Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

The A-chain of Human Relaxin Family Peptides Has Distinct Roles in the Binding and Activation of the Different Relaxin Family Peptide Receptors

2008; Elsevier BV; Volume: 283; Issue: 25 Linguagem: Inglês

10.1074/jbc.m801911200

ISSN

1083-351X

Autores

Mohammed Akhter Hossain, K. Johan Rosengren, Linda M. Haugaard‐Kedström, Soude Zhang, Sharon Layfield, Tania Ferraro, Norelle L. Daly, Geoffrey W. Tregear, John D. Wade, Ross A. D. Bathgate,

Tópico(s)

Pregnancy-related medical research

Resumo

The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1–4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent. The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1–4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent. Relaxin was first identified more than 90 years ago and subsequently shown to be a peptide hormone having a two-chain structure similar to insulin (Fig. 1) (1Schwabe C. McDonald J.K. Science. 1977; 197: 914-915Crossref PubMed Scopus (99) Google Scholar). It has since been established that relaxin is a member of the relaxin peptide family, comprising a total of seven members in the human (2Bathgate R.A.D. Hsueh A.J. Sherwood O.D. Neill J.D. Physiology of Reproduction. 3rd Ed. Elsevier, San Diego2006: 679-770Google Scholar). These are the H1, 3The abbreviations used are: H1, human relaxin-1; H2, human relaxin-2, H3, human relaxin-3; GPCR, G-protein-coupled receptor; LRR, leucine-rich repeat; RXFP, relaxin family peptide; MALDI-TOF, matrix-assisted laser desorption time-of-flight; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; HEK, human embryonic kidney. H2, and H3 relaxin peptides that are encoded by the three relaxin genes RLN1 to -3 and the insulin-like peptides INSL3 to -6 (insulin-like peptides 3–6). Phylogenetic analyses indicate that all of these relaxin family peptides evolved from a relaxin-3 (H3 relaxin equivalent) ancestral gene prior to the emergence of fish (3Wilkinson T.N. Speed T.P. Tregear G.W. Bathgate R.A.D. BMC Evol. Biol. 2005; 5: 14Crossref PubMed Scopus (171) Google Scholar). In most mammals other than humans and higher primates, there are only two relaxin genes that encode relaxin and relaxin-3. The RLN1 gene in these species is equivalent to the RLN2 gene in humans (encoding H2 relaxin) and higher primates and encodes the relaxin peptide that is expressed by the corpus luteum and/or placenta (2Bathgate R.A.D. Hsueh A.J. Sherwood O.D. Neill J.D. Physiology of Reproduction. 3rd Ed. Elsevier, San Diego2006: 679-770Google Scholar). The function of the RLN1 gene in higher primates is unknown, and an H1 relaxin peptide has not been isolated. In contrast to the receptors for insulin and insulin-like growth factors I and II, which are tyrosine kinases, the receptors for relaxin family peptides are members of two unrelated branches of the G-protein-coupled receptor (GPCR) family. LGR7 (leucine-rich repeat-containing G-protein-coupled receptor) is the receptor for relaxin and is characterized by an unusually large ectodomain that terminates with a low density lipoprotein receptor class A module (4Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Crossref PubMed Scopus (694) Google Scholar). Relaxin also has high affinity for the related receptor, LGR8, which is the receptor for INSL3 (5Kumagai 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: 31283-31286Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). The native receptor for H3 relaxin is the unrelated receptor GPCR135, also known as the somatostatin- and angiotensin-like peptide receptor (6Liu 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 (299) Google Scholar). H3 relaxin also has high affinity for the related receptor