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CELLULAR AND MOLECULAR

Neuropeptide Y Y4 Receptor Homodimers Dissociate upon Agonist Stimulation

Eli Lilly and Company, Lilly Research Laboratories, LCC, Indianapolis, Indiana

Received June 23, 2003; accepted August 28, 2003.

	Abstract

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The pancreatic polypeptide-fold family of peptides consists of three 36-amino acid peptides, namely neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP). These peptides regulate important functions, including food intake, circadian rhythms, mood, blood pressure, intestinal secretion, and gut motility, through four receptors: Y1, Y2, Y4, and Y5. Additional receptor subtypes have been proposed based on pharmacology observed in native tissues. Recent studies with other G-protein-coupled receptors have shown that homo- and heterodimerization may be important in determining receptor function and pharmacology. In the present study, the recently cloned rhesus (rh) Y4 receptor was evaluated using radioligand binding, and the pharmacological profile was found to be very similar to the human Y4 receptor. To study homo- and heterodimerization involving the Y4 receptor using bioluminescence resonance energy transfer 2 (BRET²), the carboxy termini of the rhesus Y1, Y2, Y4, and Y5 receptors were fused to Renilla luciferase, and rhY4 was also fused to green fluorescent protein. Dimerization was also studied using Western blot analysis. Using both BRET² and Western analysis, we found that the rhY4 receptor is present at the cell surface as a homodimer. Furthermore, agonist stimulation using the Y4-selective agonists PP and 1229U91 can dissociate these dimers in a concentration-dependent manner. In contrast, rhY4 did not heterodimerize with other members of the NPY receptor family or with human opioid and µ receptors. Therefore, homodimerization is an important component in the regulation of the Y4 receptor.

Lately, it has become evident that GPCRs can form homo- and heterodimers (Devi, 2001) and that dimer formation can be essential for and modify receptor function, as shown for the opioid receptors (Jordan and Devi, 1999). The most well known case is perhaps the GABA-B receptor, where two receptors (GABA-B1 and -B2) are needed to form a functional unit (Jones et al., 1998). Furthermore, opioid (Cvejic and Devi, 1997; Jordan and Devi, 1999; McVey et al., 2001) and the ₂ adrenergic (Angers et al., 2000) receptors have been found to form homodimers as well as a heterodimer (McVey et al., 2001). Some other examples of GPCR homodimers are bradykinin B2 (AbdAlla et al., 1999) and muscarinic M3 (Zeng and Wess, 1999) receptors. Heterodimers between bradykinin B2 and angiotensin AT1 (AbdAlla et al., 2000) as well as somatostatin SSTR5 and dopamine D2 receptors (Rocheville et al., 2000) have also been reported. Therefore, it is important to explore the potential for homo- and heterodimers within the NPY receptor family.

A number of methods have been used to address protein-protein interactions. One of the more recent is bioluminescence resonance energy transfer (BRET), a natural process that occurs in many organisms that emit light (Xu et al., 1999). When luciferase catalyzes the reaction coelenterazine coelenteramide, blue light [ = 410 nm for Renilla luciferase (RLUC) catalyzing the modified version of coelenterazine, DeepBlueC in this study] is emitted. If present in close proximity (within 100 Å; Xu et al., 1999), green fluorescent protein (GFP) can act as an acceptor for the blue photon and re-emit light in the green spectra ( = 515 nm for GFP²). When attached to proteins, GFP and RLUC can be used to investigate almost any protein-protein interaction, and several groups have employed it for studies of GPCR dimerization (see Angers et al., 2002 for a review) but also for other applications (Xu et al., 1999; Boute et al., 2001; Germain-Desprez et al., 2003).

Recently, we cloned the rhY4 receptor and utilized BRET² to study agonist-induced -arrestin 2 interaction in the NPY receptor family (Berglund et al., 2003b). In the present study, we explored the ability of the rhY4 receptor to form homo- and/or heterodimers with the other members of the NPY receptor family. In addition, we evaluated the effects of agonist stimulation on dimer stability.

