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

Satoko SudoDagger , Jin KumagaiDagger , Shinya NishiDagger , Sharon Layfield§, Tania Ferraro§, Ross A. D. Bathgate§, and Aaron J. W. HsuehDagger ||

From the Dagger  Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317 and the § Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Victoria 3010, Australia

Received for publication, December 6, 2002, and in revised form, December 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 orphan LGRs, LGR7 and LGR8, whereas INSL3/Leydig insulin-like 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 33P-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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 repeat-containing, 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 relaxin-related peptides to these receptors, we designed chimeric LGR7/LGR8 receptors to identify the domains in the receptor that are important for their ligand specificity. We demonstrate that both the ectodomain and exoloop 2 of LGR7 are important for ligand receptor binding and signaling.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hormones and Reagents-- Porcine relaxin was purchased from the National Hormone and Peptide Program (Torrance, CA). Recombinant human H2 relaxin was a gift from Dr. Elaine Unemori (Connectics Co., Palo Alto, CA). H3 relaxin and human INSL3 were chemically synthesized and characterized as described previously (10, 11). Anti-FLAG M1 monoclonal antibody and the FLAG peptide were purchased from Sigma Chemical Co. (St. Louis, MO). The soluble ectodomain of LGR7 was prepared as described previously (6). Briefly, cDNA for the ectodomain of human LGR7, named as 7BP, was fused in-frame with the prolactin signal peptide and the FLAG epitope at the 5'-end. At the 3'-end, the ectodomain was connected to the single transmembrane region of T cell surface antigen CD8 through a thrombin cleavage consensus region. Stable 293T cell lines expressing 7BP encoded in the pcDNA3.1-Zeo expression vector (Invitrogen Co., Carlsbad, CA) were selected using Zeocin (0.5 mg/ml). To release soluble 7BP anchored on the cell surface, cells were treated with thrombin (10 IU/ml) for 3 days under serum-free conditions. The cleaved 6-His- and FLAG epitope-tagged recombinant 7BP in serum-free conditioned media was purified by sequential nickel and anti-FLAG affinity chromatography.

Construction of Chimeric Receptor cDNAs-- Polymerase chain reaction-based, site-directed mutagenesis was performed to generate mutant receptor cDNAs (12) using cDNAs encoding human LGR7 (13) and human LGR8 (6). To predict the transmembrane helices in LGR7 and LGR8, a membrane protein topology prediction method based on a hidden Markov model (available at www.cbs.dtu.dk/services/TMHMM) was used (14). This method was also used to deduce the junctions of exoloops and transmembrane helices for the chimeric constructs. The junctional amino acid sequences for different chimeric receptors are listed in Table I. Polymerase chain reaction was performed using Vent DNA polymerase (New England BioLabs, Inc., Beverly, MA) in accordance with the manufacturer's instructions. All cDNAs were subcloned into the expression vector pcDNA3.1/Zeo (Invitrogen Co.). To allow efficient targeting of receptors to the cell surface, a lead cDNA sequence containing a prolactin signal peptide for secretion (MNIKGSPWKGSLLLLLLVSNLLLCQSVAP) and an M1 FLAG (DYKDDDDK) epitope were added to the amino terminus of the mature region of all receptors (15). The expression constructs were purified using the Plasmid Maxi kit (Qiagen, Inc., Valencia, CA). Fidelity of the PCR products was confirmed by sequencing on both strands of the final constructs before use in expression studies.

                              
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Table I
Junctional amino acid sequences for different chimeric receptors

Transfection of Cells and Analysis of Signal Transduction-- Human 293T cells derived from human embryonic kidney fibroblast were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 (Invitrogen Co.) supplemented with 10% fetal bovine serum, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Before transfection, 2 × 106 cells were seeded in 10-cm dishes (Becton Dickinson, Franklin Lakes, NJ). When cells were 70-80% confluent, transient transfection was performed using 10 µg of plasmid by the calcium phosphate precipitation method. Cells (2 × 105/ml) were placed on 24-well tissue culture plates (Corning, Corning, NY) and preincubated at 37 °C for 30 min in the presence of 0.25 mM 3-isobutyl-1-methyl xanthine (Sigma Chemical Co.) before treatment with or without hormones for 16 h. At the end of incubation, cells and medium were frozen. After thawing, samples were heated to 95 °C for 3 min to inactivate phosphodiesterase activity, and total cAMP was measured in triplicate by a specific radioimmunoassay as described previously (16). All experiments were repeated at least three times using cells from independent transfections. To monitor transfection efficiency, 0.5 µg of beta -galactosidase plasmid (17) was routinely included in the transfection mixture, and beta -galactosidase activity in the cell lysate was measured as described previously (18). Statistical analysis was performed using Student's t test.