GPCR142, which is the receptor for INSL5 (7Liu C. Kuei C. Sutton S. Chen J. Bonaventure P. Wu J. Nepomuceno D. Wilkinson T. Bathgate R. Eriste E. Sillard R. Lovenberg T.W. J. Biol. Chem. 2005; 280: 292-300Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Both receptors are classic peptide ligand GPCRs and lack a large ectodomain. Importantly, H3 relaxin has a high affinity for LGR7 and will also interact with LGR8, albeit with a significantly lower affinity (8Bathgate R.A.D. Lin F. Hanson N.F. Otvos Jr., L. Guidolin A. Giannakis C. Bastiras S. Layfield S.L. Ferraro T. Ma S. Zhao C. Gundlach A.L. Samuel C.S. Tregear G.W. Wade J.D. Biochemistry. 2006; 45: 1043-1053Crossref PubMed Scopus (135) Google Scholar) and hence will interact with all of the relaxin family receptors. Neither INSL4 (9Lin F. Otvos Jr., L. Kumagai J. Tregear G.W. Bathgate R.A. Wade J.D. J. Pept. Sci. 2004; 10: 257-264Crossref PubMed Scopus (30) Google Scholar) nor INSL6 (10Bogatcheva N.V. Truong A. Feng S. Engel W. Adham I.M. Agoulnik A.I. Mol. Endocrinol. 2003; 17: 2639-2646Crossref PubMed Scopus (156) Google Scholar) can bind to the relaxin family receptors, and their native receptors are unknown. Based on IUPHAR-NC nomenclature, the receptors were recently named relaxin family peptide (RXFP) receptors: LGR7-RXFP1, LGR8-RXFP2, GPCR135-RXFP3, and GPCR142-RXFP4 (11Bathgate R.A. Ivell R. Sanborn B.M. Sherwood O.D. Summers R.J. Pharmacol. Rev. 2006; 58: 7-31Crossref PubMed Scopus (275) Google Scholar). The determinants for H3 relaxin activity on RXFP3 and RXFP4 are probably located in the B-chain alone, since synthetic S-reduced H3 relaxin B-chain is an RXFP3 and RXFP4 agonist (12Liu 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 (210) Google Scholar, 13Liu C. Chen J. Kuei C. Sutton S. Nepomuceno D. Bonaventure P. Lovenberg T.W. Mol. Pharmacol. 2005; 67: 231-240Crossref PubMed Scopus (119) Google Scholar). It has recently been demonstrated that key residues in the H3 relaxin B-chain are responsible for both binding affinity and cAMP-inhibitory activity (14Kuei C. Sutton S. Bonaventure P. Pudiak C. Shelton J. Zhu J. Nepomuceno D. Wu J. Chen J. Kamme F. Seierstad M. Hack M.D. Bathgate R.A. Hossain M.A. Wade J.D. Atack J. Lovenberg T.W. Liu C. J. Biol. Chem. 2007; 282: 25425-25435Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Further, a series of chimeric peptides that consist of the B-chain of H3 relaxin in combination with A-chains from other members of the relaxin family demonstrated that the A-chain from H1 relaxin, H2 relaxin, INSL3, and INSL6 does not change the pharmacological properties of the H3 relaxin B-chain significantly. However, substitution of the relaxin-3 A-chain with the A-chain from INSL5 results in a chimeric peptide that selectively activates RXFP3 over RXFP1 (13Liu C. Chen J. Kuei C. Sutton S. Nepomuceno D. Bonaventure P. Lovenberg T.W. Mol. Pharmacol. 2005; 67: 231-240Crossref PubMed Scopus (119) Google Scholar). Hence, although the A-chain of H3 relaxin is not necessary for RXFP3/4 activity, it is essential for RXFP1 activity. Similarly, H2 relaxin and INSL3 require both A- and B-chains for RXFP1 and RXFP2 activation. Ligand-mediated activation of RXFP1 and RXFP2 involves a three-stage process (15Scott D.J. Layfield S. Yan Y. Sudo S. Hsueh A.J. Tregear G.W. Bathgate R.A. J. Biol. Chem. 2006; 281: 34942-34954Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Primary ligand binding occurs in the leucine-rich repeats (LRRs) of the receptor ectodomain, and there is a lower affinity secondary binding site in the transmembrane exoloops (16Sudo S. Kumagai J. Nishi S. Layfield S. Ferraro T. Bathgate R.A. Hsueh A.J. J. Biol. Chem. 2003; 278: 7855-7862Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Receptor