From the Department of Discovery Technology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033
Received for publication, March 21, 2003
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ABSTRACT |
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INTRODUCTION |
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EXPERIMENTAL PROCEDURES |
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Bioinformatic Identification of Novel RF-amide PeptidesRF-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 (20). Using a pattern-based oligopeptide homology search algorithm (PepPat) (16), 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 (16). 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 SP9155A 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 GenBankTM with BLAST, and two mouse EST sequences (BI729969 [GenBank] and BB084541 [GenBank] ) 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 PrecursorPrimers 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: 5'-GCCGAATTCGCCGCCACCATGAGGGGCTTCCGGCCTTTGCTTTCCCTA-3'; and the 3'-primer was: 5'-GCCAAGCTTTCACCGTCCAAAGCGGAAGCTGAAGCCTCC-3'. The PCR reaction was performed using human Quick-clone spleen 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 Transfection and AssayFor ligand screening assays, 5 µg of SP9155/pCDNA3.1 was transfected into HEK293SFM (Invitrogen) or CHO-DHFR cells in a 75-cm2 flask by LipofectAMINE (Invitrogen). As a negative control, the same amount of empty pCDNA3.1 plasmid was transfected into HEK293SFM or CHO cells. FLIPR assays were performed 48 h later as previously described.
Human and Mouse Tissue PanelsHuman tissue autopsy samples were purchased from Zoion Diagnostics (Shrewsbury, MA). Postmortem times for tissue collection ranged from 2 to 6 h. Total RNA was isolated from the tissues using TRI Reagent (MRC, Cincinnati, OH), and tested for quality and quantity using an Agilent 2100 Bioanalyzer (Waldbroun, Germany). Tissues from three donors were used in the analysis, and included 19 brain regions and 36 peripheral tissues. Mouse tissues were prepared from three C57/Bl6 mice. The RNAs were extracted and analyzed in the same manner as for the human tissues and included 12 brain regions and 23 peripheral tissues.
Quantitative PCRTaqman primers and probes were designed with Primer Express software (ABI), and purchased from ABI. The fluorogenic probes were labeled with 6-carboxyfluorescein as the reporter and 6-carboxy-4,7,2,7'-tetramethylrhodamine as a quencher. The sequences of the human and mouse primers and probes for SP9155 and P518 precursor are shown in Table I, with the numbers corresponding to the open reading frames for each. For the human tissues, quantitative PCR was carried out with an ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA). The PCR reactions were prepared using the components from the Invitrogen Platinum quantitative reverse transcriptase-PCR one-step kit and assembled according to the manufacturer's instructions (Invitrogen). The final concentrations of the primers and probe in the PCR reactions were 200 and 100 nM, respectively. In addition, 0.25 µl of a passive reference dye (ROX, Invitrogen) was added to each reaction, and each 12.5-µl PCR contained 2.5 µl (25 ng) of total RNA prepared as described above. The reverse transcriptase-PCR reactions for both SP9155 and P518 precursor were performed in a single 384-well plate according to the following protocol: 1 cycle for 30 min at 48 °C, followed by one 20-min cycle at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Separate plates of the same RNAs were used to quantitate 18 S RNA as an internal control for RNA quality, and a primer/probe set for the CD4 promoter was used to check the RNAs for genomic contamination.
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For the mouse tissues, the RNA from three mice/tissue was pooled and cDNAs were generated by reverse transcription using random hexamers (Promega, Madison, WI) and oligo(dT) primers (Invitrogene). For TaqMan analysis, 25 ng of tissue cDNA was used together with primers at 900 nM final concentration, and the 6-carboxyfluorescein-labeled probe at a final concentration of 250 nM. Each cDNA was run in duplicate, and ribosomal RNA primers/probe (PE Applied Biosystems, Foster City, CA) were used as an internal control. Quantitative PCR conditions were as follows: 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min.
Data AnalysisThe PCR data was quantitated based on standard curves generated using serial dilutions of either plasmid DNA or a gene-specific PCR product. For the human PCR, 4-fold dilutions began at 0.25 ng, and eight dilutions were used to generate the standard curve. The human SP9155 standard curve was generated using plasmid dilutions, whereas the P518 data was generated using dilutions of a 350-bp PCR product. The mouse standard curves were generated using plasmids containing the mouse SP9155 and P518 precursor genes, with the dilution ranging from 1 ng to 1 fg. To compare the results obtained using plasmids versus PCR products as standards, all data were averaged and converted to copy number/25 ng of cDNA, thus accounting for the differences in weight between the two.
