Identification and Characterization of a Novel RF-amide Peptide Ligand for Orphan G-protein-coupled Receptor SP9155

Abstract

Orphan G-protein-coupled receptors are a large class of receptors whose cognate ligands are unknown. SP9155 (also referred to as AQ27 and GPR103) is an orphan G-protein-coupled receptor originally cloned from a human brain cDNA library. SP9155 was found to be predominantly expressed in brain, heart, kidney, retina, and testis. Phylogenetic analysis shows that SP9155 shares high homology with Orexin, NPFF, and cholecystokinin (CCK) receptors, but identification of the endogenous ligand for SP9155 has not been reported. In this study, we have used a novel method to predict peptides from genome data bases. From these predicted peptides, a novel RF-amide peptide, P52 was shown to selectively activate SP9155-transfected cells. We subsequently cloned the precursor gene of the P52 ligand and characterized the activity of other possible peptides encoded by the precursor. This revealed an extended peptide, P518, which exhibited high affinity for SP9155 (EC50 = 7 nm). mRNA expression analysis revealed that the peptide P518 precursor gene is predominantly expressed in various brain regions, coronary arteries, thyroid and parathyroid glands, large intestine, colon, bladder, testes, and prostate. These results indicate the existence of a novel RF-amide neuroendocrine peptide system, and suggest that SP9155 is likely the relevant G-protein-coupled receptor for this peptide. Orphan G-protein-coupled receptors are a large class of receptors whose cognate ligands are unknown. SP9155 (also referred to as AQ27 and GPR103) is an orphan G-protein-coupled receptor originally cloned from a human brain cDNA library. SP9155 was found to be predominantly expressed in brain, heart, kidney, retina, and testis. Phylogenetic analysis shows that SP9155 shares high homology with Orexin, NPFF, and cholecystokinin (CCK) receptors, but identification of the endogenous ligand for SP9155 has not been reported. In this study, we have used a novel method to predict peptides from genome data bases. From these predicted peptides, a novel RF-amide peptide, P52 was shown to selectively activate SP9155-transfected cells. We subsequently cloned the precursor gene of the P52 ligand and characterized the activity of other possible peptides encoded by the precursor. This revealed an extended peptide, P518, which exhibited high affinity for SP9155 (EC50 = 7 nm). mRNA expression analysis revealed that the peptide P518 precursor gene is predominantly expressed in various brain regions, coronary arteries, thyroid and parathyroid glands, large intestine, colon, bladder, testes, and prostate. These results indicate the existence of a novel RF-amide neuroendocrine peptide system, and suggest that SP9155 is likely the relevant G-protein-coupled receptor for this peptide. G-protein coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G-protein coupled receptor; RF-amide, Arg-Phe-amide; FLIPR, fluorometric image plate reader; HEK, human embryonic kidney; VTS, virtual transcripts; CHO, Chinese hamster ovary; NPFF, neuropeptide FF; NPAF, neuropeptide AF; RFRP RF-amide-related peptide.1The abbreviations used are: GPCR, G-protein coupled receptor; RF-amide, Arg-Phe-amide; FLIPR, fluorometric image plate reader; HEK, human embryonic kidney; VTS, virtual transcripts; CHO, Chinese hamster ovary; NPFF, neuropeptide FF; NPAF, neuropeptide AF; RFRP RF-amide-related peptide. are members of a large protein family that share a common structural motif of seven transmembrane domains (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4669) Google Scholar, 2Strader C.D. Fong T.M. Graziano M.P. Tota M.R. FASEB J. 1995; 9: 745-754Crossref PubMed Scopus (327) Google Scholar). GPCRs mediate a variety of physiological functions by interacting with extracellular ligands including small chemical molecules, peptides, and proteins, then transmitting the signal to intracellular