R3(BΔ23–27)R/I5 Chimeric Peptide, a Selective Antagonist for GPCR135 and GPCR142 over Relaxin Receptor LGR7
2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês
10.1074/jbc.m701416200
ISSN1083-351X
AutoresChester Kuei, Steven W. Sutton, Pascal Bonaventure, Cindy M. Pudiak, Jonathan Shelton, Jessica Zhu, Diane Nepomuceno, Jiejun Wu, Jing‐Cai Chen, Fredrik Kamme, Mark Seierstad, Michael D. Hack, Ross A. D. Bathgate, Mohammed Akhter Hossain, John D. Wade, John Atack, Timothy W. Lovenberg, Changlu Liu,
Tópico(s)Pregnancy-related medical research
ResumoBoth relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(BΔ23–27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly23 to Arg, a truncation at the C terminus (Gly24-Trp27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(BΔ23–27)R/I5 binds to human GPCR135 (IC50 = 0.67 nm) and GPCR142 (IC50 = 2.29 nm) with high affinity and is a potent functional GPCR135 antagonist (pA2 = 9.15) but is not a human LGR7 ligand. Furthermore, R3(BΔ23–27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC50 = 0.25 nm, pA2 = 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(BΔ23–27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(BΔ23–27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system. Both relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(BΔ23–27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly23 to Arg, a truncation at the C terminus (Gly24-Trp27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(BΔ23–27)R/I5 binds to human GPCR135 (IC50 = 0.67 nm) and GPCR142 (IC50 = 2.29 nm) with high affinity and is a potent functional GPCR135 antagonist (pA2 = 9.15) but is not a human LGR7 ligand. Furthermore, R3(BΔ23–27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC50 = 0.25 nm, pA2 = 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(BΔ23–27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(BΔ23–27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system. Relaxin-3 (R3) 2The abbreviations used are:β-gal, β-galactosidase.R3relaxin-3I5INSL5 peptide125I-R3/I5125Ilabeled R3/I5GPCRG protein receptorCRFcorticotrophin-releasing factor 2The abbreviations used are:β-gal, β-galactosidase.R3relaxin-3I5INSL5 peptide125I-R3/I5125Ilabeled R3/I5GPCRG protein receptorCRFcorticotrophin-releasing factor (1Bathgate R. Samuel C. Burazin T. Layfield S. Claasz A. Reytomas I. Dawson N. Zhao C. Bond C. Summers R. Parry L. Wade J. Tregear G. J. Biol. Chem. 2002; 277: 1148-1157Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar) is the most recently identified member of the insulin-relaxin peptide family. Both relaxin-3 and its receptor, GPCR135 (2Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), are predominantly expressed in the brain (2Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 3Burazin T. Bathgate R. Macris M. Layfield S. Gundlach A. Tregear G. J. Neurochem. 2002; 82: 1553-1557Crossref PubMed Scopus (171) Google Scholar). GPCR135, an inhibitory receptor, is expressed in many regions of the rodent brain such as the superior colliculus, sensory cortex, olfactory bulb, amygdale, and paraventricular nucleus (4Sutton S. Bonaventure P. Kuei C. Chen J. Nepomuceno D. Lovenberg T.W. Liu C. Neuroendocrinology. 2004; 80: 298-307Crossref PubMed Scopus (109) Google Scholar, 5Sutton S. Bonaventure P. Kuei C. Nepomuceno D. Wu J. Zhu J. Lovenberg T. Liu C. Neuroendocrinology. 2005; 82: 139-150Crossref PubMed Scopus (39) Google Scholar, 6Ma S. Bonaventure P. Ferraro T. Shen P-J. Burazin T.C. Bathgate R.A. Liu C. Tregear G.W. Sutton S.W. Gundlach A.L. Neuroscience. 