FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2057916223> ?p ?o ?g. }

• W2057916223 endingPage "7862" @default.
• W2057916223 startingPage "7855" @default.
• W2057916223 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 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. 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. insulin-like peptide 3/Leydig insulin-like factor leucine-rich repeat-containing, G protein-coupled receptor phosphate-buffered saline ectodomain of LGR7 Relaxin and Leydig insulin-like peptide/relaxin-like factor (INSL3)1 are peptide hormones with a two-chain structure similar to that of insulin (1Bedarkar S. Turnell W.G. Blundell T.L. Schwabe C. Nature. 1977; 270: 449-451Google Scholar, 2Isaacs N. James R. Niall H. Bryant-Greenwood G. Dodson G. Evans A. North A.C. Nature. 1978; 271: 278-281Google Scholar). Relaxin is important for the function of reproductive tissues, heart, kidney, and brain (3Sherwood O.D. Knobil E. Neill J.D. 2nd Ed. The Physiology of Reproduction. 1. Raven Press, Ltd., New York1994: 861-1009Google Scholar), whereas INSL3 is essential for testis descent (4Nef S. Parada L.F. Nat. Genet. 1999; 22: 295-299Google Scholar, 5Ivell R. Bathgate R.A. Biol. Reprod. 2002; 67: 699-705Google Scholar). 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 (6Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Google Scholar). 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 (7Kumagai J. Hsu S.Y. Matsumi H. Roh J.S. Fu P. Wade J.D. Bathgate R.A. Hsueh A.J. J. Biol. Chem. 2002; 277: 31283-31286Google Scholar).In addition to the two known human relaxin genes, H1 (8Hudson P. Haley J. John M. Cronk M. Crawford R. Haralambidis J. Tregear G. Shine J. Niall H. Nature. 1983; 301: 628-631Google Scholar) andH2 (9Hudson P. John M. Crawford R. Haralambidis J. Scanlon D. Gorman J. Tregear G. Shine J. Niall H. EMBO J. 1984; 3: 2333-2339Google Scholar), 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 (10Bathgate 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. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Google Scholar). 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.DISCUSSIONRecent 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 (6Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Google Scholar, 7Kumagai J. Hsu S.Y. Matsumi H. Roh J.S. Fu P. Wade J.D. Bathgate R.A. Hsueh A.J. J. Biol. Chem. 2002; 277: 31283-31286Google Scholar). 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 (22Jiang X. Dreano M. Buckler D.R. Cheng S. Ythier A. Wu H. Hendrickson W.A. el Tayar N. Structure. 1995; 3: 1341-1353Google Scholar, 23Kajava A.V. Vassart G. Wodak S.J. Structure. 1995; 3: 867-877Google Scholar, 24Kobe B. Deisenhofer J. Nature. 1993; 366: 751-756Google Scholar), 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 (25Winslow J.W. Shih A. Bourell J.H. Weiss G. Reed B. Stults J.T. Goldsmith L.T. Endocrinology. 1992; 130: 2660-2668Google Scholar), whereas the expression of H1 relaxin is restricted to decidua, trophoblasts, and prostate (26Hansell D.J. Bryant-Greenwood G.D. Greenwood F.C. J. Clin. Endocrinol. Metab. 1991; 72: 899-904Google Scholar). 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 (27Bullesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Google Scholar). Although the paralogous INSL3 produced by the testis and ovary (4Nef S. Parada L.F. Nat. Genet. 1999; 22: 295-299Google Scholar, 5Ivell R. Bathgate R.A. Biol. Reprod. 2002; 67: 699-705Google Scholar) 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 (10Bathgate 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. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Google Scholar). The distribution of H3 relaxin in human tissues is unknown; however, the predominant site of relaxin 3 expression in rodents is the brain (10Bathgate 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. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Google Scholar,28Burazin T.C. Bathgate R.A. Macris M. Layfield S. Gundlach A.L. Tregear G.W. J. Neurochem. 2002; 82: 1553-1557Google Scholar). H3 relaxin is believed to be a neuropeptide that activates its receptor in neuronal synapses (28Burazin T.C. Bathgate R.A. Macris M. Layfield S. Gundlach A.L. Tregear G.W. J. Neurochem. 