{"type": "root", "children": [{"type": "p", "children": [{"type": "t", "text": "\nRXFP4, originally identified as GPCR142, is a G protein‐coupled receptor that is activated by members of the insulin/relaxin peptide family. Early studies demonstrated that relaxin‐3 can potently activate RXFP4/GPCR142 with subsequent inhibition of adenylate cyclase and selective modulation of G protein signaling, while later work established that insulin‐like peptide 5 (INSL5) acts as a highly specific and potent agonist for RXFP4. These findings, derived from analyses of receptor activation and tissue expression, laid the groundwork for appreciating the receptor’s diverse physiological roles."}, {"type": "fg", "children": [{"type": "fg_fs", "start_ref": "1", "end_ref": "3"}]}, {"type": "t", "text": "\n\nStudies focused on the interaction mechanisms have revealed that specific electrostatic contacts are essential for ligand recognition by RXFP4. Mutagenesis experiments identified a conserved EXXXD motif in the receptor’s extracellular domain that interacts with key positively charged residues on the B-chain of INSL5 (notably Arg residues), thereby forming a crucial receptor-binding patch. Complementary approaches, including charge-exchange and alanine-scanning mutagenesis, have further characterized these hydrophobic and electrostatic interactions and established parallels—and subtle differences—with relaxin‐3 binding."}, {"type": "fg", "children": [{"type": "fg_fs", "start_ref": "4", "end_ref": "7"}]}, {"type": "t", "text": "\n\nThe receptor’s activation determinants have also been explored via domain swapping and chimeric receptor studies, which demonstrated that while the N-terminus and extracellular loop 2 contribute to ligand binding, transmembrane domains (particularly TM2, TM3, and TM5) are critical for functional receptor activation by INSL5. Moreover, selective engineering of the INSL5 peptide has illuminated four key determinants that modulate its activity profile and distinguish RXFP4 activation from that of other related receptors such as RXFP3."}, {"type": "fg", "children": [{"type": "fg_fs", "start_ref": "8", "end_ref": "10"}]}, {"type": "t", "text": "\n\nPhysiologically, RXFP4 is prominently expressed in peripheral tissues such as the colon and even in human sperm, suggesting roles in both gastrointestinal motility and reproductive function. In the colon, activation of RXFP4 on enteroendocrine cells triggers a cascade that promotes 5-HT release and enhances propulsive activity, whereas in sperm, RXFP4 activation by INSL5 helps preserve motility and mitigate mitochondrial reactive oxygen species production. Recent cryo-electron microscopy studies further elucidate the structural basis of RXFP4 activation, offering deeper insights into the receptor’s ligand selectivity and signaling mechanisms."}, {"type": "fg", "children": [{"type": "fg_fs", "start_ref": "11", "end_ref": "14"}]}, {"type": "t", "text": "\n"}]}, {"type": "rg", "children": [{"type": "r", "ref": 1, "children": [{"type": "t", "text": "Changlu Liu, Jingcai Chen, Steven Sutton, et al. 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