©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Functional Expression of a cDNA Encoding a Human Type 2 Neuropeptide Y Receptor (*)

(Received for publication, July 11, 1995; and in revised form, July 28, 1995)

Patricia M. Rose (§) Prabhavathi Fernandes Jean S. Lynch Scott T. Frazier Susan M. Fisher Krishna Kodukula (2) Bernadette Kienzle (1) Ramakrishna Seethala

From the  (1)Departments of Microbial Molecular Biology and Metabolic Diseases, Bristol-Myers Squibb, Princeton, New Jersey 08543-4000 and the (2)Department of Biomolecular Screening, Bristol-Myers Squibb, Wallingford, Connecticut 06492

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Neuropeptide Y (NPY) is a 36-amino acid polypeptide that is widely distributed in the central nervous system and periphery. Pharmacological studies have suggested that there are at least three receptor subtypes, Y1, Y2, and Y3. Cloning of the Y1 subtype has been reported previously. Here we report the isolation by expression cloning of a cDNA encoding a human NPY receptor displaying a pharmacology typical of a Y2 receptor. COS-7 cells transfected with the cDNA express high affinity binding sites for NPY, peptide YY, and NPY, whereas [Leu,Pro]NPY binds with lower affinity. The receptor is 381 amino acids in length and has seven putative transmembrane regions typical of G-protein-coupled receptors. Comparison of the amino acid sequence of this Y2 receptor to that of the human Y1 receptor indicates that the two receptors are 31% identical at the amino acid level. Northern blot analyses reveal a single 4-kilobase mRNA species and indicate that the messenger RNA is present in many areas of the central nervous system. NPY induced calcium mobilization and inhibited forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells that stably express the Y2 receptor cDNA, indicating that the recombinant Y2 receptor is functionally coupled to second messenger systems.


INTRODUCTION

Neuropeptide Y (NPY) (^1)is a 36-amino acid peptide amide that is widely distributed in the central and peripheral nervous systems. It belongs to a family of homologous peptides including the gut peptide YY (PYY) and pancreatic polypeptide. NPY has been highly conserved throughout evolution and is therefore thought to be an important hormone/neurotransmitter. Centrally its effects include blood pressure regulation, memory enhancement, anxiolysis/sedation, and increased food intake, and in the periphery it affects vascular and other smooth muscle activity, intestinal electrolyte secretion, and urinary sodium excretion(1, 2) .

Heterogeneity among NPY receptors has been observed. Based on the rank order of potency of NPY and related peptides to displace I-NPY binding, NPY receptors have been classified into at least three receptor subtypes, Y1, Y2, and Y3(3) . Both the Y1 and Y2 subtypes have high affinity for NPY and PYY. The Y1 subtype has a high affinity for the NPY analog [Leu,Pro]NPY and low affinity for C-terminal fragments of NPY such as NPY. In contrast, the Y2 receptor subtype has high affinity for NPY and low affinity for [Leu,Pro]NPY. The Y3 subtype has a low affinity for PYY. The existence of a fourth receptor, Y1a or Y4, that is important in the feeding response has been hypothesized (2) .

The NPY Y1 receptor has been cloned from rat forebrain(4) , human fetal brain(5) , bovine hypothalamus, (^2)and murine genomic DNA(6) . It belongs to the superfamily of G-protein-coupled receptors and appears to couple to more than one second messenger systems(5, 7) . To date the cloning of a Y2 receptor has not been reported. Attempts to isolate a Y2 cDNA using homology to the Y1 cDNA have not been successful. Here we report the isolation by expression cloning (8) of a cDNA encoding a human NPY receptor displaying a pharmacology typical of a Y2 receptor. The clone has been functionally expressed in CHO cells where its activation causes mobilization of calcium and inhibition of forskolin-stimulated cAMP accumulation.


EXPERIMENTAL PROCEDURES

Expression Cloning

An oriented expression cDNA library was prepared from SMS-KAN mRNA in the pcDNA1 vector. Approximately 2.5 times 10^5 COS-7 cells were seeded on to single-well Labteks(TM) (Nunc) and transfected with 2 µg of plasmid DNA prepared from pools of bacterial clones from the library using Lipofectamine(TM) (Life Technologies, Inc.). Three days later the cells were washed twice with binding buffer A (Dulbecco's modified Eagle's medium containing 1% BSA). Following incubation with 100 pMI-PYY (2200 Ci mmol, DuPont NEN) in buffer A at 37 °C for 2 h, they were washed three times, air-dried, and autoradiographed. The one positive pool detected during the primary screening was subdivided and screened similarly until a pure clone was isolated.