2007; 144: 165-190Crossref PubMed Scopus (167) Google Scholar), suggesting potential physiological involvement in neuroendocrine and sensory processing. Recent in vivo studies have further shown that relaxin-3 and GPCR135 are involved in the stress response and in regulation of feeding. More specifically, water restraint stress or intracerebroventricular corticotrophin-releasing factor (CRF) infusion induces relaxin-3 expression in cells of the nucleus incertus, a region where CRF receptor-1 is also expressed (7Tanaka M. Iijima N. Miyamoto Y. Fukusumi S. Itoh Y. Ozawa H. Ibata Y. Eur. J. Neurosci. 2005; 21: 1659-1670Crossref PubMed Scopus (193) Google Scholar), and central administration of relaxin-3 induces feeding in rat (8McGowan B.M. Stanley S.A. Smith K.L. White N.E. Connolly M.M. Thompson E.L. Gardiner J.V. Murphy K.G. Ghatei M.A. Bloom S.R. Endocrinology. 2005; 146: 3295-3300Crossref PubMed Scopus (150) Google Scholar, 9Hida T. Takahashi E. Shikata K. Hirohashi T. Sawai T. Seiki T. Tanaka H. Kawai T. Ito O. Arai T. Yokoi A. Hirakawa T. Ogura H. Nagasu T. Miyamoto N. Kuromitsu J. J. Recept. Signal Transduct. Res. 2006; 26: 147-158Crossref PubMed Scopus (84) Google Scholar). These findings suggest that GPCR135 and relaxin-3 may be involved in multiple physiological processes, some of which might be as yet unknown.In vitro relaxin-3 activates GPCR135 (2Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), GPCR142 (10Liu C. Chen J. Sutton S. Roland B. Kuei C. Farmer N. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50765-50770Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), and LGR7 (11Sudo S. Kumagai J. Nishi S. Layfield S. Ferraro T. Bathgate R. Hsueh A.J. J. Biol. Chem. 2003; 278: 7855-7862Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) receptors. The predominant brain expression of both relaxin-3 and GPCR135, coupled with their high affinity interaction, strongly suggests that relaxin-3 is the endogenous ligand for GPCR135 (2Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Pharmacological characterization, tissue expression profile, and the evolutionary study of GPCR142 and INSL5 indicate that GPCR142 is the endogenous INSL5 receptor (10Liu C. Chen J. Sutton S. Roland B. Kuei C. Farmer N. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50765-50770Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 12Conklin D. Lofton-Day C.E. Haldeman B.A. Ching A. Whitmore T.E. Lok S. Jaspers S. Genomics. 1999; 60: 50-56Crossref PubMed Scopus (117) Google Scholar, 13Chen J. Kuei C. Sutton S.W. Bonaventure P. Nepomuceno D. Eriste E. Sillard R. Lovenberg T.W. Liu C. J. Pharmacol. Exp. Ther. 2005; 312: 83-95Crossref PubMed Scopus (75) Google Scholar, 14Liu C. Kuei C. Sutton S. Chen J. Bonaventure P. Wu J. Nepomuceno D. Wilkinson T. Bathgate S. Eriste E. Sillard R. Lovenberg T.W. J. Biol. Chem. 2005; 280: 292-300Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The high affinity interaction between relaxin and LGR7 as well as knock-out studies demonstrate that relaxin is the endogenous ligand for LGR7 (15Hsu 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: 1257-1271Crossref PubMed Scopus (306) Google Scholar, 16Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Crossref PubMed Scopus (669) Google Scholar, 17Zhao L. Roche P.J. Gunnersen J.M. Hammond V.E. Tregear G.W. Wintour E.M. Beck F. Endocrinology. 1999; 140: 445-453Crossref PubMed Scopus (173) Google Scholar, 18Krajin-Franken M.A. van Disseldorp A.J. Koenders J.E. Moselman S. van Duin M. Gossen J.A. Mol. Cell. Biol. 2004; 24: 687-696Crossref PubMed Scopus (122) Google Scholar).Despite the proposed ligand/receptor pairs mentioned above, in vivo administration of relaxin-3 could potentially activate all three receptors (GPCR135, GPCR142 and LGR7), and therefore selective pharmacological tools (agonists and antagonists) are crucial to probe the in vivo function(s) of GPCR135. Because GPCR142 is a pseudogene in the rat (13Chen J. Kuei C. Sutton S.W. Bonaventure P. Nepomuceno D. Eriste E. Sillard R. Lovenberg T.W. Liu C. J. Pharmacol. Exp. Ther. 2005; 312: 83-95Crossref PubMed Scopus (75) Google Scholar) and is not detected in the mouse brain (5Sutton S. Bonaventure P. Kuei C. Nepomuceno D. Wu J. Zhu J. Lovenberg T. Liu C. Neuroendocrinology. 