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). I-PYY binding to transiently expressed Y4 receptors was saturable (pK = 9.89) and displaceable by human PP family derivatives: PP (pK = 10.25) PP (pK = 10.06) > PYY (pK = 9.06) [Leu,Pro]NPY (pK = 8.95) > NPY (pK = 8.68) > PP (pK = 7.13) > PP (pK = 6.46) > PP free acid (pK < 5). Human PP decreased [cAMP] and increased intracellular [Ca] in Y4-transfected LMTK cells. Y4 mRNA was detected by reverse transcriptase-polymerase chain reaction in human brain, coronary artery, and ileum, suggesting potential roles for Y4 receptors in central nervous system, cardiovascular, and gastrointestinal function.

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) ϩ 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) ϩ 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, 5Ј-CGCGTGTTTCA-CAAGGGCACCTA-3Ј, and reverse, 5Ј-TGCCACTTAGCCTCAGG-GACCC-3Ј, respectively. The PCR products were analyzed by Southern blot techniques using a 5Ј end-labeled oligonucleotide (located in the carboxyl terminus: 5Ј-TCCG TATGTACTGTGGACAGGGGCAGAT-GCTCCGACTCCTCCAGG-3Ј) 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 Culture-Stocks of COS-7 (African green monkey kidney) and * 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) U35232.
Receptor Expression-The 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 (22), called EXJ.HR. 2 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 (23) or without plasmid for mock-transfection. For stable expression, plasmid hp25a/EXJ was co-transfected with a G418-resistant plasmid into LMTK Ϫ cells by the calcium phosphate method. Stably transfected cells were selected with G418.
Membrane Preparation-COS-7 cells were washed in phosphatebuffered 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 ϫ g, 10 min, 4°C) was subjected to high speed centrifugation (32,000 ϫ g, 18 min, 4°C), and the resulting membrane pellet was resuspended by sonication into ice-cold binding buffer ( 36 ]PYY (specific activity ϭ 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 air-dried before use) and counted for 125 I. Nonspecific binding was defined by 100 nM human PP. Data were analyzed by nonlinear regression.
cAMP Measurements-96-Well microtiter plates were precoated with poly-D-lysine (0.05 mg/well), dried, and seeded with stably transfected LMTK Ϫ cells. Serum, potentially desensitizing, was reduced to 1.5% 4 -16 h before the assay. Cells were pre-equilibrated in Hanks' buffered saline plus supplements (HBSP: 150 mM NaCl, 20 mM HEPES, 1 mM CaCl 2 , 5 mM KCl, 1 mM MgCl 2 , and 10 mM glucose plus 0.1% bovine serum albumin and 5 mM theophylline) for 20 min at 37°C in 5% CO 2 , then stimulated 5 min with 10 M forskolin and peptides prepared in HBSP. Intracellular cAMP was extracted with 100 mM HCl at 4°C for 30 min and quantified by radioimmunoassay. The final antigen-antibody complex was collected by filtration through a 96-well Durapore filter plate (Millipore) and counted for 125 I. Data were analyzed by nonlinear regression and reported as mean Ϯ S.E.
[Ca 2ϩ ] Measurements-Intracellular free [Ca 2ϩ ] was measured by microspectrofluorometry (25). Stably transfected LMTK Ϫ cells were seeded into 35-mm culture dishes with glass coverslip inserts. Cells were loaded with 10 M Fura-2/AM in HBS for 20 to 40 min, then equilibrated with HBS alone for an additional 10 to 20 min. Cells were visualized under a ϫ 40 microscope objective, and fluorescence emission was determined at 510 nM with excitation alternating between 340 and 380 nM. Raw fluorescence data were converted to [Ca 2ϩ ] using standard [Ca 2ϩ ] curves and reported as mean Ϯ S.E.

RESULTS AND DISCUSSION
A human genomic placenta library was screened, under reduced stringency conditions, with oligonucleotide probes directed to the first, second, third, fifth, and seventh TM regions of the rat Y1 neuropeptide receptor gene. One positive hybridizing phage clone called hp25a, whose gene product will be referred to in this report as hY4, was characterized by Southern blot analysis to reveal a 1.3-kb PstI fragment and further analyzed by subcloning and sequencing. DNA sequence analysis indicated greatest homology to the rat and human Y1 receptor genes. Since this clone represented a partial intronless gene fragment, the full-length gene was subcloned as a 2.0-kb BamHI/EcoRI hybridizing fragment into an expression vector and sequenced.