Stable Expression of NPY-Y2 Receptor in Chinese Hamster Ovary (CHO) Cells

A BamHI and XbaI fragment of the pNPYR-398 clone, which contained the entire open reading frame for the receptor, was subcloned into the pcDNA3 vector cut with the same enzymes. This resulting construct was linearized with PvuI and introduced into the CHO cells by electroporation (9) using a Bio-Rad Gene Pulser(TM). After 48 h the cells were placed in selection medium (Ham's F-12 media containing 10% fetal bovine serum, 50 units/ml penicillin G, 50 µg/ml streptomycin, 2 mML-glutamine, and 300 µg/ml G418). Clones were picked after 3 weeks, expanded, and assayed for NPY receptor expression using a radioligand binding assay.

Radioligand Binding

COS-7 cells (5 times 10^4 cells/assay), transfected with the purified pNPYR-398 clone were incubated in a 96-well microtiter plate in 100 µl of binding buffer B (50 mM Hepes, pH 7.4, 5 mM MgCl(2), 1 mM CaCl(2), 0.5% BSA, 1 mg/ml of pepstatin, leupeptin, and aprotinin, 0.1% bacitracin, 5 mg/ml N-p-tosyl-L-lysine chloromethyl ketone, and 1 mM phenylmethylsulfonyl fluoride) containing 100 pMI-NPY (2200 Ci/mmol, DuPont NEN), with or without competitor, at room temperature with shaking. The reaction was terminated with the addition of 50 µl of the reaction mix into 600 µl of ice-cold binding buffer B. The quenched reaction mixture was centrifuged at 4 °C for 20 min, the supernatant removed and the pellets were counted in a counter (Cobra, Packard Instrument Co.).

The CHO cells stably expressing the NPY receptor (CHO-hY2) in a 24-well tissue culture plate (5 times 10^5 cells/well) were incubated in 250 µl of binding buffer C (Dulbecco's modified Eagle's medium containing 0.1% BSA) with 100 pMI-PYY, with or without competitor, at room temperature for 1 h. The cells were then washed twice with 0.5 ml of ice-cold buffer C, suspended in 0.5 M NaOH, transferred to a counting tube, and counted in a counter. The competition binding and saturation binding data were analyzed by logistic.fit and hyperbola.fit programs of Sigma Plot, respectively.

Sequencing

The nucleotide sequence of both strands of the cDNA insert was determined by the dideoxy chain termination method (10) on an Applied Biosystems model 373A automated sequencer. The sequence data were analyzed using the GCG sequence analysis software package(11) .

Northern Blot Analysis

Human brain multiple tissue Northern blots (2 µg of poly(A) RNA/lane) were purchased from Clontech. The blots were probed using standard molecular biology techniques(12) . The BamHI/XbaI fragment of the pNPYR-398 cDNA clone described above was labeled to a specific activity of 5 times 10^8 cpm/µg with [alpha-P]dCTP by random priming to be used as a probe. Hybridization was at 42 °C in 5 times SSPE, 10 times Denhardt's solution, 100 µg/ml denatured salmon sperm, 50% formamide, and 2% SDS for 24 h with shaking. The membranes were washed twice in 2 times SSC and 0.05% SDS at room temperature, followed by one wash in 0.1 times SSC and 0.1% SDS at 50 °C for 40 min and autoradiographed.