2005; 82: 139-150Crossref PubMed Scopus (39) Google Scholar), activation of GPCR142 by central administration of relaxin-3 is not a great concern in these species. However, potential activation of LGR7 by relaxin-3 remains a potentially confounding issue, especially because LGR7 is expressed in the brain and is reported to play a role in drinking (8McGowan B.M. Stanley S.A. Smith K.L. White N.E. Connolly M.M. Thompson E.L. Gardiner J.V. Murphy K.G. Ghatei M.A. Bloom S.R. Endocrinology. 2005; 146: 3295-3300Crossref PubMed Scopus (150) Google Scholar, 19Thornton S.M. Fitzsimons J.T. J. Neuroendocrinol. 1995; 3: 165-169Crossref Scopus (42) Google Scholar) and potentially in other physiological functions (20Wilson B.C. Connell B. Saleh T.M. Neurosci. Lett. 2006; 393: 160-164Crossref PubMed Scopus (25) Google Scholar, 21Nistri S. Bani D. Curr. Neurovasc. Res. 2005; 2: 225-233Crossref PubMed Scopus (12) Google Scholar, 22Sherwood O.D. Endocr. Rev. 2004; 25: 205-234Crossref PubMed Scopus (445) Google Scholar).Previous studies showed that the B-chain of relaxin-3 is capable of binding to and activating GPCR135 (2Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), suggesting that the B-chain of relaxin-3 contains the receptor binding domains for GPCR135. Later studies have demonstrated that a chimeric peptide (R3/I5) composed of the relaxin-3 B-chain and the INSL5 A-chain selectively activates GPCR135 over LGR7 (23Liu C. Chen J. Kuei C. Sutton S. Nepomuceno D. Bonaventure P. Lovenberg T.W. Mol. Pharmacol. 2005; 67: 231-240Crossref PubMed Scopus (117) Google Scholar), which further support our hypothesis. Homology modeling of the relaxin-3 structure (Fig. 1) and solution structure analysis of relaxin-3 (24Rosengren K.J. Lin F. Bathgate R.A. Tregear G.W. Daly N.L. Wade J.D. Craik D.J. J. Biol. Chem. 2006; 281: 5845-5851Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) show that the middle segment of the relaxin-3 B-chain forms an α-helix. Amino acid residues Arg12, Ile15, Arg16, Ile19, and Phe20 are presented on one side of the α-helix that faces away from the A-chain, suggesting that these residues may play a role in the interactions between relaxin-3 and its receptors. In addition, although the N termini of the B-chains of the members in the insulin/relaxin family have no conservation (Fig. 1), a few different members can activate the same receptor (relaxin-1 and -2, and relaxin-3 for LGR7; relaxin-1, -2, and INSL3 for LGR8; relaxin-3 and INSL5 for GPCR142), suggesting that the N terminus of relaxin-3 may not be important for interactions between relaxin-3 and its receptors. In this report, using mutagenesis studies, we describe the identification of the GPCR135 binding domain (the α-helix region of the relaxin-3 B-chain) and the receptor activation domain (the C terminus of the relaxin-3 B-chain) of relaxin-3. In addition, we report the creation of a selective GPCR135 antagonist (R3(BΔ23–27)R/I5) that consists of the relaxin-3 B-chain with a truncation at the C terminus (Gly23–Trp27, the GPCR135 activation domain), an addition of an Arg residue in place of Gly23 of the B-chain, and the A-chain of INSL5. This novel, high affinity GPCR135 antagonist, R3(BΔ23–27)R/I5, does not interact with LGR7. In addition, we demonstrate increased feeding in satiated Wistar rats following intracerebroventricular dosing of R3/I5 (a selective GPCR135 agonist), which is blocked by prior administration of the GPCR135-specific antagonist R3(BΔ23–27)R/I5.MATERIALS AND METHODSGeneration of Relaxin-3 Mutant Peptides—Different relaxin-3 mutant peptides with various