second messengers via G proteins (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4669) Google Scholar, 2Strader C.D. Fong T.M. Graziano M.P. Tota M.R. FASEB J. 1995; 9: 745-754Crossref PubMed Scopus (327) Google Scholar). In recent years, the primary sequence of many GPCRs have been identified through searching the human genome sequence data base. Whereas these GPCRs have common structural motifs including seven transmembrane domains, often their cognate ligands and biological function are unknown (3Howard A.D. McAllister G. Feighner S.D. Liu Q. Nargund R.P. Van der Plough L.H. Patchett A.A. Trends Pharmacol. Sci. 2001; 3: 132-140Abstract Full Text Full Text PDF Scopus (234) Google Scholar, 4Civelli O. Nothacker H.-P. Saito Y. Wang Z. Lin S. Reinscheid R.K. Trends Neurosci. 2001; 4: 230-237Abstract Full Text Full Text PDF Scopus (123) Google Scholar, 5Wilson S. Bergsma D.J. Chambers J.K. Muir A.I. Fantom K.G.M. Ellis C. Murdock P.R. Herrity N.C. Stadel J.M. Br. J. Pharmacol. 1998; 125: 1387-1392Crossref PubMed Scopus (114) Google Scholar). To understand the biological functions of these so called orphan receptors, and to enable identification of pharmacological agents active at these receptors, it is helpful to first identify their endogenous cognate ligands (3Howard A.D. McAllister G. Feighner S.D. Liu Q. Nargund R.P. Van der Plough L.H. Patchett A.A. Trends Pharmacol. Sci. 2001; 3: 132-140Abstract Full Text Full Text PDF Scopus (234) Google Scholar, 4Civelli O. Nothacker H.-P. Saito Y. Wang Z. Lin S. Reinscheid R.K. Trends Neurosci. 2001; 4: 230-237Abstract Full Text Full Text PDF Scopus (123) Google Scholar, 5Wilson S. Bergsma D.J. Chambers J.K. Muir A.I. Fantom K.G.M. Ellis C. Murdock P.R. Herrity N.C. Stadel J.M. Br. J. Pharmacol. 1998; 125: 1387-1392Crossref PubMed Scopus (114) Google Scholar). Currently there are two main approaches generally used to identify orphan receptor ligands. One common approach has been to screen the orphan GPCRs against a collection of known or putative GPCR ligands. This approach has been used successfully to identify several ligand receptor pairings, including MCH, NMU, and P2Y13 etc. (6Charmers J. Ames R.S. Bergsma D. Muir A. Fitzgerald L.R. Hervieu G. Dytko G.M. Foley J.J. Martin J. Liu W.S. Park J. Ellis C. Ganguly S. Konchar S. Cluderay J. Leslie R. Wilson S. Sarau H.M. Nature. 1999; 400: 261-265Crossref PubMed Scopus (460) Google Scholar, 7Shan L. Qiao X. Crona J.H. Behan J. Wang S. Laz T. Bayne M. Gustafson E.L. Monsma Jr., F.J. Hedrick J.A. J. Biol. Chem. 2000; 275: 39482-39486Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 8Zhang F.L. Luo L. Gustafson E. Palmer K. Qiao X. Fan X. Yang S. Laz T.M. Bayne M. Monsma Jr., F. J. Pharmacol. Exp. Ther. 2002; 301: 705-713Crossref PubMed Scopus (153) Google Scholar). However, because ligand collections are somewhat limited, they often do not contain the appropriate ligand for the orphan receptor of interest. Another approach involves purification of the ligands from animal tissue extracts. This approach has also been successful in a number of cases including OFQ/Nociceptin, MCH, Orexin, and P2Y12 etc. (9Reinscheid R.K. Nothacker H.P. Bourson A. Ardati A. Henningsen R.A. Bunzow J.R. Grandy D.K. Langen H. Monsma Jr., F.J. Civelli O. Science. 1995; 270: 792-794Crossref PubMed Scopus (1752) Google Scholar, 10Saito Y. Nothacker H.P. Wang Z. Lin S.H. Leslie F. Civelli O. Nature. 1999; 400: 265-269Crossref PubMed Scopus (475) Google Scholar, 11Sakurai T. Amemiya A. Ishii M. Matsuzaki I. Chemelli R.M. Tanaka H. Williams S.C. Richardson J.A. Kozlowski G.P. Wilson S. Arch J.R. Buckingham R.E. Haynes A.C. Carr S.A. Annan R.S. McNulty D.E. Liu W.S. Terrett J.A. Elshourbagy N.A. Bergsma D.J. Yanagisawa M. Cell. 1998; 92: 573-585Abstract Full Text Full Text PDF PubMed Scopus (3018) Google Scholar, 12Zhang F.L. Luo L. Gustafson E. Lachowicz J. Smith M. Qiao X. Liu Y.