	Materials and Methods

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Generation of Expression Constructs. Based on the recently published sequence of the rhY4 receptor (Berglund et al., 2003b), one forward primer was synthesized: rhY4fH containing a HindIII site with the sequence 5'-AAGCTTAAGCTTACCATGAACACCTCTCACCTCCT-3' and two reverse primers, rhY4rKS with the sequence 5'-CCGCGGTACCTTAAATGGGATTGGACCT-3' containing a KpnI and a SacI site and rhY4rNSKS (5'-CCGCGGTACCAATGGGATTGGACCTGC-3') also containing a KpnI and a SacI site but lacking the stop codon to make the carboxy terminally tagged constructs. The primer pair rhY4fH + rhY4rKS were used in a PCR reaction using genomic rhesus DNA as template. A band of 1.1 kilobase was generated and gel purified. The PCR product and the vectors pGFP²-C3, pRLUC-C1 (PerkinElmer Life Sciences, Boston, MA), and pcDNA3.1(+) (Invitrogen, Carlsbad, CA) were cut using restriction enzymes HindIII and KpnI (10 U of each for 2 h at 37°) and gel purified. Subsequently, the cut PCR fragment was ligated into the three different vectors, cut as described above, to generate the constructs GFP²-rhY4, RLUC-rhY4, and wild-type (WT) rhY4. The primer pair rhY4fH + rhY4rNSKS and the vectors pGFP²-N2 and pRLUC-N2 were treated similarly to generate the carboxy terminally tagged constructs rhY4-GFP² and rhY4-RLUC. At least four clones of each construct were sequenced fully to confirm the correct sequence.

The Y1, Y2, and Y5 receptors from the rhesus monkey have been cloned previously (Gehlert et al., 2001). Using clones containing the coding sequence of the rhesus Y1 and Y2 receptors and the same genomic DNA as above as template for the rhY5 receptor and the primer pairs: rhY1.fH (5'-AAGCTTAAGCTTACCATGAATTCAACATTATTTTCCCAG-3') and rhY1.NSrKS (5'-CCGCGGTACCGATTCTTTCATTATCATCATTGTTG-3'); rhY2.fH (5'-AAGCTTAAGCTTACCATGGGTCCAATAGGTACAGAGG-3') and rhY2NSrKS (5'-CCGCGGTACCGACATTGGTAGCTTCTGTGAAAG-3'); rhY5.fH (5'-AAGCTTAAGCTTACCATGGATTTAGAGCTCGATGAAT-3') and rhY5.NSrKS (5'-CCGCGGTACCCATATGAAGACAGTGTATAAGGGAC), the coding sequence of these receptors were amplified by PCR and cut and ligated into plasmid pRLUC-N2 as described for rhY4, generating the constructs rhY1-RLUC, rhY2-RLUC, and rhY5-RLUC. A fragment containing human opioid receptor was amplified from a plasmid using the primers hDEL.fH (5'-AAGCTTAAGCTTACCATGGAACCGGCCCCCTCC-3') and hDEL.NSrKS (5'-CCGCGGTACCGGCGGCACGGCCACC-3'), cut and ligated into pRLUC-N2 to generate hDELTA-RLUC. Similarly, the human opioid µ receptor was amplified using the primers hMU.fH (5'-AAGCTTAAGCTTACCATGGACAGCAGCGCTGCC-3') and hMU.NSrKS (5'-CCGCGGTACCGGGCAACGGAGCAGTTTCTG-3') to generate hMU-RLUC.

Expression of NPY Receptor Family in Human Embryonic Kidney (HEK)293 Cells. HEK293 cells were grown in 90-mm dishes in a Dulbecco's modified Eagle's medium:Ham's F-12 medium (3:1) mix (Invitrogen) supplemented with 5% fetal bovine serum, 20 mM HEPES, and penicillin streptomycin (Invitrogen) at 37°C in 5% CO₂. Two micrograms of DNA from the constructs rhY4-GFP and rhY4 (WT) as well as the uncut pGFP²-N2 and the control vector BRET+ (PerkinElmer Life Sciences) were individually transfected into HEK293 cells using 5 µl of FuGENE6 (Roche Diagnostics, Indianapolis, IN) diluted in 145 µl of OptiMEM (Invitrogen) according to the instructions of the manufacturer. Seventy-two hours later, the media was replaced with media containing Zeocin (100 µg/ml; Invitrogen) for the GFP² constructs and G418 (500 µg/ml; Invitrogen) for the RLUC and WT [in pcDNA3.1(+)] constructs. After 2 to 3 weeks, individual clones were picked using a sterile pipette tip. Clones expressing rhY4-GFP² or cytosolic GFP² were selected based on fluorescence intensity using an inverted fluorescent microscope and subsequently sorted into six-well plates, whereas rhY4 (WT)-expressing clones were randomly picked from the plate. Subsequently, clones were tested for receptor expression using ¹²⁵I-hPP (PerkinElmer Life Sciences) as radioligand, as described below.