signaling through cAMP then requires the unique low density lipoprotein receptor class A module at the N terminus of the receptors (15Scott D.J. Layfield S. Yan Y. Sudo S. Hsueh A.J. Tregear G.W. Bathgate R.A. J. Biol. Chem. 2006; 281: 34942-34954Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). RXFP1 and RXFP2 receptors without this domain bind ligand normally but do not signal. A recent study has shown that the B-chain of relaxin binds to specific residues in the RXFP1 receptor LRRs (17Bullesbach E.E. Schwabe C. J. Biol. Chem. 2005; 280: 14051-14056Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). This interaction is determined by the arginine residues at B13 and B17 and an isoleucine or valine at position B20 within the B-chain forming a "relaxin binding cassette" (Arg-X-X-X-Arg-X-X-Ile/Val) (18Bullesbach E.E. Schwabe C. J. Biol. Chem. 2000; 275: 35276-35280Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Similarly, we have recently demonstrated that the INSL3 B-chain interacts with specific residues in the RXFP2 receptor LRRs (19Scott D.J. Wilkinson T.N. Zhang S. Ferraro T. Wade J.D. Tregear G.W. Bathgate R.A. Mol. Endocrinol. 2007; 21: 1699-1712Crossref PubMed Scopus (47) Google Scholar). Primary ligand binding to the LRRs is directed by the B-chain-specific residues HisB12, ArgB16, ValB19, ArgB20, and TrpB27 (20Rosengren K.J. Zhang S. Lin F. Daly N.L. Scott D.J. Hughes R.A. Bathgate R.A. Craik D.J. Wade J.D. J. Biol. Chem. 2006; 38: 28287-28295Abstract Full Text Full Text PDF Scopus (63) Google Scholar). Importantly, B-chain-only INSL3 peptides can bind to the primary binding sites in the LRRs but do not activate the receptor (21Del Borgo M.P. Hughes R.A. Bathgate R.A. Lin F. Kawamura K. Wade J.D. J. Biol. Chem. 2006; 281: 13068-13074Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). These peptides are low affinity antagonists and highlight that the A-chain is required for receptor activation. Very little is known about the residues involved in secondary binding to the receptors, although it is known that this site has a lower affinity for the ligand and is necessary for receptor activation (22Halls M.L. Bathgate R.A. Sudo S. Kumagai J. Bond C.P. Summers R.J. Ann. N. Y. Acad. Sci. 2005; 1041: 17-21Crossref PubMed Scopus (13) Google Scholar). Recently, it was demonstrated that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting RXFP2 binding affinity (23Bullesbach E.E. Schwabe C. J. Biol. Chem. 2005; 280: 14586-14590Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, this truncated peptide does not stimulate cAMP signaling and in fact acts as a high affinity antagonist. It is therefore possible that the N-terminal region of the INSL3 A-chain is somehow involved in the secondary interactions with the RXFP2 receptor, which are necessary for receptor activation. Similarly, it has previously been shown that the N terminus of the A-chain of porcine relaxin is important for its activity on the mouse pubic symphysis (24Bullesbach E.E. Schwabe C. Biochemistry. 1986; 25: 5998-6004Crossref PubMed Scopus (19) Google Scholar). Deletion of more than 3 amino acids resulted in the loss of the ability of porcine relaxin to relax the pubic symphysis. However, the role of the N-terminal A-chain residues in H2 relaxin upon RXFP1 and RXFP2 receptor binding and activation as well as the role of these residues in H3 relaxin upon RXFP1 and RXFP3 binding and activation is unknown. In this study, we have synthesized H2 and H3 relaxin peptides with A-chain truncations to examine whether the N terminus of the A-chain of the relaxin peptides is important for peptide structure, receptor binding, and/or activation. H2 relaxin A-chain-shortened analogs were tested for binding and