peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes

Neuropeptides 45 (2011) 293–300

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Neuropeptides journal homepage: www.elsevier.com/locate/npep

Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes Helena Fällmar a, Helena Åkerberg a, Hugo Gutiérrez-de-Terán b, Ingrid Lundell a, Nina Mohell a, Dan Larhammar a,⇑ a b

Department of Neuroscience, Uppsala University, 751 24 Uppsala, Sweden Fundación Pública Galega de Medicina Xenómica, Hospital Clínico Universitario de Santiago, E-15706 Santiago de Compostela, Spain

a r t i c l e

i n f o

Article history: Received 15 February 2011 Accepted 25 May 2011 Available online 21 June 2011 Keywords: Y2 receptor Y1 receptor Binding experiment Neuropeptide Y Peptide YY BIIE0246 Appetite regulation

a b s t r a c t The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T3.40 was selected based on sequence alignments both between subtypes and between species and G2.68, L4.60 and Q6.55 also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3–36), NPY(13–36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3–36) increased for the mutants T3.40I and Q6.55A. Increased affinity was also observed for PYY to Q6.55A. PYY(3– 36) displayed decreased affinity for G2.68N and L4.60A whereas binding of NPY(13–36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T3.40I, L4.60A and Q6.55A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The involvement of the neuropeptide Y (NPY) family of peptides and receptors in appetite regulation has in recent years received intense attention (Chee and Colmers, 2008). The reason for this is the escalating problem with human obesity and therewith associated diseases. A recent report in the Lancet declared that there were approximately 1.5 billion overweight adults in the world year 2008 of which approximately 500 million people were estimated clinically obese (Finucane et al., 2011). The neuropeptide Y family consist of the peptides NPY, peptide YY (PYY) and pancreatic polypeptide (PP) (Cerda-Reverter and Larhammar, 2000; Sundstrom et al., 2008). These peptides convey their effects in humans via four Abbreviations: NPY, neuropeptide Y; PYY, peptide YY; PP, pancreatic polypeptide; GPCR, G protein-coupled receptor; wt, wild type; h, human; p, porcine; PCR, polymerase chain reaction; GFP, green fluorescent protein; HEK, human embryonic kidney; PBS, phosphate-buffered saline; Kd, dissociation constant; Ki, inhibition constant; Bmax, maximal binding capacity. ⇑ Corresponding author. Tel.: +46 184714173; fax: +46 18511540. E-mail address: [email protected] (D. Larhammar). 0143-4179/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2011.05.006

G protein-coupled receptors (GPCRs), i.e. Y1, Y2, Y4 and Y5 (Michel et al., 1998). Taking into consideration also non-human vertebrates the full repertoire of receptors in the NPY family consists of the additional four receptors Y6, Y7, Y8a and Y8b (Larsson et al., 2009). According to their amino acid sequence identity the receptors can be divided into the three subfamilies Y1, Y2 and Y5: the Y1 subfamily comprises Y1, Y4, Y6, Y8a and Y8b, the Y2 subfamily includes Y2 and Y7, and the third subfamily is only Y5 (Larhammar and Salaneck, 2004). Although all NPY receptors interact with all peptides, each receptor has a unique ligand binding profile with regard to both intact and truncated peptides as well as subtypeselective non-peptide antagonists (Michel et al., 1998). The NPY/ PYY receptor Y2 is pharmacologically characterized by its ability to retain high affinity for N-terminally truncated peptide fragments, i.e. NPY(3–36), NPY(13–36), PYY(3–36) and PYY(13–36) (Michel et al., 1998). All receptors in the NPY family belong to the large superfamily of GPCRs and more specifically the rhodopsin-like clan (Larhammar and Salaneck, 2004; Lindner et al., 2008). In 2002, Batterham et al. published a study where they demonstrated that peripheral administration of PYY(3–36) in rodents and