Determination of FLAG Epitope-tagged Receptors on the Cell Surface (M1 Binding)-- The levels of cAMP production were normalized by correcting for varying expressions of receptors to monitor the levels of their tagged epitope. Transfected cells were washed twice with Dulbecco's PBS, and resuspended cells (2 × 106/tube) were incubated with the FLAG M1 antibody (50 mg/ml) (Sigma Chemical Co.) in Tris-buffered saline (pH 7.4) containing 5 mg/ml bovine serum albumin and 2 mM CaCl2 (assay buffer) for 4 h at room temperature in siliconized tubes. Cells were then washed twice with 1 ml of assay buffer after centrifugation at 14,000 × g for 15 s. The 125I-labeled second antibody (anti-mouse IgG from sheep: ~400,000 cpm/tube) was added to the resuspended cell pellet and incubated for 1 h at room temperature. Cells were washed twice with 1 ml of assay buffer by repeated centrifugation before determination of radioactivity in cell pellets. Background binding was determined by adding excess amounts of the synthetic FLAG peptide (Sigma Chemical Co.) at a concentration of 100 µg/ml.

Ligand Binding Analysis-- Transiently transfected 293T cells were plated in 24-well plates for whole cell binding assays. Media were removed, and cells were washed with PBS before preincubation in 300 µl of binding buffer (20 mM HEPES, 50 mM NaCl, 1.5 mM CaCl2, 1% bovine serum albumin, 0.1 mg/ml lysine, 0.01% NaN4, pH 7.5) (19). Binding studies were performed with 100 µl of 33P-labeled H2 relaxin, labeled as previously described (20), and 100 µl of competitor or blank in binding buffer at 25 °C for 60 min. Saturation binding was performed using increasing concentrations of 33P-labeled H2 relaxin, whereas competition experiments were performed with 100 pM 33P-labeled H2 relaxin in the absence or presence of increasing concentrations of unlabeled peptides. Nonspecific binding was determined by an excess of H2 relaxin (1 µM). After incubation, the cells were washed with PBS followed by their recovery from the plates using 500 µl of 1 M NaOH before transfer to scintillation vials. Liquid scintillation mixture (Ultima Gold, Packard, Meriden, CT) was added to these vials for counting in a liquid scintillation analyzer (Packard 1900 TR). Data are expressed as mean ± S.E. of the percentage of specific binding of triplicate determinations performed at least three times. Furthermore, data were analyzed using the LIGAND program (21). All Scatchard plots were linear, and the best fit to the data, given by LIGAND, was obtained with a one-binding site model. IC50 values, determined from the competition curves, were analyzed by one-way analysis of variance followed by a Newman-Keuls multiple comparison test.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LGR7 Is Activated by Porcine Relaxin, H2 Relaxin, and H3 Relaxin, Whereas LGR8 Is Activated by Porcine Relaxin, H2 Relaxin, and INSL3-- Earlier studies indicated that porcine relaxin activates both LGR7 and LGR8, whereas different INSL3 preparations activate only LGR8 (6, 7). Furthermore, H3 relaxin, like H2 relaxin, stimulates cAMP production in the THP-1 cells and competes for 33P-relaxin binding to its receptors (10). Based on these results, we tested the ability of these peptides in the activation of human LGR7 and LGR8. 293T cells were transiently transfected with LGR7 and LGR8 plasmids and ligand signaling was estimated based on total cAMP production (Fig. 1). To correct for varying receptor expression, all data for this and subsequent experiments were normalized based on cell surface M1 antibody binding to the tagged FLAG epitope of the recombinant receptors (Table II). The EC50 values and maximal levels of cAMP production for different dose-response curves are shown in Table III.