-H. Chen G. Pramanik B. Laz T.Z. Palmer K. Bayne M. Monsma Jr., F.J. J. Biol. Chem. 2001; 276: 8608-8615Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 13Hollopeter G. Jantzen H.-M. Vincent D. Li G. England L. Ramakrishnan V. Yang R.-B. Nurden P. Julius D. Conley P.B. Nature. 2001; 409: 202-207Crossref PubMed Scopus (1267) Google Scholar). However, purification of ligands from animal tissue extracts is labor-intensive and is complicated by the potential for background activity, the low abundance of ligands, and stability of ligands in animal tissue extracts. In the present study we utilized an alternative approach to identifying ligands for orphan receptors. Because many orphan receptors are predicted to be peptide receptors, and peptide ligands must be encoded by DNA sequences, it should be possible using a variety of methods to predict peptide ligands from sequences stored in the human genome data bases. Peptides predicted in this manner can then be synthesized and tested for biological activity of GPCRs. In the present study, we have used a novel peptide homology algorithm (PepPat) to search the human genome for potential bio-active peptides (16Jiang Y. Gao G. Fang G. Gustafson E.L. Laverty M. Yin Y. Zhang Y. Luo J. Greene J.R. Bayne M.L. Hedrick J. Murgolo N.J. et al.Mamm. Genome. 2003; 14: 341-349Crossref PubMed Scopus (16) Google Scholar). Using this approach, we have successfully identified a novel ligand for an orphan receptor designated as SP9155. Reagents and Materials—All chemicals were obtained from Sigma except as otherwise noted. Fluo-3-AM and pluronic acid were from Molecular Probes. Cell culture media and reagents were from Invitrogen. All synthetic peptides were custom synthesized at Invitrogen. All cloning work was performed according to standard procedures in Ref. 24. Chimeric Gα proteins (Gαq/z, Gαq/s, Gαq/12, Gαq/i, Gαq/i3, Gαq/o, Gαq/16) were constructed by replacing the five C-terminal residues of human Gαq with the five amino acid residues of the corresponding G protein (14Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (599) Google Scholar), and were cloned into the mammalian expression vector pCDNA3. 1 (Invitrogen). Bioinformatic Identification of Novel RF-amide Peptides—RF-amide peptides share a common C-terminal RFG[KR] motif. First, a virtual transcripts protein data base (VTS) was generated from human genomic sequence using GenScan (20Burge C. Karlin S. J. Mol. Biol. 1997; 268: 78-94Crossref PubMed Scopus (2983) Google Scholar). Using a pattern-based oligopeptide homology search algorithm (PepPat) (16Jiang Y. Gao G. Fang G. Gustafson E.L. Laverty M. Yin Y. Zhang Y. Luo J. Greene J.R. Bayne M.L. Hedrick J. Murgolo N.J. et al.Mamm. Genome. 2003; 14: 341-349Crossref PubMed Scopus (16) Google Scholar), we searched the VTS protein data base for proteins or exons containing this RFG[KR] motif. Proteins or exons that contain RFG[KR] motifs were further analyzed for the presence of a leader peptide or transmembrane domains (16Jiang Y. Gao G. Fang G. Gustafson E.L. Laverty M. Yin Y. Zhang Y. Luo J. Greene J.R. Bayne M.L. Hedrick J. Murgolo N.J. et al.Mamm. Genome. 2003; 14: 341-349Crossref PubMed Scopus (16) Google Scholar). Those genes containing at least one RFG[KR] motif, with a predicted leader sequence but without predicted TM domain, were regarded as putative RF-amide precursors. These predicted peptides were then synthesized and put into our in-house peptide collection for screening. Cloning and Expression of Human and Mouse SP9155—A full-length cDNA of SP9155 (referred to as AQ27) was originally isolated from a human brain cDNA library and was disclosed in patent WO2001016316. Based on the AQ27 sequence two primers were designed and the full-length PCR product was obtained from human brain cDNA library. This clone was designated as SP9155. The open reading frame of SP9155 was then subcloned into the expression vector pCDNA3.1 (Invitrogen) and used for cell transfection experiments. To identify the mouse homologue of SP9155, the protein sequence of human SP9155 