Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m308995200
ISSN1083-351X
AutoresChanglu Liu, Elo Eriste, Steven W. Sutton, Jing‐Cai Chen, Barbara Roland, Chester Kuei, Niven Farmer, Hans Jörnvall, Rannar Sillard, Timothy W. Lovenberg,
Tópico(s)Pregnancy-related medical research
ResumoGPCR135, publicly known as somatostatin- and angiotensin-like peptide receptor, is expressed in the central nervous system and its cognate ligand(s) has not been identified. We have found that both rat and porcine brain extracts stimulated 35S-labeled guanosine 5′-O-(3-thiotriphosphate) (GTPγS) incorporation in cells over-expressing GPCR135. Multiple rounds of extraction, purification, followed by N-terminal sequence analysis of the ligand from porcine brain revealed that the ligand is a product of the recently identified gene, relaxin-3 (aka insulin-7 or INSL7). Recombinant human relaxin-3 potently stimulates GTPγS binding and inhibits cAMP accumulation in GPCR135 overexpressing cells with EC50 values of 0.25 and 0.35 nM, respectively. 125I-Relaxin-3 binds GPCR135 at high affinity with a Kd value of 0.31 nM. Relaxin-3 is the only member of the insulin/relaxin superfamily that can activate GPCR135. In situ hybridization showed that relaxin-3 mRNA is predominantly expressed in the dorsomedial ventral tegmental nucleus of the brainstem (aka nucleus incertus), as well as in discrete cells in the lateral periaqueductal gray and in the central gray nucleus. GPCR135 is expressed abundantly in the hypothalamus with discrete expression in the paraventricular nucleus of the hypothalamus and supraoptic nucleus, as well as in the cortex, septal nucleus, and preoptical area. Relaxin-3 has previously been shown to bind and activate the LGR7 relaxin receptor. However, we believe that neuroanatomical colocalization of GPCR135 and relaxin-3, coupled with a clear high affinity interaction, suggest that GPCR135 is the receptor for relaxin-3. The identification of relaxin-3 as the ligand for GPCR135 provides the framework for the discovery of a new brainstem/hypothalamus circuitry. GPCR135, publicly known as somatostatin- and angiotensin-like peptide receptor, is expressed in the central nervous system and its cognate ligand(s) has not been identified. We have found that both rat and porcine brain extracts stimulated 35S-labeled guanosine 5′-O-(3-thiotriphosphate) (GTPγS) incorporation in cells over-expressing GPCR135. Multiple rounds of extraction, purification, followed by N-terminal sequence analysis of the ligand from porcine brain revealed that the ligand is a product of the recently identified gene, relaxin-3 (aka insulin-7 or INSL7). Recombinant human relaxin-3 potently stimulates GTPγS binding and inhibits cAMP accumulation in GPCR135 overexpressing cells with EC50 values of 0.25 and 0.35 nM, respectively. 125I-Relaxin-3 binds GPCR135 at high affinity with a Kd value of 0.31 nM. Relaxin-3 is the only member of the insulin/relaxin superfamily that can activate GPCR135. In situ hybridization showed that relaxin-3 mRNA is predominantly expressed in the dorsomedial ventral tegmental nucleus of the brainstem (aka nucleus incertus), as well as in discrete cells in the lateral periaqueductal gray and in the central gray nucleus. GPCR135 is expressed abundantly in the hypothalamus with discrete expression in the paraventricular nucleus of the hypothalamus and supraoptic nucleus, as well as in the cortex, septal nucleus, and preoptical area. Relaxin-3 has previously been shown to bind and activate the LGR7 relaxin receptor. However, we believe that neuroanatomical colocalization of GPCR135 and relaxin-3, coupled with a clear high affinity interaction, suggest that GPCR135 is the receptor for relaxin-3. The identification of relaxin-3 as the ligand for GPCR135 provides the framework for the discovery of a new brainstem/hypothalamus circuitry. The recent completion of the sequencing of the human genome revealed thousands of new genes. 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As part of a directed effort to identify novel receptors and ligands in the central nervous system, we cloned the full-length cDNAs for nearly all GPCR-like sequences from public and in-house data bases. One of these receptors, GPCR135, was used to fish out a ligand from brain extracts. GPCR135, publicly known as somatostatin- and angiotensin-like peptide receptor (SALPR) (31.Matsumoto M. Kamohara M. Sugimoto T. Hidaka K. Takasaki J. Saito T. Okada M. Yamaguchi T. Furuichi K. Gene (Amst.). 