Cloning and Functional Expression of a Human Y4 Subtype Receptor for Pancreatic Polypeptide, Neuropeptide Y, and Peptide YY

The pancreatic polypeptide family includes pancreatic polypeptide (PP), neuropeptide Y (NPY), and peptide YY (PYY). Members of the PP family regulate numerous physiological processes, including appetite, gastrointestinal transit, anxiety, and blood pressure. Of the multiple Y-type receptors proposed for PP family members, only the Y1 subtype has been cloned previously. We now report the cloning of an additional Y-type receptor, designated Y4, by homology screening of a human placental genomic library with transmembrane (TM) probes derived from the rat Y1 gene. The Y4 genomic clone encodes a predicted protein of 375 amino acids that is most homologous to Y1 receptors from human, rat, and mouse (42% overall; 55% in TM). 125I-PYY binding to transiently expressed Y4 receptors was saturable (pKd = 9.89) and displaceable by human PP family derivatives: PP (pKi = 10.25) ~ PP2-36 (pKi = 10.06) > PYY (pKi = 9.06) ~ Leu31,Pro34NPY (pKi = 8.95) > NPY (pKi = 8.68) > PP13-36 (pKi = 7.13) > PP31-36 (pKi = 6.46) > PP31-36 free acid (pKi PYY (pKi = 9.06) ~ Leu31,Pro34NPY (pKi = 8.95) > NPY (pKi = 8.68) > PP13-36 (pKi = 7.13) > PP31-36 (pKi = 6.46) > PP31-36 free acid (pKi PP and COOH-terminal fragments; 2) Y2 binds NPY, PYY, and COOH-terminal fragments > PP and Leu31,Pro34NPY; 3) Y3 binds NPY > PYY; 4) the PP receptor binds PP > NPY, PYY; and 5) the putative Y1-like feeding receptor is activated by NPY, PYY, Leu31,Pro34NPY, and NPY2-36 > COOH-terminal fragments. Only the Y1 subtype has been cloned previously(12Larhammar D. Blomqvist A.G. Yee F. Jazin E. Yoo H. Wahlestedt C. J. Biol. Chem. 1992; 267: 10935-10938Abstract Full Text PDF PubMed Google Scholar, 13Herzog H. Hort Y.J. Ball H.J. Hayes G. Shine J. Selbie L.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5794-5798Crossref PubMed Scopus (394) Google Scholar, 14Krause J. Eva C. Seeburg P.H. Sprengel R. Mol. Pharmacol. 1992; 41: 817-821PubMed Google Scholar, 15Eva C. Oberto A. Sprengel R. Genazzani E. FEBS Lett. 1992; 314: 285-288Crossref PubMed Scopus (79) Google Scholar, 16Blomqvist A.G. Roubos E.W. Larhammar D. Martens G.J.M. Biochim. Biophys. Acta. 1995; 1261: 439-441Crossref PubMed Scopus (34) Google Scholar). Here we describe homology cloning of an additional human Y-type receptor, Y4, which is functionally activated by PP.MATERIALS AND METHODSCloning and SequencingA human placenta genomic library (Stratagene) was screened using overlapping TM oligonucleotide probes derived from the rat Y1 neuropeptide receptor gene (17; GenBank™ accession number Z11504). Overlapping oligomers were labeled with 32PdATP and 32PdCTP by synthesis with the large fragment of DNA polymerase: TM1, nucleotides 198-251; TM2, nucleotides 269-328; TM3, nucleotides 401-478; TM5, nucleotides 716-778; and TM7, nucleotides 971-1045. Hybridization was performed at low stringency conditions, as described previously(18Weinshank R.L. Zgombick J.M. Macchi M. Adham N. Lichtblau H. Branchek T.A. Hartig P.R. Mol. Pharmacol. 1990; 38: 681-688PubMed Google Scholar). For subcloning and further Southern blot analysis, DNA was cloned into pUC18 (Pharmacia Biotech Inc.)