mutations at the B-chain were created, including truncations at the N terminus (mutants Δ1–6, Δ1–7, Δ1–8, and Δ1–9), different point mutations at residues Arg8, Arg12, Ile15, Arg16, Ile19, Phe20, Arg26, and Trp27 as well as truncations at the C terminus (R3(BΔ23–27), R3(BΔ24–27), R3(BΔ25–27), R3(BΔ26–27), and R3(BΔ23–27)/I5). All peptides (except as specified) were generated recombinantly in mammalian cells similar to the production of relaxin-3 as described previously (2Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). All relaxin-3 mutant coding regions were created by a two-step PCR using primers shown in supplemental Table 1. In the first round PCR, overlapping 5′-end and 3′-end coding regions for each mutant were PCR-amplified. The human relaxin-3 cDNA construct (2Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) was used as the template for the first-step PCR reactions for all mutants except for the chimeric peptides R3(BΔ23–27)/I5, for which R3/I5 expression cDNA (23Liu C. Chen J. Kuei C. Sutton S. Nepomuceno D. Bonaventure P. Lovenberg T.W. Mol. Pharmacol. 2005; 67: 231-240Crossref PubMed Scopus (117) Google Scholar) was used as the templates. The first-step PCR products (5′-end and 3′-end) were then mixed and used as the templates for the respective second-round PCR reactions using primers P1 and P2 (Pi5 for R3(BΔ23–27)/I5; listed in supplemental Table 1). All PCRs were run under the conditions of 94 °C for 20 s, 65 °C for 20 s, and 72 °C for 1 min for 20 cycles. The final PCR products were cloned into a modified pCMV-SPORT1 vector containing a coding region for an α-signal peptide for secretion, which was followed by a FLAG peptide coding region for affinity purification (2Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). All mutant peptides (except for R3(BΔ23–27)/I5) have the intact A-chain of the wild type relaxin-3 but different B-chains. R3(BΔ23–27)/I5 has an A-chain of human INSL5. The B-chain sequences of the mutant peptides with truncations at the C terminus of relaxin-3 B-chain are shown in Table 1. B-chain sequences of other mutants are shown in supplemental Table 2. All recombinant peptides were co-expressed with furin protease in COS-7 cells for efficient removal of the C-chain (2Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 25Hosaka M. Nagahama M. Kim W.S. Watanabe T. Hatsuzawa K. Ikemizu J. Murakami K. Nakayama K. J. Biol. Chem. 1991; 266: 12127-12130Abstract Full Text PDF PubMed Google Scholar). The N-terminal FLAG-tagged peptides were first purified using an anti-FLAG affinity column, and then the tag was removed by enterokinase (Novagen, Madison, WI) digestion. The peptides, free of the tag, were then further purified by reversed phase high pressure liquid chromatography (HPLC). The purified peptides were analyzed by mass spectrometry as described (2Liu C. Eriste E. Sutton S. Chen J. Roland B. Kuei C. Farmer N. Jornvall H. Sillard R. Lovenberg T.W. J. Biol. Chem. 2003; 278: 50754-50764Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) to verify the peptide identities. R3(BΔ23–27)R and R3(BΔ23–27)R/I5, which are derivative of R3(BΔ23–27) and R3(BΔ23–27)/I5 respectively, have an extra Arg residue at the C terminus of the B-chain because of incomplete processing (Table 1). Mutant peptides R12K, R12K,R16K, and W27R were made by solid phase peptide synthesis using methods described previously (1Bathgate R. Samuel C. Burazin T. Layfield S. Claasz A. Reytomas I. Dawson N. Zhao C. Bond C. Summers R. Parry L. Wade J. Tregear G. J. Biol. Chem. 2002; 277: 1148-1157Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 26Bathgate R. Lin F. Hanson N. Otvos L. Guidolin A. Giannakis C. Bastiras S. Layfield S. Ferraro T. Ma S. Zhao C. Gundlach A. Samuel C. Tregear G. Wade J. Biochemistry. 