2000; 248: 183-189Crossref PubMed Scopus (67) Google Scholar), shares significant homology to somatostatin receptor SSTR5 and angiotensin II receptor AT1 with 35 and 31% identity, respectively. GPCR135 mRNA is expressed in various regions in the brain, particularly in the substantia nigra and pituitary as determined by RT-PCR (31.Matsumoto M. Kamohara M. Sugimoto T. Hidaka K. Takasaki J. Saito T. Okada M. Yamaguchi T. Furuichi K. Gene (Amst.). 2000; 248: 183-189Crossref PubMed Scopus (67) Google Scholar). GPCR135 mRNA can also be detected in the peripheral tissues, albeit at low levels (31.Matsumoto M. Kamohara M. Sugimoto T. Hidaka K. Takasaki J. Saito T. Okada M. Yamaguchi T. Furuichi K. Gene (Amst.). 2000; 248: 183-189Crossref PubMed Scopus (67) Google Scholar). The predominant expression of GPCR135 mRNA in the brain suggests a role in central nervous system function. However, the endogenous ligand(s) for GPCR135 had not been identified. In the present studies, we report the purification, identification, and characterization of relaxin-3, a new member of the insulin/relaxin peptide superfamily (27.Bathgate R.A. Samuel C.S. Burazin T.C. Layfield S. Claasz A.A. Reytomas I.G. Dawson N.F. Zhao C. Bond C. Summers R.J. Parry L.J. Wade J.D. Tregear G.W. J. Biol. Chem. 2002; 277: 1148-1157Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), as the endogenous ligand for GPCR135. Relaxin-3 binds to and activates GPCR135 at high affinity. No other members of the insulin peptide family bind to or activate GPCR135. Because GPCR135 is expressed in the hypothalamus and relaxin-3 is expressed in the brainstem nuclei with known projections to the hypothalamus, we conclude that relaxin-3 is the endogenous ligand for GPCR135. Reagents—Human insulin, IGF1, and IGF2 were purchased from Sigma. Human INSL3, INSL4, INSL6, oxidized relaxin-3 A-chain and B-chain peptides were purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA). Porcine relaxin was purchased from the National Hormone & Peptide Program (Torrance, CA). All other peptides were produced recombinantly. α-Cyano-4-hydroxycinnamic acid (Sigma) was recrystallized from ethanol/water, and dissolved in 0.3% trifluoroacetic acid, 50% acetonitrile/water at 10 mg/ml. Carboxypeptidase Y (Roche Applied Sciences) was dissolved in 30 mm citrate buffer, pH 6.0, at 0.5 μg/μl, and methylated trypsin (sequencing grade, Promega, Madison, WI) was dissolved at 0.2 μg/μl in 50 mm acetic acid, both were stored at -20 °C until use. Calibration solution was prepared in 0.1% trifluoroacetic acid, water from [Arg8]vasopressin (Sigma) at 0.28 pmol/μl and human insulin (Sigma) at 18 pmol/μl. Molecular Cloning of GPCR135—The human GPCR135 coding region was identified from a human genome sequence (GenBank™ accession number NT_023085) based on its homology to somatostatin receptors. The complete coding region of human GPCR135 was PCR amplified from human genomic DNA (Clontech, Palo Alto, CA) using two primers with the forward primer: 5′-ACAGCTCGAGGCCACCATGCAGATGGCCGATGCAGCCACG-3′ and the reverse primer: 5′-ACATCATCTAGATCAGTAGGCAGAGCTGCTGGGCAGCAG-3′. The PCR was performed at 94 °C for 40 s, 65 °C for 40 s, and 72 °C for 3 min for 35 cycles. The PCR products were cloned into pCIneo and the insert region was sequenced to confirm the identity of the sequence. Initial Identification and Characterization of the GPCR135 Ligand Activity from the Rat Brain—Five grams of frozen rat brain were homogenized in 50 ml of -30 °C ethanol, 0.8 m HCl (3:1 ratio). The homogenate was extracted at 4 °C for 2 h and then centrifuged at 4 °C at 20,000 × g for 30 min. The supernatant was loaded onto a 2-ml SP-Sephadex C-25 (Amersham Biosciences) column. The column was washed with 20 ml of 1 m acetic acid and eluted with 2 m pyridine and 1 m acetic acid. The eluted peptides were loaded onto a 500-mg C-18 Bond Elut column (Varian, Harbor City, CA), washed with 0.1% trifluoroacetic acid, and eluted with 60% acetonitrile and 0.1% trifluoroacetic acid. The eluted peptides were lyophilized, reconstituted in water, and tested for ligand activity in GPCR135-expressing cell membranes using GTPγS binding assays. The reconstituted rat brain extract was also run on a HPLC gel filtration column (BioSep-SEC-S2000, Phenomenex, Torrance, CA) in 1 m acetic acid to characterize the molecular weight of the ligand. Briefly, fractions of 1 ml were collected, lyophilized, and tested for GPCR135 ligand activity using GTPγS binding assays. In a parallel experiment, proteins