Receptor Binding Studies. HEK293 cells stably expressing the rhesus Y4 (WT) receptor and a cell line expressing the recombinant rhesus Y4-GFP² receptor protein were washed once with phosphate-buffered saline (PBS) and pelleted in fresh PBS. Radioligand binding assays were conducted on isolated crude membrane homogenates as previously described (Gehlert et al., 1992) using ¹²⁵I-hPP as radioligand. Nonspecific binding was defined as the amount of radioactivity remaining on the filter after incubating in the presence of 0.1 µM human (h)PP (American Peptide Co., Inc., Sunnyvale, CA). Various concentrations of peptides and peptide analogs (American Peptide Co., Inc.) or 1229U91 (Eli Lilly & Co., Indianapolis, IN) were added to the incubations to determine binding affinity. For saturation binding analysis, HEK293 cell homogenates containing the wild-type and GFP²-tagged Y4 receptors were incubated with 12 different concentrations of ¹²⁵I-hPP for 2 h at room temperature. The results were analyzed using the Prism software package (GraphPad Software Inc., San Diego, CA). Protein concentrations were measured using Coomassie Plus protein assay reagent (Pierce Biotechnology, Rockford, IL) using bovine serum albumin standards.

Agonist-Induced GTPS Binding. Binding of [³⁵S]GTPS (PerkinElmer Life Sciences) was determined as described previously (Xu et al., 2001). Briefly, 96-well Costar plates received 50 µl of buffer, 50 µl of drug, 50 µl of [³⁵S]GTPS (final concentration 100 pM), and 50 µl of cell membranes (50 µg of protein). The final concentrations of reagents in the [³⁵S]GTPS-binding assays were 50 mM Tris-HCl, pH 7.4, containing 100 mM NaCl, 10 mM MgCl₂, and 0.1% bovine serum albumin. Incubations were conducted for 3 h at 25°C (steady state). A scintillation proximity assay using wheat germ agglutinin beads was used to detect [³⁵S]GTPS binding. Basal activity was defined as binding in the absence of agonist.

BRET² Studies of Coexpressed GFP- and RLUC-Tagged Receptors. HEK293 cells of the stable cell line rhY4-GFP²were plated out in six-well dishes (30-mm diameter; Fisher Scientific Co., Pittsburgh, PA). At about 40 to 50% confluence, the cells were transfected with 1 µg of each of the following constructs: rhY1-RLUC, rhY2-RLUC, rhY4-RLUC, rhY5-RLUC, hDELTA-RLUC, hMU-RLUC, or 250 ng of pRLUC-N2 using 3 µl of FuGENE6 diluted in 97 µl of OptiMEM (Invitrogen) according to the instructions of the manufacturer. After 72 h, the cells from each well were detached by washing with 1 ml of ice-cold PBS using a pipette tip, transferred to a 1.5-ml eppendorff tube, spun in a microcentrifuge at 5000 rpm for 2 min, and resuspended in 150 µl of PBS (about 10,000 cells/µl). Cell suspension (25 µl) was dispensed into each well of a 96-well plate (OptiPlate96, white; PerkinElmer Life Sciences). Immediately before counting the plate in a Fusion instrument (PerkinElmer Life Sciences), 25 µl of modified coelenterazine (DeepBlueC; PerkinElmer Life Sciences) diluted 1:100 in PBS was added (final concentration 5 µm). The emission from each well was counted at = 410 nm (RLUC optimum) and = 515 nm (GFP² optimum). The BRET² ratio for each sample was calculated as follows: (sample_{515 nm} – BG_{515 nm})/(sample_{410 nm} – BG_{410 nm}) – baseline. The baseline signal was defined as the BRET² ratio from the stable rhY4-GFP² cell line cotransfected with pRLUC-N2 (i.e., Renilla luciferase expressed in the cytosol). The average of four wells with untransfected HEK293 cells was used to define background (BG). Each sample was run in quadruplicate, and the ratio was calculated for each replicate. As a positive control, cell extract from a stable cell line expressing a cytosolic fusion protein (BRET+; PerkinElmer Life Sciences) consisting of luciferase in the amino terminus and GFP² in the carboxy terminus was run in every experiment. Two non-NPY receptors (human opioid and µ) were also tagged with RLUC and tested for dimer formation with the rhY4 receptor. The levels of rhY4-GFP² in each assay were determined by adding 25 µl of cell suspension to a black 96-well polypropylene plate (Nalge Nunc International, Naperville, IL), and the fluorescence was assessed using the Fusion instrument (excitation = 425 ± 20 nm, detection = 515 ± 30 nm).