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humans inhibits food intake (Batterham et al., 2002). This finding was later confirmed in obese humans (Batterham et al., 2003) and resulted in increased interest in the Y2 receptor as a potential anti-obesity target. However, the efforts to design pharmaceuticals are hampered by the fact that the three-dimensional receptor structures of the NPY receptors are still unknown. For almost 7 years the bovine receptor rhodopsin (bRho) was the only available structural model of a GPCR based on crystallography (Palczewski et al., 2000). Recently, several publications describe successful crystallization of other related GPCRs, i.e. the human b2-adrenergic receptor (hb2R) in inactive state (Cherezov et al., 2007; Rasmussen et al., 2007; Rosenbaum et al., 2007) and in active state (Rasmussen et al., 2011; Rosenbaum et al., 2011), the turkey b1-adrenergic receptor (tb1R) (Warne et al., 2008, 2011), rhodopsin without ligand (Park et al., 2008), the human adenosine 2A receptor (hA2AAR) (Jaakola et al., 2008), the human chemokine receptor type 4 (hCxCR4) (Wu et al., 2010) and the human dopamine D3 receptor (hD3R) (Chien et al., 2010). To date only three mutagenesis studies on the human Y2 receptor (hY2) have been published involving in total nine amino acid positions (Akerberg et al., 2010; Berglund et al., 2002; Merten et al., 2007). Here we present a site-directed mutagenesis study of four additional amino acids in the hY2 receptor (Fig. 1). The human NPY family of receptors is of particular interest because the subtypes Y1, Y2 and Y5 share only approximately 30% sequence identity but nevertheless interact with the same two conserved NPY and PYY ligands (Larhammar and Salaneck, 2004). The recently reported structural models cited above in combination with mutagenesis studies will help to build more accurate models of the NPY receptor subtypes and their sites for ligand interaction.

2. Material and methods 2.1. Numbering of the mutated positions The positions of the mutated amino acid residues herein are numbered according to Ballesteros and Weinstein (Ballesteros and Wein-

stein, 1995) where the first number indicates the transmembrane (TM) region of the position investigated (or the closest TM region for residues in a loop). The second number indicates the mutated position relative to the most conserved position, denoted 50, in each TM region. The amino acids are shown with either single-letter or triple-letter code with the native residue before the position number and the introduced residue after the number. 2.2. Sequence comparison of Y2 receptors The NPY family receptor sequences from all species available to date were downloaded from the Ensembl database (www.ensembl.org), GenBank (www.ncbi.nlm.nih.gov) and the elephant shark genome project (esharkgenome.imcb.a-star.edu.sg). Multiple sequence alignments of the sequences were made using Clustal W (Thompson et al., 1994) with standard settings as implemented in Jalview 2.4 (Clamp et al., 2004). 2.3. Generation of expression vectors with mutant hY2 receptors using the Gateway™ system The hY2 receptor gene mutants were created by a two-step PCR using a wt hY2 receptor with Gateway™ compatible sites and restriction sites added to the ends as template. Each PCR was run with the proofreading enzyme PLATINIUMÒ Pfx DNA polymerase (Invitrogen) and the products were purified with the QIAquick Gel Extraction kit (Qiagen). The PCR product was cloned using the Gateway™ system according to the manufacturer’s instruction (Invitrogen). Briefly, the GatewayTM system consists of a two-step cloning method using a donor vector before transfer into an expression vector, pcDNA-GFP vector (Invitrogen). The cloning was done via recombination by modified phage lambda Pfx enzyme (Invitrogen) and resulted in specific positioning of the PCR product regarding both direction and place of insertion in the vector. The expression vector carried a gene for green fluorescent protein (GFP), positioned after the recombination site for the cloning product, resulting in a final receptor protein with C-terminal GFP-tag. The cloned hY2 recep-

Fig. 1. Snake plot of the hY2 receptor. The residues investigated by site directed mutagenesis in this article are highlighted in yellow. Grey residues indicate the most conserved position in each transmembrane region.