2002; 82: 1553-1557Google Scholar), 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 andCaenorhabditis elegans, whereas the second subgroup consists of mammalian orphan receptors LGR4, LGR5, and LGR6, as well as fly LGR2 (29Hsu S.Y. Liang S.G. Hsueh A.J. Mol. Endocrinol. 1998; 12: 1830-1845Google Scholar, 30Nishi S. Hsu S.Y. Zell K. Hsueh A.J. Endocrinology. 2000; 141: 4081-4090Google Scholar). The third group of LGRs, including mammalian LGR7 and LGR8 as well as snail LGR (13Hsu S.Y. Kudo M. Chen T. Nakabayashi K. Bhalla A. van der Spek P.J. van Duin M. Hsueh A.J. Mol. Endocrinol. 2000; 14: 1257-1271Google Scholar), 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 (31Nakabayashi K. Kudo M. Kobilka B. Hsueh A.J. J. Biol. Chem. 2000; 275: 30264-30271Google Scholar).At least three steps are involved in the ligand signaling of glycoprotein hormone receptors, each probably requiring unique but overlapping domains (31Nakabayashi K. Kudo M. Kobilka B. Hsueh A.J. J. Biol. Chem. 2000; 275: 30264-30271Google Scholar, 32Duprez L. Parma J. Costagliola S. Hermans J. Van Sande J. Dumont J.E. Vassart G. FEBS Lett. 1997; 409: 469-474Google Scholar, 33Zeng H. Phang T. Song Y.S. Ji I. Ji T.H. J. Biol. Chem. 2001; 276: 3451-3458Google Scholar, 34Nishi S. Nakabayashi K. Kobilka B. Hsueh A.J. J. Biol. Chem. 2002; 277: 3958-3964Google Scholar). 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 α-subunit of the glycoprotein hormones interacts with the leucine-rich repeats of the receptor ectodomains, whereas the unique β-subunits of these ligands stabilize the ligand-receptor complex by binding to the exoloops (33Zeng H. Phang T. Song Y.S. Ji I. Ji T.H. J. Biol. Chem. 2001; 276: 3451-3458Google Scholar). 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. 7 A). 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. 7 B). Binding of H3 relaxin to both regions of LGR7 evokes efficient receptor activation by interacting with the Gs protein and stimulating cAMP production (Fig.7 C). 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.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. Relaxin and Leydig insulin-like peptide/relaxin-like factor (INSL3)1 are peptide hormones with a two-chain structure similar to that of insulin (1Bedarkar S. Turnell W.G. Blundell T.L. Schwabe C. Nature. 1977; 270: 449-451Google Scholar, 2Isaacs N. James R. Niall H. Bryant-Greenwood G. Dodson G. Evans A. North A.C. Nature. 1978; 271: 278-281Google Scholar). Relaxin is important for the function of reproductive tissues, heart, kidney, and brain (3Sherwood O.D. Knobil E. Neill J.D. 2nd Ed. The Physiology of Reproduction. 1. Raven Press, Ltd., New York1994: 861-1009Google Scholar), whereas INSL3 is essential for testis descent (4Nef S. Parada L.F. Nat. Genet. 1999; 22: 295-299Google Scholar, 5Ivell R. Bathgate R.A. Biol. Reprod. 2002; 67: 699-705Google Scholar). 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 (6Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Google Scholar). 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 (7Kumagai J. Hsu S.Y. Matsumi H. Roh J.S. Fu P. Wade J.D. Bathgate R.A. Hsueh A.J. J. Biol. Chem. 2002; 277: 31283-31286Google Scholar). In addition to the two known human relaxin genes, H1 (8Hudson P. Haley J. John M. Cronk M. Crawford R. Haralambidis J. Tregear G. Shine J. Niall H. Nature. 1983; 301: 628-631Google Scholar) andH2 (9Hudson P. John M. Crawford R. Haralambidis J. Scanlon D. Gorman J. Tregear G. Shine J. Niall H. EMBO J. 1984; 3: 2333-2339Google Scholar), 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 (10Bathgate 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. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Google Scholar). 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. DISCUSSIONRecent 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 (6Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Google Scholar, 7Kumagai J. Hsu S.Y. Matsumi H. Roh J.S. Fu P. Wade J.D. Bathgate R.A. Hsueh A.J. J. Biol. Chem. 2002; 277: 31283-31286Google Scholar). 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 (22Jiang X. Dreano M. Buckler D.R. Cheng S. Ythier A. Wu H. Hendrickson W.A. el Tayar N. Structure. 1995; 3: 1341-1353Google Scholar, 23Kajava A.V. Vassart G. Wodak S.J. Structure. 1995; 3: 867-877Google Scholar, 24Kobe B. Deisenhofer J. Nature. 1993; 366: 751-756Google Scholar), 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 (25Winslow J.W. Shih A. Bourell J.H. Weiss G. Reed B. Stults J.T. Goldsmith L.T. Endocrinology. 