Identification and Characterization of Two G Protein-coupled Receptors for Neuropeptide FF
2000; Elsevier BV; Volume: 275; Issue: 50 Linguagem: Inglês
10.1074/jbc.m004385200
ISSN1083-351X
AutoresJames A. Bonini, Kenneth A. Jones, Nika Adham, Carlos Forray, Roman Artymyshyn, Margaret M. Durkin, Kelli E. Smith, Joseph A. Tamm, Lakmal W. Boteju, Parul P. Lakhlani, Rita Raddatz, Wen-Jeng Yao, Kristine L. Ogozalek, Noel Boyle, Evguenia Kouranova, Yong Quan, Pierre J.‐J. Vaysse, John M. Wetzel, Theresa A. Branchek, Christophe Gerald, Beth Borowsky,
Tópico(s)Regulation of Appetite and Obesity
ResumoThe central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (Kd = 1.13 nm) and NPFF2 (Kd = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions. The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (Kd = 1.13 nm) and NPFF2 (Kd = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions. neuropeptide FF human NPFF receptor 1/2 rat NPFF receptor 1/2 central nervous system G protein-coupled receptor polymerase chain reaction reverse transcription-PCR [125I]d-Tyr-Leu-(N-methyl)Phe-Gln-Pro-Glu-Arg-Phe-NH2 rapidamplification of cDNA ends Stanford Human Genome Center base pair(s) pancreatic polypeptide phosphatidyl-inositol pertussis toxin (R)-N2- (diphenylacetyl-N-[4-hydroxyphenyl)methyl]-argininamide The octapeptide neuropeptide FF (NPFF1 or F-8-F-amide) and the related octadecapeptide neuropeptide AF (NPAF or A-18-F-amide) were originally isolated from bovine brain (1Yang H.Y. Fratta W. Majane E.A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7757-7761Crossref PubMed Scopus (567) Google Scholar) and later determined to be encoded by the same gene and cleaved from a common precursor protein (2Vilim E.S. Ziff E. Soc. Neurosci. 1995; 21: 760Google Scholar). There is a large body of evidence suggesting that NPFF is involved in nociception and in the modulation of opiate-induced analgesia, morphine tolerance, and morphine abstinence (3Panula P. Aarnisalo A.A. Wasowicz K. Prog. Neurobiol. 1996; 48: 461-487Crossref PubMed Scopus (203) Google Scholar, 4Vilim F.S. Aarnisalo A.A. Niemenen M.-L. Lintunen M. Karlstedt K. Kontinen V.K. Kalso E. States B. Panula P. Ziff E. Mol. Pharmacol. 1999; 55: 804-811PubMed Google Scholar, 5Gouarderes C. Mathieu Tafani J.-A. Zajac J.-M. Peptides. 1998; 19: 727-730Crossref PubMed Scopus (35) Google Scholar, 6Panula P. Kalso E. Niemenen M.-L. Kontinen V.K. Brandt A. Pertovaara A. Brain Res. 1999; 848: 191-196Crossref PubMed Scopus (151) Google Scholar, 7Payza K. Akar C.A. Yang H.-Y. J. 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Conversely, intrathecal administration of NPFF analogs induces a long-lasting, opioid-induced analgesia and potentiates morphine-ionduced analgesia (12Roumy M. Zajac J.-M. Eur. J. Pharmacol. 1998; 345: 1-11Crossref PubMed Scopus (212) Google Scholar). Other reports have also implicated NPFF in physiological processes such as insulin release, food intake, memory, blood pressure regulation, and electrolyte balance (3Panula P. Aarnisalo A.A. Wasowicz K. Prog. Neurobiol. 1996; 48: 461-487Crossref PubMed Scopus (203) Google Scholar). Binding of the NPFF analog [125I]YLFQPQRF-amide to rat spinal cord membranes has revealed a high affinity binding site for which opioid receptor ligands do not compete (13Allard M. Geoffre S. Legendre P. Vincent J.D. Simonnet G. Brain Res. 1989; 500: 169-176Crossref PubMed Scopus (154) Google Scholar), and the autoradiographic distribution of [125I]YLFQPQRF-amide binding sites indicates high density binding in various regions throughout the rat CNS (14Allard M. Zajac J.-M. Simonnet G. Neuroscience. 1992; 49: 101-116Crossref PubMed Scopus (113) Google Scholar). The exact mechanism underlying the anti- and pro-opioid effects of NPFF is currently unknown, but these seemingly opposing physiological effects could be accounted for by the existence of multiple receptor subtypes. Until now, the cloning of NPFF receptors has remained elusive. NPFF has been shown to activate adenylyl cyclase in mouse olfactory bulb membranes (15Gherardi N. Zajac J.