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Originally published In Press as doi:10.1074/jbc.M304056200 on August 22, 2003

J. Biol. Chem., Vol. 278, Issue 43, 42115-42120, October 24, 2003
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Functional Annotation of the Putative Orphan Caenorhabditis elegans G-protein-coupled Receptor C10C6.2 as a FLP15 Peptide Receptor*

Teresa M. Kubiak{ddagger}, Martha J. Larsen§, Marjorie R. Zantello§, Jerry W. Bowman§, Susan C. Nulf§, and David E. Lowery§

From the Animal Health Discovery Research, Pharmacia Corp., Kalamazoo, Michigan 49001

Received for publication, April 17, 2003 , and in revised form, August 21, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
This report describes the cloning and functional annotation of a Caenorhabditis elegans orphan G-protein-coupled receptor (GPCR) (C10C6.2) as a receptor for the FMRFamide-related peptides (FaRPs) encoded on the flp15 precursor gene, leading to the receptor designation FLP15-R. A cDNA encoding C10C6.2 was obtained using PCR techniques, confirmed identical to the Worm-pep-predicted sequence, and cloned into a vector appropriate for eucaryotic expression. A [35S]guanosine 5'-O-(thiotriphosphate) (GTP{gamma}S) assay with membranes prepared from Chinese hamster ovary (CHO) cells transiently transfected with FLP15-R was used as a read-out for receptor activation. FLP15-R was activated by putative FLP15 peptides, GGPQGPLRF-NH2 (FLP15-1), RGPSGPLRF-NH2 (FLP15-2A), its des-Arg1 counterpart, GPSGPLRF-NH2 (FLP15-2B), and to a lesser extent, by a tobacco hornworm Manduca sexta FaRP, GNSFLRFNH2 (F7G) (potency ranking FLP15-2A > FLP15-1 > FLP15-2B >> F7G). FLP15-R activation was abolished in the transfected cells pretreated with pertussis toxin, suggesting a preferential receptor coupling to Gi/Go proteins. The functional expression of FLP15-R in mammalian cells was temperature-dependent. Either no stimulation or significantly lower ligand-evoked [35S]GTP{gamma}S binding was observed in membranes prepared from transfected FLP15-R/CHO cells cultured at 37 °C. However, a 37 to 28 °C temperature shift implemented 24 h post-transfection consistently resulted in an improved activation signal and was essential for detectable functional expression of FLP15-R in CHO cells. To our knowledge, the FLP15 receptor is only the second deorphanized C. elegans neuropeptide GPCR reported to date.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
FMRFamide-related peptides (FaRPs)1 are important neurotransmitters and/or neuromodulators in the neuromuscular systems of invertebrates (1–6). More than 50 FaRPs have been isolated from parasitic helminths, insects, and other invertebrates (4–9). At present, a total of 59 distinct FaRPs, encoded on 22 flps (precursor genes), have been predicted from the Caenorhabditis elegans genome (10, 11). This number might be greater, since despite extensive bioinformatic searches, all family members have probably not been identified (10–12). In addition to FaRPs, 23 C. elegans neuropeptide-like protein (nlp) genes have been identified defining at least 11 families of 134 unique and 151 total putative neuropeptides (13). This brings the total number of isolated and putative C. elegans flp-and nlp-encoded neuropeptides to over 200.

There have been over 50 genes encoding putative neuropeptide receptors identified in the C. elegans genome (12). The much higher number of putative flp- and nlp-encoded neuropeptides than the number of the predicted neuropeptides GPCRs implies that a single receptor might be activated by multiple peptides. In C. elegans (10, 11, 13) and other invertebrates (14–17), multiple forms of related peptides are often encoded on a single precursor protein gene, and these peptide families can indeed recognize the same GPCR as it was recently shown by us and others for the Drosophila allatostatin type A receptors (18, 19). Despite the publication of the C. elegans genome more than 4 years ago (20), there has been only one orphan C. elegans neuropeptide GPCR matched with its cognate ligand to date (21, 22). This is in contrast to numerous orphan vertebrate GPCRs, which have been successfully paired to their natural ligands using approaches employing heterologous receptor expression and reverse pharmacology (23).