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RESULTS |
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Identification of a Novel RF-amide Peptide as a Ligand for SP9155RF-amides are a well known family of GPCR peptide ligands in which the C terminus is arginine-phenylalanineamide (17). Recently, novel RF-amide peptides such as hRFP-1, hRFP-2, and KiSS have been identified as ligands for orphan GPCRs (17, 18). To search for novel RF-amide peptides, we used a novel computational approach to search the human genome sequence data base. First, a VTS data base was generated from human genomic sequence using GenScan (20). Using a novel proprietary pattern-based oligopeptide homology search algorithm (PepPat), the protein VTS data base was searched for genes or exons containing RFG[KR] as a motif, where G[KR] is the peptidase digestion and amidation signal. VTS sequences containing at least one RFG[KR] motif were further analyzed for the presence of a signal peptide and for transmembrane domains. The VTS sequences containing a signal peptide and no transmembrane domain were used to predict a variety of peptides for synthesis and inclusion in our in-house peptide ligand collection for screening against different orphan GPCRs. These orphan GPCRs were transiently transfected into HEK293SFM cells, and screened by FLIPR assay, which measures intracellular calcium mobilization. As a result of the screening, a peptide called P52 was found to specifically activate the orphan GPCR SP9155-transfected HEK293SFM cells (Fig. 3A). The same specificity of P52 was also observed in SP9155-transfected CHO cells (data not shown). However, the EC50 of P52 in both cell lines was only about 250 nM (Tables II and III). Examination of the genomic sequence corresponding to P52 revealed the presence of an uninterrupted open reading frame of 381 bp nucleotides, yielding a 126-residue predicted protein (designated as P518 precursor protein) (Fig. 2). The mouse homolog of the precursor was identified by BLAST search of the GenBankTM data base. Both human and mouse precursor genes were cloned from cDNA libraries by using the primers designed based on the predicted precursor genes. Analysis of the predicted protein by the PSORT and SignalP programs indicated that the first 22 amino acid residues could serve as a signal peptide, and that no potential transmembrane domains were present, suggesting that the predicted human protein (or cleavage products) could be secreted.
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Because the EC50 of P52 was relatively low, a series of peptides were synthesized that included residues extending upstream to other potential peptide processing sites. These longer versions of P52 including P513, P517, and P518 were then tested for activity at SP9155 (Tables II and III). As shown in Table III and Fig. 3, the activities for P52, P513, and P517 were similar, with EC50 values for SP9155 around 250 nM. However, peptide P518 was found to more potently activate SP9155 with an EC50 of 7 nM, whereas no activity was detected in the vector-transfected HEK293SFM cells (Fig. 3C). The high potency of P518 suggests that this could be an endogenous ligand for SP9155. The mouse homolog of the P518 peptide (designated as P550) has similar activity for SP9155 as the P518 peptide, and its EC50 is about 6 nM. N-terminal extended peptides from the other RFGR motif of P518 preproprotein (P51, P242, and P552) were also tested for agonist activity for the SP9155 (Fig. 3B). The activities of P51 and P242 for SP9155 are very weak, with EC50 values greater than 1 µM. However, the activity of P552 is comparable with those of P52, P513, and P517 with an EC50 of 600 nM.
The activation of SP9155 by P518 in the absence of added G proteins,
suggests that SP9155 probably couples to Gq. To further determine
the effect of G-protein coupling on SP9155, different single chimeric
G-proteins including Gq/12, G
q/16,
G
q/i, G
q/z, G
q/i3,
G
q/s, G
16, and G
q/o were
co-transfected with SP9155 into HEK293SFM cells, and the potency to
P518 was then measured by FLIPR
(12,
14). It was found that with or
without chimeric G proteins the potency of P518 for SP9155 is similar (data
not shown). Moreover, the ability of P518 to mobilize intracellular
Ca2+ via SP9155 was not affected by pertussis toxin
treatment (data not shown). Therefore, we conclude that SP9155 is coupled to
the G
q signaling pathway.
Expression Profile of P518 Precursor mRNA and SP9155 in Human and Mouse TissuesThe standard curves generated from the plasmids and PCR products indicated that all the Taqman primer and probe sets behaved in the expected manner, with approximately equivalent efficiencies (data not shown). In addition, the 18 S and CD4 control probe sets demonstrated that the RNAs were of good quality and without genomic contamination.