was used to search GenBank™ with BLAST, and two mouse EST sequences (BI729969 and BB084541) were identified that were highly homologous to the 5′ and 3′ ends of human SP9155. Primers were designed based on the two EST sequences to clone the full-length of mouse SP9155; the 5′-primer was: 5′-CACCATGCAGGCGCTCAACATCACCGCGGA-3′, and the 3′-primer was: 5′-GGCTTACAGTTCATGTCCACTGCCGAAAGT-3′. PCR reactions were performed using Marathon mouse brain cDNA (Clontech), and the resulting PCR product was then cloned into TOPO-pCDNA3.1 vector (Invitrogen) and sequence confirmed. Cloning of Human and Mouse Peptide P518 Precursor—Primers for the human were designed based on the human genomic sequence for the peptide coding VTS; the 5′-primer was: 5′-GCCGAATTCGCCGCCACCATGGTAAGGCCTTACCCCCTGATCTACTTC-3′, and the 3′-primer was: 5′-GCCTCACCGCCGACCGAAGCGGAAGCTGAAGCC-3′. The PCR reaction was performed using the human Quick-clone kidney cDNA (Clontech) with the Advantage HF 2 kit. The cycling conditions were 1 cycle at 94 °C for 1 min, 35 cycles at 95 °C for 15 s, 68 °C for 3 min. The PCR product was first cloned into PCR blunt vector (Invitrogen), then subcloned to pCDNA3.1(–) (Invitrogen) vector and the sequence was confirmed. Primers for mouse were designed based on the suggested mouse precursor sequence, the 5′-primer was: and the 3′-primer was: The PCR reaction was performed using human Quick-clone cDNA (Clontech) with the Advantage HF 2 kit. The cycling conditions were 1 cycle at 94 °C for 1 min, 35 cycles at 95 °C for 15 s, 68 °C for 3 min. The PCR product was cloned into TOPO-pCDNA3.1 vector and sequence confirmed. Cell and ligand of was into (Invitrogen) or in a by (Invitrogen). a the of pCDNA3.1 was into or cells. were performed as Human and Mouse tissue were from for tissue collection from 2 to was isolated from the using and tested for and using an from were used in the and brain and Mouse were from The were and analyzed in the manner as for the human and brain and primers and were designed with and from The were with as the and as a The sequences of the human and mouse primers and for SP9155 and P518 precursor are shown in with the corresponding to the open reading for the human PCR was with an sequence The PCR reactions were using the from the and according to the (Invitrogen). The of the primers and in the PCR reactions were and In of a was to and PCR of as The reactions for SP9155 and P518 precursor were performed in a plate according to the 1 cycle for at by one cycle at 95 by cycles at 95 °C for 15 and °C for 1 min. of the were used to as an for and a for the was used to the for genomic and sequences used in the PCR in a the mouse the from was and were generated by using and primers of tissue cDNA was used with primers at and the at a of cDNA was in and were used as an PCR conditions were as °C for 2 95 °C for cycles of 95 °C for 15 s, °C for 1 min. PCR data was based on standard generated using of DNA or a PCR the human at and were used to the standard The human SP9155 standard was generated using the P518 data was generated using of a PCR The mouse standard were generated using containing the mouse SP9155 and P518 precursor with the from 1 to 1 To the results obtained using PCR as data were and to of for the in the Cloning and full-length human cDNA of SP9155 was first identified from WO2001016316. a open reading frame amino acid residues and was to a orphan GPCR receptor D.K. T. R. O. T. 2001; 275: PubMed Scopus Google Scholar). However, of sequence in the and of sequence in the of the gene with SP9155. analysis of these sequences that they were and these sequences in revealed that was the as SP9155 D.K. T. R. O. T. 2001; 275: PubMed Scopus Google Scholar). of the of the peptide sequence revealed the presence of seven in with a of G receptors C.D. Fong T.M. Graziano M.P. Tota M.R. FASEB J. 1995; 9: 745-754Crossref PubMed Scopus (327) Google Scholar). Using the human SP9155 protein sequence to search EST sequences with protein