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tors were fully sequenced to confirm the introduced mutation and exclude unwanted changes. Created mutants were G2.68N, T3.40I, L4.60A and Q6.55A. 2.4. Transfection for transient protein expression Human embryonic kidney (HEK) 293 cells were transfected with the expression vectors using Lipofectamine 2000 Transfection reagent (Invitrogen), diluted in OptiMem medium (Gibco BRL) according to the manufacturer’s instructions and subsequently grown in serum free DMEM (Dulbecco’s modified Eagle’s medium)/Nut Mix F-12 medium with Glutamax containing 100 units of penicillin/100lg of Streptomycin/ml (Gibco BRL) and 2.5 lg of Amphotericin/ml (Gibco BRL) for 24 h. To obtain cells with transient expression, this was followed by growth for 24 h in DMEM medium with 10% (v/v) fetal calf serum (Biotech Line AS). The cell cultures were harvested after a wash in phosphate-buffered saline (PBS), collected by centrifugation and resuspended in binding buffer (25 mM Hepes buffer pH 7.4 containing 2.5 mM CaCl2 and 1.0 mM MgCl2). The transfected cells were stored at 80 °C until further use. 2.5. Ligands [125I]pPYY with a specific activity of 2200 Ci/mmol from PerkinElmer was used as radioligand. The other peptide ligands, pNPY, hPYY(3–36) and pNPY(13–36) were purchased from Bachem. The Y2-selective non-peptide antagonist BIIE0246 was provided by Boehringer Ingelheim. 2.6. Binding studies Ligand binding studies to the hY2 receptors were performed as previously described (Sjodin et al., 2006), with minor modifications. In short, cells were resuspended in binding buffer with 0.2 g/l bacitracin and homogenized with an Ultra-Turrax homogenizer. Binding experiments were performed in a final volume of 100 ll followed by 3 h incubation, saturation studies in duplicate and competition studies in triplicate. Serial dilutions were made of the radioligand and competing ligands for saturation and competition experiments, respectively. Non-specific binding was defined in the presence of 1 lM hPYY. The incubation was terminated by filtration with 50 mM Tris (pH 7.4) at 4 °C through Filtermat A, GF/C filters (PerkinElmer) pre-soaked in 0.3% polyethyleneimine (Sigma–Aldrich) using a Tomtec cell harvester (Orange, CT, USA). The filters were dried at 50 °C and covered with MeltiLex A melt on scintillator sheets (PerkinElmer). The radioactivity was counted in a Wallac 1450 Microbeta counter. Protein concentrations were determined by the Bradford method using the Bio-Rad Dc Protein Assay (Bio-Rad) with BSA as standard. 2.7. Statistical analyses The data from the binding experiments were analyzed with nonlinear regression curve-fitting in the GraphPad Prism 4.0 software package. Saturation experiments were also analyzed with linear regression using Scatchard transformation and in competition experiments the Hill coefficient was calculated for each individual competition binding curve. All competition experiments were tested for one- and two-site curve fitting. The two-site model was accepted if each site accounted for >20% of the receptors and it significantly improved the curve fit (p < 0.05; F test). The pKdand pKi- values for the receptor mutants were compared with those for wt by one-way ANOVA followed by Dunnett’s multiple comparison test for groups.

