…For instance, the binding mechanism between INSL5 and RXFP4, as reveals by cryo-electron microscopy, demonstrates a novel peptide binding mode. In this mode, the B-chain of INSL5 adopts a single α-helix that penetrates the orthosteric pocket, and the A-chain is positioned above this pocket [ 148 ]. Furthermore, the cryo-electron microscopy structure of active-state human RXFP1, bound to a single-chain version of the endogenous agonist RLN and the heterotrimeric Gs protein, has been elucidated.…”