sequence to the 5′ and 3′ ends of the human open reading frame were identified The full-length mouse SP9155 was cloned using the primers based on these 5′ and 3′ EST The sequence for human and mouse SP9155 is shown in Phylogenetic analysis shows that SP9155 shares homology with NPFF, Orexin, and cholecystokinin (CCK) receptors known receptors are and receptors Identification of a Novel RF-amide Peptide as a for are a known family of GPCR peptide ligands in which the is S. Y. S. Y. M. R. T. K. Y. T. T. Y. Y. M. C. T. H. O. Tanaka M. Y. M. Cell Biol. 2000; PubMed Scopus Google Scholar). novel RF-amide peptides as and have been identified as ligands for orphan GPCRs S. Y. S. Y. M. R. T. K. Y. T. T. Y. Y. M. C. T. H. O. Tanaka M. Y. M. Cell Biol. 2000; PubMed Scopus Google Scholar, T. Y. S. H. A. K. Y. S. Y. Y. Y. T. M. T. M. C. S. T. H. O. M. Nature. 2001; PubMed Scopus Google Scholar). To search for novel RF-amide peptides, we used a novel approach to search the human genome sequence data base. First, a VTS data base was generated from human genomic sequence using GenScan (20Burge C. Karlin S. J. Mol. Biol. 1997; 268: 78-94Crossref PubMed Scopus (2983) Google Scholar). Using a novel pattern-based oligopeptide homology search algorithm the protein VTS data base was searched for genes or exons containing RFG[KR] as a motif, is the and VTS sequences containing at least one RFG[KR] motif were further analyzed for the presence of a signal peptide and for transmembrane The VTS sequences containing a signal peptide and transmembrane were used to predict a variety of peptides for and in our in-house peptide ligand collection for against orphan GPCRs. These orphan GPCRs were into and by which intracellular a of the a peptide called P52 was found to activate the orphan GPCR SP9155-transfected The of P52 was also in SP9155-transfected not However, the of P52 in cell was and of the genomic sequence corresponding to P52 revealed the presence of an open reading frame of a predicted protein as P518 precursor The mouse of the precursor was identified by search of the GenBank™ data base. human and mouse precursor genes were cloned from cDNA by using the primers designed based on the predicted precursor of the predicted protein by the and that the first amino acid residues as a signal peptide, and that potential transmembrane domains were that the predicted human protein be peptides from peptide P52 mouse of in a of peptides for in a sequence of human peptide P518 precursor and mouse peptide P518 precursor. to the to amino the RF-amide image Because the of P52 was a of peptides were synthesized that residues to other potential peptide These of P52 including and P518 were then tested for activity at SP9155 and shown in and the for and were with for SP9155 However, peptide P518 was found to activate SP9155 with an of 7 activity was in the The high of P518 that this be an endogenous ligand for SP9155. The mouse of the P518 peptide as has activity for SP9155 as the P518 peptide, and is extended peptides from the other motif of P518 and were also tested for activity for the SP9155 The of and for SP9155 are with 1 However, the activity of is with of and with an of The of SP9155 by P518 in the of G proteins, that SP9155 to To further the of G-protein on SP9155, including Gαq/12, Gαq/i, Gαq/i3, Gαq/s, and were with SP9155 into and the to P518 was then by F.L. Luo L. Gustafson E. Lachowicz J. Smith M. Qiao X. Liu Y.