(19Southern E.M. J. Mol. Biol. 1975; 98: 503-517Crossref PubMed Scopus (21364) Google Scholar, 20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 9.31-9.62Google Scholar). Nucleotide sequence analysis was accomplished by the Sanger dideoxy nucleotide chain termination method (21Sanger F. Nicklen S. Coulsen A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52360) Google Scholar) on denatured double-stranded plasmid templates, using Sequenase (U. S. Biochemical Corp.) and confirmed on both strands.Tissue Localization/Expression (Reverse Transcriptase-PCR)Human tissues, obtained from National Disease Research Interchange (Philadelphia, PA), were homogenized and total RNA extracted using guanidine isothiocyanate/CsCl cushion method. In some instances, poly(A)+ mRNA was isolated with oligo(dT) affinity chromatography, using standard protocols. RNA was treated with DNase to remove any contaminating genomic DNA. cDNA was prepared from total RNA with random hexanucleotide primers using reverse transcriptase (Superscript II; Life Technologies, Inc.). An aliquot of the first strand cDNA (corresponding to 250 ng of total RNA or 5 ng of poly(A)+ RNA) was amplified using a program consisting of 30 cycles of 94°C for 2 min, 68°C for 2 min, and 72°C for 3 min, with a pre- and postincubation of 95°C for 5 min and 72°C for 10 min, respectively. PCR primers were designed against the human Y4 sequence in the third intracellular loop and carboxyl-terminal regions: Forward, 5′-CGCGTGTTTCACAAGGGCACCTA-3′, and reverse, 5′-TGCCACTTAGCCTCAGGGACCC-3′, respectively. The PCR products were analyzed by Southern blot techniques using a 5′ end-labeled oligonucleotide (located in the carboxyl terminus: 5′-TCCG TATGTACTGTGGACAGGGGCAGATGCTCCGACTCCTCCAGG-3′) under high stringency. Under the above conditions, PCR products were identified on Southern blots using the subtype-specific probes, and no cross-reactivity was observed with other NPY receptor subtypes (data not shown). Similar PCR and Southern blot analysis were conducted with primers and probe directed to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (Clontech), except 22 cycles were used. In order to control for the amplification of contaminating genomic DNA, control PCR reactions were run in parallel with RNA which had not been converted to cDNA (i.e. minus reverse transcriptase).Cell CultureStocks of COS-7 (African green monkey kidney) and LMTK- (mouse fibroblast) cells were grown on 150-mm plates in Dulbecco's modified Eagle's medium with supplements (10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin) at 37°C and 5% CO2.Receptor ExpressionThe coding region of hp25a (1127 bp) plus 680 bp upstream and 205 bp downstream was cloned into the BamHI and EcoRI sites of the polylinker-modified eukaryotic expression vector pCEXV-3(22Miller J. Germain R.N. J. Exp. Med. 1986; 164: 1478-1489Crossref PubMed Scopus (191) Google Scholar), called EXJ.HR. 2J. Bard, unpublished data. COS-7 cells were transiently transfected with plasmid hp25a/EXJ (expression vector containing the hp25a gene) or with human Y1 receptor (hY1) DNA (cloned at Synaptic Pharmaceutical Corp.) by the DEAE-dextran method (23Larsson L.-I. Boder G.B. Shaw W.N. Lab. Invest. 1977; 36: 398-593Google Scholar) or without plasmid for mock-transfection. For stable expression, plasmid hp25a/EXJ was co-transfected with a G418-resistant plasmid into LMTK- cells by the calcium phosphate method. Stably transfected cells were selected with G418.Membrane PreparationCOS-7 cells were washed in phosphate-buffered saline 48 h after transfection and lysed by sonication on ice in 20 mM Tris-HCl, 5 mM EDTA, pH 7.7. The supernatant remaining after low speed centrifugation (200 × g, 10 min, 4°C) was subjected to high speed centrifugation (32,000 × g, 18 min, 4°C), and the resulting membrane pellet was resuspended by sonication into ice-cold binding buffer (10 mM NaCl, 20 mM HEPES, 0.22 mM KH2PO4, 1.26 mM CaCl2, 0.81 mM MgSO4, pH 7.4). Protein was measured by the Bradford method (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213462) Google Scholar) using Bio-Rad reagent.125I-PYY BindingMembrane suspensions, porcine 125I-Tyr36PYY (specific activity = 2200 Ci/mmol) and peptides were diluted in binding buffer supplemented with 0.1% bovine serum albumin (Sigma) and distributed into 