Cloning, functional expression, and developmental regulation of a neuropeptide Y receptor from Drosophila melanogaster.

Neuropeptide Y, peptide YY, and pancreatic polypeptide are homologous 36-amino acid peptides that differ from most other peptide transmitters by having a relatively rigid conformation in aqueous solutions, defined as the pancreatic polypeptide fold, and a critical C-terminal tyrosine amide. These peptides serve as gastrointestinal hormones and neurotransmitters. A cDNA encoding a novel G protein-coupled receptor activated by neuropeptide Y was cloned from Drosophila by use of degenerate oligonucleotide primers and polymerase chain reaction amplification of cDNA prepared from transcripts expressed early in embryogenesis. The cDNA encodes a protein of 449 amino acids with the characteristics of a G protein-coupled receptor and shares significant amino acid identity with mammalian tachykinin receptors. When expressed in Xenopus oocytes, the PR4 protein is activated by mammalian neuropeptides in the order: peptide YY greater than neuropeptide Y much greater than pancreatic polypeptide. Northern analysis showed that PR4 receptor is expressed at equivalent levels in adult Drosophila head and body and that the expression of the PR4 receptor is regulated during development. The molecular characterization of this receptor should lead to a better understanding of the functional role of this important family of hormone receptors in adult organisms and during development.

analysis showed that PR4 receptor is expressed at equivalent levels in adult Drosophila head and body and that the expression of the PR4 receptor is regulated during development. The molecular characteriztion of this receptor should lead to a better understanding of the functional role of this important family of hormone receptors in adult organisms and during development.
Neuropeptide Y (NPY),l peptide YY (PYY), and pancreatic polypeptide are homologous peptides that function as gastrointestinal hormones and neurotransmitters (1-4). NPY is often co-localized in nerves with noradrenaline (5, 6) and markedly potentiates vasoconstriction by noradrenaline (7), suggesting an important role in the control of blood pressure. NPY and PYY binding sites are particularly dense in limbic * This work was supported by the National Institutes of Health.
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The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M81490. structures of the mammalian brain (8,9), and NPY has been shown to affect memory and food intake (10,ll). Some of the cellular actions of NPY are mediated through G proteins (25) indicating that its receptors will likely belong to the family of G protein-coupled receptors that have seven transmembrane domains.

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We have been interested in the role of G protein-coupled signaling during Drosophila development (26) and, particularly, the transient expression of G protein-coupled receptors in early embryogenesis. For example, we have identified a Drosophila tachykinin receptor whose expression coincides with the beginning of neural development during early stages of embryogenesis (16). Here, we report the isolation of a cDNA clone that encodes a G protein-coupled receptor activated by NPY and PYY when expressed in Xenopus oocytes.

EXPERIMENTAL PROCEDURES
cDNA Cloning-First strand cDNA for PCR reaction was generated from poly(A)+ RNA samples using murine leukemia virus reverse transcriptase (Bethesda Research Laboratories). The 25-pl reaction contained 1 pg of poly(A)+ RNA, 10 pmol of oligo-dT, 10 units of RNasin (Promega), 300 units of murine leukemia virus reverse transcriptase, and 0.5 mM deoxynucleotide triphosphates. Following incubation at 37 "C for 1 h, 0.5 pl of each reaction was amplified in a 50-pl reaction containing 50 mM KC1, 10 mM Tris (pH 8.3), 1.5 mM MgCI', 200 p~ dNTPs, 10 mM dithiothreitol, 10 units of TaqI polymerase (Bethesda Research Laboratories), and 10 pmol of both the reverse and forward primers. Two degenerate oligonucleotides corresponding to amino acids relatively conserved in TM 111 and TM VI were used in the PCR. The sequence of the forward primer (TM 111) was GA GTC GAC CTG TGT(C) GC(T)C(G) ATG A(G)CT ITT(G) GAC A(C)GG(C) TAC. The sequence of the reverse primer (TM VI) was CAG AAT TCA GT(A)A GGG CAI CCA GCA GAI G(C)G(A)T(C) GAA. Drosophila embryonic cDNA served as template in 35 cycles of PCR with 1 min denaturing at 96 "C, 1 min annealing at 45 "C, and 1 min extension a t 72 "C. The PCR products were then digested with EcoRI and SalI, and a portion from 400-800 base pairs was extracted (Geneclean) and subcloned into vector pBS ("13) for sequence analysis. One clone, PR4, possessed a sequence characteristic of G protein-coupled receptors. Random-primed PR4 probe was then used to screen a Drosophila head XcDNA library at high stringency. Two positive clones were isolated. Partial sequence analysis and the digestion map showed they were identical. The whole sequence of one clone (PR4) was then obtained using Sequenase (U. S. Biochemical Corp.) following subcloning of restriction fragments into vector pBS ("13).
Oocyte Expression-RNA was transcribed and capped in uitro from PR4 cDNA clones using T7 RNA polymerase. Stage V and VI oocytes from mature female Xenopus laeuis were removed and prepared for experiments as described (21). Approximately 50 ng of RNA was injected into each oocyte. After incubation for 3-5 days at 18 "C, membrane currents were recorded at room temperature with a twoelectrode voltage clamp. Individual oocytes were placed in a chamber (500 p l ) and perfused with saline solution (ND96) (21). Electrodes contained potassium chloride (3 M; 500 k0). NPY and related compounds (obtained from commercial sources, except C2NPY and [ P~O~~I N P Y which were given by T. Schwartz, Novo Pharmaceuticals) were applied by superfusion.
Northern Analysis-Poly(A)+ RNA was isolated from adult head, body, and the indicated developmental stages using MagnaSpheres (Promega) according to the manufacturer's protocols. RNAs (10 pg/ lane) were separated on denaturing gels, transferred to nylon supports, and hybridized as described previously. Probes were generated by random priming of the entire cDNA for PR4.