In the human tissue RNA panel (Fig. 4A), both the SP9155 receptor and the P518 precursor mRNAs exhibited the highest expression in brain. In most brain regions, there was coordinate expression of receptor and ligand mRNAs, however, in peripheral tissues the receptor was nonexistent or expressed at low levels. In brain, the receptor was most abundant in retina, trigeminal ganglion, hypothalamus, and vestibular nucleus, whereas the peptide mRNA was most abundant in cerebellum, medulla, retina, and vestibular nucleus. In peripheral tissues, significant expression of the SP9155 receptor was found only in heart, kidney, and testes RNAs. The P518 precursor mRNA was found in prostate, testes, colon, thyroid, parathyroid, coronary artery, and bladder.
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In murine tissues, SP9155 mRNA was highly expressed in brain, particularly in hypothalamus, cortex, and spinal cord (Fig. 4B). In contrast to human brain, the murine version of the P518 precursor was expressed at low levels relative to the receptor. In mouse peripheral tissues, both SP9155 and P518 precursor were found in low abundance, although as in human peripheral tissues the P518 precursor mRNA was expressed at higher levels than the receptor.
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DISCUSSION |
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Many RF-amide peptides have been previously identified in lower organisms: in Caenorhabditis elegans, more than 50 distinct RF-amide peptides have been found (21, 22). In such lower organisms these peptides exhibit diverse functions including cardioexitation, control of muscle contraction, and neuromodulation (21, 22). However, in mammals, only a few RF-amide peptides have been reported so far. These include neuropeptide FF (NPFF), neuropeptide AF (NPAF), RFRP (RF-amide-related peptide), all of which have been shown to interact with one or more specific GPCRs (17, 19). Interestingly, the two NPFF receptors are the receptors most homologous to SP9155.
Multiple ligands have been identified for NPFF receptors. In addition to NPFF and NPAF peptides, two RFRP peptides have also been shown to act as ligands for NPFF receptors (17, 19). The RFRP peptides are peptides predicted from a precursor identified through a bioinformatics approach, in a similar manner used to predict the RF-amide peptide P518. A recent survey of GPCR sequences suggested that NPAF and/or NPFF are agonist ligands for SP9155 (23). We found that NPAF, NPFF, and other known peptide ligand GPCR agonists were not functional agonists for SP9155 (data not shown). Additionally, P518 possessed no appreciable agonist activity for other peptide ligand GPCRs, including NPFF and mestastin.
Previously, Met-Enkephalin-Arg-Phe-amide was shown to activate SP9155, but its EC50 is greater than 40 µM, and Met-enkephalin-Arg-Phe-amide has not been shown to be an endogenous peptide (patent WO2001016316). In the present study, the high potency of P518 for SP9155 suggests that P518 may be the endogenous ligand for SP9155. There are several lines of evidence to support this possibility. First, the precursor for P518 is derived from genomic sequence, and its physical clones have been obtained from cDNA libraries. Moreover, P518 precursor was shown to be expressed in human tissues under normal physiological conditions. Second, because SP9155 shares high homology to NPFF receptors, and the ligands for NPFF receptors are RF-amide peptides, phylogenetic analysis suggests that the ligand for SP9155 is likely to be an RF-amide peptide. Third, P518 peptide is a potent ligand for SP9155 at low nanomolar and the potency is in the range as expected for an endogenous ligand. Although P518 is most likely the endogenous ligand for SP9155, the current study does not exclude the possibility that there exist additional endogenous peptides similar to or distinctly different from the structure of P518. Confirmation of P518 as the sole or one of the multiple endogenous ligands await studies using natural tissue sources for ligand isolation.
Although a potent ligand for SP9155 has been identified, the biological functions for both the ligand and SP9155 are still not clear. Further studies utilizing animal models such as knockout mice with deficient P518 or SP9155 will be useful in dissecting the functions of the peptide and receptor. Nevertheless, identification of the ligand will provide a research tool to study the functions of SP9155 and allow new agonists and antagonists to be identified by setting up screening assays for SP9155.
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FOOTNOTES |
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To whom all correspondence should be addressed: K-15-1/1945, Schering-Plough
Research Institute, Kenilworth, NJ 07033. Tel.: 908-740-4704; Fax:
908-740-7101; E-mail:
fang.zhang{at}spcorp.com.
1 The 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.
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REFERENCES |
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