Effect of Agonist Stimulation on Cells Coexpressing Y4-GFP² and Y4-RLUC. Cells in six-well plates were transfected as described above. These experiments were carried out 48 to 72 h post-transfection. One hour prior to the experiment, 1 ml of media containing hPP, 1229U91, hNPY, hPYY, or h[D-Trp³²]NPY was added to the cells. The Y4 agonists hPP and 1229U91 were tested at final concentrations ranging from 1 pM to 1 µM, whereas hNPY, hPYY, or h[D-Trp³²]NPY were all tested at a single 100 nM concentration. Subsequently, the cells were detached, spun at 5000 rpm, resuspended in PBS, and the luminescence and fluorescence were assessed using the Fusion instrument as described above. A time course study was also performed by adding 100 nM hPP at various times before the BRET² ratio was assayed. The effects of concanavalin A type III (0.5 mg/ml; Sigma-Aldrich, St. Louis, MO), an inhibitor of clathrin-mediated internalization, and cycloheximide (100 µM; Sigma-Aldrich), a protein synthesis inhibitor, were also investigated by addition to the media 30 min prior to BRET² assay.

Chemical Cross-Linking and Western Blot Analysis. GFP²-tagged Y4 receptors stably expressed in HEK293 cells were grown to confluence in six-well plates. Prior to cross-linking, some cells were treated with various concentrations of hPP for1hat37°C. Following agonist stimulation, the cells were washed twice with ice-cold PBS. Subsequently, the cells were resuspended in ice-cold PBS containing 2 mg/ml bis[sulfosuccinimidyl]suberate (BS³; Pierce Biotechnology) and incubated at 8°C on a rocking platform. After 1 h, the crosslinker was quenched with the addition of 50 mM Tris for 15 min. The cells were pelleted, resuspended in NuPAGE lauryl dodecyl sulfate sample loading buffer (141 mM Tris Base, 106 mM Tris HCl, 2% lauryl dodecyl sulfate, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, 0.175 mM Phenol Red, and 10% glycerol, pH 8.5) (Invitrogen), and heated for 5 min at 95°C. Electrophoresis was conducted using a Xcell II mini cell (Invitrogen) with 7% NuPAGE Tris-acetate SDS-polyacrylamide gel electrophoresis gels (Invitrogen). Proteins were transferred onto polyvinylidene difluoride membranes using an Xcell II Blot Module (Invitrogen). The presence of GFP²-tagged rhY4 receptors was detected using a 1:4000 dilution of a mouse monoclonal GFP antibody (Roche Diagnostics) and a mouse WesternBreeze chemiluminescent kit (Invitrogen). The gel was opposed to a Hyperfilm (high-performance chemiluminescence film; Amersham Biosciences Inc., Piscataway, NJ) for 30 s. The film was developed, and images were evaluated by densitometry using the MCID Elite 6.0 software (Imaging Research, St. Catherines, ON, Canada).

	Results

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Sequence Analyses of the rhY4 Receptor. The rhY4 receptor was cloned using PCR with primers based on the flanking sequences of the hY4 gene, and the full-length sequence has been submitted to GenBank with accession number AY149475 [GenBank] . The coding nucleotide sequence differs from that of the hY4 gene in 45 to 47 positions and encodes a 375-amino acid protein (i.e., the same length as the human receptor) that differs from the human amino acid sequence in 15 or 16 positions. Four polymorphic positions were found. Six clones contained the sequence variant (C⁷⁰,T⁹⁶,T³⁴⁸, and G⁷¹⁶), and six contained the sequence variant (T⁷⁰, C⁹⁶, C³⁴⁸, and T⁷¹⁶). One of the polymorphisms resulted in a replacement of a polar amino acid to a highly hydrophobic amino acid; Arg²³⁹ (the same amino acid as in human Y4) leucine. This position is located at the beginning of the third intracellular loop, right after transmembrane domain 5. The impact of this replacement in the third intracellular loop was tested both in binding and in dimerization assays but did not appear to cause a change in function of the receptor (data not shown). The pharmacology and dimerization data presented in this paper are derived from the variant that is most similar to the human receptor (Arg²³⁸ = C⁷⁰, T⁹⁶, T³⁴⁸, and G⁷¹⁶).