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2.8. Detection of receptor-protein expression HEK-293 cells were grown on cover slips coated with 0.01 mg/ ml Poly-D-lysine (Sigma) in DMEM medium with 10% (v/v) fetal calf serum for 24 h. This was followed by transfection with Lipofectamine 2000 Transfection reagent (Invitrogen) as described above. Cover slips were washed twice with PBS and cells were fixed with 4% paraformaldehyde for 10 min. After two additional PBS washes the cells were incubated in dark with 0.5 lg/ml DAPI (2-6-indolecarbamidine dihydrochloride purchased from Sigma) for 10 min. Two final PBS washes preceded mounting of the cover slips upside down with 2.5% DABCO (1,4-Diazobicyclo(2,2,2)octane purchased from Sigma) in TRIS–glycerol buffer (pH 8.6) on glass slides. Non-transfected HEK-293 cells served as a negative control. Fluorescence images were acquired using an inverted laser scanning confocal microscope (Zeiss LSM 510 Meta) with a 63x oil objective (NA = 1.4) and the LSM software. The lasers used were argon (514 nm) and diode (405 nm) at 9.9% and 11.9% transmission, respectively. Images were captured using a 2048  2048-pixel frame, gain settings between 700–800 for argon and 760–850 for diode and scan speed set to 7. The mean of four lines was detected and zoom function was set to 1. The pinhole was set to 1 airy unit for all experiments. 2.9. Homology modeling of the hY2 receptor The modeling of the hY2 receptor was adapted from our previous reported models of the Y receptors (Akerberg et al., 2010). Briefly, the sequence of hY2 was retrieved from the UniProt database (Boeckmann et al., 2003) (accession number: P49146), and aligned with the sequences for all the GPCRs crystallized to date in the inactive form (i.e., hA2AAR, hb2R, tb1R, bovine and squid rhodopsin, hCxCR4, hD3R), with the GPCR modeling toolkit (http:// gpcr.usc.es). This web server uses the program Clustal W as an alignment tool (Thompson et al., 1994), and provides additional information about sequence identity for each of the secondary structural elements. The A2A adenosine receptor (Jaakola et al., 2008) was selected as the template, and the A2A/Y2 pairwise alignment was used as input for the model generation with the program Modeller v9.7 (Sali and Blundell, 1993). An initial pool of 15 homology models was generated, and the best initial model was subject to extracellular loop refinement with the LOOPMODEL routine, as implemented in Modeller. Only EL1 and EL3 were included in this refinement, while EL2 was removed at this step because of its short size and very low sequence identity with any template. 15 loop refined models were generated for each initial model at this stage, and a best final model was selected. At each step of model selection we took into account several criteria. The Modeller objective function and the DOPE assessment score (Shen and Sali, 2006) were used for consensus scoring of the models. Additionally, the stereochemical quality of the modeled receptor was validated and approved by the program and PROCHECK (Laskowski et al., 1993). Addition of hydrogens was performed with PDB2PQR software (Dolinsky et al., 2004), which includes a routine for assignment of protonation states and hydrogen-bond network optimization. Images in Fig. 4 were made in PyMOL (DeLano, 2002). 3. Results 3.1. Sequence alignments and selection of positions for mutation The mutants G2.68, L4.60 and Q6.55 were selected based on the results from previous mutagenesis studies of the corresponding positions in the hY1 receptor (Kanno et al., 2001; Sautel et al., 1996;

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Walker et al., 1994) in combination with evolutionary comparisons of sequences between the NPY receptor subtypes and between species. The latter approach was used to identify amino acids of particular interest, either by detection of conserved residues in all receptors or those that deviate in Y2 compared to the rest of the family. In total, 108 sequences from a broad range of vertebrates were available for analysis. Position T3.40 was selected for mutagenesis based exclusively on these alignment comparisons. Position 2.68 is a well conserved Asp in all NPY receptor subtypes except in the Y2 receptors where it is a Gly. However, in two fishes, namely three-spined stickleback Gasterosteus aculeatus and spotted green pufferfish Tetraodon nigroviridis, Y2 displays an Asp. This variation appears to be a lineage specific mutation for these species. Position 3.40 is an Ile in the majority of Y1, Y4 and Y8 receptors. A Val is present in Y1 in chicken Gallus gallus and elephant shark Callorhinchus milii and in Y4 in zebrafish Danio rerio and pufferfish Takifugu rubripes. All Y5 receptors carry a Thr at this position like many of the Y2 and Y7 receptors. Exceptions are Y2 in G. aculeatus and T. nigroviridis and Y7 in D. rerio, T. rubripes and rainbow trout Oncorhynchus mykiss, where it is an Ile. Leu at position 4.60 is well conserved in all Y2, Y5 and Y7 receptors. An exception is Y2 in C. milii where it is a Gln. The Y1 subfamily is occupied by a Phe at this position except the frog Xenopus laevis which has a Leu in Y1. The amino acids at position 6.55 in the NPY receptors show subtype specific conservation for the Y1-, Y2- and Y5-subfamily with Asn, Gln and His, respectively. The only two exceptions are the Y5 receptors in the coelacanth Latimeria chalumnae with Gln, and C. milii with Ile, reflecting two independent mutations. 3.2. Binding characteristics of [125I]pPYY to wt and mutant hY2 receptors