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Fig. 1.   LGR7 is activated by porcine relaxin and H2 relaxin as well as H3 relaxin, whereas LGR8 is activated by porcine relaxin, H2 relaxin, and INSL3. A, ligand-stimulated cAMP production mediated by LGR7. B, ligand-mediated cAMP production mediated by LGR8. Purified porcine relaxin, recombinant H2 relaxin, synthetic H3 relaxin, and synthetic INSL3 were used.

                              
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Table II
Cell surface expression of LGR7, LGR8, and different chimeric receptors
Expression of different receptors was estimated based on the cell surface binding of the labeled M1 antibody against the FLAG epitope appended to the amino terminus of individual receptors. Values are mean ± S.E.

                              
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Table III
Kinetic parameters for the hormonal stimulation of cAMP production by wild type and mutant receptors and the competition for labeled H2 binding to the same receptors by different peptides
Data represent the mean ± S.E. of three to four separate experiments with triplicate cultures for each experiment. Estimations of EC50 values for all peptides were based on maximal cAMP production (Max) induced by H2. EC50 and IC50 values are in nM.

In cells expressing LGR7, treatment with porcine relaxin and H2 relaxin led to dose-dependent increases in cAMP production. Although with lower efficacy, treatment with H3 relaxin also stimulated a dose-dependent increase in cAMP levels, whereas treatment with INSL3 was ineffective (Fig. 1A). In contrast, cells expressing LGR8 responded to treatment with INSL3, porcine relaxin, and H2 relaxin with increases in cAMP production (Fig. 1B). Moreover, treatment with H3 relaxin was ineffective in activating LGR8.

To demonstrate the receptor binding of relaxin-related peptides, cells expressing LGR7 and LGR8 were incubated with 33P-labeled H2 relaxin with or without increasing competing ligands. As shown in Fig. 2A, Scatchard plot analyses of saturation binding studies indicated that 33P-labeled H2 relaxin shows a higher affinity for LGR7 (Kd: 0.209 ± 0.025, n = 4) than LGR8 (1.062 ± 0.127; n = 4) (p < 0.05). Consistent with these results and the observed potencies in stimulating cAMP production, H2 relaxin was most potent in competing for LGR7 binding by 33P-labeled H2 relaxin, whereas H3 relaxin has lower binding affinity than H2 relaxin. In contrast, INSL3 showed minimal affinity (Fig. 2B). For LGR8, the displacement data showed that INSL3 and H2 relaxin, but not H3 relaxin, could compete for 33P-labeled H2 relaxin binding to this receptor (Fig. 2C). The IC50 values for different peptides are shown in Table III. These results demonstrate that H3 relaxin binds to LGR7, but not LGR8, and stimulates cAMP production mediated by LGR7. Thus, H3 relaxin is a specific ligand for LGR7, but not LGR8. In contrast, INSL3 binds to LGR8 and activates cAMP production, acting as a specific ligand for LGR8.


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Fig. 2.   Direct binding of 33P-labeled H2 relaxin to LGR7 and LGR8 and competition by relaxin-related peptides. A, Scatchard plot analyses of 33P-labeled H2 binding to LGR7 and LGR8. B, competition of 33P-labeled H2 relaxin binding to LGR7 by relaxin-related peptides. C, competition of 33P-labeled H2 relaxin binding to LGR8 by relaxin-related peptides. D, the soluble ectodomain of LGR7 (7BP) blocks H3 relaxin stimulation of cAMP production by LGR7-expressing cells.

We have used an anchored receptor approach to generate soluble ectodomains of the gonadotropin and thyrotropin receptors (15), as well as the ectodomain of LGR7. The soluble ectodomain of LGR7, designated as 7BP, was able to block relaxin actions in vitro and in vivo (6). To test whether the ectodomain of LGR7 is capable of interacting with H3 relaxin, we treated 293T cells expressing LGR7 with H3 relaxin and H2 relaxin together with 7BP. As shown in Fig. 2D, co-treatment with 7BP completely blocked the stimulatory effects of H3 and H2 relaxin in a dose-dependent manner, thus demonstrating the ability of the ectodomain of LGR7 to bind these ligands.