activation on RXFP1 and RXFP2 only, since H2 relaxin does not bind to RXFP3 (6Liu 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 (299) Google Scholar). H3 relaxin A-chain shortened analogs were tested for binding and activation on RXFP1 and RXFP3 only, since H3 relaxin has a poor affinity for, and does not activate, RXFP2 (22Halls M.L. Bathgate R.A. Sudo S. Kumagai J. Bond C.P. Summers R.J. Ann. N. Y. Acad. Sci. 2005; 1041: 17-21Crossref PubMed Scopus (13) Google Scholar). Interestingly, we found that there are significant differences in the mechanisms by which the various relaxin family peptides activate their receptors. These data provide important new biochemical insights into the action of relaxin peptides. Solid Phase Peptide Synthesis—Appropriately regioselectively S-protected individual A- and B-chains of H2, H3, INSL3, and their analogs were prepared as their C-terminal amide forms using either continuous flow or microwave-assisted solid phase methodology on an automated PerSeptive Biosystems peptide synthesizer and a CEM Liberty peptide synthesizer, respectively. After simultaneous cleavage, side chain deprotection, and purification of the individual chains, use of a previously reported sequential disulfide bond formation strategy (8Bathgate R.A.D. Lin F. Hanson N.F. Otvos Jr., L. Guidolin A. Giannakis C. Bastiras S. Layfield S.L. Ferraro T. Ma S. Zhao C. Gundlach A.L. Samuel C.S. Tregear G.W. Wade J.D. Biochemistry. 2006; 45: 1043-1053Crossref PubMed Scopus (135) Google Scholar) led to the production of the following A-chain-truncated relaxin analogs: A-(5–24) H3, A-(7–24) H3, A-(8–24) H3, A-(9–24) H3, A-(10–24) H3, Ala-4 A-(9–24) H3, Ala-5 A-(9–24) H3, A-(5–24) H2, A-(7–24) H2, A-(9–24) H2, Ala-4 A-(9–24) H2, Ala-5 A-(9–24) H2 (Fig. 1). Additionally, the A-chain-truncated INSL3 analogs A-(9–26) INSL3 and A-(10–24) INSL3 were also prepared. The native B-chains of H2, H3, and INSL3 were used in each case. The overall yield was ∼5% for H3 analogs, 6% for the INSL3 analogs, and ∼10–15% for H2 analogs relative to the starting B-chain peptide. Peptide Characterization—The purity of each synthetic peptide was assessed by analytical reverse phase HPLC and MALDI-TOF mass spectrometry using a Bruker Autoflex II instrument (Bremen, Germany) in the linear mode at 19.5 kV. Peptides were quantitated by amino acid analysis of a 24-h acid hydrolysate using a GBC instrument (Melbourne, Australia). Circular Dichroism Spectroscopy—CD spectra were recorded on a JASCO (J-185; Tokyo, Japan) spectrophotometer at 25 °C using a 1-mm path length cell. The peptides were dissolved in 10 mm phosphate buffer (pH 7.5) at a concentration of 0.01 or 0.1 mg/ml. NMR Structural Analysis—The truncated analogues A-(9–24) H2, A-(10–24) H3, and A-(10–24) INSL3 were each analyzed by solution NMR spectroscopy. For each of the peptides, 0.5-ml samples containing 1, 0.5, and ∼1 mg, respectively, in the solvent system 90% H2O, 10% D2O at pH ∼4 were prepared. Two-dimensional homonuclear data, including TOCSY, NOESY, and DQF-COSY, were recorded at 600 and 900 MHz on Bruker Avance spectrometers. All two-dimensional spectra were generally recorded with 4000 data points in the direct dimension and 512 increments in the indirect dimension, which was zero-filled to 1000 data points prior to transformation. For A-(9–24) H2 relaxin, a series of TOCSY spectra were recorded at 288, 293, 298, 303, and 308 K in order to determine the amide temperature dependence. A temperature coefficient of >-4.6 ppb/K was considered indicative of a hydrogen bond (25Cierpicki T. Zhukov I. Byrd R.A. Otlewski J. J. Magn. Reson. 