-M. Peptides. 1997; 18: 577-583Crossref PubMed Scopus (28) Google Scholar), and NPFF binding to rat brain and spinal cord membranes is inhibited by guanine nucleotides (16Payza K. Yang H.-Y. J. Neurochem. 1993; 60: 1894-1899Crossref PubMed Scopus (32) Google Scholar), suggesting that NPFF elicits its actions through a G protein-coupled receptor (GPCR). A peptide related to NPFF, FMRF-amide, activates a cation channel (FaNaCh) in the mollusc Helix aspersa (17Lingueglia E. Champigny G. Lazdunski M. Barbry P. Nature. 1995; 378: 730-733Crossref PubMed Scopus (359) Google Scholar), which is a member of the DEG/ENaC family of channels. Although an FMRF-amide-gated channel homologous to FaNaCh has not been identified in vertebrates, both FMRF-amide and to a lesser extent, NPFF, can potentiate responses to acid at members of the related ASIC (acid-sensing ion channel) family of acid-sensing channels (34Askwith C.C. Cheng C. Ikuma M. Benson C. Price M.P. Welsh M.J. Neuron. 2000; 26: 133-141Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). This action of NPFF is clearly distinct from the effects observed in the CNS which exhibit a considerably higher potency. Utilizing a GPCR-targeted degenerate PCR methodology, we have identified a novel GPCR that can specifically bind and be activated by neuropeptide FF and the related peptides PQRF-amide and A-18-F-amide, which we have named NPFF1. In addition, we have identified and isolated a second GPCR, structurally related to NPFF1, that can also bind and be activated by NPFF. We have named this second receptor NPFF2. Although NPFF binding sites have been identified in the literature in isolated membranes or in situ, this is the first report identifying a specific receptor system for NPFF. NPFF and other commercially available peptides were purchased from Bachem (Torrance, CA). All other peptides and peptoids were synthesized manually or by using an Advanced Chemtech 396-9000 automated peptide synthesizer (Advanced Chemtech, Louisville, KY). Oligonucleotides were synthesized on an Expedite 8909 oligonucleotide synthesizer (PerkinElmer Life Sciences). 100 ng of rat genomic DNA was subjected to PCR with primers corresponding to the sixth (5′-GYNTWYRYNNTNWSNTGGHTNCC-3′) and seventh (5′-AVNADNGBRWAVANNANNGGRTT-3′) transmembrane domains of the rhodopsin GPCR family. Conditions were as follows: 94 °C for 3 min; 10 cycles of 94 °C for 1 min, 44 °C for 1 min, 45 s, and 72 °C for 2 min; 30 cycles of 94 °C for 1 min, 49 °C for 1 min, 45 s, and 72 °C for 2 min; 72 °C for 4 min. Products were subcloned into the TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced using the ABI Big Dye cycle sequencing protocol and ABI 377 sequencers (Applied Biosystems Inc., Foster City, CA). Nucleotide and amino acid sequence analyses were performed using the Wisconsin Package (GCG, Genetics Computer Group, Madison, WI). To determine the full-length coding sequence of AA449919, 5′/3′ RACE was performed on human spleen Marathon Ready cDNA (CLONTECH, Palo Alto, CA). Nested primers specific to AA449919 were used according to the manufacturer's instructions. The products were sequenced as described above. The Wisconsin Package and Sequencher 3.0 (Gene Codes Corp., Ann Arbor, MI) were used to assemble the full-length contiguous sequence of human NPFF2 (hNPFF2) from the AA449919 EST and the RACE products. The full-length clone was amplified from human spinal cord cDNA using primers flanking the initiating methionine and the stop codon in six independent PCR reactions with the Expand Long Template PCR System (Roche Molecular Biochemicals), and subcloned into pcDNA3.1(+). Each of the six products was fully sequenced, and the construct that agreed 100% with the consensus of the six reactions was used for pharmacological