-H. Chen G. Pramanik B. Laz T.Z. Palmer K. Bayne M. Monsma Jr., F.J. J. Biol. Chem. 2001; 276: 8608-8615Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (599) Google Scholar). was found that with or without G proteins the of P518 for SP9155 is not the of P518 to intracellular via SP9155 was not by not we that SP9155 is coupled to the Gαq Expression of P518 mRNA and SP9155 in Human and Mouse standard generated from the and PCR that the and in the with not In the and that the were of and without genomic In the human tissue the SP9155 receptor and the P518 precursor exhibited the expression in In brain regions, there was expression of receptor and ligand in the receptor was or expressed at low In brain, the receptor was in retina, and the peptide mRNA was in retina, and In expression of the SP9155 receptor was found in heart, kidney, and The P518 precursor mRNA was found in testes, colon, coronary and In SP9155 mRNA was highly expressed in brain, in and In to human brain, the of the P518 precursor was expressed at low to the In mouse SP9155 and P518 precursor were found in low as in human the P518 precursor mRNA was expressed at the In the present study, we have used a approach to identify novel RF-amide peptide precursor genes from human and mouse genomic data bases. One of these genes has been shown to several potential peptides, one of which an orphan we have that the gene for this peptide precursor protein is expressed as mRNA in various human and mouse The peptides P518, and have been shown to be for the orphan G-protein-coupled receptor SP9155, and in peptide P518 has been shown to activate SP9155 with an of 7 Peptides and P518 have a common C-terminal sequence, but they in the of The for peptides and are and are that the in and not have on the of However, the for P518 is 7 which is and that the of P518 also with SP9155 RF-amide peptides have been identified in in RF-amide peptides have been found M.L. Li C. Science. 1998; PubMed Scopus Google Scholar, C. K. 1999; PubMed Scopus Google Scholar). In these peptides functions including of and M.L. Li C. Science. 1998; PubMed Scopus Google Scholar, C. K. 1999; PubMed Scopus Google Scholar). However, in a RF-amide peptides have been so These neuropeptide neuropeptide RFRP of which have been shown to with one or GPCRs S. Y. S. Y. M. R. T. K. Y. T. T. Y. Y. M. C. T. H. O. Tanaka M. Y. M. Cell Biol. 2000; PubMed Scopus Google Scholar, N.A. Ames R.S. Fitzgerald L.R. Foley J.J. Chambers J.K. N.A. Dytko G.M. Murdock P.R. G. P. Wang Wilson S. Bergsma D.J. Sarau H.M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). the two receptors are the receptors homologous to SP9155. ligands have been identified for receptors. In to and peptides, two RFRP peptides have also been shown to as ligands for receptors S. Y. S. Y. M. R. T. K. Y. T. T. Y. Y. M. C. T. H. O. Tanaka M. Y. M. Cell Biol. 2000; PubMed Scopus Google Scholar, N.A. Ames R.S. Fitzgerald L.R. Foley J.J. Chambers J.K. N.A. Dytko G.M. Murdock P.R. G. P. Wang Wilson S. Bergsma D.J. Sarau H.M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The RFRP peptides are peptides predicted from a precursor identified through a approach, in a manner used to predict the RF-amide peptide recent of GPCR sequences suggested that are ligands for SP9155 P. A. Biol. 2002; 3: Google Scholar). We found that NPAF, NPFF, and other known peptide ligand GPCR were not for SP9155 not P518 activity for other peptide ligand including and was shown to activate SP9155, but is and has not been shown to be an endogenous peptide In the present study, the high of P518 for SP9155 that P518 be the endogenous ligand for SP9155. are several of to this First, the precursor for P518 is from genomic sequence, and have been obtained from cDNA P518 precursor was shown to be expressed in human physiological because SP9155 shares high homology to receptors, and the ligands for receptors are RF-amide peptides, analysis that the ligand for SP9155 is likely to be an RF-amide peptide. P518 peptide is a ligand for SP9155 at low and the is in the as for an endogenous P518 is likely the endogenous ligand for SP9155, the study not the that there endogenous peptides to or from the of of P518 as the or one of the endogenous ligands using tissue for ligand a ligand for SP9155 has been the biological functions for the ligand and SP9155 are not animal as with P518 or SP9155 be in the functions of the peptide and identification of the ligand a to study the functions of SP9155 and and to be identified by for SP9155.