Southern Blot Analysis

Approximately 12 µg of human genomic DNA digested with BamHI or EcoRI were loaded on an 0.8% agarose gel in TAE, electrophoresed at about 1.75-2.0 V/cm for approximately 36 h and transferred to a nitrocellulose membrane. Probe was prepared as described for the Northern blot. Hybridization was overnight at 37 °C in 4 times SSC, 1 times Denhardt's solution, 7 mM Tris-Cl, pH 7.4, 40% formamide, 10% dextran sulfate, and 100 µg/ml sheared herring sperm DNA with shaking. The membrane was washed thoroughly in 2 times SSC and 0.1% SDS at room temperature, followed by two 15-min washes in 0.1 times SSC and 0.1% SDS at 65 °C and autoradiography.

cAMP Assays

CHO-hY2 in six-well plates (1 times 10^6 cells/well) were preincubated for 10 min at 37 °C in media (Ham's F-12 containing 10% fetal bovine serum) containing 100 µM 3-isobutyl-1-methylxanthine. They were then treated with media (basal), forskolin (10 µM), peptide, or forskolin plus peptide (100 nM) for 10 min at 37 °C. The reaction was quenched by incubation in 1 N HCl for 30 min at 4 °C. Cellular debris was removed by centrifugation, and the samples were lyophilized. The cAMP content was determined by radioimmune assay (DuPont NEN). For experiments in which Galpha(i) was inhibited, cells were incubated overnight in media containing 100 ng/ml pertussis toxin.

Calcium Mobilization Assay

CHO-hY2 cells (1 times 10^6 cells/ml) were washed twice with phosphate-buffered saline and loaded with Fura-2AM (Molecular Devices) in buffer D (Hank's balanced salt solution, 1 mM CaCl(2), 1 mM MgCl(2), 25 mM Hepes, pH 7.5, and 0.2% BSA) for 45 min at 37 °C. The cells were then washed with buffer D, suspended at 3 times 10^5 cells/ml, and placed in a Spex spectrofluorometer with submersible magnetic stirrer attachment. A stable base line was established at 340 nm and 490 nm before the NPY (1 µM), NPY (1 µM), MCP-1 (5 µM), or galanin (5 µM) was added and the fluorescence measured. Fluorescence maxima and minima were determined using Triton X-100 (1%) and EGTA (10 mM), respectively. Calcium mobilized was calculated according to McCormack and Cobbold(13) ; the K(d) value used was 224 nmol/liter.


RESULTS AND DISCUSSION

Neuropeptide Y is a peptide hormone with diverse and important physiological effects in the central nervous system and periphery that are mediated through multiple receptor subtypes. In order to gain insight into the molecular mechanisms associated with these effects, we undertook the cloning of a human NPY Y2 receptor. A plasmid cDNA expression library was prepared from poly(A) RNA isolated from SMS-KAN cells, a human neuroblastoma cell line that expresses high levels of Y2 receptor(14) . Plasmid library DNA was transfected into COS-7 cells that are null for NPY binding; the cells were assayed for NPY receptor expression by I-PYY binding. Detection was by autoradiography. A single pool (pool 398) was identified that had increased levels of I-PYY binding. This pool was subdivided and the process was repeated until a pure cDNA clone (pNPYR-398) was isolated.

Binding of I-NPY and I-PYY to COS-7 cells transfected with pNPYR-398 was saturable and high affinity, with K(d) values of 0.58 and 0.27 nM, respectively. Specific binding to COS-7 cells transfected with vector (pcDNA1) was negligible. Scatchard analysis suggests that I-NPY bound to a single class of binding sites in COS-7 cells transfected with pNPYR-398 cDNA (data not shown).

To characterize the pharmacology of the NPY receptor encoded by pNPYR-398 cDNA, I-NPY (100 pM) binding to COS-7 cells transfected with the recombinant plasmid was competed with various NPY analogs (Fig. 1). PYY, NPY, and NPY were the most effective competitors of I-NPY binding, followed by the Y2-specific agonist NPY (Table 1). The Y1-specific agonist [Leu,Pro]NPY inhibited I-NPY binding only at high concentration with an IC of 0.5 µM. [D-Trp]NPY, a competitive antagonist of NPY in rat hypothalamus(15) , inhibited I-NPY binding with an IC of 2.9 µM. The rank order of potency of NPY and related peptides to COS-7 cells expressing the NPY receptor cDNA is PYY geq NPY geq NPY > NPY > NPY [Leu,Pro]NPY > [D-Trp]NPY. To further confirm the characteristics of pNPYR-398 NPY receptor, the potency of the NPY analogs to this receptor in competing for I-PYY to COS-7 cells transfected with pNPYR-398 was studied. The results were similar to those described for I-NPY binding (Table 1). These data are consistent with the previously reported pharmacology for the Y2 subtype of NPY receptors(3) .