Expression and Pharmacological Characterization. The rhY4-GFP² and rhY4 (WT) receptors expressed stably in HEK293 cells bound ¹²⁵I-hPP according to a saturable onesite model with dissociation constants (K_d) of 65 ± 2.6 and 31 ± 1.7 pM, respectively, and B_max values of 17709 ± 326 and 1593 ± 31 fmol/mg protein, respectively. A very high rhY4-GFP²-expressing cell line was selected to maximize the fluorescent signal for subsequent BRET² studies. Eight different peptides were tested for inhibition of ¹²⁵I-hPP binding at the rhY4-GFP² and rhY4 (WT) receptor constructs, and the results are presented in Table 1. In brief, the peptides bound to the rhY4 (WT) receptor with affinities and a relative rank order of potencies indistinguishable from that of the cloned hY4 receptor (Lundell et al., 1995). The correlation (r²) between the GFP²-tagged receptor to the wild type was 0.97 (Fig. 1), but the affinities were slightly lower than the rhY4 (WT) receptor. The difference was smaller for the endogenous high-affinity agonist hPP about two-fold in agreement with the difference in K_d values seen for the radioligand. Functional coupling to G-proteins for the rhY4 (WT) receptor stably expressed in HEK293 cells was confirmed by [³⁵S]GTPS binding. Human PP increased binding in a concentration-dependent manner with a pEC₅₀ value of 8.01 ± 0.16 nM (n = 3; Fig. 2). HEK293 cells expressing the rhY4-GFP² chimeric protein were subjected to the same conditions but no agonist effect on [³⁵S]GTPS binding could be monitored in these cells.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Correlation between rhY4-GFP2 receptors expressed in HEK293 cells and the rhY4 (WT) receptors expressed in HEK293 cells. The correlation (r2) between the pKi for rhY4-GFP2 and the pKi for rhY4 (WT) was 0.97.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Agonist-induced G-protein coupling of the rhY4 receptor stably expressed in HEK293 cells. Various concentrations of hPP were incubated with 100 pM [35S]GTPS and 50 µg of tissue for 60 min. Following this incubation, 1 mg of wheat germ agglutinin-coated scintillation beads was added, and the plate was counted on a Wallac MicroBeta (PerkinElmer Wallac, Gaithersburg, MD). The data are presented as mean ± S.E.M. from three experiments performed in duplicate.

BRET² Studies of Coexpressed GFP²- and RLUC-Tagged Receptors. The BRET² ratios were assayed for the cloned rhY4 receptor coexpressed with the rhesus Y1, Y2, and Y5 receptors as well as the human - and µ-opioid receptors. The expression of the GFP²-tagged rhY4 receptor (as determined by GFP² fluorescence) remained the same no matter which RLUC-tagged construct was coexpressed with it. The expression levels of each of the RLUC-tagged receptors were assayed by chemiluminescence. The Y2, Y4, , and µ receptors tagged with RLUC achieved equivalent expression levels, whereas the RLUC-tagged Y1 and Y5 receptors were routinely expressed at levels two- to three-fold lower than the Y2, Y4, , and µ receptors. The Y1, Y2, and Y5 receptor subtypes showed similar BRET² ratios as the less related opioid and µ receptors of 0.035 to 0.065, whereas coexpression of rhY4-GFP² and rhY4-RLUC gave a value of 0.169 ± 0.009 (Fig. 3). As a positive control, BRET+, a cytosolic fusion protein with RLUC in the N terminus and GFP² in the C terminus, was used. Homogenate from a cell line stably expressing BRET+ gave a ratio of 0.50 ± 0.01 (n = 9).

View larger version (11K): [in this window] [in a new window]   Fig. 3. BRET2 ratios for the rhY4 receptor homodimer compared with heterodimers between rhY4 and rhesus Y1, Y2, and Y5 and human opioid receptors and µ. One microgram of each of the constructs expressing the RLUC-tagged receptors indicated were transiently transfected into HEK293 cells that stably expressed the rhY4-GFP2 receptor. Ratios are expressed as mean ± S.E.M. from five to nine experiments performed in quadruplicate.