2002; 157: 178-180Crossref PubMed Scopus (70) Google Scholar). Based on this analysis, the following hydrogen bond donors were identified: A15, B5, B9, B10, B12, B19, B21, and B22. Additional data for structural restraints were also recorded on a lyophilized sample redissolved in 100% D2O. Structural restraints for structure calculations included interproton distances derived from a NOESY spectrum recorded at 900 MHz with a mixing time of 150 ms. Dihedral angle restraints of -100 ± 80° were introduced for ϕ angles, where a positive angle could be excluded based on a stronger interresidual Hαi- 1-HNi than intra-Hαi-HNi NOE. Hydrogen bond restraints were introduced where hydrogen bonds could be identified based on temperature coefficients in combination with preliminary structures. Preliminary structures were calculated using torsion angle dynamics within the program CYANA (26Guntert P. Methods Mol. Biol. 2004; 278: 353-378Crossref PubMed Scopus (1187) Google Scholar). The final structures were generated by torsion angle dynamics within CNS followed by Cartesian dynamics and refinement in explicit water as described in detail previously (27Rosengren K.J. Daly N.L. Plan M.R. Waine C. Craik D.J. J. Biol. Chem. 2003; 278: 8606-8616Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Binding Assays—Human embryonic kidney (HEK)-293T cells stably transfected with RXFP1 and RXFP2 (8Bathgate R.A.D. Lin F. Hanson N.F. Otvos Jr., L. Guidolin A. Giannakis C. Bastiras S. Layfield S.L. Ferraro T. Ma S. Zhao C. Gundlach A.L. Samuel C.S. Tregear G.W. Wade J.D. Biochemistry. 2006; 45: 1043-1053Crossref PubMed Scopus (135) Google Scholar) were grown in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine and plated into 24-well poly-l-lysine-coated plates for whole cell binding assays. Competition binding experiments were performed as previously described with either 100 pm 33P-labeled H2 relaxin (8Bathgate R.A.D. Lin F. Hanson N.F. Otvos Jr., L. Guidolin A. Giannakis C. Bastiras S. Layfield S.L. Ferraro T. Ma S. Zhao C. Gundlach A.L. Samuel C.S. Tregear G.W. Wade J.D. Biochemistry. 2006; 45: 1043-1053Crossref PubMed Scopus (135) Google Scholar) or 125I-labeled INSL3 (28Muda M. He C. Martini P.G. Ferraro T. Layfield S. Taylor D. Chevrier C. Schweickhardt R. Kelton C. Ryan P.L. Bathgate R.A. Mol. Hum. Reprod. 2005; 11: 591-600Crossref PubMed Scopus (55) Google Scholar) in the absence or presence of increasing concentrations of unlabeled hormones. Nonspecific binding was determined with an excess of unlabeled peptides (500 nm H2 relaxin or INSL3). CHO-K1 cells stably expressing RXFP3 (29Van Der Westhuizen E.T. Sexton P.M. Bathgate R.A. Summers R.J. Ann. N. Y. Acad. Sci. 2005; 1041: 332-337Crossref PubMed Scopus (31) Google Scholar) were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 5% (v/v) fetal calf serum, 2 mm l-glutamine, 100 μg/ml penicillin, and 100 μg/ml streptomycin. Crude membrane preparations were prepared for competition binding curves using 100 pm 125I-labeled H3 relaxin B chain-INSL5 A-chain chimeric peptide (kindly labeled by Dr. Steve Sutton) as previously described (13Liu C. Chen J. Kuei C. Sutton S. Nepomuceno D. Bonaventure P. Lovenberg T.W. Mol. Pharmacol. 2005; 67: 231-240Crossref PubMed Scopus (119) Google Scholar). Nonspecific binding was determined by the addition of 500 nm H3 relaxin. All data are presented as the mean ± S.E. of the percentage of the total specific binding of triplicate wells, repeated in at least three separate experiments, and curves were fitted using onesite binding curves in Graphpad Prism 4 (Graphpad Software). Statistical differences in pIC50 values were analyzed using Student's t tests in Graphpad Prism 4. Inhibition of Forskolin-induced Intracellular cAMP Accumulation—The influence of the various ligands on cAMP signaling in cells expressing RXFP receptors was assessed using a cAMP reporter gene assay as previously described (15Scott D.J. Layfield S. Yan Y. Sudo S. Hsueh A.J. Tregear G.W. Bathgate R.A. J. Biol. Chem. 2006; 281: 34942-34954Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Briefly, HEK-293T cells in 96-well plates were co-transfected with either RXFP1 or RXFP2 and a pCRE-β-galactosidase reporter plasmid (30Chen W. Shields T.S. Stork P.J. Cone R.D. Anal. Biochem. 