In this report, we describe the cloning and matching of the putative C. elegans orphan GPCR (Wormpep designation C10C6.2) with its cognate ligands. We propose to name this GPCR the FLP15 receptor (FLP15-R), because the receptor-activating peptides are encoded on the C. elegans precursor gene flp15 (10).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The chicken brain peptide was purchased from the American Peptide Co. (Sunnyvale, CA). NPAF (human and bovine) and bovine NPFF were from Sigma. Other mammalian RFamides (listed in Table I) were made in house by the Pharmacia Research Operation Unit using routine peptide synthetic methods. The FLP15 peptides and the other invertebrate FaRPs used for screening were custom-made at Auspep Pty. Ltd. (Parkville, Australia). Chinese hamster ovary cells CHO-10001A (referred to as CHO cells in the text), cell culture media, and transfection and assay reagents were as described previously (18, 24). The stable cell line for the rat dopamine type 2 receptor, rD2-CHO-10001A-L6, used for control GTP{gamma}S experiments, was a gift from Dr. Rita Huff (CNS Genomics, Pharmacia Corp.). Plasmids for chimeric G{alpha}q proteins, the G{alpha}q5o,G{alpha}q5i1–2,G{alpha}q5s, and G{alpha}q5z chimeras, in pLEC1 vector for expression in mammalian cells were purchased from Molecular Devices (Sunnyvale, CA). Cell culture media and supplements, fetal bovine serum, antibiotics, and transfection reagents were purchased from Invitrogen.


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  		TABLE I
Functional activation of FLP15-R by FLP15 and selected related peptides assessed in the [35S]GTP{gamma}S assay

[35S]GTP{gamma}S assay was carried out in the presence of 10 µM GDP with plasma membranes prepared from CHO cells transiently transfected with the flp15-R/pCR3.1 plasmid and incubated at 28°C for 24 h prior to cell harvesting. See Experimental Procedures for more details. NA, not active when tested up to 5—10 µM.

 

Cloning and Plasmid Preparation—Molecular biological techniques followed either manufacturer's recommendations or general protocols (25). PCR primers were designed using the cDNA sequence C10C6.2 predicted within Wormpep (release 13). The only significant modification was the addition of an optimized translational initiation sequence immediately preceding the authentic initiation codon ("GCC GCC") (26). Utilizing cDNA prepared from C. elegans strain N2 total RNA, a PCR product containing the open reading frame for C10C6.2 was obtained and cloned directly into the eucaryotic expression vector pCR3.1 (Invitrogen) to produce the C10C6.2(flp15-R)/pCR3.1 plasmid. The DNA sequence of the final open reading frame was confirmed to be identical to the Wormpep-predicted C10C6.2 gene.

Cell Cultures, Cell Transfection, and Membrane Preparation—CHO-10001A cells (referred to as CHO cells) were cultured and transfected essentially as described previously (18, 24) with some modifications. The cells were cultured on 10-cm cell culture plates at 37 °C in a humidified atmosphere of 5% CO2 in low glucose Dulbecco's modified Eagle's medium, containing 2 mM glutamine, sodium pyruvate (110 mg/liter), 10% heat-inactivated fetal bovine serum, gentamicin (10 µg/ml), and additionally supplemented with 10 mM HEPES to improve media buffering capacity in temperature-lowering experiments. The flp15-R1/pCR3.1 plasmid (2.5 µg DNA/10-cm plate) was used for transfections via the LipofectAMINE PLUSTM (Invitrogen) method. The transfected cells were incubated at 37 °C for 24 h post-transfection and then were moved to a humidified 28 °C/3% CO2 incubator for an additional 16–24 h before harvesting for membrane preparation. To test pertussis toxin (PTX) effects, the transfected cells were treated with PTX (100 ng/ml) 16–24 h prior to harvesting. Membranes were prepared as described previously (18, 24).