2006; 45: 1043-1053Crossref PubMed Scopus (132) Google Scholar).TABLE 1Amino acid sequence of relaxin-3 and mutant peptidesPeptidesB-chainaCompared with R3(BΔ23-27) and R3(BΔ23-27)/I5, R3(BΔ23-27)R and R3(BΔ23-27)R/I5 peptides have an extra Arg residue at the C terminus of the B-chain because of incomplete processing of the peptide by carboxypeptidase-B during peptide maturation. The extra Arg residue is highlighted in bold and underlined.A-chain23 27* *Relaxin-3RAAPYGVRLCGREFIRAVIFTCGGSRWDVLAGLSSSCCKWGCSKSEISSLCR3(BΔ23-27)RAAPYGVRLCGREFIRAVIFTCDVLAGLSSSCCKWGCSKSEISSLCR3(BΔ23-27)RRAAPYGVRLCGREFIRAVIFTCRDVLAGLSSSCCKWGCSKSEISSLCR3(BΔ24-27)RAAPYGVRLCGREFIRAVIFTCGDVLAGLSSSCCKWGCSKSEISSLCR3(BΔ25-27)RAAPYGVRLCGREFIRAVIFTCGGDVLAGLSSSCCKWGCSKSEISSLCR3(BΔ26-27)RAAPYGVRLCGREFIRAVIFTCGGSDVLAGLSSSCCKWGCSKSEISSLCR3/I5RAAPYGVRLCGREFIRAVIFTCGGSRW<EDLQTLCCTDGCSMTDLSALCR3(BΔ23-27)/I5RAAPYGVRLCGREFIRAVIFTC<EDLQTLCCTDGCSMTDLSALCR3(BΔ23-27)R/I5RAAPYGVRLCGREFIRAVIFTCR<EDLQTLCCTDGCSMTDLSALCa Compared with R3(BΔ23-27) and R3(BΔ23-27)/I5, R3(BΔ23-27)R and R3(BΔ23-27)R/I5 peptides have an extra Arg residue at the C terminus of the B-chain because of incomplete processing of the peptide by carboxypeptidase-B during peptide maturation. The extra Arg residue is highlighted in bold and underlined. Open table in a new tab Radioligand Binding Assays—125I-Relaxin-3/INSL5 (125I-R3/I5), a radiolabeled chimeric peptide with the human relaxin-3 B-chain and the human INSL5 A-chain (23Liu C. Chen J. Kuei C. Sutton S. Nepomuceno D. Bonaventure P. Lovenberg T.W. Mol. Pharmacol. 2005; 67: 231-240Crossref PubMed Scopus (117) Google Scholar) was used at a final concentration of 50 pm as the tracer to characterize the binding properties of GPCR135 and GPCR142 for the mutant relaxin-3 peptides. 125I-H2 (human gene-2) relaxin (PerkinElmer Life Sciences) was used at a final concentration of 50 pm to characterize the binding properties of the relaxin receptor LGR7 for the peptides. COS-7 cells in 24-well tissue culture plates that transiently expressed GPCR135, GPCR142, or LGR7 were used in radioligand binding assays as described (13Chen J. Kuei C. Sutton S.W. Bonaventure P. Nepomuceno D. Eriste E. Sillard R. Lovenberg T.W. Liu C. J. Pharmacol. Exp. Ther. 2005; 312: 83-95Crossref PubMed Scopus (75) Google Scholar). The results were analyzed by GraphPad Prism 4.0 software (San Diego). The IC50 values, which are the ligand concentrations that inhibited 50% of the maximum binding, were calculated and then converted to Ki values using the Cheng-Prusoff formula (31Whittingham J.L. Edward D.J. Antson A.A. Clarkson J.M. Dodson G.G. Biochemistry. 1998; 37: 11516-11523Crossref PubMed Scopus (99) Google Scholar) with Kd values of 0.41, 0.89, and 0.18 nm for binding of 125I-R3/I5 to GPCR135, 125I-R3/I5 to GPCR142, and 125I-H2 relaxin to LGR7, respectively.Agonist and Antagonist Analysis for Mutant Relaxin-3 Peptides—All peptides were tested for their agonist activities against GPCR135, GPCR142, and LGR7 expressed in SK-NMC/CRE cells as described previously (23Liu C. Chen J. Kuei C. Sutton S. Nepomuceno D. Bonaventure P. Lovenberg T.W. Mol. Pharmacol. 2005; 67: 231-240Crossref PubMed Scopus (117) Google Scholar). SK-N-MC/CRE-βgal cells harbor a β-galactosidase (β-gal) gene under the control of a CRE promoter. An increase in cAMP concentration in these cells is associated with increased β-gal expression, which can be measured using chlorophenol red-β-d-galactopyranoside as a substrate and reading the optical absorbance at 570 nm. GPCR135 and GPCR142 are coupled with Gαi proteins; therefore agonists inhibit forskolin-stimulated β-gal expression in GPCR135- or GPCR142-expressing cells. LGR7 is Gs linked, and therefore agonists stimulate expression in LGR7-expressing cells. R3(BΔ23–27)R/I5 was tested for its ability to produce a rightward-shift in the relaxin-3 