analysis. Primers specific to the rat receptor fragment were used to isolate a clone representing the full-length BN6 (rNPFF1) receptor from a rat hypothalamic cDNA library (18Smith K.E. Walker M.W. Artymyshyn R. Bard J. Borowsky B. Tamm J.A. Yao W.J. Vaysse P.J. Branchek T.A. Gerald C. Jones K.A. J. Biol. Chem. 1998; 273: 23321-23326Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) using the following PCR protocol: 94 °C, hold for 3 min; 40 cycles of 94 °C for 1 min, 68 °C for 2 min; 4-min hold at 68 °C. Positive library pools were subsequently diluted and rescreened by PCR using the same protocol. Positive sub-pools were plated for colony hybridization with 32P-labeled oligonucleotide probes. Isolated positive colonies were chosen, and the respective plasmids were sequenced as described above. Similarly, the full-length hNPFF1 receptor was isolated by PCR screening of pools of a human spinal cord cDNA library. Chimeric Gαq/i3, Gαq/z, and Gαq/s were generated by PCR using primers encoding human Gαq and the C-terminal five amino acids of Gαi3, Gαz, and Gαq/s (19Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (624) Google Scholar). Xenopus oocytes were prepared and injected with mRNA as described (18Smith K.E. Walker M.W. Artymyshyn R. Bard J. Borowsky B. Tamm J.A. Yao W.J. Vaysse P.J. Branchek T.A. Gerald C. Jones K.A. J. Biol. Chem. 1998; 273: 23321-23326Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 20Quick M.W. Simon M.I. Davidson N. Lester H.A. Aragay A.M. J. Biol. Chem. 1994; 269: 30164-30172Abstract Full Text PDF PubMed Google Scholar). Unless otherwise specified, oocytes were voltage clamped at −80 mV. Drugs were applied by superfusion in a solution containing 96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES, pH 7.5. Membranes from COS-7 or HEK-293 cells expressing hNPFF1 or hNPFF2 were isolated and subjected to equilibrium binding assays. In equilibrium saturation binding assays, isolated membranes were incubated in binding buffer (50 mmTris-HCl, 60 mm NaCl, 1 mm MgCl, 33 μm EDTA, 33 μm EGTA, pH 7.4, supplemented with 0.2% bovine serum albumin, 2 μg/ml aprotinin, and 20 μm bestatin) with increasing concentrations of [125I]d-Tyr-Leu-(N-methyl)Phe-Gln-Pro-Glu-Arg-Phe-NH2([125I]1DMeNPFF). In equilibrium competition binding assays, isolated membranes were incubated with 50 pm[125I]1DMeNPFF in the presence of 10–12 different concentrations of competing ligand for 2 h at 25 °C, after which the reaction was stopped by filtration through a double layer of glass fiber filters treated with 0.1% polyethyleneimine using a cell harvester. Radioactivity was measured by scintillation counting. Nonspecific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of 1 μm final concentration of unlabeled NPFF. COS-7 cells were transiently transfected in 96-well plates with Gαq/z5 and either hNPFF1 or hNPFF2. The day before the assay, the growth medium was changed to 100 μl of medium containing 1% serum and 0.5 μCi of [3H]myo-inositol, and the plates were incubated overnight at 37 °C in 5% CO2. Immediately before the assay, the medium was removed and replaced with 200 μl of phosphate-buffered saline containing 10 mm LiCl. The [3H]inositol phosphate accumulation from inositol phospholipid metabolism was started by the addition of increasing concentrations of NPFF, after which the plates were incubated for 1 h in a CO2 incubator. Reactions were terminated by addition of 15 μl of 50% v/v trichloroacetic acid, followed by a 40-min incubation at 4 °C. After neutralizing trichloroacetic acid with 40 μl of 1 m Tris, the contents of the wells were transferred to a multiscreen HV filter plate (Millipore, Bedford, MA) containing Dowex AG1-X8 (200–400 mesh, formate form). Each well was washed two times with 200 μl water, followed by 2× 200 μl of 5 mm sodium tetraborate/60 mm ammonium formate. The [3H]inositol phosphates were eluted with 200 μl of 1.2 m ammonium formate/0.1 m formic