Effect of Agonist Stimulation on Cells Expressing rhY4-GFP² and rhY4-RLUC. To assess the effects of agonist stimulation, cells expressing rhY4-GFP² and rhY4-RLUC were incubated in the presence of the Y4 agonists hPP and 1229U91. The addition of hPP into the growth media produced a decrease in the BRET² ratio. A time course study revealed that the maximal decrease was reached within 15 min after addition of hPP at a final concentration of 100 nM (Fig. 4a). Both hPP and 1229U91 lowered the BRET² ratio in a concentration-dependent fashion (Fig. 5), with EC₅₀ values of 2.9 and 14 nM, respectively. In these experiments, the top BRET² ratio for hPP was 0.167 ± 0.010 and the bottom was 0.077 ± 0.005 (n = 9), whereas the corresponding values for 1229U91 were 0.163 ± 0.016 and 0.072 and 0.008 (n = 6), respectively. In contrast, no inhibition was detected when 100 nM hNPY, hPYY, or the Y5-selective agonist h[D-Trp³²]NPY was added to the growth media (Fig. 5). Concanavalin A, an inhibitor of clathrin-mediated internalization, and cycloheximide, a protein synthesis inhibitor, did not affect the agonist-induced reduction in BRET² ratio (Fig. 4b). With both inhibitors, the reduction in BRET² ratio was similar to that observed with the nontreated cells (45–49% reduction). A small increase in both the unstimulated and agonist-stimulated BRET² ratios was observed with cycloheximide pretreatment and a decrease with concanavalin A pretreatment compared with untreated cells (Fig. 4b, columns 1 and 2), possibly due to cell toxicity.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Dissociation of rhY4 homodimers by agonist stimulation. A, the effect of 100 nM hPP added to growth media 15, 30, 60, and 180 min prior to BRET2 assay. Maximal effect was observed within 15 min of stimulation. Data are presented as mean ± S.E.M. (n = 3). B, effect of inhibitors on agonist-induced dissociation of Y4 homodimers. Concanavalin A, an inhibitor of clathrin-mediated internalization (0.5 mg/ml; columns 3 and 4) and cycloheximide, a protein synthesis inhibitor (100 µM; columns 5 and 6) were added to the growth media 30 min before agonist (hPP at 100 nM). BRET2 ratio was assayed 60 min after agonist stimulation. Data are presented as mean ± S.E.M. (n = 3).

View larger version (16K): [in this window] [in a new window]   Fig. 5. Concentration-response effect of agonist-induced dissociation of the rhY4 receptor homodimer. Normalized BRET2 ratios after 60-min stimulation by the Y4 agonists hPP (n = 9) and 1229U91 (n = 6) demonstrate a concentration-dependent dissociation of the rhY4 homodimer. In contrast, 100 nM hPYY, hNPY, and h[D-Trp32]NPY did not reduce the BRET2 signal. Data are presented as mean ± S.E.M.

Western Blot Analysis of rhY4-GFP² Receptors. Rhesus Y4 receptors tagged with GFP² were chemically crosslinked using the membrane-impervious cross-linker, BS³. Subsequent analysis of the unstimulated BS³ treated cells by nonreducing SDS-polyacrylamide gel electrophoresis and Western blotting using a GFP-specific antibody revealed two large molecular weight protein complexes, 80- and 160-kDa with relative optical densities of 0.93 and 0.86, respectively (Fig. 6, lane 1). The 80-kDa complex was the only band detected when the cross-linking reagent was omitted (Fig. 6, lane 2). The addition of 1 µM hPP produced almost a 2-fold decrease in the 160-kDa band (relative optical density 0.54) (Fig. 6, lane 4).

View larger version (39K): [in this window] [in a new window]   Fig. 6. Western blot analyses of HEK293 cells stably expressing rhY4-GFP2. The cells were detached, cross-linked with the membrane-impermeable agent BS3 (lanes 1, 3, and 4 and the controls). Two bands, one at MW = 160 kDa (dimerized and glycosylated rhY4-GFP2) and one of 80 kDa (monomer), are visual after BS3 incubation. Without BS3, the 80-kDa band is the only visible band. Three controls were run on the same gel: HEK293 cells stably expressing the rhY4 receptor without GFP (negative control) (lane 5), GFP2 without receptor (lane 6), and the positive control RLUC-GFP2 construct (BRET+) (lane 7). Western blot analysis was performed, and the rhY4-GFP2 receptors were detected using a GFP antisera.

	Discussion

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Our group has recently cloned the Y1, Y2, Y5 (Gehlert et al., 2001), and Y4 (Berglund et al., 2003b) receptors from the rhesus monkey. In this paper, we discuss the detailed characterization of the rhY4 receptor with regards to pharmacology and dimerization. In agreement with what is known about the Y4 receptor in other mammals (Eriksson et al., 1998), the rhY4 receptor has evolved much more rapidly than the other NPY receptors as the protein sequence differed from the hY4 receptor in 15 positions compared with 2, 4, and 2 for the Y1, Y2, and Y5 receptors, respectively. Four positions of the rhY4 gene were found to be polymorphic. One of the polymorphisms resulted in an amino acid replacement (Arg²³⁹/Leu²³⁹), located in the third intracellular loop, whereas the other three were silent. Interestingly, the hY4 gene has also been found to be highly polymorphic, as no less than seven positions in the coding region can differ between individuals (GenBank accession number XM_011916).