1992; 130: 2660-2668Google Scholar), whereas the expression of H1 relaxin is restricted to decidua, trophoblasts, and prostate (26Hansell D.J. Bryant-Greenwood G.D. Greenwood F.C. J. Clin. Endocrinol. Metab. 1991; 72: 899-904Google Scholar). 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 (27Bullesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Google Scholar). Although the paralogous INSL3 produced by the testis and ovary (4Nef S. Parada L.F. Nat. Genet. 1999; 22: 295-299Google Scholar, 5Ivell R. Bathgate R.A. Biol. Reprod. 2002; 67: 699-705Google Scholar) 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 (10Bathgate 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. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Google Scholar). The distribution of H3 relaxin in human tissues is unknown; however, the predominant site of relaxin 3 expression in rodents is the brain (10Bathgate 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. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Google Scholar,28Burazin T.C. Bathgate R.A. Macris M. Layfield S. Gundlach A.L. Tregear G.W. J. Neurochem. 2002; 82: 1553-1557Google Scholar). H3 relaxin is believed to be a neuropeptide that activates its receptor in neuronal synapses (28Burazin T.C. Bathgate R.A. Macris M. Layfield S. Gundlach A.L. Tregear G.W. J. Neurochem. 2002; 82: 1553-1557Google Scholar), 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 andCaenorhabditis elegans, whereas the second subgroup consists of mammalian orphan receptors LGR4, LGR5, and LGR6, as well as fly LGR2 (29Hsu S.Y. Liang S.G. Hsueh A.J. Mol. Endocrinol. 1998; 12: 1830-1845Google Scholar, 30Nishi S. Hsu S.Y. Zell K. Hsueh A.J. Endocrinology. 2000; 141: 4081-4090Google Scholar). The third group of LGRs, including mammalian LGR7 and LGR8 as well as snail LGR (13Hsu S.Y. Kudo M. Chen T. Nakabayashi K. Bhalla A. van der Spek P.J. van Duin M. Hsueh A.J. Mol. Endocrinol. 2000; 14: 1257-1271Google Scholar), 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 (31Nakabayashi K. Kudo M. Kobilka B. Hsueh A.J. J. Biol. Chem. 2000; 275: 30264-30271Google Scholar).At least three steps are involved in the ligand signaling of glycoprotein hormone receptors, each probably requiring unique but overlapping domains (31Nakabayashi K. Kudo M. Kobilka B. Hsueh A.J. J. Biol. Chem. 2000; 275: 30264-30271Google Scholar, 32Duprez L. Parma J. Costagliola S. Hermans J. Van Sande J. Dumont J.E. Vassart G. FEBS Lett. 1997; 409: 469-474Google Scholar, 33Zeng H. Phang T. Song Y.S. Ji I. Ji T.H. J. Biol. Chem. 2001; 276: 3451-3458Google Scholar, 34Nishi S. Nakabayashi K. Kobilka B. Hsueh A.J. J. Biol. Chem. 2002; 277: 3958-3964Google Scholar). 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 α-subunit of the glycoprotein hormones interacts with the leucine-rich repeats of the receptor ectodomains, whereas the unique β-subunits of these ligands stabilize the ligand-receptor complex by binding to the exoloops (33Zeng H. Phang T. Song Y.S. Ji I. Ji T.H. J. Biol. Chem. 2001; 276: 3451-3458Google Scholar). 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. 7 A). 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. 7 B). Binding of H3 relaxin to both regions of LGR7 evokes efficient receptor activation by interacting with the Gs protein and stimulating cAMP production (Fig.7 C). 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.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. 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 (6Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Google Scholar, 7Kumagai J. Hsu S.Y. Matsumi H. Roh J.S. Fu P. Wade J.D. Bathgate R.A. Hsueh A.J. J. Biol. Chem. 2002; 277: 31283-31286Google Scholar). 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 (22Jiang X. Dreano M. Buckler D.R. Cheng S. Ythier A. Wu H. Hendrickson W.A. el Tayar N. Structure. 1995; 3: 1341-1353Google Scholar, 23Kajava A.V. Vassart G. Wodak S.J. Structure. 1995; 3: 867-877Google Scholar, 24Kobe B. Deisenhofer J. Nature. 1993; 366: 751-756Google Scholar), 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 (25Winslow J.W. Shih A. Bourell J.H. Weiss G. Reed B. Stults J.T. Goldsmith L.T. Endocrinology. 