acid, and samples were counted by liquid scintillation counting. COS-7 cells were transiently transfected with Gαz and either hNPFF1 or hNPFF2 and incubated in phosphate-buffered saline supplemented with 10 mm HEPES, 5 mm theophylline, 2 μg/ml aprotinin, 0.5 mg/ml leupeptin, and 10 μg/ml phosphoramidon for 20 min at 37 °C, in 5% CO2. Test compounds were added, and the cells were incubated for an additional 10 min at 37 °C, after which the reaction was stopped by the addition of 100 mmHCl. The plates were then incubated at 4 °C for 15 min, and the cAMP content in the stopping solution was measured by radioimmunoassay. Radioactivity was measured using a gamma counter equipped with data reduction software. COS-7 cells expressing the chimeric G protein Gαq/z5, Gαq/i3, or Gαq/s and either rNPFF1 or hNPFF2 were plated in 96-well plates and grown to confluence. After incubation with Fluo 3-AM, cells were washed with Hanks' balanced salt solution and equilibrated for 20 min. The fluorescence emission caused by intracellular calcium mobilization elicited by agonists of the expressed receptor was determined with a fluorescence imaging plate reader (FLIPRTM, Molecular Devices Corp., Sunnyvale, CA). These methods have been described in detail previously (21Dupuy V. Zajac J.-M. Synapse. 1996; 24: 282-296Crossref PubMed Scopus (41) Google Scholar). [125I]1DMeNPFF (specific activity, 2200 Ci/mmol) was synthesized by iodination with chloramine-T (PerkinElmer Life Sciences). Adjacent tissue sections were incubated in the presence of 300 nm BIBP3226 (Research Biochemicals Inc., Natick, MA), to selectively displace NPFF1 receptor binding sites or 300 nm frog pancreatic polypeptides (PP) (frog PP, Peninsula, Belmont, CA) to selectively displace binding to the NPFF2 receptor binding sites. Nonspecific binding was determined in the presence of 1 μm NPFF in the incubation buffer. For the detection of RNA encoding NPFF receptors, quantitative RT-PCR was performed on mRNA extracted from multiple tissue samples. RNA was prepared using Trizol (Life Technologies, Inc.) or was purchased (CLONTECH). Reverse transcription and PCR reactions were carried out in 50-μl volumes using rTth DNA polymerase (PerkinElmer Life Sciences). The following primer sets were synthesized: hNPFF1 forward, 5′-CTGGTCACCGTCTACGCCTT-3′, reverse, 5′-CCGCGGCGGAAGTTCT-3′; hNPFF2 forward, 5-CCTGATTGTGGCCCTGCT-3′, reverse, 5′-CATTTGGAGAAAGGTCAGCGTAG-3′; rNPFF1 forward, 5′-GCTGTGGAAAGGTTCCGCT-3′, reverse, 5′-CGCCTTCCGAAGGGTCA-3′; rNPFF2 forward, 5′-GAGGATCTACACCACCGTGCTATT-3′, reverse, 5′-GAAGCCCCAATCCTTGCATAC-3′. Fluorogenic probes were synthesized using 6-carboxyfluorescein as the reporter at the 5′ end and 6-carboxy-4,7,2,7′-tetramethylrhodamine as a quencher at the 3′ end of the oligonucleotide (Synthegen, LLC). Each RT-PCR reaction contained 100 ng of total RNA. RNA was quantified using spectroscopy (A260) and RiboGreen (Molecular Probes) assays. All reagents for RT-PCR (except mRNA and oligonucleotide primers) were obtained from PerkinElmer Life Sciences, and the manufacturer's protocols were used for RT-PCR. Each 96-well plate contained RNA extracted from tissue (in triplicate), controls, and standard curves to facilitate relative quantification of NPFF1 and NPFF2 RNA. Standard curves for quantification of human and rat NPFF1 and NPFF2 were constructed using varying amounts of RNA extracted from whole brain. To confirm that RNA was not contaminated with genomic DNA, PCR reactions were carried out without reverse transcription using Taq DNA polymerase. The integrity of the RNA was assessed by amplification of RNA coding for cyclophilin or glyceraldehyde 3-phosphate dehydrogenase. Following reverse transcription and PCR amplification, data were analyzed using PerkinElmer sequence detection software. The fluorescent signal from each well was normalized using an internal passive reference, and data were fitted to a standard curve to obtain the relative quantities of NPFF RNA expression. Chromosomal localization for humanNPFF1 and NPFF2 receptor genes was established using a panel of radiation hybrids prepared by the Stanford Human Genome Center (SHGC) and distributed by Research Genetics, Inc. The "Stanford G3" panel of 83 radiation hybrids was analyzed by PCR using the same primers, probes, and thermal cycler profiles as used for localization. 