H3 Activates Chimeric Receptor LGR7/8, Whereas INSL3 Activates Chimeric Receptor LGR8/7-- To further confirm the importance of the ectodomain of LGR7 and LGR8 for ligand binding, we constructed chimeric receptors with their extracellular regions switched. LGR7/8 is comprised of the extracellular region from LGR7 and the transmembrane region to the carboxyl terminus from LGR8. In contrast, LGR8/7 has the extracellular region from LGR8 and the transmembrane region and C-tail from LGR7. As shown in Fig. 3, H2 relaxin stimulated cAMP production in transfected 293T cells expressing LGR7/8 (Fig. 3A) or LGR8/7 (Fig. 3C), whereas treatment with INSL3 resulted in a dose-dependent cAMP increase only in cells expressing LGR8/7 (Fig. 3C). Even though H3 relaxin stimulates cells expressing LGR7/8 to produce cAMP in a dose-dependent manner (Fig. 3A), the efficacy of cAMP production is lower compared with the H3 relaxin stimulation of wild type LGR7 (Fig. 1A). H3 was 30-fold less potent than H2 in stimulating wild type LGR7 (Table III). In contrast, H3 was 60-fold less potent than H2 in activating the chimeric LGR7/8. Receptor binding analyses also showed that H3 relaxin exhibited a decreased ability to compete for 33P-H2 relaxin binding to the chimeric receptor LGR7/8 (Fig. 3B) as compared with the H3 relaxin competition for 33P-H2 relaxin binding to the wild type LGR7 (Fig. 2B). Although the ability of H3 relaxin to bind and stimulate the LGR7 receptor could be blocked by the soluble ectodomain of LGR7, these results suggested that additional regions of LGR7 might participate in receptor signaling by the H3 relaxin. In contrast, the chimeric receptor LGR8/7 responded to INSL3 and H2 relaxin but was not stimulated by H3 relaxin. Furthermore, INSL3 and H2 relaxin competed effectively for 33P-H2 relaxin binding to LGR8/7 (Fig. 3D).


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Fig. 3.   H3 relaxin activates the chimeric receptor LGR7/8 with a lower efficacy than H2 relaxin, whereas INSL3 activates the chimeric receptor LGR8/7. A, H2 relaxin and H3 relaxin, but not INSL3, stimulate cAMP production mediated by LGR7/8. B, H2 relaxin and H3 relaxin, competed for 33P-H2 relaxin binding to LGR7/8. C, INSL3 and H2 relaxin stimulate cAMP production mediated by LGR8/7. D, INSL3 and H2 relaxin competed for 33P-H2 relaxin binding to LGR8/7.

Replacement of Exoloop 2, but Not Exoloop 1 or 3, of LGR7 in the Chimeric Receptor LGR7/8 Restores Ligand Binding and Signaling by H3 Relaxin-- We hypothesized that exoloops in LGR7, in addition to the ectodomain, are important for interaction with H3 relaxin and designed additional chimeric constructs by replacing the individual exoloop of LGR7 into the chimeric receptor LGR7/8. The chimeric receptor LGR7/8(EL2) with exoloop 2 and the ectodomain from LGR7 responded to H3 relaxin treatment with an EC50 value comparable to the H3 stimulation of wild type LGR7 (Figs. 1A versus 4B and Table III). In contrast, LGR7/8(EL1) and LGR7/8(EL2), chimeric receptors with the other exoloops replaced, responded to H3 relaxin treatment with an ED50 similar to the H3 stimulation of LGR7/8 (Figs. 4A and 4C versus Fig. 3A and Table III). Likewise, replacement of exoloop 2, but not exoloop 1 or 3, in the chimeric receptor LGR7/8 restores receptor binding by H3 relaxin (Fig. 5).


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Fig. 4.   Replacement of exoloop 2, but not exoloop 1 or 3, of LGR7 in the chimeric receptor LGR7/8 restores ligand signaling by H3 relaxin. A, LGR7/8(EL1); B, LGR7/8(EL2); C, LGR7/8(EL3).