The genomic full-length construct contains an open reading frame of 1125 bp (intronless), with 680 bp upstream and 205 bp downstream of the coding region. The gene would be predicted to encode a protein of 375 amino acids, with a predicted molecular mass of Ϸ41 kDa. Hydropathy analysis of the protein is consistent with a putative topography of seven transmembrane domains, indicative of the G-protein-coupled receptor family.
Initial sequence analysis revealed that hY4 contained several structural features/residues (see Fig. 1) found among the members of the neuropeptide receptor family, including two glycines and an asparagine in TM1 (positions 55, 58, and 59, respectively), an asparagine, leucine, and aspartic acid in TM2 (positions 82, 83, and 87, respectively), a serine and leucine in TM3 (positions 128 and 132, respectively), a tryptophan and proline in TM4 (positions 164 and 173, respectively), a tyrosine and proline in TM5 (positions 223 and 226, respectively), a phenylalanine, tryptophan, and proline in TM6 (positions 274, 278, and 280, respectively), and a serine, threonine, asparagine, and proline in TM7 (positions 314, 315, 318, and 319, respectively). Other features of hY4 are the presence of three potential sites for N-linked glycosylation in the amino terminus (asparagine residues 2, 19, and 29; Fig. 1) and the presence of several serines and threonines in the carboxyl terminus and intracellular loops, which may serve as sites for potential phosphorylation by protein kinases. It is interesting that the sequence ERH (Glu-Arg-His), which is immediately downstream of TM3, is also contained in the hY1 sequence; this is distinct from the DRY (Asp-Arg-Tyr) sequence which occurs in most members of the G-protein-coupled receptor superfamily. Additionally, two of the three potential N-linked glycosylation sites found in hY4 are also present in the corresponding positions in the hY1 receptor.
A comparison of nucleotide and peptide sequences of hY4 with sequences contained in the GenBank™/EMBL data bases reveals that the clone is most related to the rat, mouse, Xenopus, and human Y1 receptor genes and proteins. At the nucleotide level there is 58% identity between hY4 and hY1; at the amino acid level there is 42% identity overall, 55% in TM domains, with the greatest identity of 71% in TM7 (Fig. 1). A similar comparison of hY4 with the cloned human Y2 (hY2) 2 J. Bard, unpublished data. gene (see accompanying paper by Gerald et al. (36)) reveals lower homology; hY4 versus hY2 nucleotide identity is 57% and amino acid identity is 34% overall, with 43% in TM domains (see also Fig. 1). Southern blot analysis on human genomic DNA suggest that the genome contains a single Y4 gene (data not shown).
Human Y4 mRNA was detected by reverse transcriptase-PCR using specific hY4 primers in a broad range of human tissues (Fig. 2). Relatively intense hybridization signals were detected in total brain (including the hypothalamus), coronary artery, and ileum. Liver failed to express hY4 mRNA, whereas pancreas and kidney exhibited very low levels of expression. Interestingly, the endometrium and myometrium of the uterus displayed differential expression, with the former containing higher levels of Y4 mRNA than the latter. No signal was observed when either the RNA was absent (distilled H 2 0 control) or reverse transcriptase was omitted from the first strand cDNA conversion (data not shown); the latter suggests that the signals observed not due to any genomic DNA contaminating the RNA. We also demonstrated that equal amounts of cDNA from the different tissues were assayed for NPY expression by conducting control reverse transcriptase-PCR with primers for the moderately high level constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (Clontech) (Fig. 2). 125 I-PYY (0.06 nM) bound specifically to membranes from hY4-transfected COS-7 cells (but not from mock-transfected cells) with an observed association rate (K obs ) ϭ 0.12 Ϯ 0.02 min Ϫ1 , t1 ⁄2 ϭ 6 min, and 100% complete equilibrium binding within 50 min at 30°C (n ϭ 3). Human Y1-transfected COS-7 cell membranes, when studied under the same conditions, yielded a K obs ϭ 0.06 Ϯ 0.02 min Ϫ1 , t1 ⁄2 ϭ 12 min, and 100% complete equilibrium binding within 90 min (n ϭ 3). Subsequent 125 I-PYY binding assays involving both hY1 and hY4 receptors were conducted for 120 min. 125 I-PYY binding to the transiently expressed hY4 receptor was specific and saturable at 125 I-PYY concentrations ranging from 0.003 to 2.5 nM. Binding data were fit to a one-site model with an apparent pK d ϭ 9.89 Ϯ 0.04 (0.13 nM) and B max ϭ 1.9 Ϯ 0.3 pmol/mg membrane protein (n ϭ 8). The transiently expressed hY1 receptor bound 125 I-PYY with an apparent pK d ϭ 10.19 Ϯ 0.04 (0.065 nM) and B max ϭ 4.0 Ϯ 0.7 pmol/mg membrane protein (n ϭ 9).