[35S]GTP{gamma}S Binding Assays—The routine GTP{gamma}S assays in a 96-well filter plate format were carried out in the presence of 10 µM GDP as described previously (18, 24). Additionally a set of experiments, depicted in Fig. 4, A and B, was performed with 1, 5, or 10 µM GDP for direct comparisons. In cell-cooling experiments, the cells transfected at 37 °C were moved 24 h post-transfection to a humidified 28 °C/3% CO2 incubator and incubated at 28 °C for an additional 16–24 h before cell harvesting for membrane preparations.



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  		FIG. 4.
GDP- and PTX-dependent stimulation of [35S]GTP{gamma}S binding for FLP15-2 (A) and FLP15-1 (B). The EC50 values (nM) and EC50 95% confidence limits (nM) were as follows: FLP15-2A: 108.0 (82.0–142.3), 177.1 (140.1–229.9), 214.7 (180.0–256.1) with 1, 5, or 10 µM GDP, respectively; FLP15-1: 139.3 (102.0–190.2), 312.4 (221.1–441.3), 337.2 (254.4–446.9) with 1, 5, or 10 µM GDP, respectively. Peptide-driven stimulation of [35S]GTP{gamma}S binding was almost completely abolished by PTX pretreatment indicating predominant involvement of Gi/Go signaling pathways in FLP15-R activation.

 

Intracellular Ca2+ Mobilization—The assay was run using a 96-well fluorescence imaging plate reader (FLIPR) (Molecular Devices) essentially as described previously (18, 24), except that the CHO cells were transfected using either the flp15-R/pCR3.1 plasmid alone (2.5 µg/10-cm plate) or in a combination with plasmids for individual G-protein chimeras (2.5 µg of receptor DNA and 2.5 µg of DNA of G{alpha}q5o, G{alpha}q5i1–2, G{alpha}q5s, or G{alpha}q5z). In the 28 °C shift experiments, the transfected cells were split and plated in 96-well FLIPR plates 24 h post-transfection, followed by an additional 24-h incubation at 37 °C and then an additional 16–24-h incubation at 28 °C prior to peptide testing.

Data Analysis—Dose-response curves for ligand-induced stimulation of [35S]GTP{gamma}S binding were analyzed by nonlinear regression using Prism (GraphPad Software, Inc., San Diego, CA) based on specific binding (total minus nonspecific binding) expressed as mean counts/min values ± S.E. for each treatment run in triplicate. The calculated EC50 values were considered statistically different (p = 0.05) when their 95% confidence limits did not overlap.

Bioinformatic Analyses—BLAST searches (27) were performed at the NCBI BLAST site (www.ncbi.nlm.nih.gov/BLAST/) on the nonredundant protein data base. For the purposes of this paper, a representative set of vertebrate/invertebrate GPCRs was chosen from a more comprehensive analysis containing ~300 representative peptidergic/aminergic GPCRs. For all of the C. elegans sequences noted in the subgroup shown in Fig. 1, actual cloned full-length sequences were used in the alignment. Sequences were aligned using ClustalX (28), followed by manual editing. The neighbor-joining phylogenetic tree was then assembled using ClustalX, and the correction for multiple substitutions provided by the software. The graphic shown in Fig. 1 was generated using Treeview (29).



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  		FIG. 1.
Neighbor-joining phylogenetic tree of a representative group of C. elegans and related peptide GPCRs. C. elegans receptors use the Wormpep designation. The other accession numbers are as follows: CG1147, AAK50050 [GenBank] ; GPRnpy2, XP_313000 [GenBank] ; NPYR4, NP_005963 [GenBank] ; NPYR5, Q15761 [GenBank] ; NPY1R, NP_000900 [GenBank] ; NPY6R, BAA13103 [GenBank] ; NPY3R, A45747 [GenBank] : NPFF-R, AAK94197 [GenBank] ; CCK-A receptor, L19315 [GenBank] ; NPY2R, NP000901; CG18729, NP_652717 [GenBank] ; GPRnpy3, XP_320180 [GenBank] ; NPR-1 (flatworm), AAK58856 [GenBank] . The matched and annotated receptors, C39E6.6 (NPR-1/AF9-R) (21, 22) and C10C6.2/FLP15-R, (this study) are boxed.