receptors, T3.40I (p < 0.05), L4.60A (p < 0.01) and Q6.55A (p < 0.01) when compared to the wt hY2. A Hill plot was created for each competition curve and the Hill coefficients were close to 1 for the majority of the competition curves (range: 0.65 to 1.21) and the results were consistent with one-site curve fitting. 3.4. Expression of hY2 receptor protein The receptor expression of both wt and mutant hY2 receptors in transfected HEK-293 cells was confirmed using confocal microscopy. The receptors were mainly located in the cell surface membranes but they were also present in lower amounts in the intracellular space of the cells (Fig. 3). 3.5. Modeling of the wt hY2 receptor The 2.6 Å crystal structure of the A2A adenosine receptor (Jaakola et al., 2008) was selected as the template to model the hY2 receptor on the basis of our previous experience and preliminary docking with C-terminus fragments of the peptide agonists (Akerberg et al., 2010). The derived three dimensional model of the hY2 receptor is presented in Fig. 4, with the amino acids selected for the mutagenesis studies highlighted in spheres. Three of these positions are located close to the extracellular side, clearly oriented towards the potential binding crevice of the receptor (2.68, 4.60 and 6.55). The fourth position (3.40) is located deeper in the transmembrane cavity, in the interface between helices 3, 5 and 6, surrounded by hydrophobic residues. 4. Discussion 4.1. Previous mutagenesis studies in the hY2 receptor

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[ I]pPYY bound with a high affinity and saturability to the wt Y2 receptors, with a Kd value of 0.026 ± 0.005 nM and a Bmax value of 83 ± 32 fmol/mg protein (mean ± S.E.M.). The Scatchard analysis of the specific radioligand binding resulted in a linear plot consistent with a non-cooperative apparently single class of binding sites. These results are in good agreement with previously published results (Akerberg et al., 2010; Berglund et al., 2002). Results from the radioligand binding studies to the wt and various mutant receptors are summarized in the Table 1. As can be seen radioligand [125I]pPYY displayed an increased affinity for the mutant Q6.55A with a Kd value of 0.011 ± 0.002 nM (p < 0.05). No significant differences in Kd values for the other mutant receptors were observed. 3.3. Binding characteristics of the three peptide agonists and the nonpeptide antagonist BIIE0246 The results from the competition studies with the various ligands are summarized in Table 2 and representative competition curves of the ligands that were affected by mutation are shown in Fig. 2. All three peptides had an affinity for the wt hY2 receptor in the nanomolar range with a Ki value of 0.81 ± 0.16 nM for pNPY, 0.32 ± 0.03 nM for hPYY(3–36) and 4.03 ± 0.61 nM for pNPY(13– 36). pNPY showed an increased affinity for T3.40I and Q6.55A (p < 0.01). The truncated agonist hPYY(3–36) also displayed an increased affinity for T3.40I and Q6.55A (p < 0.01) but had a decreased affinity for both G2.68N (p < 0.01) and L4.60A (p < 0.05). The affinity of pNPY(13–36) for any of the mutant receptors did not differ significantly from the affinity for wt hY2. The antagonist BIIE0246 also displayed a nanomolar affinity for the wt hY2 receptor with a Ki value of 1.15 ± 0.12 nM. BIIE0246 showed a significantly decreased affinity for three of the mutant

To date only three mutagenesis studies of the hY2 receptor have been published. In the first one, Berglund et al. presented a reciprocal mutagenesis study between hY2 and chicken Y2 receptors of three positions and concluded that they all were of importance in BIIE0246 binding (Berglund et al., 2002). 5 years later, Merten et al. used a functional assay to study the effects of mutation in three additional positions and presented a docking model of NPY to the Y2 receptor (Merten et al., 2007). The third study investigated the probability of three new residues participating in a hydrophobic pocket in hY2 interacting with Tyr36 in the NPY ligands, using both mutagenesis and computer modeling (Akerberg et al., 2010). Hence, none of the positions investigated in this study have previously been subjected to mutagenesis and tested in binding experiments in the hY2 receptor. However, three of the corresponding amino acids at the positions 2.68, 4.60 and 6.55 in the hY1 receptor have been analyzed by mutagenesis in independent Table 1 Kd and Bmax values of [125I]pPYY binding to wt and mutant hY2 receptors. The results are shown as means ± S.E.M for n independent experiments performed in duplicates. The data were analyzed using non-linear regression (GraphPad Prism 4.0 software). Mutant

wt G2.68N T3.40I L4.60A Q6.55A

125

I-pPYY

Kd (nM)

Kd/Kd (wt)

Bmax (fmol/mg of protein)

n

0.026 ± 0.005 0.022 ± 0.003 0.015 ± 0.004 0.024 ± 0.004 0.011 ± 0.002*

1 0.85 0.58 0.92 0.42

83 ± 32 140 ± 40 18 ± 5 15 ± 2 36 ± 3

5 4 3 4 3

Abbreviations: wt = wild type, Kd/Kd (wt) = Kd mutant/Kd wt. Kd values were converted to pKd values prior to statistical analysis by the formula log Kd. * A pKd value significantly different from the pKd value of the wt receptor by p < 0.05.