The pharmacology of the rhY4 receptor (Arg²³⁹) was found to be indistinguishable from that of the hY4 (Bard et al., 1995; Lundell et al., 1995), suggesting similar ligand recognition domains. The wild-type rhY4 receptor was also found to couple to G-protein (Fig. 2), demonstrating that the receptor is functional when stably expressed in HEK293 cells. On the other hand, the GFP²-tagged rhY4 receptor bound ¹²⁵I-hPP with slightly lower affinity than the wild-type receptor, and there was some reduction in potency for the peptide inhibitors tested. Nevertheless, these peptides had the same rank order of potencies as observed with the native receptor, suggesting that the presence of GFP² at the C terminus of the receptor may affect agonist affinity, but the pharmacological properties remain intact. However, the lack of agonist effect on [³⁵S]GTPS at the rhY4-GFP²-expressing cells suggests that the G-protein coupling is impaired in these cells. One possibility is that the presence of the 239-amino acid GFP² molecule attached to the C terminus of the receptor affects coupling to G-proteins and thus shifts the equilibrium of the receptor-pool toward more receptors being in low-affinity conformation with regards to agonist binding and with a low ability to couple to G-proteins. Similarly, the very high expression of the rhY4-GFP² receptor may lead to a high receptor-to-G-protein ratio, which may also result in uncoupled receptors.

Some of the first convincing pieces of evidence for dimerization or oligomerization of GPCRs came from studies using "domain swapping" between receptors (Maggio et al., 1993). Dimers between receptors of most of the major GPCR families have now been found (Gomes et al., 2001). Thus, it appears that dimerization of GPCRs is a general and important feature among these receptors. In the present study, we evaluated the ability of the rhY4 receptor to form dimers using BRET² technology. The main advantage of BRET² over its predecessor is the larger gap between the optimal wavelength for the luminescence from DeepBlueC and fluorescence from GFP² (105 nm) compared with earlier versions of BRET. High BRET² ratios, suggesting the close proximity (i.e., receptor dimerization) of GFP² and RLUC-tagged rhY4 receptors, were observed. This homodimerization was confirmed by cross-linking and subsequent Western blot analysis. In these studies, dimer formation was demonstrated by the detection of a high-molecular weight band of 160 kDa after incubating with cross-linking reagent BS³. Previously, the molecular weight of the hY4 receptor expressed in Chinese hamster ovary cells has been shown to be 60 kDa (Voisin et al., 2000). Based on the molecular weights we observed on the gel, the 80-kDa protein probably represents a glycosylated Y4-GFP² monomer, whereas the 160-kDa protein is a homodimer. Similar to what has been shown for the opioid homodimer (Cvejic and Devi, 1997), very little of the 160-kDa band was seen without cross-linking (Fig. 6). The fact that the integrity of the Y4 homodimer does not withstand the nonreducing gel conditions suggests that covalent disulphide bonds are not involved in Y4 receptor dimerization, contrary to what is known for the metabotropic glutamate receptor 5 and the muscarinic acetylcoline receptor M3 homodimers (Zeng and Wess, 1999; Romano et al., 2001), indicating that the biochemical properties between different GPCR homodimers can vary extensively.

The rhY4 receptor did not form heterodimers with any of the other members of the NPY receptor family or with the human - and µ-opioid receptors (Fig. 3). However, using fluorescence resonance energy transfer and fluorescence microscopy, it was very recently shown that the Y1, Y2, and Y5 receptors, when expressed individually in baby hamster kidney cells, form homodimers (Dinger et al., 2003). In the present study, the corrected BRET² ratio for the rhY4 homodimer (0.17) was almost three times higher than that found when rhY4-GFP² was coexpressed with rhY2-RLUC. Still, all receptors (Y1, Y2, Y5, , and µ) generated a signal that was significantly above zero (0.035–0.064; Fig. 3) possibly representing a baseline for membrane-bound proteins as the baseline in these studies was defined by rhY4-GFP² coexpressed with cytosolic RLUC.