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Fig. 5.   Replacement of exoloop 2, but not exoloop 1 or 3, of LGR7 in the chimeric receptor LGR7/8 restores receptor binding by H3 relaxin. A, LGR7/8(EL1); B, LGR7/8(EL2); C, LGR7/8(EL3).

When H3-stimulated maximal cAMP production mediated by wild type and mutant receptors was normalized as 100% (Fig. 6A), it became apparent that LGR7/8(EL2) is capable of responding to H3 relaxin treatment with cAMP production comparably to that of the wild type LGR7 (Table III). In contrast, cAMP production in cells expressing LGR7/8(EL1) or LGR7/8(EL3) showed higher EC50 values similar to that of LGR7/8 (EC50: LGR7/8, 66.2 nM; LGR7/8(EL1), 69.7 nM; LGR7/8(EL3), 71.8 nM; p < 0.01 versus LGR7 or LGR7/8(EL2)). Likewise, the competition of 33P-labeled H2 relaxin binding to LGR7 and LGR7/8(EL2) by relaxin H3 (IC50: 33 and 12 nM, respectively) displayed a >10-fold reduction in the IC50 value as compared with those for LGR7/8, LGR7/8(EL1), and LGR7/8(EL3) (IC50: 557, 167, and 315 nM, respectively; p < 0.01 versus LGR7 or LGR7/8(EL2)) (Fig. 6B). These data demonstrate the importance of exoloop 2 in the H3 relaxin binding and activation of LGR7.


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Fig. 6.   Comparison of H3 relaxin stimulation of cAMP production and receptor binding inhibition by wild type LGR7 and different chimeric receptors. A, H3 relaxin stimulation of cAMP production mediated by different receptors is standardized based on maximal cAMP production induced by H3 relaxin (100%). B, H3 relaxin competition of 33P-labeled H2 relaxin binding to different receptors is compared with binding in the absence of competing ligands (set at 100%).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent studies demonstrated that porcine relaxin activates LGR7, and, with lower efficacy, LGR8. In addition, INSL3 is a specific and more potent ligand for LGR8 than relaxin (6, 7). The present data indicate that relaxin H3 is a specific ligand for LGR7 but not LGR8. However, relaxin H3 is less potent than porcine relaxin or H2 relaxin in activating LGR7.

Taking advantage of the structural similarity between LGR7 and LGR8, and because leucine-rich repeats in the ectodomain have been postulated to be important for protein-protein interactions (22-24), we designed chimeric constructs to investigate the importance of the ectodomain of these receptors in ligand signaling. Similar to wild type LGR7, LGR7/8 responded to porcine relaxin and H2 relaxin stimulation, whereas neither LGR7 nor LGR7/8 responded to treatment with INSL3. Although treatment with H3 relaxin in cells expressing LGR7/8 led to dose-dependent increases in cAMP production, this chimeric receptor was less responsive to H3 relaxin as compared with the wild type LGR7. Further analyses of competition data based on ligand-receptor binding also indicated that the chimeric receptor LGR7/8 showed lower affinity for H3 relaxin. These results suggest that, in addition to the ectodomain, the transmembrane region of LGR7 plays an important role for optimal H3 relaxin binding and activation of LGR7. We further demonstrated that replacement of exoloop 2 of LGR7 in the chimeric receptor LGR7/8 completely restored receptor binding and cAMP production. In contrast, LGR7/8(EL1) or LGR7/8(EL3) showed similar EC50 and IC50 values for LGR7/8. These results indicate that exoloop 2, but not exoloop 1 or 3, is responsible for H3 relaxin binding to the receptor and optimal signal transduction.