1992; 130: 2660-2668Google Scholar), whereas the expression of H1 relaxin is restricted to decidua, trophoblasts, and prostate (26Hansell D.J. Bryant-Greenwood G.D. Greenwood F.C. J. Clin. Endocrinol. Metab. 1991; 72: 899-904Google Scholar). 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 (27Bullesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Google Scholar). Although the paralogous INSL3 produced by the testis and ovary (4Nef S. Parada L.F. Nat. Genet. 1999; 22: 295-299Google Scholar, 5Ivell R. Bathgate R.A. Biol. Reprod. 2002; 67: 699-705Google Scholar) 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 (10Bathgate 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. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Google Scholar). The distribution of H3 relaxin in human tissues is unknown; however, the predominant site of relaxin 3 expression in rodents is the brain (10Bathgate 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. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Google Scholar,28Burazin T.C. Bathgate R.A. Macris M. Layfield S. Gundlach A.L. Tregear G.W. J. Neurochem. 2002; 82: 1553-1557Google Scholar). H3 relaxin is believed to be a neuropeptide that activates its receptor in neuronal synapses (28Burazin T.C. Bathgate R.A. Macris M. Layfield S. Gundlach A.L. Tregear G.W. J. Neurochem. 2002; 82: 1553-1557Google Scholar), 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 andCaenorhabditis elegans, whereas the second subgroup consists of mammalian orphan receptors LGR4, LGR5, and LGR6, as well as fly LGR2 (29Hsu S.Y. Liang S.G. Hsueh A.J. Mol. Endocrinol. 1998; 12: 1830-1845Google Scholar, 30Nishi S. Hsu S.Y. Zell K. Hsueh A.J. Endocrinology. 2000; 141: 4081-4090Google Scholar). The third group of LGRs, including mammalian LGR7 and LGR8 as well as snail LGR (13Hsu S.Y. Kudo M. Chen T. Nakabayashi K. Bhalla A. van der Spek P.J. van Duin M. Hsueh A.J. Mol. Endocrinol. 2000; 14: 1257-1271Google Scholar), 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 (31Nakabayashi K. Kudo M. Kobilka B. Hsueh A.J. J. Biol. Chem. 2000; 275: 30264-30271Google Scholar). At least three steps are involved in the ligand signaling of glycoprotein hormone receptors, each probably requiring unique but overlapping domains (31Nakabayashi K. Kudo M. Kobilka B. Hsueh A.J. J. Biol. Chem. 2000; 275: 30264-30271Google Scholar, 32Duprez L. Parma J. Costagliola S. Hermans J. Van Sande J. Dumont J.E. Vassart G. FEBS Lett. 1997; 409: 469-474Google Scholar, 33Zeng H. Phang T. Song Y.S. Ji I. Ji T.H. J. Biol. Chem. 2001; 276: 3451-3458Google Scholar, 34Nishi S. Nakabayashi K. Kobilka B. Hsueh A.J. J. Biol. Chem. 2002; 277: 3958-3964Google Scholar). 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 α-subunit of the glycoprotein hormones interacts with the leucine-rich repeats of the receptor ectodomains, whereas the unique β-subunits of these ligands stabilize the ligand-receptor complex by binding to the exoloops (33Zeng H. Phang T. Song Y.S. Ji I. Ji T.H. J. Biol. Chem. 2001; 276: 3451-3458Google Scholar). 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. 7 A). 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. 7 B). Binding of H3 relaxin to both regions of LGR7 evokes efficient receptor activation by interacting with the Gs protein and stimulating cAMP production (Fig.7 C). 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. 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. 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." @default.
• W2057916223 created "2016-06-24" @default.
• W2057916223 creator A5006681163 @default.
• W2057916223 creator A5014646087 @default.
• W2057916223 creator A5019497246 @default.
• W2057916223 creator A5052200323 @default.
• W2057916223 creator A5073068749 @default.
• W2057916223 creator A5074997845 @default.
• W2057916223 creator A5081497816 @default.
• W2057916223 date "2003-03-01" @default.
• W2057916223 modified "2023-10-18" @default.
• W2057916223 title "H3 Relaxin Is a Specific Ligand for LGR7 and Activates the Receptor by Interacting with Both the Ectodomain and the Exoloop 2" @default.
• W2057916223 cites W1559988016 @default.
• W2057916223 cites W1970953993 @default.
• W2057916223 cites W1981984074 @default.
• W2057916223 cites W1985937682 @default.
• W2057916223 cites W1997862028 @default.
• W2057916223 cites W1999854623 @default.
• W2057916223 cites W2005241408 @default.