RESULTS AND DISCUSSION
Two degenerate oligonucleotide primers corresponding to amino acids conserved in TM I11 and VI of most G protein-

ATG TIC TAC ATA GCT CAC CAG CAG CCG ATG CTG CGG M C GAG GAT GAT M C TAC CAG GW2 GGG TAC TTC ATC AGG CCG GAC CCC GCA TCC
ATT TAC M T ACC ACC GCA CTG CCA GCG GAC GAT G M GOO TCC M E TAT GGA TAT GGC TCC ACC ACA ACG CTC AGT GGC CTC CAG TTC GAG ACC 106

TAT M T ATC ACT GTG ATG ATG M C TTT AGC TCT GAC GAC TAT GAC CTT CTA TCG GAG GAC ATG TGG TCT'AGT GCC TAC TTT
I TAc ATG mc TIC ATT ccc A?c TTT ATc nc cEc CTG GGc M c GGA AcG &c xic TAT ATc mc TAT K c AcA ccT C o c k CGC mc 312
ATT  The numbers beside each curue indicate the number of oocytes tested at that concentration. Holding potential was -60 mV. Each oocyte was tested with only one concentration in this experiment because of the marked and variable desensitization that was observed with repeated applications. PP, pancreatic polypeptide. coupled receptors were used to amplify members of this receptor family in cDNA prepared from transcripts expressed in 2-6-h Drosophila embryos. Sequence analysis of one subcloned PCR product showed that it represented a novel G protein-coupled receptor. This fragment was then used to probe a XcDNA library made from adult Drosophila heads. Restriction mapping and partial sequence analysis indicated that the two clones identified were identical. One clone, XPR4, was further characterized. Fig. 1 shows the restriction map, nucleotide sequence, and derived amino acid sequence of the XPR4 cDNA and protein.