96-well polypropylene microtiter plates. Samples were incubated at 30°C with shaking for 120 min. Membranes were collected by filtration over Whatman GF/C filters (precoated with 0.5% polyethyleneimine and and for binding was by 100 human were analyzed by microtiter plates were with and with transfected LMTK- cells. was to h the were in buffered saline plus supplements mM NaCl, 20 mM HEPES, mM CaCl2, 5 mM mM and 10 mM plus 0.1% bovine serum albumin and 5 mM for 20 min at 37°C in 5% 5 min with 10 and peptides prepared in was extracted with 100 mM at for 30 min and by The was collected by filtration a 96-well and for were analyzed by and as free Ca2+ was measured by J.M. L.A. S.A. R.L. Branchek T.A. Mol. Pharmacol. 1993; Google Scholar). Stably transfected LMTK- cells were into with were with 10 in for 20 to min, with for an additional 10 to 20 min. were under a × and was at with and were converted to Ca2+ using standard Ca2+ and as AND human genomic placenta library was under stringency conditions, with oligonucleotide probes directed to the and TM of the rat Y1 neuropeptide receptor gene. clone called gene to in report as was characterized by Southern blot analysis to a fragment and further analyzed by subcloning and DNA sequence analysis homology to the rat and human Y1 receptor clone a gene the gene was as a fragment into an expression vector and genomic an of bp with 680 bp upstream and 205 bp downstream of the coding The gene predicted to a protein of 375 amino with a predicted of analysis of the protein is with a putative of transmembrane of the receptor sequence analysis that 1) among the of the neuropeptide receptor family, including and an in and an and acid in and a and in and a and in and a and in and a and in and and a and in and of are the of potential sites for in the amino and 1) and the of and in the carboxyl and intracellular which as sites for potential by protein is that the sequence which is downstream of TM3, is in the is from the sequence which in most of the receptor of the potential sites in are in the in the of nucleotide and peptide of with in the that the clone is most to the rat, and human Y1 receptor and the nucleotide is and at the amino acid is 55% in TM with the of in A of with the cloned human Y2 gene by C. C. Branchek T.A. R.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google nucleotide is and amino acid is with in TM Southern blot analysis on human genomic DNA that the a Y4 gene (data not Y4 mRNA was detected by reverse using primers in a of human were detected in total the coronary artery, and to whereas and low of the and of the expression, with the containing of Y4 mRNA the was observed the RNA was or reverse transcriptase was from the first strand cDNA (data not the that the observed not to any genomic DNA contaminating the We that of cDNA from the were for NPY expression by control reverse with primers for the high expressed gene, glyceraldehyde-3-phosphate dehydrogenase of Y4 receptor mRNA in human amplification of human Y4 cDNA derived from RNA of human RNA was for all except and poly(A)+ RNA was used. Southern blots of the PCR products were prepared and with oligonucleotide probes for the Y4 receptor subtype or glyceraldehyde-3-phosphate dehydrogenase to from COS-7 cells not from with an observed = = min, and binding min at 30°C = Human COS-7 under the conditions, a = = min, and binding min = 125I-PYY binding assays both and receptors were conducted for 120 min. 125I-PYY binding to the transiently expressed receptor was and saturable at 125I-PYY from to were to a with an = and = membrane protein = The transiently expressed receptor 125I-PYY with an = and = membrane protein = Y4 human PP family in 125I-PYY membrane binding assays with a order PP > PYY > NPY > NPY free Human Y4 PP from