 


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Molecular Cloning of C10C6.2/flp15-R and Phylogenetic Analysis—The DNA sequence of the cloned C10C6.2 gene was identical to the predicted cDNA sequence within Wormpep. The corresponding amino acid sequence of the FLP15-R protein is typical of a seven-transmembrane receptor in the class A rhodopsin-type family. When the C10C6.2 amino acid sequence was used to query the NCBI nonredundant protein data base using BLAST (27), the highest BLAST scores were to a group of ~25 invertebrate "NPY-like" GPCRs. Following those, the highest scoring vertebrate sequences were neuropeptide Y receptors. The phylogenetic analysis depicted in Fig. 1 shows the most highly related C. elegans GPCRs. Most of these GPCRs have not yet been linked with their cognate ligands, with the protein encoded by C39E6.6 as the sole exception. This gene has been reported to encode a neuropeptide Y-like receptor (NPR-1) implicated in feeding behaviors (30). Our group recently matched NPR-1 with AF9, GLGPRGPLRF-NH2, the peptide encoded on the C. elegans precursor gene flp21 (21, 22).

Temperature-dependent Receptor Expression and Identification of Cognate Ligands for FLP15-R—The receptor-ligand matching was achieved by functionally expressing FLP15-R in mammalian cells using special cell culturing conditions. The cloned C10C6.2 (flp15-R gene) was transiently transfected into CHO cells, and a GTP{gamma}S assay was utilized as a read-out for detection of receptor function based on agonist-driven stimulation of [35S]GTP{gamma}S binding to membranes prepared from the transfected cells. The routine [35S]GTP{gamma}S assays were run in a 96-well filter plate mode in the presence of 10 µM GDP and 0.1 nM [35S]GTP{gamma}S (18). A collection of over 150 synthetic peptides representing a variety of C. elegans and other invertebrate FMRFamide-related peptides (based both on the isolated and predicted peptide sequences) was used as a source of potential activating ligands. The collection included nematode FaRPs (6, 9–11, 31), arthropod FaRPs (31), and vertebrate RF-amides (32–35).

In initial experiments, no stimulation of [35S]GTP{gamma}S binding could be detected in response to peptide treatments (5 µM) when membranes were prepared from the transfected cells cultured at 37 °C. However, when the cells were moved to 28 °C 24 h post transfection, and incubated at this lower temperature for an additional 16–24 h prior to cell harvesting for membrane preparation, distinct signals could be detected for several peptides as exemplified in Fig. 2 for GNSFLRF-NH2 (MasFLRF-amide II or F7G), a myotropic peptide, originally isolated from the tobacco hornworm M. sexta (36). The other actives, identified with the 28 °C membranes, were GGPQGPLRF-NH2, RGPSGPLRF-NH2, and GPSGPLRF-NH2. These peptides represent C. elegans FaRPs, which we designated FLP15-1, FLP15-2A, and FLP15-2B peptides, respectively, because their sequences are encoded on the C. elegans flp-15 precursor gene (10). In subsequent experiments, the active peptide responses were found to be dose-dependent. As shown in Fig. 3A, in one of the representative transient transfection experiments with the 28 °C cooling step implemented, FLP15-2A was the most potent peptide in the series (EC50 162.4 nM), followed by FLP15-1 (EC50 250.6 nM), FLP15-2B (EC50 598.6 nM), and F7G (EC50 4.8 µM) (Table I). Without the temperature-lowering step, significantly lower [35S]GTP{gamma}S responses were recorded in cells from the same transfection but incubated only at 37 °C (Fig. 3B). At 37 °C, dose-response curves were poorly defined (r2 0.569–0.794), and the maximal responses (Emax) were only 20–25% of those observed at 28 °C Fig. 3B). This made the ligand-stimulated [35S]GTP{gamma}S binding barely above the basal levels even at the highest peptide concentrations used (10 µM) and, therefore, difficult to positively identify the actives (Fig. 3B). It should be noted that the observed activity for the FLP15 peptides was receptor-specific since it was detected only in the FLP15-R/CHO membranes, while there was no response to the same peptides when membranes from the untransfected CHO cells or CHO cells transfected with an unrelated C. elegans (22) or Drosophila neuropeptide GPCRs (18, 24) were used as controls.