The most common critique against the concept of GPCRs forming dimers is that overexpression of a recombinant protein in a cell line may force the receptors together in a non-natural fashion (Devi, 2001). In the present study, the cell line used for the BRET² studies displayed a very high expression level of the rhY4-GFP². However, the expression levels of the RLUC-tagged human opioid and µ receptors as well as the rhY2 receptor did not differ significantly from that of the rhY4 receptor, whereas the rhY1 and rhY5 receptors displayed two- to three-fold lower expression. Yet, the BRET² signals of these receptors were less than a third of that of the unstimulated rhY4 homodimer, providing strong evidence that this is a specific feature of the Y4 receptor and not an artifact provoked by overexpression. However, it is possible that the high expression level might account for the high basal BRET² signal seen for rhY4 together with the other receptors. Interestingly, the Y2 receptor had, after Y4 itself, the highest level of interaction with Y4. It has been proposed that the higher the sequence identity, the higher likelihood for dimerization (Ramsay et al., 2002). However, of the NPY receptor family, Y2 shows the lowest sequence identity to Y4, whereas the Y1 receptor is highly similar to the Y4 receptor and displayed the lowest BRET² ratio for heterodimerization with the Y4 receptor.

In contrast to the agonist-induced homodimers observed with the ₂-adrenergic (Angers et al., 2000), bradykinin B2 (AbdAlla et al., 1999), thyrotropin-releasing hormone (Kroeger et al., 2001; Zhu et al., 2002), and gonadotropin-releasing hormone (Kroeger et al., 2001) receptors, the rhY4 receptor complex dissociated in a concentration-dependent fashion when stimulated by the endogenous agonist, hPP, or by the bridged antiparallel dipeptide compound 1229U91 (Daniels et al., 1995; Schober et al., 1998). The symmetric structure of 1229U91 and its high affinity for Y1 and Y4 receptors has sometimes been used as an argument favoring a dimeric structure for the receptors (Daniels et al., 1995; Dinger et al., 2003). PP was more potent than 1229U91 in lowering the dimer/monomer ratio, in agreement with their affinity for the receptor (Fig. 5; Table 1). 1229U91 has been suggested to be a partial agonist at Y4 receptors. Studies of agonist induced -arrestin 2 translocation at the rhY4 receptor showed that the maximal response of hPP was 30% higher than that from 1229U91 (Berglund et al., 2003b). However, in the present study, the maximum and minimum levels were indistinguishable, consistent with full agonism for both peptides. In contrast, a 100 nM concentration of the low-affinity Y4 agonists hNPY and hPYY or the selective Y5 agonist h[D-Trp³²]NPY did not affect dimer formation. Therefore, agonist-induced dissociation of the dimer is a receptor-mediated response and not a nonspecific action of these peptides. The agonist effect on dimer formation remained after incubation with concanavalin A or cycloheximide. These compounds are used to block clathrin-mediated receptor internalization and protein synthesis, respectively. Thus, the effect of PP on Y4 receptor dimerization may be independent of internalization. Previously, agonist-induced dimer dissociation has been shown for the -opioid receptor (Cvejic and Devi, 1997) and for the cholecystokinin-A receptor (Cheng and Miller, 2001).

In agreement with the results from the BRET² studies, Western blot with antibodies directed against GFP revealed that the 160-kDa protein (dimer) decreases after a 1-h stimulation by hPP (Fig. 6). It has been suggested that a change in BRET signal may merely reflect conformational changes in the RLUC- and GFP-tagged receptors rather than dimer formation (Devi, 2001). This is not the case in the present study, because a change in Y4 receptor dimer/monomer could also be detected by Western blot analysis (Fig. 6).

In conclusion, we have used BRET² and Western blot to show that the rhY4 receptor exists at the cell surface as a dimer that dissociates upon agonist stimulation. This suggests that homodimerization is important for the activation and/or down-regulation of this receptor.

	Footnotes

 
This work was supported by Eli Lilly and Company.

Parts of this work have previously been presented as an abstract at the annual Neuroscience Meeting in Orlando, FL in 2002 (Abstract 544:11).

DOI: 10.1124/jpet.103.055673.

ABBREVIATIONS: NPY, neuropeptide Y; PP, pancreatic polypeptide; PYY, peptide YY; GPCR, G-protein-coupled receptor; BRET, bioluminescence resonance energy transfer; RLUC, Renilla luciferase; GFP, green fluorescent protein; HEK, human embryonic kidney; rh, rhesus monkey; h, human; BRET², bioluminescence resonance energy transfer 2; PCR, polymerase chain reaction; WT, wild-type; PBS, phosphate-buffered saline; [³⁵S]GTPS, guanosine 5'-O-(3-[³⁵S]thio)triphosphate; BG, background; BS³, bis[sulfosuccinimidyl]suberate.

¹ Present address: Department of Assay Development and Screening, Biovitrum AB, SE-11276 Stockholm, Sweden.

Address correspondence to: Donald R. Gehlert, Eli Lilly and Company, Lilly Research Laboratories, LCC, Indianapolis, IN 46285. E-mail: gehlert_donald_r{at}lilly.com

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