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Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) represent a unique subgroup of G protein-coupled receptors with a large ectodomain. Recent studies demonstrated that relaxin activates two orphanLGRs, LGR7 and LGR8, whereas INSL3/Leydig insulinlike peptide specifically activates LGR8. Human relaxin 3 (H3 relaxin) was recently discovered as a novel ligand for relaxin receptors. Here, we demonstrate that H3 relaxin activates LGR7 but not LGR8. Taking advantage of the overlapping specificity of these three ligands for the two related LGRs, chimeric receptors were generated to elucidate the mechanism of ligand activation of LGR7. Chimeric receptor LGR7/8 with the ectodomain from LGR7 but the transmembrane region from LGR8 maintains responsiveness to relaxin but was less responsive to H3 relaxin based on ligand stimulation of cAMP production. The decreased ligand signaling was accompanied by decreases in the ability of H3 relaxin to compete for 33 P-relaxin binding to the chimeric receptor. However, replacement of the exoloop 2, but not exoloop 1 or 3, of LGR7 to the chimeric LGR7/8 restored ligand binding and receptor-mediated cAMP production. These results suggested that activation of LGR7 by H3 relaxin involves specific binding of the ligand to both the ectodomain and the exoloop 2, thus providing a model with which to understand the molecular basis of ligand signaling for this unique subgroup of G protein-coupled receptors.Relaxin and Leydig insulin-like peptide/relaxin-like factor (INSL3) 1 are peptide hormones with a two-chain structure similar to that of insulin (1, 2). Relaxin is important for the function of reproductive tissues, heart, kidney, and brain (3), whereas INSL3 is essential for testis descent (4, 5). We have recently demonstrated that two orphan leucine-rich repeatcontaining, G protein-coupled receptors (LGRs) with homology to gonadotropin and thyrotropin receptors, are capable of mediating the action of relaxin through a cAMP-dependent pathway (6). These two receptors, LGR7 and LGR8, share 50% sequence identity to each other, and contain a unique low density lipoprotein receptor-like cysteine-rich motif at the amino terminus. However, LGR7 and LGR8 do not have the consensus hinge region found in gonadotropin and thyrotropin receptors. In contrast to relaxin, INSL3 activates LGR8 but not LGR7; interactions between INSL3 and LGR8 were demonstrated by ligand-receptor cross-linking (7).In addition to the two known human relaxin genes, H1 (8) and H2 (9), another related gene, designated H3 relaxin (H3), was identified recently. A synthetic peptide with a design based on this gene was found to possess relaxin activity in bioassays using the human monocyte cell line, THP-1 (10). Here, we demonstrate that H3 relaxin activates recombinant LGR7 but not LGR8. Taking advantage of the structural similarity of LGR7 and LGR8, and the differential specificity of relaxinrelated peptides to these receptors, we designed chimeric LGR7/LGR8 receptors to identify the domains in the receptor that are ...

Relaxin family peptide 1 (RXFP1) receptor (LGR7) and RXFP2 receptor (LGR8) were recently identified as the receptor targets for H2 relaxin and insulin-like peptide 3 (INSL3), respectively. In this study, we define the pharmacology of these two receptors by using a number of receptor chimeras and relaxin family peptides. We have identified two binding sites on these receptors: one primary, high-affinity site within the ectodomain and a secondary, lower affinity site within the transmembrane region. The primary site was found to dictate receptor binding characteristics, although the lower affinity site also exerts some influence and modulates ligand affinity for the primary site in a manner dependent upon the peptide in question. Not all relaxin peptides were able to bind to the RXFP2 receptor, indicating that the relaxin-RXFP2 receptor interaction is species-specific. INSL3 was found to exhibit characteristics of a partial agonist at the RXFP2 and chimeric RXFP1/2 receptors, with low maximal cAMP responses but high potency in coupling to this pathway. cAMP accumulation studies also revealed that the binding sites couple to cAMP signaling pathways with differing efficiency: the high-affinity site signals with high efficiency, whereas the lower affinity site signals with little to no efficiency. Comparisons between RXFP1, RXFP2, the chimeric receptors, and the truncated receptors revealed that the interaction between receptor sites is critical for optimal ligand binding and signal transduction and that the ectodomain is essential for signaling. Evidence obtained in this study supports a two-stage binding model of receptor activation: binding to the primary site allows a conformational change and interaction with the low-affinity transmembrane site.Relaxin is a two-chain peptide that was discovered after the observation that serum from pregnant guinea pigs caused relaxation of the pubic ligament (Hisaw, 1926). It is structurally closely related to insulin: both peptides have an A and B chain joined by two interchain disulfide bonds, and one intra-A-chain disulfide bond. This discovery established the concept of the insulin-relaxin superfamily (Schwabe and McDonald, 1977). The high degree of similarity between insulin and relaxin precipitated a search for additional members of the insulin-relaxin superfamily with the same structural motif. To date, this peptide family includes insulin-like growth factor-1 and insulin-like growth factor-II (Humbel, 1990), INSL3 (Adham et al., 1993), INSL4 (Koman et al., 1996, INSL5 (Conklin et al., 1999), and INSL6 (Lok et al., 2000).Relaxin itself has many paralogs: in humans, three nonallelic genes produce H1 relaxin (Hudson et al., 1983), H2 relaxin (Hudson et al., 1984, and the recently identified H3 relaxin . H2 relaxin is the major circulating form of relaxin in the human and has equivalent orthologs in other species, including porcine relaxin (Hudson et al., 1981;Haley et al., 1982), rhesus monkey relaxin (Crawford et al., 1989), and rat relaxin (Hudson et al., 1981)...