GCC CGT TTC AGG AGC GGA TTC GTC CAG CTG AT0 CAC COT ATG CCC GGC CTG CGT CcC TGG TGC TGC CTG CGG AGC GTC GGC GAT CGC ATG M C 1209
The longest open reading frame encodes a 449-amino acid protein with a relative molecular mass of 49.3 kDa. Hydropathy analysis of this protein indicated seven stretches of hydrophobic amino acids forming potential transmembrane domains characteristic of G protein-coupled receptors (data not shown). Four potential asparagine-linked glycosylation sites (Fig. 1B) are found at the N terminus preceding TM I. Several serine and threonine residues are present in the third intracellular loop and in the C-terminal cytoplasmic tail that are potential phosphorylation sites (12). The PR4 protein also possesses a number of residues conserved in most G proteincoupled receptors (Fig. IC), such as the Asp-Arg-Tyr sequence in the second intracellular loop, cysteine residues in the first and second extracellular loops that may participate in a structurally important disulfide bonds, and prolines in TM VI and VII. PR4 protein shares significant homology with the tachykinin receptors (Fig. 1C). From residue 79 to 390, PR4 is 32-34.5% identical to mammalian (13-15) and Drosophila (16) tachykinin receptors. Unlike tachykinin receptors, PR4 protein does not have a His residue in TM VI; instead, PR4 possesses two His residues in the TM VII. Moreover, unlike most G protein-coupled receptors, the PR4 protein does not have a cysteine in the C-terminal intracellular domain that could serve as a substrate for palmitoylation.
These considerations suggested that the PR4 cDNA encodes a G protein-coupled receptor that is activated by a peptide. The cRNA was injected into Xenopus oocytes, and voltage clamp recordings were made 3-5 days later; of several peptides applied (Fig. 2), only NPY and PYY had any effect. These peptides evoked inward currents typical of those induced by other agonists that activate a calcium-dependent chloride current. No responses were obtained in the same oocytes with NPY free acid; this analog, which lacks the Cterminal amide of NPY, does not share the biological activity of NPY. PYY was more potent than NPY, and pancreatic polypeptide had almost no effect (Fig. 2); this order of potency is typical for the actions of these peptides on mammalian tissues (17). Peptides that had no effect on oocytes expressing PR4 protein included ( n = 3 or 5) neuromedin K (4 p~) , kassinin ( (18), was as effective as NPY in activating the PR4 receptor (Fig. 2), and the analog NPY   (1 p~) also caused an inward current of 500 pA in three of four oocytes tested; these two analogs are somewhat Y2selective (17). On the other hand, the substitution of Pro for Gln in NPY [ P~o~~] N P Y reduces binding to Y2 receptors but has no effect on the binding of this compound to Y1 receptors; as shown in Fig. 2, [ P~O~~I N P Y was a relatively poor agonist at the PR4 receptor. These data suggest that the PR4 receptor is more similar to the Y2 than the Y1 mammalian receptor, but it is possible that differences between Drosophila and mammalian receptor may confound the pharmacological classification. The inward current evoked by activation of the PR4 receptor in the oocyte is similar to that elicted by other transmitters known to stimulate phosphatidylinositol metabolism (e.g. . Activation of NPY receptors in mammalian cells has also been shown to increase phosphatidylinositol turnover (19,20). However, it is not clear whether the Y1 or Y2 receptor type is normally associated with this transduction pathway.
Expression of the PR4 receptor was assessed by Northern analysis of poly(A)+ RNA prepared from adult head and body. A dominant 2.4-kilobase transcript appeared to be equally abundant in both head and body (Fig. 3A). Two weakly hybridizing bands (about 2.8 and 1.5 kilobases) were also present. These transcripts may represent alternative processing of a common RNA precursor for PR4 or transcripts from distinct yet highly related receptor genes. During development, the level of PR4 is subject to precise regulation. Northern blot (Fig. 3B) and PCR analysis (not shown) indicate that PR4 is expressed at low levels during early embryonic stages (0-10 h). The expression of PR4 receptor increases later (10-14 h) and reaches the highest level during late stages of embryogenesis (14-18 h). This pattern of expression during embryogenesis corresponds to periods of rapid neuronal proliferation and differentiation. Subsequently, PR4 levels are reduced during larval stages and increased again during pupal (13) (or substance K, SKR), and NK3 (14) (or neuromedin K, NKR) receptors. Identical residues are boxed. Dashes indicate deletion of amino acid residues. Dots indicate the position of amino acid residues conserved in most G protein-coupled receptors. Likely transmembrane domains I-VI1 are indicated. bp, base pairs. stages. The location of the gene encoding PR4 was identified by in situ hybridization to salivary gland chromosomes. Hybridization was present a t position 9731,2 on the right arm of the third chromosome (data not shown).

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Most G protein-coupled receptors of Drosophila show quite high homology to their mammalian counterparts (see for example, Refs. 22-24); therefore, the PR4 cDNA should be useful for the isolation of mammalian receptors for NPY and related peptides. It has been very difficult to resolve subclasses of the family of receptors activated by NPY and PYY, and transfected cells expressing individual molecular species of NPY receptor will be particularly helpful in the development of selective ligands. Such ligands, particularly receptor antagonists, are needed to determine the functional role of this class of the peptides. Recently, the development and anatomic distribution of specific populations of neurons that contain NPY immunoreactive peptides has been described in Drosophila.2 Thus, the genetic approaches possible in Drosophila will also facilitate the study of the functional role of NPYrelated peptides and their receptors in development.