1995; 226: 349-354Crossref PubMed Scopus (183) Google Scholar). 24 h later, co-transfected cells were treated with increasing concentrations of H2 relaxin, H3 relaxin, or INSL3 analogs in parallel with 10 nm H2 relaxin or INSL3 for RXFP1- or RXFP2-transfected cells, respectively. In addition, INSL3 analogs were tested for their ability to block 1 nm INSL3 stimulation over 6 h. CHO-K1 cells in 96-well plates were co-transfected with RXFP3 and the pCRE-β-galactosidase reporter plasmid, and 24 h later, they were treated with 5 μm forskolin together with increasing concentrations of H3 relaxin analogs. 10 nm H3 relaxin was used for maximal stimulation, whereas untreated cells were used as controls. After 6 h, the cell medium was aspirated, and the cells were frozen at -80 °C overnight. The amount of cAMP-driven β-galactosidase expression in each well was determined as previously described. Ligand-induced stimulation of cAMP was expressed as a percentage of the maximum H2 relaxin, INSL3, and H3 relaxin response for RXFP1, RXFP2, and RXFP3 cells, respectively. Data points were measured in triplicate, and each experiment was repeated at least three times. A number of INSL3, H2, and H3 relaxin analogues as outlined in Fig. 1 were prepared for functional and structural studies. In all cases, a highly efficient solid phase peptide synthesis strategy using regioselectively S-protected relaxin or INSL3 A- and B-chains followed by sequential disulfide bond formation was utilized (8Bathgate R.A.D. Lin F. Hanson N.F. Otvos Jr., L. Guidolin A. Giannakis C. Bastiras S. Layfield S.L. Ferraro T. Ma S. Zhao C. Gundlach A.L. Samuel C.S. Tregear G.W. Wade J.D. Biochemistry. 2006; 45: 1043-1053Crossref PubMed Scopus (135) Google Scholar). This method assures a good overall yield and avoids a tedious and labor-intensive random oxidation method. Each peptide was comprehensively chemically characterized, including by MALDI-TOF mass spectrometry, and high purity was confirmed. Analogs of INSL3 were first prepared to confirm the reported effects of A-chain shortening on the INSL3 peptide in our RXFP2-expressing cell line. As previously reported (23Bullesbach E.E. Schwabe C. J. Biol. Chem. 2005; 280: 14586-14590Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), A-(9–26) INSL3 demonstrated equivalent binding affinity to native INSL3 (Fig. 2, A and B). Additionally, a further truncated analog A-(10–24) INSL3 also showed high binding affinity, although it was significantly lower (p < 0.001) than both native INSL3 and A-(9–26) INSL3 (Fig. 2, A and B). Neither peptide was able to stimulate cAMP in RXFP2-expressing cells (data not shown); however, they were both able to dose-dependently inhibit INSL3-meditated cAMP signaling (Fig. 2C). Analogs of H3 relaxin that had been sequentially shortened by 7, 8, or 9 amino acids at the N terminus of their A-chains were synthesized and first tested for their ability to bind to and activate RXFP3. Analogs A-(8–24) H3, A-(9–24) H3, and A-(10–24) H3 relaxin all demonstrated high affinity binding to RXFP3-expressing cells (Fig. 3A and Table 1). Additionally, they were all able to inhibit forskolin-stimulated cAMP production with a potency similar to that of native H3 relaxin (Fig. 3B). Hence, A-chain shortening had no effect on H3 relaxin activity on RXFP3.TABLE 1Pooled binding (pKi) and cAMP (pEC50) data from H2 and H3 relaxin A-chain-truncated peptides n values in parentheses.RXFP1RXFP3RXFP2Ligand33P-relaxin binding pKicAMP activity pEC50125I-H3/INSL5 binding pKicAMP activity pEC5033P-Relaxin binding pKicAMP activity pEC50H37.69 ± 0.04 (3)9.36 ± 0.21 (4)8.48 ± 0.04 (3)8.26 ± 0.09 (3)NAaNA, no activityNAA-(5–24) H38.18 ± 0.11 (3)8.34 ± 0.06 (3)bp < 0.001 versus H3 relaxinNDcND, not determinedNDNANAA-(7–24) H36.87 ± 0.03 (3)dp < 0.01 versus H3 relaxin6.88 ± 0.13 (5)bp < 0.001 versus H3 relaxinNDNDNANAA-(8–24) H36.30 ± 0.02 (3)bp < 0.001 versus H3 relaxin6.58 ± 0.31 (3)bp < 0.001 versus H3 relaxin8.34 ± 0.08 (3)7.96 ± 0.07 (2)NANAA-(9–24) H35.84 ± 0.16 (3)bp < 0.001 versus H3 relaxin7.14 ± 0.01 (3)bp < 0.001 versus H3 relaxin8.02 ± 0.12 (3)8.75 ± 0.12 (3)NANAA-(10–24) H35.58 ± 0.16 (3)bp < 0.001 versus H3 relaxin6.45 ± 0.09 (3)bp < 0.001 versus H3 relaxin8.22 ± 0.08 (3)7.98 ± 0.11 (3)NANAAla-4 A-(9–24) H37.86 ± 0.11 (3)ep < 0.001 versus H3 A-(9–24)7.23 ± 0.13 (3)bp < 0.001 versus H3 relaxinNDNDNANAAla-5 A-(9–24) H37.69 ± 0.16 (3)ep < 0.001 versus H3 A-(9–24)6.99 ± 0.30 (4)bp < 0.001 versus H3 relaxinNDNDNANAH29.24 ± 0.16 (3)10.37 ± 0.04 (4)NANA8.48 ± 0.22 (3)9.13 ± 0.06 (3)A-(5–24) H28.85 ± 0.09 (3)10.37 ± 0.12 (5)NANA7.78 ± 0.16 (3)fp < 0.01 versus H2 relaxin7.64 ± 0.30 (3)gp < 0.001 versus H2 relaxinA-(7–24) H28.62 ± 0.06 (3)9.92 ± 0.24 (4)NANA7.01 ± 0.09 (3)gp < 0.001 versus H2 relaxin7.06 ± 0.34 (3)gp < 0.001 versus H2 relaxinA-(9–24) H26.55 ± 0.10 (3)gp < 0.001 versus H2 relaxin9.12 ± 0.20 (5)gp < 0.001 versus H2 relaxinNANA6.77 ± 0.23 (3)gp < 0.001 versus H2 relaxin<6Ala-4 A-(9–24) H28.43 ± 0.04 (3)fp < 0.01 versus H2 relaxin,hp < 0.01 versus H2 A-(9–24)9.88 ± 0.16 (5)hp < 0.01 versus H2 A-(9–24)NANA7.34 ± 0.13 (3)gp < 0.001 versus H2 relaxin7.52 ± 0.43 (3)fp < 0.01 versus H2 relaxinAla-5 A-(9–24) H28.49 ± 0.06 (3)hp < 0.01 versus H2 A-(9–24),ip < 0.05 versus H2 relaxin9.79 ± 0.19 (4)hp < 0.01 versus H2 A-(9–24)NANA7.59 ± 0.12 (3)fp < 0.01 versus H2 relaxin,jp < 0.05 versus H2 A-(9–24)7.19 ± 0.12 (3)gp < 0.001 versus H2 relaxinINSL3NANANANA9.01 ± 0.08 (3)ip < 0.05 versus H2 relaxin10.35 ± 0.12 (3)fp < 0.01 versus H2 relaxina NA, no activityb p < 0.001 versus H3 relaxinc ND, not determinedd p < 0.01 versus H3 relaxine p < 0.001 versus H3 A-(9–24)f p < 0.01 versus H2 relaxing p < 0.001 versus H2 relaxinh p < 0.01 versus H2 A-(9–24)i p < 0.05 versus H2 relaxinj p < 0.05 versus H2 A-(9–24) Open table in a new tab These peptides were then tested for their ability to bind to and activate RXFP1. In stark contrast to the effects of A-chain shortening on the INSL3 peptide, the H3 relaxin analogs demonstrated a progressive loss of binding affinity with A-chain shortening by 7, 8, or 9 amino acids (Fig. 4A and Table 1). Most importantly, the peptides were able to stimulate cAMP activity in RXFP1-expressing cells with activities matching their binding affinities (Fig. 4C and Table 1). Hence, A-(8–24) H3, A-(9–24) H3, and A-(10–24) H3 all had a lower affinity and potency than H3 relaxin (p < 0.001; Table 1), and additionally the affinity of A-(10–24) H3 for RXFP1 was significantly lower than A-(8–24) H3 (p < 0.05; Table 1). Two additional A-chain-shortened peptides, A-(5–24) H3 and A-(7–24) H3 relaxin, were synthesized to determine the minimum length of A-chain required for maximum binding affinity and potency. A-(7–24) H3 relaxin also demonstrated a significantly reduced affinity to RXFP1 compared with H3 relaxin, which was similarly reflected in a decreased potency (both p < 0.001; Table 1). In comparison with the shorter H3 truncates, A-(7–24) H3 relaxin was found to have a significantly higher affinity for RXFP1 (all p < 0.01 or p < 0.001), but interestingly, its potency in

Referência(s)