Relaxin is a hormone important for the growth and remodeling of reproductive and other tissues during pregnancy. Although binding sites for relaxin are widely distributed, the nature of its receptor has been elusive. Here, we demonstrate that two orphan heterotrimeric guanine nucleotide binding protein (G protein)-coupled receptors, LGR7 and LGR8, are capable of mediating the action of relaxin through an adenosine 3',5'-monophosphate (cAMP)-dependent pathway distinct from that of the structurally related insulin and insulin-like growth factor family ligand. Treatment of antepartum mice with the soluble ligand-binding region of LGR7 caused parturition delay. The wide and divergent distribution of the two relaxin receptors implicates their roles in reproductive, brain, renal, cardiovascular, and other functions.

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 ...

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Lutropin (LH) and follitropin (FSH) receptors belong to a group of leucine-rich repeat-containing, G proteincoupled receptors (LGRs) found in vertebrates and flies.We fused the ectodomain of human LH or FSH receptors to the transmembrane region of fly LGR2. The chimeric human/fly receptors, unlike their wild type counterparts, exhibited ligand-independent constitutive activity. Because ectodomains likely interact with exoloops to constrain the receptors, individual exoloops of the chimeric receptor containing the ectodomain of the LH receptor and transmembrane region of fly LGR2 was replaced with LH receptor sequences. Chimeric receptors with the ectodomain and exoloop 2, but not exoloop 1 or 3, from LH receptors showed decreases in constitutive activity, but ligand treatment stimulated cAMP production. Furthermore, substitution of key resides in the hinge region of fly LGR2 with LH receptor sequences led to constitutive receptor activation; however, concomitant substitution of the homologous exoloop 2 of the LH receptor decreased G s coupling. These results suggest that the hinge region of the LH receptor interacts with exoloop 2 to constrain the receptor in an inactive conformation whereas ligand binding relieves this constraint, leading to G s activation.The receptors for lutropin (LH), 1 follitropin (FSH), and thyrotropin (TSH) belong to the large G protein-coupled receptor (GPCR) family with seven-transmembrane (TM) helices, but are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interaction with the glycoprotein ligands (1, 2). Recent studies indicate the evolution of a large family of the leucine-rich repeat-containing, G protein-coupled receptors (LGRs) with at least seven members in mammals, including the well studied glycoprotein hormone receptors (3, 4) and LGR4 -7 (5, 6). In addition, homologous LGRs were found in fly (LGR1 and LGR2) (7, 8), nematode (nLGR) (9), sea anemone (10), and snail (11). These genes can be divided into three subgroups, each with unique structural characteristics (6). Understanding of the evolutionary relationship of these receptors and the availability of recombinant proteins provided the opportunity to elucidate their ligand-signaling mechanisms.The allosteric ternary complex model proposes the isomerization of GPCRs from an inactive to an active state capable of coupling to G proteins (12). This isomerization involves conformational changes that may be induced or can occur spontaneously, thus allowing the constrained receptor to relax into an active conformation. The active conformation could be achieved following ligand activation of GPCRs or by gain-of-function mutations discovered in constitutively activated GPCRs. Although most of the gain-of-function mutations were found in the TM region of different , studies using thyrotropin and gonadotropin receptors indicate that point mutations in the ectodomain could also confer conformational changes in their TM regions. These point mutations were found in the hinge...