or R3/I5 dose-response curve in the presence of 10 nm, 100 nm,or 1 μm R3(BΔ23–27)R/I5 to demonstrate functional antagonism. Wild type relaxin-3 peptide was used as positive control in all experiments. The results were analyzed using GraphPad Prism 4.0 software. The EC50 values, which are the ligand concentrations that stimulate 50% of the maximum responses, were then calculated.The agonism and antagonism of R3(BΔ23–27)R/I5 for rat GPCR135 was tested in the same way as the human GPCR135 using SK-N-MC/CRE-β-gal cells stably expressing rat GPCR135. The agonism and antagonism of peptides for rat LGR7 was assayed using a cAMP luminescence assay. Briefly, HEK293 cells were transiently transfected with a cDNA construct expressing rat LGR7 (4Sutton S. Bonaventure P. Kuei C. Chen J. Nepomuceno D. Lovenberg T.W. Liu C. Neuroendocrinology. 2004; 80: 298-307Crossref PubMed Scopus (109) Google Scholar). Two days post-transfection, cells were detached with phosphate-buffered saline plus 10 mm EDTA and plated at a density of 25,000 cells/well in 96-well white opaque plates (Thermo Electron Corp., catalog no. 7571). To test the agonism of R3(BΔ23–27)R/I5, cells expressing rat LGR7 were stimulated with different concentrations of R3(BΔ23–27)R/I5 with relaxin-3 as the positive control. To test the antagonism of R3(BΔ23–27)R/I5 for rat LGR7, different concentrations of relaxin-3 were added to cells expressing rat LGR7 in the presence of 10 nm, 100 nm,or 1 μm of R3(Δ23–27)R/I5. Cells were then incubated at room temperature for 1 h. The cAMP in the cells was measured with a cAMP detection kit (DiscoveRx HitHunter, catalog no. 90–0041) according to the manufacturer's protocol. The results were analyzed using GraphPad Prism 4.0 software.Autoradiographic Studies—R3(BΔ23–27)R/I5 peptide was evaluated pharmacologically using endogenous GPCR135 from rat brain slices in autoradiographic studies as described previously (4Sutton S. Bonaventure P. Kuei C. Chen J. Nepomuceno D. Lovenberg T.W. Liu C. Neuroendocrinology. 2004; 80: 298-307Crossref PubMed Scopus (109) Google Scholar). Briefly, 125I-R3/I5 was applied in a binding buffer to rat brain slices. Unlabeled human relaxin-3 or R3(BΔ23–27)R/I5 was used at various concentrations as competitors to displace GPCR135 binding of 125I-R3/I5. The specific binding of 125I-R3/I5 to the rat brain slices was quantitated using a Fuji Bio-Imaging analyzer system (BAS-5000).In Vivo Studies—Experimentally naive male Wistar rats (Charles River, Wilmington, MA), weighing 200–225 g at the time of arrival, were used. The animals were initially housed at two/cage and given a 1-week acclimation period to the vivarium prior to intracerebroventricular cannula implantation. All animals had free access to food and water throughout the experiment. The animal colony was maintained at 22 ± 2 °C during a 12-h light/12-h dark illumination cycle with lights on from 6:00 a.m. to 6:00 p.m. All behavioral testing occurred during the light phase between 8:00 a.m. and 4:00 p.m. All studies were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U. S. National Institutes of Health.Surgical Preparation—Following the acclimation period, the animals were anesthetized with 4% isoflurane and surgically implanted with a 20-gauge guide cannula aimed at the lateral ventricle. Guide cannulae (Plastics One, Roanoke, VA) were unilaterally implanted using a stereotaxic apparatus (David Kopf, Tujunga, CA) using the following coordinates relative to Bregma (flat skull): AP =+1.0 mm, ML =–1.3 mm, DV =–3.8 mm from the top of the skull (25Hosaka M. Nagahama M. Kim W.S. Watanabe T. Hatsuzawa K. Ikemizu J. Murakami K. Nakayama K. J. Biol. Chem. 1991; 266: 12127-12130Abstract Full Text PDF PubMed Google Scholar). Three screws mounted in the skull and covered with dental cement served as an anchor