Figure 1: Pharmacological characterization of a human NPY receptor expressed in COS-7 cells. Displacement of specific binding of I-NPY to membranes from COS-7 cells transfected with pNPYR-398. Binding was determined in the presence of the indicated concentrations of PYY (bullet), NPY (), NPY (box), NPY (circle), [Leu,Pro]NPY (), and [D-Trp]NPY (up triangle).





Fig. 2shows the nucleotide sequence of the pNPYR-398 cDNA and the deduced amino acid sequence for the encoded NPY Y2 receptor. The nucleotide sequence surrounding the initiation codon agrees well with Kozak's consensus sequences(16) . The receptor is 381 amino acids in length and has a molecular mass of approximately 42 kDa. Hydrophobicity analyses determined according to the method of Kyte and Doolittle (17) suggest the existence of seven transmembrane-spanning regions. There is a single putative N-linked glycosylation site in the N-terminal region. There is an aspartic acid in the putative second transmembrane region that is conserved in many G-protein-coupled receptors and frequently involved with signal transduction(18) . In the C-terminal tail there is a cysteine residue that might be involved in palmitolyation (19) as well as several serine residues that might be involved in regulatory phosphorylation. A long polyadenylation signal was found at the 3` end of the clone, suggesting that the cDNA clone encompasses most of the mRNA species observed in the Northern blot (Fig. 3).


Figure 2: Nucleic acid and amino acid sequence of a human Y2 receptor. The nucleotide sequence of clone pNPYR-398 and the deduced amino acid sequence for the encoded human NPY receptor, subtype 2 (Y2). The deduced amino acid sequence is shown under the nucleic acid sequence. The putative transmembrane regions I-VII are underlined; the borders of the regions were assigned based on hydrophobicity profile and comparison to the Y1 sequence. The asterisk (*) indicates a potential N-glycosylation site; the ``+'' indicates a potential palmitolyation site.




Figure 3: Northern hybridization. Northern blot analysis of human brain sections probed with a 1.9-kb fragment of pNPYR-398 cDNA that contained the entire open reading frame of the Y2 receptor. A, lane1, amygdala; lane2, caudate nucleus; lane3, corpus callosum; lane4, hippocampus; lane5, hypothalamus; lane 6, substantia nigra; lane7, subthalamic nucleus, lane 8, thalamus. B, lane1, cerebellum; lane2, cortex; lane 3, medulla; lane4, spinal cord; lane 5, occipital pole; lane 6, frontal lobe; lane7, temporal lobe; lane8, putamen. Molecular size markers are indicated in kb. The blots were also probed with P-labeled actin cDNA as a standard.



Comparison of the amino acid sequence of this NPY receptor to that of the human Y1 receptor (5) reveals significant differences. Overall, the two receptors are 31% identical at the amino acid level. Their sequences show the greatest similarity in the first intracellular loop and the second and sixth transmembrane regions, being 63%, 61%, and 50% identical, respectively. On the nucleotide level, the coding regions of the two NPY receptors were 47% identical. This relatively low homology to the Y1 receptor may explain why cloning with Y1 probes has not been fruitful. A search of the GenBank(TM) data base revealed that the Y2 nucleic acid sequence showed some similarity to the NK-2R neurokinin A receptor and two putative opioid receptors(20, 21, 22) .

The Y2 receptor is believed to be a common presynaptic receptor (2) and to be the predominant NPY receptor in the brain(23) . To study the distribution of the mRNA corresponding to the Y2 receptor encoded by pNPYR-398, Northern blots of poly(A) RNA from several parts of the brain were hybridized with a 1.9-kb fragment of the Y2 cDNA that contained the entire receptor open reading frame. A single 4-kb mRNA transcript was detected in the amygdala, caudate nucleus, corpus callosum, hippocampus, hypothalamus, and subthalamic nucleus (Fig. 3A), cortex, medulla, occipital pole, and frontal lobe (Fig. 3B). No hybridization was detected in RNA from peripheral tissues (data not shown). This suggests that either another form of the Y2 receptor is found in the periphery or that the Y2 mRNA transcript is present at levels undetectable by Northern analysis. In situ hybridization studies are in progress to address this question.

Southern blot analysis to human genomic DNA (Fig. 4) suggests that there is a single Y2 receptor gene. Since the Y2 receptor is known to be expressed in the periphery (1) these data support the hypothesis that either there is another form of the Y2 receptor in the periphery that is very different from the described here or the Y2 mRNA is present at very low levels.