Human Y4 bound human PP family members in 125 I-PYY membrane binding assays with a distinctive rank order (Table  I): PP Ͼ PYY Ͼ NPY Ͼ NPY free acid. Human Y4 also bound PP from bovine, rat, salmon, and frog with a wide range of K i values consistent with PP species diversity. PYY and NPY binding to hY4 could be enhanced by inserting PP residues into positions 31 and 34 to make [Pro 34 ]PYY and [Leu 31 ,Pro 34 ]NPY. However, the corresponding modifications of PP to reflect the NPY peptide (e.g. [Ile 31 ,Gln 34 ]PP) had no effect on binding affinity to hY4 (see Table I), suggesting that for the favored ligand PP there are significant contributions to binding affinity from other peptide regions. Human PP could be truncated to PP 2-36 but further NH 2 -terminal deletion was disruptive for hY4 binding. The shortest COOH-terminal fragment studied (PP [31][32][33][34][35][36] ) was rendered inactive by hydrolysis of the carboxyl amide. A comparison of structure/activity profiles for hY4 and hY1 suggests a common mechanism of peptide interaction optimized for either PP or NPY/PYY, respectively.
Incubation of intact LMTK Ϫ cells with 10 M forskolin pro-   (Fig. 3B). These data add complexity to existing notions of PP-dependent Ca 2ϩ regulation centered around inhibition of voltage-dependent Ca 2ϩ channels in rat superior cervical ganglia (26,27).
We have cloned the gene for a novel human Y-type receptor, Y4, which is functionally activated by PP. The human Y4 pharmacological profile is similar to rat PP receptor binding profiles derived from cell and tissue models. For example, a PP receptor in rat PC-12 cells was reported to bind PP Ͼ PP 24 -36 (7) and also PP Ͼ [Leu 31 (7), although rat hepatocyte receptors bound both with equally high affinity (3). A receptor on intact rat cells from liver, pancreas, and elsewhere was reported to bind all PP family members with similar K d values (28). Whether the rat receptors described in these reports represent the same gene product analyzed under different assay conditions or multiple subtypes and what their relationship is to hY4 is not yet clear.
The human Y4 could conceivably be designated a PP receptor. We propose the name Y4, however, as a direct extension of the Y-type nomenclature previously established for Y1, Y2, and Y3 receptors. Arguments are as follows: 1) Y4 was cloned by virtue of its homology with the Y1 receptor and is most similar in sequence with Y1 receptors from several species. As such, the Y4 is structurally linked to receptors currently entrenched in Y-type nomenclature; 2) the human Y4 displayed a narrow range of K i values for human PP, PYY, and NPY in 125 I-PYY binding assays (10.25 Ն pK i Ն 8.68); 3) The letter "Y" encodes the conserved COOH-terminal Tyr in pancreatic polypeptide family members and is therefore a unifying symbol for the entire ligand family; and 4) the Y4 designation represents a reasonable nomenclature in the event that peptide rank order should change with assay conditions or with the discovery of additional PP family members.
The tissue distribution of the Y4 mRNA is consistent with reports of PP binding and function in brain and peripheral tissues and further suggests potential Y4 involvement in gastrointestinal, cardiovascular, and central nervous system function. As there appears to be an inverse correlation between pancreatic or circulating PP levels and obesity in human, rat, and mouse (i.e. circulating PP levels are increased in anorexia nervosa), one may speculate a role for Y4 in appetite and body weight control (23, 29 -31). Circulating PP has access to central sites through penetration of fenestrated capillaries (8) and through transport past the blood brain barrier (32). Additional routes of Y4 receptor activation may also exist, as PP mRNA has been recently identified by PCR in rat brain, and in particular, hypothalamus (33). Localization of Y4 receptor mRNA in hypothalamus and potential receptor activation are intriguing in that not only NPY and PYY, but also PP, can enhance food intake when injected into rat brain intracerebroventricular (34,35). One hypothesis is that the Y4 receptor is involved in hypothalamic control of feeding. This and other aspects of Y4 function can be further explored using techniques based on the Y4 receptor clone, such as expression of species homologs, selective antagonist design, receptor antisense, and transgenic animal models.