H3 Relaxin Is a Specific Ligand for LGR7 and Activates the Receptor by Interacting with Both the Ectodomain and the Exoloop 2

Abstract

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 ...

Negative cooperativity in H2 relaxin binding to a dimeric relaxin family peptide receptor 1

“…There is no competition with INSL3 M a n u s c r i p t 13 at the highest concentration used, also consistent with previous findings (Sudo et al, 2003) (Figure 4 constructs close to GFP 2 constructs. We used neurokinin type 1 receptor as a negative control ( Figure 25 7 A).…”

“…The K d of H3 relaxin towards RXFP1 was much lower than the affinity of H2 relaxin (18.55 ± 3.9 26 nM) (Figure 4 B), as has been shown before (Sudo et al, 2003). There is no competition with INSL3 M a n u s c r i p t 13 at the highest concentration used, also consistent with previous findings (Sudo et al, 2003) (Figure 4 constructs close to GFP 2 constructs.…”

Relaxin receptors in hepatic stellate cells and cirrhotic liver

“…Insulin-like peptide 3 (InsL3), also known as relaxin-like factor, is a selective activator of LGR8 [22]. Another relaxin family member, relaxin-3 (also known as InsL7), is a selective activator of LGR7 [23]. Treatment of activated HSC with either relaxin-3 or InsL3 significantly increased cellular cAMP to a level comparable to that induced by relaxin (Fig.…”

The NMR Solution Structure of the Relaxin (RXFP1) Receptor Lipoprotein Receptor Class A Module and Identification of Key Residues in the N-terminal Region of the Module That Mediate Receptor Activation

“…Simplistically, ligand binding stimulates cAMP production through the G s stimulatory pathway. The molecular details of how relaxin binding induces signaling are not yet clear; however, relaxin has been shown to bind primarily to the LRRs and the second extracellular loop of the transmembrane region (27). There is no evidence to suggest that the LDLa module is involved in the binding of relaxin.…”