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  		FIG. 2.
Temperature-dependent functional expression of FLP15-R in CHO cells. Stimulation of [35S]GTP{gamma}S binding to the FLP15-R/CHO membranes by the 5 µM M. sexta FaRP, F7G.

 


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  		FIG. 3.
Temperature-dependent functional activation of the FLP15-R receptor expressed in CHO cells by the FLP15 peptides and related FaRPs as measured by peptide-stimulated [35S]GTP{gamma}S binding. Assays were run in the presence of 10 µM GDP with membranes prepared either from the FLP15-R-transfected cells shifted to 28 °C (A) or cultured only at 37 °C (B). The calculated EC50 values for the experiment shown in A are listed in Table I.

 

Since the cooling step consistently resulted in significant increases of the ligand-stimulated [35S]GTP{gamma}S binding, all subsequent experiments were carried out with the 37 to 28 °C shift. Except for the F7G and the three FLP15 peptides, no activity could be detected for any other invertebrate FaRPs from our collection of over 150 peptides, tested at 5 µM in the GTP{gamma}S assay. This also included GLGPRGPLRF-NH2 (AF9), a related FaRP with the same C terminus, PLRF-NH2, as in the FLP15 peptides. AF9 was originally isolated from a parasitic nematode, Ascaris suum (37), but is also encoded on the C. elegans flp21 precursor gene (11). There was also a lack of functional activation of FLP15-R by the chicken brain peptide, LPLRF-NH2 (32), and a set of recently identified vertebrate RFamides (32–35) possessing C-terminal sequences, P(L/Q)RFNH2, similar to that present in the FLP15 peptides (Table I). Collectively, the presented data point to a high selectivity and specificity of the FLP15-R receptor for the FLP15 peptides.

GDP Concentration-dependent Activity of the FLP-15 Peptides in the GTP{gamma}S Assay—We were concerned that the EC50 values determined for the FLP-15 peptides under routine assay conditions in the presence of 10 µM GDP were in a relatively high nanomolar range, as shown in Table I. Similar values and the same potency order were obtained in a repeated experiment, run also in the presence of 10 µM GDP, with membranes from an independent transfection: EC50 values of 152 nM, 422 nM, and 7.8 µM for FLP15-1, FLP15-2B, and F7G, respectively (data not shown). Routinely higher GDP concentrations are used to afford better signal/noise ratios even though GDP inhibits GTP{gamma}S binding, and higher agonist concentrations are needed to stimulate [35S]GTP{gamma}S binding. To evaluate the effects of GDP, the FLP15 peptides were tested in a GTP{gamma}S assay run with membranes originated from the same transfected cell pool but incubated with 1, 5, or 10 µM GDP. As shown in Fig. 4, A (for FLP15-2A) and B (for FLP15-1), the EC50 values obtained for 1 µM GDP were 2–2.4-fold lower as compared with those obtained in the presence of the 10 µM GDP, but still remained in the relatively high nanomolar range (108–139 and 215–337 nM with 1 and 10 µM GDP, respectively). This might be the inherent characteristic of FLP15-R expressed in CHO cells, comparable with the rat dopamine D2 receptor expressed in the same host cells for which EC50 values in the 300–500 nM range were obtained in response to dopamine using the same GTP{gamma}S assay conditions in the presence of 10 µM GDP (data not shown). The observed differences in the absolute EC50 values (but not in the potency order) in repeated experiments run with the FLP15-R/CHO membranes could be attributed to variations due to the transient nature of transfections and the time of incubation at 28 °C, which affect both receptor expression levels and protein folding efficiency during the cooling step. Attempts to create stable cell lines to overcome the variability problem originated from transient transfections were unsuccessful because the receptor-transfected CHO, COS-7, or HEK293 started dying 48–72 h post-transfection.