rat, and with a of with PP PYY and NPY binding to by PP into and to and the of PP to the NPY peptide (e.g. had no on binding affinity to suggesting that for the PP are to binding affinity from other peptide Human PP to PP2-36 but further was for The COOH-terminal fragment was by of the carboxyl A of for and a of peptide for PP or in a of LMTK- cells with 10 an increase in over a = with human PP decreased the cAMP with an of in LMTK- cells transfected with but not in cells. The for human PP was in with the from 125I-PYY binding The of Y-type receptors to of and to C. Reis D.J. Annu. Rev. Pharmacol. Toxicol. 1993; 32: 309-352Crossref Google in LMTK- cells the of are of 20 of intracellular free Human PP was at the by the are selected from 10 in free Ca2+ was increased by 100 human PP in LMTK- cells transfected with the = = but not in cells. The detected 30 of PP to of of in rat superior cervical S. D. J. Pharmacol. Exp. 1993; 265: Google Scholar, M.S. B. J. 1995; PubMed Scopus Google have cloned the gene for a human Y-type receptor, Y4, which is functionally activated by The human Y4 is to rat PP receptor binding derived from and For a PP receptor in rat PC-12 cells was to PP > T.W. Sheikh S.P. O'Hare M.M.T. FEBS Lett. 1987; 225: 209-214Crossref PubMed Scopus (50) Google Scholar) and PP > Leu31,Pro34NPY, NPY J.C. Fuhlendorff J. Schwartz T.W. Eur. J. Pharmol. 1990; 186: 105-114Crossref PubMed Scopus (44) Google Scholar). on PC-12 cells bovine PP > rat T.W. Sheikh S.P. O'Hare M.M.T. FEBS Lett. 1987; 225: 209-214Crossref PubMed Scopus (50) Google Scholar), rat receptors both with high T.D. Wolfe M.S. Heintz G.G. Am. J. Physiol. 1995; 268: G215-G223PubMed Google Scholar). A receptor on rat cells from and was to all PP family with H. H. H. Biochem. Biophys. 1990; PubMed Scopus Google Scholar). the rat receptors described in the gene analyzed under or multiple subtypes and is to is not human Y4 designated a PP We the Y4, as a of the Y-type for and Y3 are as 1) Y4 was cloned by of homology with the Y1 receptor and is most in sequence with Y1 receptors from the Y4 is to receptors in Y-type 2) the human Y4 a of for human PYY, and NPY in 125I-PYY binding assays 3) The encodes the conserved COOH-terminal in pancreatic polypeptide family and is a for the and 4) the Y4 a in the that peptide order with or with the of additional PP family of the Y4 mRNA is with of PP binding and in and and further potential Y4 in cardiovascular, and central nervous function. to an pancreatic or PP and in human, rat, and mouse (i.e. PP are increased in a for Y4 in and L.-I. Boder G.B. Shaw W.N. Lab. Invest. 1977; 36: 398-593Google Scholar, B. PubMed Google Scholar, 1977; PubMed Scopus (50) Google Scholar, J. Am. 1992; PubMed Scopus Google Scholar). PP has to central sites of D.C. Taylor I.L. Vigna S.R. Am. J. Physiol. 1990; 259: G687-G691Crossref PubMed Google Scholar) and the blood Pharmacol. Biochem. 1995; PubMed Scopus (44) Google Scholar). of Y4 receptor as PP mRNA has been identified by PCR in rat brain, and in S. C. 20 Scholar). of Y4 receptor mRNA in and potential receptor are in that not only NPY and PYY, but food intake into rat W.R. S.P. PubMed Scopus Google Scholar, S.P. PubMed Scopus Google Scholar). is that the Y4 receptor is in control of and other of Y4 further using techniques on the Y4 receptor as expression of receptor and INTRODUCTIONThe pancreatic polypeptide family includes pancreatic polypeptide (PP), 1The abbreviations used are: PPpancreatic polypeptideNPYneuropeptide YPYYpeptide YYTMtransmembranePCRpolymerase chain reactionCa2+calcium concentrationHBSHanks' buffered salinekbkilobase pair(s)bpbase pair(s). neuropeptide Y (NPY), and peptide YY (PYY), all of which are 36 amino acid peptides characterized by a hairpin loop(1Wahlestedt C. Reis D.J. Annu. Rev. Pharmacol. Toxicol. 