Figure 4: Southern hybridization. Southern blot of human genomic DNA probed with the 1.9-kb fragment of Y2 receptor cDNA described in Fig. 3and washed under high stringency conditions. Each lane has approximately 12 µg of DNA. Molecular size markers are indicated in kb.



The truncated fragment of the pNPYR-398 clone containing the Y2 open reading frame described above was subcloned into pcDNA3 and transfected into CHO cells for stable expression. Competition binding experiments demonstrated a pharmacology identical to that seen in the COS-7 cells (Table 1). In neuroblastoma cells, the Y2 receptor appears to couple to multiple second messenger systems including the inhibition of forskolin-stimulated cAMP accumulation and calcium mobilization(24, 25) . To examine the functional coupling of the recombinant Y2 receptor to second messenger systems, the effect of NPY, [Leu,Pro]NPY, and NPY on cAMP and calcium mobilization was studied in the CHO-hY2 cells. Activation with 100 nM NPY, [Leu,Pro]NPY, and NPY inhibited forskolin (10 µM) stimulated cAMP accumulation by 72%, 7%, and 71%, respectively (Fig. 5A). When the cells were pretreated with pertussis toxin (100 ng/ml), the peptides did not inhibit the accumulation of forskolin-stimulated cAMP, suggesting that the Y2 receptor couples to Galpha(i). In addition, NPY did not inhibit the accumulation of forskolin-stimulated cAMP in CHO cells transfected with the pcDNA3 vector (data not shown).


Figure 5: Functional coupling of the recombinant Y2 receptor to second messenger systems. A, inhibition of forskolin (10 µM)-stimulated cAMP accumulation by NPY analogs in CHO-hY2 cells: lane 1, vehicle; lane 2, forskolin (10 µM); lane 3, NPY; lane 4, [Leu,Pro]NPY; lane 5, NPY, (lanes 3-5, peptides at 1 µM); lane 6, forskolin plus NPY; lane 7, forskolin plus [Leu,Pro]NPY; lane 8, forskolin plus NPY (lanes 6-8, forskolin at 10 µM and peptides at 100 nM). The cAMP values are expressed as a percentage of the cAMP level observed in the presence of forskolin (100%). B. Calcium mobilization by NPY analogs in CHO-hY2 cells: lane1, NPY (1 µM); lane2, NPY (1 µM); lane3, MCP-1 (5 µM); lane 4, galanin (5 µM). Three independent assays were performed for each ligand tested. The data represent an average of the two best experiments.



Activation of the CHO-hY2 cells with the NPY analogs NPY (1 µM) and NPY (1 µM) resulted in a 13-14-fold increase in intracellular calcium (Fig. 5B). Neither MCP-1 (5 µM) or galanin (5 µM) elicited a change in intracellular calcium levels. Although calcium mobilization is frequently associated with G-protein-coupled receptors linked to phospholipase C(26) , this is not necessarily the case(27) . The specific G-protein mediating the increase of intracellular calcium described here is not known at this time.

In summary, while NPY appears to be an important mediator of a wide variety of physiological functions, a clear understanding of how these functions are mediated has been hampered by the lack of cloned NPY receptors. The present cloning and pharmacological characterization of pNPYR-398 that encodes a human NPY Y2 receptor should facilitate the elucidation of the structure, function, regulation, and diversity of neuropeptide Y receptors as well as the identification of Y2 receptor-specific antagonists.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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) U32500[GenBank].

§
To whom correspondence should be addressed: Dept. of Microbial Molecular Biology, Bristol-Myers Squibb, Box 4000, Princeton, NJ 08543-4000. Tel.: 609-252-4238; Fax: 609-252-6813.

(^1)
The abbreviations used are: NPY, neuropeptide Y; Y1-Y3, NPY receptors, subtype 1-3; CHO, Chinese hamster ovary; MCP-1, monocytic chemotactic protein-1; kb, kilobase pair(s); BSA, bovine serum albumin; PYY, peptide YY.

(^2)
R. Seethala, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Michele Agler, Kenneth Carlson, Zahra Fathi, Stanley Krystek, Eric Parker, and Maria Webb for critical discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.