or peptides with various molecular weights were run in the same conditions to serve as the molecular weight standards. Purification of GPCR135 Ligand from Porcine Brain—Porcine brains (1.8 kg) were extracted with 18 liters of cold ethanol, 0.8 m HCl, mixed at 3:1. Portions of 300 g of frozen brains were homogenized in a blender (Waring, Winster, CT) for 1–2 min in 3 liters of the liquid cooled to -30 °C. The homogenate was then stirred at 4 °C for 2 h. The mixture was centrifuged at 5000 × g for 20 min and filtered through Whatman 541 filter paper (Whatman, Maidstone, UK) and then through 0.22-μm Millipore GP Express Plus filter (Millipore, Bedford, MA). Aliquots of 2.5 liters of the extract were mixed with 600 ml of Express-Ion™ Exchanger S (Whatman) equilibrated with 20 mm sodium acetate, 20% acetonitrile, pH 5.2, stirred for 20 min, and filtered. The ion exchanger was washed 3 times with 500 ml of the same buffer and then eluted with 500 ml of 20 mm sodium acetate, 0.5 m NaCl, 20% acetonitrile, pH 5.2, and the eluate was separated from the ion exchanger by filtration. The elution procedure was repeated twice, first with 500 ml and then with 400 ml of liquid, and the eluates were pooled. The remaining extract was processed identically. To the pooled eluate, an equal volume of 0.1% trifluoroacetic acid/water was added, and 10 liters of this liquid was mixed with 300 g of Amberlite XAD-16 HP (Supelco, Bellefonte, PA) and stirred for 20 min. Amberlite was washed on a filter with 0.1% trifluoroacetic acid/water, and eluted by mixing with 700 ml of 50% acetonitrile, 0.1% trifluoroacetic acid/water. The eluate was separated from the Amberlite by filtration. Such elution from Amberlite was repeated twice, and the rest of the ion exchanger eluate was processed in the same manner. The eluates from Amberlite were pooled, and acetonitrile was removed with a rotary evaporator. The remaining liquid was lyophilized, and 0.52 g of dry material was obtained. The material was dissolved in 400 ml of 20 mm sodium acetate, 20% acetonitrile, pH 5.2, and filtered through a 0.22-μm Millipore GP Express Plus filter. The following chromatographies were carried out on ÄKTA Explorer™ and Purifier™ HPLC systems (Amersham Biosciences). The filtrate (78 ml per run) was pumped onto a Resource S (6-ml column, Amersham Biosciences) equilibrated with 20 mm sodium acetate, 20% acetonitrile, pH 5.2, and eluted with a 0–20% linear gradient in 35 column volumes of the same buffer containing 2 m NaCl. The active fractions were pooled, diluted with 5 volumes of water, pH was adjusted to 2.5, and loaded onto the same column equilibrated with 20 mm sodium phosphate, 20% acetonitrile, pH 2.5. Peptides were eluted with a 0–25% linear gradient in 35 column volumes of this buffer containing 2 m NaCl. Active fractions were collected, mixed with an equal volume of 0.1% trifluoroacetic acid/water loaded onto a Source 5RPC (ST 4.6/150, Amersham Biosciences), and eluted with a 25–40% linear gradient in 12 column volumes of 0.1% trifluoroacetic acid, 95% acetonitrile/water. The pooled active fractions were mixed with 750 μl of 0.1% trifluoroacetic acid/water, and chromatographed on a μRPC C2/C18 column (ST 4.6/100, Amersham Biosciences) using a 23–40% linear gradient in 15 column volumes of 0.1% trifluoroacetic acid, 95% acetonitrile/water. At this stage the active fractions appeared as a single peak and the material was subjected to structural analysis. S-Carboxyamidomethylation—A part of the lyophilized peptide was dissolved in 200 μl of reaction buffer consisting of 0.4 m Tris-HCl, pH 8.4, 6 m guanidinium hydrochloride (Sigma), 2 mm EDTA (Merck), and 4 μl of 0.5 m dithiothreitol (Pierce) was added and kept at 40 °C for 2 h. 12 μl of 0.5 m iodoacetamide (Sigma) was added to the sample, and the sample was incubated at 40 °C for 35 min followed by 30 min at room temperature in the dark. The sample was mixed with 2 volumes of 0.1% trifluoroacetic acid/water, pumped onto the μRPC C2/C18 ST 4.6/100 column, and the peptides were separated with a linear gradient of 0.1% trifluoroacetic acid, 95% acetonitrile/water. Mass Spectrometry and Sequence Analysis—Positive-ion MALDI TOF mass spectra were recorded on a Voyager DE Pro instrument (Applied Biosystems, Foster City, CA). For mass determination 1 μl of sample from an HPLC fraction was mixed on a stainless steel MALDI target plate with 1 μl of calibration solution and 1 μl of α-cyano-4-hydroxycinnamic acid solution and allowed to dry at