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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)...

Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.

The receptors for the peptide hormones relaxin and insulinlike peptide 3 (INSL3) are the leucine-rich repeat-containing G-protein-coupled receptors LGR7 and LGR8 recently renamed as the relaxin family peptide (RXFP) receptors, RXFP1 and RXFP2, respectively. These receptors differ from other LGRs by the addition of an N-terminal low density lipoprotein receptor class A (LDLa) module and are the only human G-protein-coupled receptors to contain such a domain. Recently it was shown that the LDLa module of the RXFP1 and RXFP2 receptors is essential for ligand-stimulated cAMP signaling. The mechanism by which the LDLa module modulates receptor signaling is unknown; however, it represents a unique paradigm in understanding G-protein-coupled receptor signaling. Here we present the structure of the RXFP1 receptor LDLa module determined by solution NMR spectroscopy. The structure is similar to other LDLa modules but shows small differences in side chain orientations and inter-residue packing. Interchange of the module with the second ligand binding domain of the LDL receptor, LB2, results in a receptor that binds relaxin with full affinity but is unable to signal. Furthermore, we demonstrate via structural studies on mutated LDLa modules and functional studies on mutated full-length receptors that a hydrophobic surface within the N-terminal region of the module is essential for activation of RXFP1 receptor signal in response to relaxin stimulation. This study has highlighted the necessity to understand the structural effects of single amino acid mutations on the LDLa module to fully interpret the effects of these mutations on receptor activity.Relaxin and INSL3 (insulin-like peptide 3) are small twochain peptide hormones that belong to the relaxin-insulin superfamily. Relaxin was observed in the 1920s to induce the widening of the birth canal (1) and subsequently has been studied for the roles that it plays during pregnancy. More recently it has become apparent that relaxin is a pleiotropic hormone involved in numerous nonreproductive processes such as vasodilatation and neoangiogenesis, embryo implantation (2), collagen turnover (3, 4), and cancer progression (5). INSL3 was more recently discovered in the 1990s (6 -8), and although understanding of this hormone is still ongoing, in rodents it is clearly involved in testis descent (9, 10) and involved in oocyte maturation and male germ cell survival (11).The receptors for relaxin and INSL3 are LGR7 and LGR8, recently renamed the relaxin family peptide 1 (RXFP1) and the relaxin family peptide 2 (RXFP2) receptors, respectively (12-14). These receptors are leucine-rich repeat-containing G-protein-coupled receptors (LGR) 3 and thus belong to the LGR family. The discovery of the RXFP1 and RXFP2 receptors resulted in the addition of a third class of LGRs (15), and at present there are three subclasses of LGRs, type A, B, and C. Type A LGRs include receptors for the glycoprotein hormones follicle-stimulating hormone, thyrotropin, and luteinizing hormone and consist of a rhodops...

The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide-and receptor-dependent.Relaxin was first identified more than 90 years ago and subsequently shown to be a peptide hormone having a two-chain structure similar to insulin ( Fig. 1) (1). It has since been established that relaxin is a member of the relaxin peptide family, comprising a total of seven members in the human (2). These are the H1, 3 H2, and H3 relaxin peptides that are encoded by the three relaxin genes RLN1 to -3 and the insulin-like peptides INSL3 to -6 (insulin-like peptides 3-6). Phylogenetic analyses indicate that all of these relaxin family peptides evolved from a relaxin-3 (H3 relaxin equivalent) ancestral gene prior to the emergence of fish (3). In most mammals other than humans and higher primates, there are only two relaxin genes that encode relaxin and relaxin-3. The RLN1 gene in these species is equivalent to the RLN2 gene in humans (encoding H2 relaxin) and higher primates and encodes the relaxin peptide that is expressed by the corpus luteum and/or placenta (2). The function of the RLN1 gene in higher primates is unknown, and an H1 relaxin peptide has not been isolated.In contrast to the receptors for insulin and insulin-like growth factors I and II, which are tyrosine kinases, the receptors for relaxin family peptides are members of two unrelated branches of the G-protein-coupled recepto...

The SOX (sex-determining region [SRY]-type high mobility group [HMG] box) family of transcription factors play key roles in determining cell fate during organ development. In this study, we have identified a new human SOX gene, S O X 1 3, as encoding the type 1 diabetes autoantigen, islet cell antigen 12 (ICA12). Sequence analysis showed that SOX13 belongs to the class D subgroup of SOX transcription factors, which contain a leucine zipper motif and a region rich in glutamine. SOX13 autoantibodies occurred at a significantly higher frequency among 188 people with type 1 diabetes (18%) than among 88 with type 2 diabetes (6%) or 175 healthy control subjects (4%). Deletion mapping of the antibody epitopes showed that the autoantibodies were primarily directed against an epitope requiring the majority of the protein. S O X 1 3 R N A was detected in most human tissues, with the highest levels in the pancreas, placenta, and kidney. Immunohistochemistry on sections of human pancreas identified SOX13 in the islets of Langerhans, where staining was mostly cytoplasmic. In mouse pancreas, Sox13 was present in the nucleus and cytoplasm of -cells as well as other islet cell types. Recombinant SOX13 protein bound to the SOX consensus DNA motif AACAAT, and binding was inhibited by homodimer formation. These observations-along with the known molecular interactions of the closely related protein, rainbow trout Sox23-suggest that SOX13 may be activated for nuclear import and DNA binding through heterodimer formation. In conclusion, we have identified ICA12 as the putative transcription factor SOX13 and demonstrated an increased frequency of autoantibody reactivity in sera from type 1 diabetic subjects compared with type 2 diabetic and healthy control subjects. T ype 1 diabetes results from the destruction of pancreatic islet -cells by an autoimmune response to -cell constituents, which is initiated by an unknown environmental agent acting on a susceptible genetic background. Identification of GAD (1), a family of tyrosine phosphatase-like proteins variously designated islet cell antigen 512 (ICA512)/IA-2/IA-2 (2,3), and insulin (4) as autoantigens in type 1 diabetes has led to the proposal of several mechanisms for the initiation and progression of islet autoimmunity. These include molecular mimicry between GAD and coxsackievirus B (5) or between ICA512 and rotavirus (6), hyperexpression of GAD in response to metabolic stress (7), and lack of tolerance to (pro)insulin as a consequence of genetic variation of proinsulin expression levels in the thymus (8,9). The multifactorial nature of type 1 diabetes and the association of numerous environmental agents with the disease (10) suggest that there may be multiple pathways leading to -cell autoimm u n i t y. To attain a better understanding of the pathogenesis of type 1 diabetes, the autoimmune response to islet -c e l l s needs to be fully characterized.In a search for novel diabetes-associated autoantigens, the screen of an islet cDNA expression library with type 1 diabetes sera tha...

LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.