FLP15-R Signals via PTX-sensitive Go/Gi Proteins—FLP15-1 and FLP15-2A were tested in the GTP{gamma}S assay with the membranes prepared from the same pool of transfected cells, which were incubated either without PTX or treated with PTX (100 ng/ml) for 24 h during the 28 °C cooling step. Without the PTX treatment, well defined full responses to treatments with FLP15-2A or FLP15-1 were obtained, while PTX almost completely abolished receptor activation with only some residual signal remaining (Fig. 4A). These results indicate that the FLP15-R receptor expressed in CHO cells predominantly activates PTX-sensitive, Gi/Go-coupled signaling pathways.

It is not uncommon that the same GPCR can couple to more than one signaling pathway. Therefore, we evaluated the FLP15-R receptor for possible ligand-induced intracellular Ca2+ mobilization using FLIPR. No increases in Ca2+ signal were seen in response to any of the over 150 peptides from the collection, including the FLP15-peptides, in CHO cells transiently transfected with the FLP215-receptor cDNA and cultured either at 28 or 37 °C. This result argues against Gq/G11 involvement in the FLP15-R signaling cascade via activation by the FLP15 peptides or the possibility that peptides other than the FLP15 peptides could also be recognized by this receptor.

In an attempt to better define G-proteins that could potentially recognize FLP15-R in response to FLP15-2A, we performed redirected Ca2+ signaling experiments (38, 39). As shown in Fig. 5, no Ca2+ signal in response to FLP15-2A (up to 10 µM) was detected in CHO cells either transfected only with FLP15-R or co-transfected with FLP15-R and the chimeric G{alpha}q5s or G{alpha}q5o proteins. A dose-dependent signal was obtained in the FLP15-R/G{alpha}q5i1–2-transfected cells, but the EC50 was rather high (2.4 µM), and also only a very weak redirection was observed at the highest (10 µM) peptide concentration in the cells co-transfected with FLP15-R and G{alpha}q5z. The low efficiency of the redirection via G{alpha}q5i1–2 most likely reflects a poor compatibility for activation of the worm FLP15-R receptor with the CHO cells G-proteins and, additionally, a compromised recognition of FLP15-R by only the five C-terminal residues of the mammalian G{alpha}i1–2 present in the G-protein chimera. The weak redirection with the G{alpha}q5z explains some residual GTP{gamma}S activity recorded in the PTX-treated FLP15-R/CHO cells (Fig. 4, A and B), which was most likely due to the weak coupling of FLP15-R to the PTX-insensitive G{alpha}z protein. Overall, these results confirm the preferential coupling of FLP15-R expressed in CHO cells to the inhibitory Gi protein.



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  		FIG. 5.
Ca2+ redirection assay via FLIPR in response to FLP15-2A in cells transfected either with the FLP15-R receptor alone or co-transfected with FLP15-R and individual chimeric G-proteins G{alpha}q5o, G{alpha}q5i1–2, G{alpha}q5s, or G{alpha}q5z. All experiments were performed with the transfected cells shifted to 28 °C prior to treatments (see "Experimental Procedures" for more details).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This report describes the successful cloning and identification of ligands activating the putative C. elegans peptide GPCR designated C10C6.2. The cloned cDNA was identical with the Wormpep-predicted sequence, representing, at the protein level, a typical seven-transmembrane receptor. Using reverse pharmacology and a GTP{gamma}S assay as a read-out, we matched C10C6.2 with the peptides encoded on the flp15 (ZK525.1) precursor gene and, therefore, propose the designation FLP15-R for this GPCR.