20 ng of DNA was used in each PCR reaction. Data were submitted to the Radiation Hybrid Server (SHGC), which linked the NPFF1 and NPFF2 gene sequences to specific markers. NCBI LocusLink and NCBI GeneMap '99 were used in further analyses of gene localization. Utilizing a GPCR-targeted degenerate PCR methodology on rat genomic DNA, we identified a novel GPCR fragment most closely related to several peptide-ligand GPCRs. The full-length rat receptor, BN6, was isolated from a rat hypothalamic cDNA library, and the human ortholog, BO102, was subsequently isolated from a human spinal cord cDNA library. Sequence analysis of the rat and human receptors revealed coding sequences of 1296 bp and 1290 bp, and predicted proteins of 432 and 430 amino acids, respectively, which share 87% identity (Fig. 1). Amino acid comparison of BO102 with known GPCRs indicates that it is most similar to human orexin1 (37% identity), human orexin2 (35%), human neuropeptide Y (NPY) Y2 (34%), human cholecystokinin A (CCKA) (34%), human NPY Y1 (32%), mouse GIR (32%), human prolactin-releasing hormone receptor (32%), and human NPY Y4 (31%). To determine the ligand specificity of this receptor,Xenopus oocytes expressing BN6 and a Gαq/z5chimeric Gα G-protein were used to screen a library of peptidic neurotransmitters. Within this collection only NPFF (1 μm) elicited reliable and robust responses (Fig. 2). Current amplitudes averaged 459 ± 81 nA (n = 13) and these exhibited a concentration dependence with an EC50 of 163 nm(n = 8 oocytes; data not shown). The NPFF-related ligands, A-18-F-amide, Y-18-F-amide, Y-8-F- amide (1 μm) and the C-terminal tetrapeptide PQRF-amide (10 μm), also activated the receptor (data not shown). These results suggested that BN6 encoded a receptor for NPFF, herein called rNPFF1. A search of Genbank data bases revealed a related human expressed sequence tag (EST) fragment of 532 bp (accession #AA449919), which encoded an amino acid sequence with 59% identity to hNPFF1 and 50% identity to rNPFF1. RACE was used on human spleen cDNA to clone the 5′ and 3′ ends of AA449919, and the full-length receptor, BO89, was amplified from spinal cord cDNA. The rat ortholog of BO89 was cloned by PCR, and named BO119. The coding regions of BO89 and BO119 are 1260 and 1251 bp, encoding proteins with predicted lengths of 420 and 417 amino acids, respectively (Fig. 1). BO89 and BO119 share 78% amino acid identity and are 49–50% identical to rat and human NPFF1. Oocytes expressing BO89 were robustly activated by NPFF (1 μm, Fig. 2). Mean current amplitudes were 528 ± 99 nA (n = 18). This finding suggested that BO89 was an additional member of the NPFF receptor family; therefore, it was named hNPFF2. Both rNPFF1 and hNPFF2 receptor responses were dependent upon co-expression of the chimeric Gαq/z5 G-protein. Subsequent to the identification of BO89 as NPFF2, a report was published describing the cloning of an orphan receptor sequence named NPGPR (22Cikos S. Gregor P. Koppel J. Biochem. Biophys. Res. Commun. 