In the human genome, there are seven peptide hormones belonging to the relaxin family, all with the putative two-chain, three cysteine-bounded structure. The homology between the A- and B-chain of H1 and H2 relaxin is 62 and 85%, respectively. These two hormones show similar biological activity. H2 relaxin is expressed in the corpus luteum and is the major circulating form (25), whereas the expression of H1 relaxin is restricted to decidua, trophoblasts, and prostate (26). The distinctive RXXXRXX(I/V) motif in the B-chain of H1 and H2 relaxin is believed to be the contact site for receptor binding (27). Although the paralogous INSL3 produced by the testis and ovary (4, 5) is a specific ligand for LGR8, another human paralog relaxin 3, designated as H3 relaxin, has the RXXXRXXI motif in the B-chain and stimulates cAMP production by THP-1 cells expressing the relaxin receptors (10). The distribution of H3 relaxin in human tissues is unknown; however, the predominant site of relaxin 3 expression in rodents is the brain (10, 28). H3 relaxin is believed to be a neuropeptide that activates its receptor in neuronal synapses (28), thus being consistent with its lower efficacy in activating recombinant LGR7 as compared with the more potent endocrine hormone H2 relaxin.

The known LGRs from vertebrates and invertebrates can be divided into three distinct subgroups based on phylogenetic analysis. The first subgroup contains the mammalian gonadotropin and thyroid-stimulating hormone receptors, fly LGR1, and LGRs from sea anemone and Caenorhabditis elegans, whereas the second subgroup consists of mammalian orphan receptors LGR4, LGR5, and LGR6, as well as fly LGR2 (29, 30). The third group of LGRs, including mammalian LGR7 and LGR8 as well as snail LGR (13), is distinct from the other groups in that it has the unique low density lipoprotein receptor-like cysteine-rich motifs in the amino terminus but is missing the typical hinge region known to be important for gonadotropin and thyroid-stimulating hormone receptor activation (31).

At least three steps are involved in the ligand signaling of glycoprotein hormone receptors, each probably requiring unique but overlapping domains (31-34). First, the heterodimeric ligands interact with the ectodomain of the receptor, consisting of leucine-rich repeats that could form a 1/3-donut structure important for ligand-interaction. Second, ligand binding leads to the disruption of the constraint on the transmembrane region exerted by the interactions between the ectodomain (likely the hinge region) and exoloop 2. Third, the relaxed transmembrane region, as the result of ligand binding, interacts with the Gs protein to activate the adenyl cyclase. In this model, it is likely that the common alpha -subunit of the glycoprotein hormones interacts with the leucine-rich repeats of the receptor ectodomains, whereas the unique beta -subunits of these ligands stabilize the ligand-receptor complex by binding to the exoloops (33). Due to the lack of a hinge region in LGR7 and LGR8 comparable to those found in glycoprotein hormone receptors, it is unclear whether the ectodomains of these relaxin receptors are capable of constraining their transmembrane region similar to glycoprotein hormone receptors.

Although the present studies using chimeric receptors and the soluble ectodomain of LGR7 suggest an important role for the ectodomain in ligand-receptor binding and signal transduction, our data demonstrate that H3 relaxin binds to both the ectodomain and exoloop 2 of LGR7 to induce maximal signal transduction. We propose a model for the activation of LGR7 by H3 relaxin (Fig. 7). First, H3 relaxin binds to the ectodomain of LGR7 through the putative contact motif RXXXRXX(I/V) (Fig. 7A). This interaction could be blocked by the soluble ectodomain of LGR7. Subsequently, H3 relaxin also binds to exoloop 2 of LGR7 to stabilize the ligand-receptor complexes (Fig. 7B). Binding of H3 relaxin to both regions of LGR7 evokes efficient receptor activation by interacting with the Gs protein and stimulating cAMP production (Fig. 7C). Because LGR7 and LGR8 show 59% homology in exoloop 2, H2 relaxin could interact with the consensus sequence of these receptors and the present model could apply to the H2 relaxin.


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Fig. 7.   Proposed model for the ligand activation of LGR7. A, H3 relaxin binds to the ectodomain of LGR7. The unliganded receptor is in a constrained conformation and incapable of activating the Gs protein. B, H3 relaxin binds to exoloop 2 of LGR7 and stabilizes a relaxed conformation of the LGR7 transmembrane region capable of activating Gs. C, the liganded and activated receptor stimulates cAMP production.