Two of the C. elegans flp15 FaRPs predicted by Li and coworkers (10) are GGPQGPLRF-NH2 and RGPSGPLRF-NH2, which we designate FLP15-1 and FLP15-2A, respectively. FLP15-1 and FLP15-2A show a high degree homology, possessing a similar octapeptide core, GPXGPLRF-NH2 (where X = Q or S), and differing in their N-terminal amino acids (G versus R). In our study, we also included GPSGPLRF-NH2, an analog of FLP15-2A lacking the N-terminal arginine, which we designated FLP15-2B. None of the FLP15 peptides have yet been isolated even though sequences for flp15 have been found within EST databases, indicating that the flp15 gene is transcribed in C. elegans (10). Therefore, the presence or absence of the N-terminal arginine in the FLP15-2 peptide series is questionable because the exact processing sites in the putative precursor protein encoding the mature peptide are not known. The core GPSGPLRF sequence of FLP15-2 in the precursor protein is preceded by three basic amino acid residues, KRR (10). Each of these three residues, or all of them, might be targets for prohormone-processing enzymes. Thus, a true sequence of this FaRP will be known only when all the FLP15 peptides are isolated and sequenced.

In functional receptor activation assays, measured by peptide-evoked stimulation of [35S]GTP{gamma}S binding to the FLP15-R receptor expressing CHO membranes, FLP15-2A was the most potent ligand, followed by ~1.5-fold less potent FLP15-1 and the least potent FLP15-2B peptide (potency order FLP15-2A > FLP15-1>> FLP15-2B). The fact that FLP15-2A was ~4-fold more potent than its des-Arg1 counterpart (FLP15-2B) indicates that FLP15-2A rather than FLP15-2B might represent the processed, mature ligand for FLP15-R.

Although the determined EC50 values for the FLP15 peptides in the GTP{gamma}S assay have been in the high nanomolar range, several lines of evidence support the hypothesis that the FLP15 peptides are indeed the authentic cognate ligands for FLP15-R. First, it is well recognized that in GTP{gamma}S assays, higher GDP concentrations are used to afford better signal/noise ratios even though GDP inhibits GTP{gamma}S binding. Therefore, higher agonist concentrations are needed to stimulate [35S]GTP{gamma}S binding in the presence of high GDP concentrations (40). As reported by others for previously matched and well characterized GPCRs, the potency in the GTP{gamma}S assay could be one to 3 orders of magnitude lower than the affinity of the same ligand-receptor systems determined in a classical binding assay (41). This is also supported by our own data on the recently de-orphaned C. elegans AF9 receptor, which bound AF9 with nanomolar affinity but was functionally activated by the same peptide with the EC50 values in the high nanomolar range in the GTP{gamma}S assay (21, 22). Moreover, while using the same assay conditions as those for the FLP15-R receptor system, EC50 values in the 300–500 nM nanomolar range were obtained for dopamine-stimulated [35S]GTP{gamma}S binding to the rat dopamine rD2 receptor expressed in CHO cells used as a control. Therefore, these findings, similar for the nematode (FLP15-R) and mammalian (rD2) receptors, could reflect the intrinsic properties of some GPCRs. Second, the FLP15-R receptor appears to be highly specific for its activating ligands, since the FLP15 peptides showed no activity in the untransfected or mock-transfected CHO cells (18, 24). The receptor selectivity was also high and almost exclusive for the FLP15 peptides, since FLP15-R was not activated by more than 150 other invertebrate FaRPs and related vertebrate RFamides. The inactive ligands included even such homologous ligands as GLGPRPLRF-NH2 (AF9), the C.elegans/A. suum peptide (10, 11), having the same C-terminal sequence PLRF-NH2 as in the FLP15 peptides. Also inactive were vertebrate RFamides (32–35) with the similar C termini, P(L/Q)RF-NH2, such as the chicken brain peptide (LPLRFamide) (32), NPFF (FLFQPQRFNH2) and NPAF (AGEGLSSPFWSLAAPQRF-NH2) (33), quail gonadotropin inhibitory hormone, GnIH (SIKPSAYLPLRF-amide) (34), and human RFamide-related peptides, hRFRPs (35) (see Table I for individual sequences). GNSFLRF-NH2 (Mas-FLRFamide II or F7G), a M. sexta (tobacco hornworm) FaRP (36), was the only peptide other than the FLP15 peptides that activated FLP15-R, although with the 25–50-fold lower potency as compared with the FLP15 peptides, and thus not likely biologically significant. Interestingly, F7G might have been recognized by FLP15-R via its active conformation rather than due to the strict sequence homology because F7G and the FLP15 peptides share only the common C-terminal LRFamide motif, while their N termini are quite different.