1999; 256: 352-356Crossref PubMed Scopus (24) Google Scholar). NPGPR is nearly identical to NPFF2 except that the N terminus of NPGPR is longer by 102 amino acids. Although it is possible that the more N-terminal initiating methionine could be used for translation of this receptor, the second methionine (the initiating methionine of NPFF2) is surrounded by a good kozak consensus sequence (atcATGaat) and would code for a protein of approximately the same length as rNPFF2, rNPFF1, and hNPFF1. To further assess the pharmacological identity of the human NPFF1 and NPFF2 receptors, the binding properties of the cloned receptors were explored using [125I]1DMeNPFF as a radioligand. The specific binding of [125I]1DMeNPFF with membranes harvested from COS-7 cells transfected with NPFF1 or NPFF2 receptors at 25 °C reached a maximum by 60 min and remained unchanged for up to 120 min (data not shown). Membranes from transiently transfected COS-7 cells exhibited high affinity, saturable [125I]1DMeNPFF binding for both NPFF1 and NPFF2 receptors (Fig. 3, A and B). Nonlinear analysis of [125I]1DMeNPFF saturation data yielded an equilibrium dissociation constant (Kd) of 1.13 ± 0.16 and 0.37 ± 0.03 nm (S.D.n = 2) for NPFF1 and NPFF2 receptors, respectively (Fig. 3, C and D). Untransfected host cells did not display specific [125I]1DMeNPFF binding. Transient expression of hNPFF1 and hNPFF2 receptors in 293 human embryonic kidney cells (HEK-293 cells) yielded similarKd values from saturation studies and a more robust expression (Bmax = 1592 and 510 fmoles/mg protein for hNPFF1 and hNPFF2, respectively) as compared with the COS-7 cells (Bmax = 543 and 47 fmol/mg protein for hNPFF1 and hNPFF2, respectively). Therefore, the HEK-293 cells were used to measure the binding affinities (pKi) of various NPFF-related peptides in a competition binding assay using [125I]1DMeNPFF as the radioligand. The C-terminal RF-amide peptide, PQRF-amide, displaced [125I]1DMeNPFF binding to both NPFF1 and NPFF2. In addition, NPFF receptors showed high binding affinity for FMRF and lower binding affinity for itsd-Met analog, suggesting that the binding domain of the receptors recognizes the C-terminal RF-amide of NPFF. Other C-terminal RF-amide peptides such as frog PP, an NPY Y4 receptor agonist (23Hoyle C.H. Brain Res. 1999; 848: 1-25Crossref PubMed Scopus (155) Google Scholar), showed greater affinity for the rat (125-fold) and human (300-fold) NPFF2 receptors compared with the rat and human NPFF1 receptors (see Table I). Conversely, human PP, human NPY, and peptide YY, which contain a C-terminal RY-amide (24Boel E. Schwartz T.W. Norris K.E. Fiil N.P. EMBO J. 1984; 3: 909-912Crossref PubMed Scopus (44) Google Scholar, 25Takeuchi T. Gumucio D.L. Yamada T. Meisler M.H. Minth C.D. Dixon J.E. Eddy R.E. Shows T.B. J. Clin. Invest. 1986; 77: 1038-1041Crossref PubMed Scopus (34) Google Scholar, 26Kohri K. Nata K. Yonekura H. Nagai A. Konno K. Okamoto H. Biochim. Biophys. Acta. 1993; 1173: 345-349Crossref PubMed Scopus (21) Google Scholar), did not bind to either NPFF1 or NPFF2 (data not shown). Interestingly, the synthetic C-terminal RY-amide peptoid BIBP3226, an NPY Y1-selective compound (27Doods H.N. Wieland H.A. Engel W. Eberlein W. Willim K.D. Entzeroth M. Wienen W. Rudolf K. Regul. Pept. 1996; 65: 71-77Crossref PubMed Scopus (119) Google Scholar), displayed 10–60-fold higher affinity for the human and rat NPFF1 receptor as compared with NPFF2 receptors. These findings question the pharmacological selectivity of this peptoid for NPY Y1 receptors, suggesting that BIBP3226 and related compounds may mediate some of their in vivo effects through NPFF receptors rather than through NPY Y1 receptors (28Morgan D.G. Small C.J. Abusnana S. Turton M. Gunn I. Heath M. Rossi M. Goldstone A.P. O'Shea D. Meeran K. Ghatei M. Smith D.M. Bloom S. Regul. Pept. 1998; 75–76: 377-382Crossref PubMed Scopus (48) Google Scholar, 29Iyengar S. Li D.L. Simmons R.M. J. Pharmacol. Exp. Ther. 1999; 289: 1031-1040PubMed Google Scholar).Table IPharmacology of cloned NPFF receptorsHumanRatNPFF1KiNPFF2 KiNPFF1KiNPFF2 Kinm(d-Tyr1-(NMe)Phe3)NPFF7.9 ± 1.03.2 ± 0.61.6 ± 0.052.0 ± 0.62Frog PP1259 ± 23640 ± 113980 ± 82050 ± 2.1FMRF-amide0.79 ± 0.284.0 ± 0.22.0 ± 0.058.0 ± 9.5d-Met-FMRF-amide251 ± 106398 ± 26631 ± 118630 ± 41A-18-F-amide63 ± 141.3 ± 0.3732 ± 9.06.3 ± 0.14PQRF-amide40 ± 2625 ± 3.025 ± 3.025 ± 0.60BIBP3226126 ± 111259 ± 11125 ± 2.01585 ± 71Affinities, expressed as Ki were obtained from competition binding data using [125I]1DMeNPFF as a ligand at the cloned human and rat NPFF1 and NPFF2 receptors expressed in HEK-293 cells. Results are means ± S.E. obtained from at least three separate experiments. Open table in a new tab Affinities, expressed as Ki were obtained from competition binding data using [125I]1DMeNPFF as a ligand at the cloned human and rat NPFF1 and NPFF2 receptors expressed in HEK-293 cells. Results are means ± S.E. obtained from at least three separate experiments. The ability of NPFF1 and NPFF2 receptors to couple functionally to heterotrimeric G proteins was tested using intact COS-7 cells transiently expressing these receptors. NPFF (1 μm) had no effect on either basal or forskolin-stimulated cAMP formation or PI turn-over in untransfected COS-7 cells, indicating that endogenous adenylate cyclase- or PI-coupled NPFF receptors are not expressed in untransfected cells. In COS-7 cells transfected with the rat NPFF1 receptor, NPFF elicited a small (2-fold) increase in total inositol phosphate release with an EC50 of 239 nm (Fig. 4 A), which most likely reflects a minor activation of this pathway. Pretreatment of cells expressing the rat NPFF1 receptor with 100 ng/ml pertussis toxin (PTX) for 18 h prevented the NPFF-mediated activation of PI turn-over, suggesting that the activation of the phospholipase C pathway in native cells transiently expressing NPFF1 is most likely secondary to the activation of endogenous PTX-sensitive G proteins and not Gαq. When NPFF1 was co-expressed with the Gαq/z5 chimera, NPFF stimulation resulted in a much more robust inositol phosphate release response, which was not sensitive to PTX treatment, with an EC50 that was left-shifted 2 log units relative to transfection with NPFF1 alone (Fig. 4 B). The PTX insensitivity of the response in cells co-expressing NPFF1 and the Gαq/z5 chimera suggests that the PI response in cells co-expressing the chimera is mediated by the activation of phospholipase C by the Gαq domain of the chimera and not secondary to activation of endogenous PTX-sensitive G proteins. In COS-7 cells expressing the human NPFF2 alone, we could not detect a PI turn-over response to NPFF. To further characterize the functional activity of the receptors, the ability of rNPFF1 and hNPFF2 to stimulate intracellular Ca2+ mobilization when co-expressed with different chimeric G proteins was tested in COS-7 cells. Co-transfection of rat NPFF1 or human NPFF2 receptors with either Gαq/i3 or Gαq/z5 led, in both cases, to the activation by NPFF of intracellular Ca2+ mobilization in a concentration-dependent manner (Fig. 5). The EC50 values for the NPFF-mediated stimulation of intracellular Ca2+ release were in good agreement with the binding affinities of NPFF at NPFF1 and NPFF2 receptors as measured in equilibrium binding assays. However, when Gαq/s was co-transfected with NPFF1, the activation of intracellular Ca2+ mobilization by NPFF was right-shifted and displayed a weaker maximal response (Fig. 5 A). Furthermore, co-transfection of Gαq/s with NPFF2 did not permit intracellular Ca2+ mobilization by NPFF (Fig. 5 B). No response was detected in cells expressing Gαq/s or Gαq/z alone. These results would suggest that although NPFF1 can couple to cyclase-stimulatory G proteins, NPFF1 and NPFF2 may couple more efficiently to cyclase-inhibitory G proteins in this heterologous system. Subsequent functional studies monitoring intracellular Ca2+fluxes with hNPFF1 and hNPFF2 were conducted, using a fluorescence imaging plate reader, with transiently transfected COS-7 cells co-expressing either NPFF1 or NPFF2 and Gαq/z5. NPFF elicited an increase in intracellular Ca2+ when either hNPFF1 or hNPFF2 were transfected, whereas there was no response observed in cells transfected with only the Gαq/zchimera. As show
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