• W2057916223 cites W2006132563 @default.
• W2057916223 cites W2008681847 @default.
• W2057916223 cites W2014703753 @default.
• W2057916223 cites W2017303308 @default.
• W2057916223 cites W2026038641 @default.
• W2057916223 cites W2031604686 @default.
• W2057916223 cites W2033883110 @default.
• W2057916223 cites W2043813981 @default.
• W2057916223 cites W2044129398 @default.
• W2057916223 cites W2059111116 @default.
• W2057916223 cites W2061073198 @default.
• W2057916223 cites W2061689421 @default.
• W2057916223 cites W2063019860 @default.
• W2057916223 cites W2078190500 @default.
• W2057916223 cites W2083891358 @default.
• W2057916223 cites W2091020913 @default.
• W2057916223 cites W2105605178 @default.
• W2057916223 cites W2111341171 @default.
• W2057916223 cites W2125686850 @default.
• W2057916223 cites W2125873842 @default.
• W2057916223 cites W2131874815 @default.
• W2057916223 cites W2136091185 @default.
• W2057916223 cites W2152770371 @default.
• W2057916223 cites W2159294289 @default.
• W2057916223 cites W307624243 @default.
• W2057916223 doi "https://doi.org/10.1074/jbc.m212457200" @default.
• W2057916223 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12506116" @default.
• W2057916223 hasPublicationYear "2003" @default.
• W2057916223 type Work @default.
• W2057916223 sameAs 2057916223 @default.
• W2057916223 citedByCount "250" @default.
• W2057916223 countsByYear W20579162232012 @default.
• W2057916223 countsByYear W20579162232013 @default.
• W2057916223 countsByYear W20579162232014 @default.
• W2057916223 countsByYear W20579162232015 @default.
• W2057916223 countsByYear W20579162232016 @default.
• W2057916223 countsByYear W20579162232017 @default.
• W2057916223 countsByYear W20579162232018 @default.
• W2057916223 countsByYear W20579162232019 @default.
• W2057916223 countsByYear W20579162232020 @default.
• W2057916223 countsByYear W20579162232021 @default.
• W2057916223 countsByYear W20579162232022 @default.
• W2057916223 countsByYear W20579162232023 @default.
• W2057916223 crossrefType "journal-article" @default.
• W2057916223 hasAuthorship W2057916223A5006681163 @default.
• W2057916223 hasAuthorship W2057916223A5014646087 @default.
• W2057916223 hasAuthorship W2057916223A5019497246 @default.
• W2057916223 hasAuthorship W2057916223A5052200323 @default.
• W2057916223 hasAuthorship W2057916223A5073068749 @default.
• W2057916223 hasAuthorship W2057916223A5074997845 @default.
• W2057916223 hasAuthorship W2057916223A5081497816 @default.
• W2057916223 hasBestOaLocation W20579162231 @default.
• W2057916223 hasConcept C1009742 @default.
• W2057916223 hasConcept C116569031 @default.
• W2057916223 hasConcept C170493617 @default.
• W2057916223 hasConcept C185592680 @default.
• W2057916223 hasConcept C2777121736 @default.
• W2057916223 hasConcept C55493867 @default.
• W2057916223 hasConcept C86803240 @default.
• W2057916223 hasConcept C95444343 @default.
• W2057916223 hasConceptScore W2057916223C1009742 @default.
• W2057916223 hasConceptScore W2057916223C116569031 @default.
• W2057916223 hasConceptScore W2057916223C170493617 @default.
• W2057916223 hasConceptScore W2057916223C185592680 @default.
• W2057916223 hasConceptScore W2057916223C2777121736 @default.
• W2057916223 hasConceptScore W2057916223C55493867 @default.
• W2057916223 hasConceptScore W2057916223C86803240 @default.
• W2057916223 hasConceptScore W2057916223C95444343 @default.
• W2057916223 hasIssue "10" @default.
• W2057916223 hasLocation W20579162231 @default.
• W2057916223 hasOpenAccess W2057916223 @default.
• W2057916223 hasPrimaryLocation W20579162231 @default.
• W2057916223 hasRelatedWork W1523748284 @default.
• W2057916223 hasRelatedWork W1972879499 @default.
• W2057916223 hasRelatedWork W1980377213 @default.
• W2057916223 hasRelatedWork W2020034592 @default.
• W2057916223 hasRelatedWork W2023498325 @default.
• W2057916223 hasRelatedWork W2046666360 @default.
• W2057916223 hasRelatedWork W2070290044 @default.

• next