for the guide cannula. Animals were then housed individually and given a 7-day recovery period from surgery. During the surgical recovery period, the animals were handled 2–3 times to minimize stress effects that might occur because of being handled at the time of behavioral testing.Apparatus—The testing apparatus consisted of a plastic cage (containing no bedding) in which a wire grid was placed on the floor of the cage. A food hopper and drinking spout were located on opposite walls of the cage. The drinking spout was connected to an automated watering system and thereby delivered water to the animal on demand throughout the session(s).A predetermined amount of standard rat chow (Formulab Diet no. 5008) was placed in the food hopper at the start of the 4-h session(s). The amount of food remaining in the food hopper was determined by subtracting the weight of the food at 1 and 4 h from the initial food weight (i.e. weight of the food at the start of the session). Food crumbs detected on the floor of the apparatus were included in the determination of food weights.Drugs—The peptides (i.e. R3/I5, R3(BΔ23–27)R/I5) were dissolved in vehicle (sterile physiological saline plus 0.1% bovine serum albumin). All solutions were infused in a 5-μl volume. R3/I5 and R3(BΔ23–27)R/I5 were infused at a concentration of 2 μg/μl.Feeding Procedure—Following the surgical recovery period, the animals were randomly assigned to one of the four treatment conditions: vehicle (5 μl) + vehicle; vehicle + R3/I5 (10 μg); R3(BΔ23–27)R/I5 (10 μg) + vehicle; R3(BΔ23–27)R/I5 (10 μg) + R3/I5 (10 μg).Testing consisted of a two-day protocol. Day 1 served as the base-line session. No injections were administered during this session, and it served as a habituation period to the testing apparatus, while also providing a base-line measure of food intake. Day 2 served as the test session. Immediately prior to this session, all animals were removed from their home cage, and two infusions were administered directly into the lateral ventricle. Test substances were given via a preloaded catheter without removing the catheter between injections. A 0.5-μl air bubble separated each injection to prevent mixing. The animals were first infused with vehicle or R3(BΔ23–27)R/I5, followed by a second infusion that consisted of vehicle or R3/I5. The infusions were separated by 10 min, and the injection needle remained in the guide cannula for 1 min following the termination of the final infusion. Following the second infusion, the animals were placed in the testing apparatus, and food intake was measured at 1 and 4 h during a 4-h session. Food intake measured at the end of the session served as a measure of total food intake. All animals were euthanized with carbon dioxide, and cannula placements were verified at the end of behavioral testing.RESULTSProbing the Receptor Binding and Activation Domains of Relaxin-3—The functions of the N terminus of the relaxin-3 B-chain were evaluated by removing residues Arg1–Gly6 (Δ1–6), Arg1–Val7 (Δ1–7), Arg1–Arg8 (Δ1–8), or Arg1–Leu9 (Δ1–9) from the N-terminal region of the relaxin-3 B-chain. The A-chains of those mutant peptides were left unchanged. The RΔ1–6 and Δ1–7 mutants were expressed in mammalian expression system with the similar yield to that of the wild type relaxin-3 peptide (5Sutton S. Bonaventure P. Kuei C. Nepomuceno D. Wu J. Zhu J. Lovenberg T. Liu C. Neuroendocrinology. 2005; 82: 139-150Crossref PubMed Scopus (39) Google Scholar). Δ1–8 mutant had a significantly reduced expression level (∼100 μg/liter), but the expression level for Δ1–9 mutant was too low to generate enough peptide for functional testing. Δ1–6 and Δ1–7 mutants retained high binding affinity and agonist potency for GPCR135, GPCR142, and LGR7.
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