FLP15-R can be classified as an inhibitory receptor because it preferentially couples to Gi proteins. This was concluded based on the PTX-sensitive activation of FLP15-R (Fig. 4A) and by its redirection to calcium signaling via the G{alpha}q5i1–2 chimeric protein (Fig. 5). It remains to be established whether the same pattern of receptor coupling to G-protein(s) will be seen in C. elegans in vivo.

It is worth noting that all the experiments described in this study were performed with CHO cells transiently transfected with the FLP15-R encoding DNA. Multiple attempts to generate stable cell lines for this receptor in CHO, COS-7, and HEK293 cells failed because the cells started dying shortly after transfection.

It is also important to stress that a temperature shift from 37 to 28 °C, implemented 24 h after transfection, was absolutely critical for functional expressions of FLP15-R in mammalian cells. Without this temperature-lowering step, no significant peptide-invoked receptor activation could be observed. A similar pattern was found for the recently matched C. elegans AF9 receptor, AF9-R1 (21, 22), also known as NPR-1 (30). Since optimal temperatures for the free living nematode C. elegans are 15–19 °C, it might be that FLP15-R and other worm peptide GPCRs require lower temperature and/or different accessory proteins than those present in mammalian cells for proper folding and functional receptor expression. This poor worm receptor-mammalian host compatibility could be responsible for a lack of progress in finding cognate ligands for over 50 other C. elegans peptide GPCRs still waiting to be de-orphaned.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Veterinary Medicine R & D, Pfizer Inc., 7000 Portage Rd., Kalamazoo, MI 49001. Back

{ddagger} To whom correspondence should be addressed: Veterinary Medicine R & D, Pfizer Inc., 7000 Portage Rd., Kalamazoo, MI 49001. Tel.: 269-833-7978; Fax: 269-833-3581; E-mail: teresa.m.kubiak{at}pfizer.com.

1 The abbreviations used were: FaRPs, FMRFamide-related peptides; flp, FMRFamide-like precursor protein gene; FLP15-R, FLP15 peptide receptor; FLIPR, fluorescence imaging plate reader; hRFRP, human RFamides-related peptide; GPCR, G-protein-coupled receptor; NPAF, neuropeptide AF; NPFF, neuropeptide FF; nlp, neuropeptide-like precursor protein gene; PTX, pertussis toxin; CHO, Chinese hamster ovary; GTP{gamma}S, guanosine 5'-O-(thiotriphosphate). Back


   ACKNOWLEDGMENTS
 
We thank Dr. Rita Huff (CNS Genomics, Pharmacia Corp.) for the gift of the CHO-10001A and the rD2-CHO-10001A-L6 stable cell lines as well as for valuable advice, Carol Bannow (Research Operations, Pharmacia Corp.) for the synthesis of the vertebrate RFamides, Ron Hiebsch and Heidi Fidler (CNS Genomics, Pharmacia) for advice with cell cultures and membrane preparations, and John Davis (Pharmacia Animal Health) for technical assistance.



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 ABSTRACT
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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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