In conclusion, we demonstrate that H3 relaxin is a specific ligand for LGR7, and using chimeric receptors, H3 relaxin is shown to bind both the ectodomain and the exoloop 2 for the activation of its receptor. Further studies using chimeric LGR7 and LGR8 receptors could provide useful information regarding the mechanism of receptor activation by H2 relaxin and INSL3 and aid in the understanding the structural-functional relationship between ligands and receptors for this unique group of G protein-coupled receptors.

    ACKNOWLEDGEMENTS

We thank Drs. John Wade, Ping Fu, and Feng Lin for peptide synthesis and Professor Geoffrey Tregear for support and encouragement. We also thank C. Spencer for editorial assistance, Dr. Elaine Unemori (Connectics Co., Palo Alto, CA) for human H2 relaxin, and the National Hormone & Peptide Program for the cAMP antiserum.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant HD23273 (to A. J. H.) and by Institute Block Grant 983001 to the Howard Florey Institute from the National Health and Medical Research Council (NHMRC) of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of an NHMRC R. D. Wright Fellowship.

|| To whom correspondence should be addressed. Tel.: 650-725-6802; Fax: 650-725-7102; E-mail: aaron.hsueh@stanford.edu.

Published, JBC Papers in Press, December 27, 2002, DOI 10.1074/jbc.M212457200

    ABBREVIATIONS

The abbreviations used are: INSL3, insulin-like peptide 3/Leydig insulin-like factor; LGR, leucine-rich repeat-containing, G protein-coupled receptor; PBS, phosphate-buffered saline; 7BP, ectodomain of LGR7.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Bedarkar, S., Turnell, W. G., Blundell, T. L., and Schwabe, C. (1977) Nature 270, 449-451[Medline] [Order article via Infotrieve]
2. Isaacs, N., James, R., Niall, H., Bryant-Greenwood, G., Dodson, G., Evans, A., and North, A. C. (1978) Nature 271, 278-281[Medline] [Order article via Infotrieve]
3. Sherwood, O. D. (1994) in The Physiology of Reproduction (Knobil, E. , and Neill, J. D., eds), 2nd Ed., Vol. 1 , pp. 861-1009, Raven Press, Ltd., New York
4. Nef, S., and Parada, L. F. (1999) Nat. Genet. 22, 295-299[CrossRef][Medline] [Order article via Infotrieve]
5. Ivell, R., and Bathgate, R. A. (2002) Biol. Reprod. 67, 699-705[Abstract/Free Full Text]
6. Hsu, S. Y., Nakabayashi, K., Nishi, S., Kumagai, J., Kudo, M., Sherwood, O. D., and Hsueh, A. J. (2002) Science 295, 671-674[Abstract/Free Full Text]
7. Kumagai, J., Hsu, S. Y., Matsumi, H., Roh, J. S., Fu, P., Wade, J. D., Bathgate, R. A., and Hsueh, A. J. (2002) J. Biol. Chem. 277, 31283-31286[Abstract/Free Full Text]
8. Hudson, P., Haley, J., John, M., Cronk, M., Crawford, R., Haralambidis, J., Tregear, G., Shine, J., and Niall, H. (1983) Nature 301, 628-631[Medline] [Order article via Infotrieve]
9. Hudson, P., John, M., Crawford, R., Haralambidis, J., Scanlon, D., Gorman, J., Tregear, G., Shine, J., and Niall, H. (1984) EMBO J. 3, 2333-2339[Abstract]
10. Bathgate, R. A., Samuel, C. S., Burazin, T. C., Layfield, S., Claasz, A. A., Reytomas, I. G., Dawson, N. F., Zhao, C., Bond, C., Summers, R. J., Parry, L. J., Wade, J. D., and Tregear, G. W. (2002) J. Biol. Chem. 277, 1148-1157[Abstract/Free Full Text]