1993; 32: 309-352Crossref Google Scholar). PP is released from pancreatic endocrine cells post-prandially(2Schwartz T.W. Gastroenterology. 1983; 85: 1411-1425Abstract Full Text PDF PubMed Scopus (367) Google Scholar). PP receptors have been characterized by binding or functional assays in liver(3Nguyen T.D. Wolfe M.S. Heintz G.G. Am. J. Physiol. 1995; 268: G215-G223PubMed Google Scholar), intestine(4Gilbert W.R. Frank B.H. Gavin III, J.R. Gingerich R.L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4745-4749Crossref PubMed Scopus (33) Google Scholar), vas deferens(5Jorgensen J.C. Fuhlendorff J. Schwartz T.W. Eur. J. Pharmol. 1990; 186: 105-114Crossref PubMed Scopus (44) Google Scholar), spinal cord(6Wager-Page S.A. Ghazali B. Anderson W. Veale W.L. Davison J.S. Peptides (Elmsford). 1992; 13: 807-813Crossref PubMed Scopus (15) Google Scholar), adrenal gland, superior cervical ganglia(7Schwartz T.W. Sheikh S.P. O'Hare M.M.T. FEBS Lett. 1987; 225: 209-214Crossref PubMed Scopus (50) Google Scholar), brainstem nuclei(8Whitcomb D.C. Taylor I.L. Vigna S.R. Am. J. Physiol. 1990; 259: G687-G691Crossref PubMed Google Scholar), and PC-12 cells(9Whitcomb D.C. Vigna S.R. McVey D.C. Taylor I.L. Am. J. Physiol. 1992; 262: G532-G536PubMed Google Scholar). PP exerts regulatory effects on pancreatic exocrine secretion, gall bladder contraction, gastrointestinal motility, gastric acid secretion, and corticosterone secretion(2Schwartz T.W. Gastroenterology. 1983; 85: 1411-1425Abstract Full Text PDF PubMed Scopus (367) Google Scholar, 10McTigue D.M. Edwards N.K. Rogers R.C. Am. J. Physiol. 1993; 265: G1169-G1176Crossref PubMed Google Scholar, 11Andreis P.G. Tortorella C. Nussdorfer G.G. Life Sci. 1993; 53: 1353-1356Crossref PubMed Scopus (12) Google Scholar). PYY is localized primarily in intestinal endocrine cells, whereas NPY is localized primarily in the nervous system(1Wahlestedt C. Reis D.J. Annu. Rev. Pharmacol. Toxicol. 1993; 32: 309-352Crossref Google Scholar). NPY and PYY act similarly in a majority of biological models (e.g. to increase food intake and vasocontraction), but exceptions have been noted(1Wahlestedt C. Reis D.J. Annu. Rev. Pharmacol. Toxicol. 1993; 32: 309-352Crossref Google Scholar). Within the PP family, NPY is most conserved among species, whereas PP is least conserved(1Wahlestedt C. Reis D.J. Annu. Rev. Pharmacol. Toxicol. 1993; 32: 309-352Crossref Google Scholar).At least five receptor subtypes have been proposed for PP family members(1Wahlestedt C. Reis D.J. Annu. Rev. Pharmacol. Toxicol. 1993; 32: 309-352Crossref Google Scholar) : 1) Y1 binds NPY, PYY, and Leu31,Pro34NPY > PP and COOH-terminal fragments; 2) Y2 binds NPY, PYY, and COOH-terminal fragments > PP and Leu31,Pro34NPY; 3) Y3 binds NPY > PYY; 4) the PP receptor binds PP > NPY, PYY; and 5) the putative Y1-like feeding receptor is activated by NPY, PYY, Leu31,Pro34NPY, and NPY2-36 > COOH-terminal fragments. Only the Y1 subtype has been cloned previously(12Larhammar D. Blomqvist A.G. Yee F. Jazin E. Yoo H. Wahlestedt C. J. Biol. Chem. 1992; 267: 10935-10938Abstract Full Text PDF PubMed Google Scholar, 13Herzog H. Hort Y.J. Ball H.J. Hayes G. Shine J. Selbie L.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5794-5798Crossref PubMed Scopus (394) Google Scholar, 14Krause J. Eva C. Seeburg P.H. Sprengel R. Mol. Pharmacol. 1992; 41: 817-821PubMed Google Scholar, 15Eva C. Oberto A. Sprengel R. Genazzani E. FEBS Lett. 1992; 314: 285-288Crossref PubMed Scopus (79) Google Scholar, 16Blomqvist A.G. Roubos E.W. Larhammar D. Martens G.J.M. Biochim. Biophys. Acta. 1995; 1261: 439-441Crossref PubMed Scopus (34) Google Scholar). Here we describe homology cloning of an additional human Y-type receptor, Y4, which is functionally activated by