room temperature. Carboxypeptidase Y digests were prepared by pipetting 1 μl of samples from HPLC on the MALDI target, mixed with 1 μl of carboxypeptidase Y solution and allowed to dry at room temperature. Calibration solution (1 μl) and α-cyano-4-hydroxycinnamic acid solution (1 μl) were added to the same spot and allowed to dry again. Acquired mass spectra were calibrated using monoisotopic peaks of singly or doubly charged ions of [Arg8]vasopressin and human insulin (m/z 1084.4457 Th, m/z 2902.8266 Th, m/z 5804.6455 Th). On-target tryptic digests were prepared by pipetting 1 μl each of sample, trypsin, and 0.2% ammonium bicarbonate solutions, and allowed to dry at room temperature. Finally, 1 μl of matrix solution was added and allowed to dry again. The spectra were calibrated using monoisotopic peaks of fragments from self-digestion of trypsin (m/z 515.3306 Th, m/z 842.5100 Th, and m/z 2211.1046 Th). Sequence analysis of tryptic fragments using tandem mass spectrometry was carried out on a Q-TOF Ultima instrument (Waters/Micromass, Manchester, UK) equipped with the standard Z-spray source. The samples were sprayed in 60% acetonitrile, 1% acetic acid/water from gold-coated borosilicate capillaries (Protana, Odense, Denmark) using a capillary voltage of 1.5 kV, and positive-ion spectra were recorded. Tryptic fragments were prepared from carboxyamidomethylated peptides by incubation with methylated trypsin in 0.2% ammonium bicarbonate at 37 °C for 6 h, and the samples were lyophilized. Tandem MS/MS experiments were carried out at collision energies optimized within 25–35 eV, using 5-s scans during 5 min. Data were analyzed with a Biolynx peptide sequencing module of Masslynx 3.5. N-terminal sequence analysis was carried out on an Applied Biosystems Procise HT sequencer. GTPγS Binding Assays—The GPCR135 expression vector described above was transfected into CHO-K1 cells using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. Two days after transfection, the cells were harvested and the cell membranes were prepared by homogenizing the cells in 50 mm Tris-HCl, 5 mm EDTA followed by centrifugation at 20,000 × g at 4 °C for 30 min. GTPγS binding buffer (50 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 10 μm GDP, 1 mm EDTA, pH 8.0, and 100 mm NaCl) was added to the pellet and the pellet was homogenized using a Polytron tissue homogenizer. Protease inhibitors were added to the buffer at concentrations of 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstain A, 10 μg/ml leupeptin. Cell membranes and different concentrations of ligands were added to 96-well plates and incubated at room temperature for 20 min. 35S-GTPγS (PerkinElmer Life Sciences) was then added to each well at a final concentration of 200 pm in a final volume of 200 μl. The reactions were allowed to proceed at room temperature for 1 h, filtered though a 96-well GFC filter plate (Packard Instrument Co.), and washed with cold washing buffer: 50 mm Tris-HCl, pH 7.4, 10 mm MgCl2. 50 μl of Microscint-40 (PerkinElmer Life Sciences) was added to each well and the plate was counted on a top counter (TopCount NTX, Packard). Expression and Purification of Human Recombinant Relaxin-3—Human relaxin-3 complete coding region was PCR amplified from human brain cDNA (Clontech) using two primers with the forward primer: 5′-ACGATCGTCGACGCCACCATGGCCAGGTACATGCTGCTGCTGCTC-3′ and the reverse primer: 5′-ACGATAAAGCTTCTAGCAAAGGCTACTGATTTCACTTTTGC-3′. The PCR products were cloned into a mammalian expression vector pCMV-sport1 (Invitrogen) between SalI and HindIII sites. The cloned cDNAs were sequenced to confirm the identities of human relaxin-3. The expression vector was transfected into COS-7 cells using LipofectAMINE (Invitrogen). Three days after the transfection, cell culture medium from the transfected cells was collected, adjusted to pH 3.0, and loaded onto a Sephadex C-25 cation exchange column. The column was washed with 1 m acetic acid and eluted with 2 m pyridine and 1 m acetic acid. The eluted proteins were loaded on a C-18 Bond Elut column (Varian), washed with 0.1% trifluoroacetic acid, and eluted with 60% acetonitrile and 0.1% trifluoroacetic acid. The eluted proteins were lyophilized, reconstituted in 50 mm Tris-HCl, pH 7.5, and used for GTPγS binding assay. In a parallel experiment, we cloned the human relaxin-3 pro-peptide coding region into a modified pCMV-sport1 vector with the polycloning sites modified by replac
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