11. Smith, K. J., Wade, J. D., Claasz, A. A., Otvos, L., Jr., Temelcos, C., Kubota, Y., Hutson, J. M., Tregear, G. W., and Bathgate, R. A. (2001) J. Pept. Sci. 7, 495-501[CrossRef][Medline] [Order article via Infotrieve]
12. Kudo, M., Osuga, Y., Kobilka, B. K., and Hsueh, A. J. (1996) J. Biol. Chem. 271, 22470-22478[Abstract/Free Full Text]
13. Hsu, S. Y., Kudo, M., Chen, T., Nakabayashi, K., Bhalla, A., van der Spek, P. J., van Duin, M., and Hsueh, A. J. (2000) Mol. Endocrinol. 14, 1257-1271[Abstract/Free Full Text]
14. Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E. L. (2001) J. Mol. Biol. 305, 567-580[CrossRef][Medline] [Order article via Infotrieve]
15. Osuga, Y., Kudo, M., Kaipia, A., Kobilka, B., and Hsueh, A. J. (1997) Mol. Endocrinol. 11, 1659-1668[Abstract/Free Full Text]
16. Davoren, J. B., and Hsueh, A. J. (1985) Biol. Reprod. 33, 37-52[Abstract]
17. Hall, C. V., Jacob, P. E., Ringold, G. M., and Lee, F. (1983) J. Mol. Appl. Genet. 2, 101-109[Medline] [Order article via Infotrieve]
18. Su, J. G., and Hsueh, A. J. (1992) Biochem. Biophys. Res. Commun. 186, 293-300[Medline] [Order article via Infotrieve]
19. Parsell, D. A., Mak, J. Y., Amento, E. P., and Unemori, E. N. (1996) J. Biol. Chem. 271, 27936-27941[Abstract/Free Full Text]
20. Tan, Y. Y., Wade, J. D., Tregear, G. W., and Summers, R. J. (1999) Br. J. Pharmacol. 127, 91-98[Abstract/Free Full Text]
21. Munson, P. J., and Rodbard, D. (1980) Anal. Biochem. 107, 220-239[Medline] [Order article via Infotrieve]
22. Jiang, X., Dreano, M., Buckler, D. R., Cheng, S., Ythier, A., Wu, H., Hendrickson, W. A., and el Tayar, N. (1995) Structure 3, 1341-1353[Medline] [Order article via Infotrieve]
23. Kajava, A. V., Vassart, G., and Wodak, S. J. (1995) Structure 3, 867-877[Medline] [Order article via Infotrieve]
24. Kobe, B., and Deisenhofer, J. (1993) Nature 366, 751-756[CrossRef][Medline] [Order article via Infotrieve]
25. Winslow, J. W., Shih, A., Bourell, J. H., Weiss, G., Reed, B., Stults, J. T., and Goldsmith, L. T. (1992) Endocrinology 130, 2660-2668[Abstract]
26. Hansell, D. J., Bryant-Greenwood, G. D., and Greenwood, F. C. (1991) J. Clin. Endocrinol. Metab. 72, 899-904[Abstract]
27. Bullesbach, E. E., and Schwabe, C. (2000) J. Biol. Chem. 275, 35276-35280[Abstract/Free Full Text]
28. Burazin, T. C., Bathgate, R. A., Macris, M., Layfield, S., Gundlach, A. L., and Tregear, G. W. (2002) J. Neurochem. 82, 1553-1557[CrossRef][Medline] [Order article via Infotrieve]
29. Hsu, S. Y., Liang, S. G., and Hsueh, A. J. (1998) Mol. Endocrinol. 12, 1830-1845[Abstract/Free Full Text]
30. Nishi, S., Hsu, S. Y., Zell, K., and Hsueh, A. J. (2000) Endocrinology 141, 4081-4090[Abstract/Free Full Text]
31. Nakabayashi, K., Kudo, M., Kobilka, B., and Hsueh, A. J. (2000) J. Biol. Chem. 275, 30264-30271[Abstract/Free Full Text]
32. Duprez, L., Parma, J., Costagliola, S., Hermans, J., Van Sande, J., Dumont, J. E., and Vassart, G. (1997) FEBS Lett. 409, 469-474[CrossRef][Medline] [Order article via Infotrieve]
33. Zeng, H., Phang, T., Song, Y. S., Ji, I., and Ji, T. H. (2001) J. Biol. Chem. 276, 3451-3458[Abstract/Free Full Text]
34. Nishi, S., Nakabayashi, K., Kobilka, B., and Hsueh, A. J. (2002) J. Biol. Chem. 277, 3958-3964[Abstract/Free Full Text]


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