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Table 2 Inhibition of [125I]pPYY binding to wt and mutants hY2 receptors by four ligands. The results are shown as means ± S.E.M for n independent experiments performed in triplicate. The data were analyzed using non-linear regression (GraphPad Prism 4.0 software). The results were analyzed by one-way ANOVA followed by Dunnett’s test. Mutant

wt G2.68N T3.40I L4.60A Q6.55A

pNPY

hPYY(3–36)

pNPY(13–36)

BIIE0246

Ki (nM)

Ki/Ki (wt)

n

Ki (nM)

Ki/Ki (wt)

n

Ki (nM)

Ki/Ki (wt)

n

Ki (nM)

Ki/Ki (wt)

n

0.81 ± 0.16 1.09 ± 0.20 0.10 ± 0.04** 1.91 ± 0.68 0.11 ± 0.01**

1 1.4 0.12 2.3 0.14

5 3 4 5 4

0.32 ± 0.03 0.87 ± 0.15** 0.14 ± 0.02** 0.67 ± 0.07* 0.03 ± 0.01**

1 2.7 0.44 2.1 0.09

4 4 3 3 4

4.03 ± 0.61 7.83 ± 4.89 1.77 ± 0.69 7.23 ± 0.35 5.44 ± 1.49

1 1.9 0.44 1.8 1.3

6 4 6 3 4

1.15 ± 0.12 0.71 ± 0.12 2.68 ± 0.58* 53.2 ± 8.7** 3.30 ± 0.60**

1 0.62 2.3 46 2.9

5 3 3 3 3

Abbreviations: wt = wild type, Ki/Ki (wt) = Ki mutant/Ki wt. Ki values were converted to pKi values prior to statistical analysis by the formula A pKi value significantly different from that of the wt receptor at p < 0.05. ** p < 0.01.

log Ki.

*

Fig. 2. Ligand binding characteristics of pNPY, hPYY(3–36) and BIIE0246 to the wt hY2 and the mutant receptors. Represented in each graph are the wt hY2 and only the mutant receptors for which the respective ligands displayed a changed affinity. Results shown are from one typical experiment per ligand and receptor. The figure legends are listed in the order of appearance in the graph from right to left.

investigations (Kanno et al., 2001; Sautel et al., 1996; Walker et al., 1994). These positions correspond to D104 (D2.68), F173 (F4.60) and N283 (N6.55) in hY1. The aim with our study was to investigate if the four positions, G2.68, T3.40, L4.60 and Q6.55 contribute to ligand binding in hY2 and to identify receptor subtype-specific interactions. 4.2. Position 2.68 Results from mutagenesis studies of the corresponding amino acid in the hY1 receptor, D2.68, have identified this position as important for binding of peptide agonists. A mutation of the Asp to an Ala resulted in a non-detectable specific binding of [125I]NPY (Walker et al., 1994), a considerably reduced [125I]NPY affinity (Sautel et al., 1996) and a loss of [125I]PYY binding (Kanno et al., 2001). The affinities of the three Y1-selective antagonists [3H]BIBP3226, [125I]1229U91 and [3H]J-104870 were unaffected by this change (Kanno et al., 2001; Sautel et al., 1996). The hY2 receptor has an amino acid residue with very different properties at this position, namely a Gly lacking the side chain present in Asp, implying a different interaction than in the hY1 receptor. We chose to change the Gly to an Asn residue in order

to investigate if a longer side chain would still allow binding or perhaps repel the ligand due to absence of an attracting negative charge of an Asp. The result shows that the hY2 receptor mutant G2.68N only affected the binding of one out of five tested ligands, i.e. truncated hPYY(3–36). This peptide had a nearly 3-fold decrease in binding affinity for G2.68N as compared to the wt hY2 (p < 0.01). In contrast to the hY1 receptor, mutation of this position did not affect the binding affinities of pNPY or [125I]pPYY. This and the fact that G2.68 is an Y2 subtype-specific amino acid deviating from the common Asp present in all other NPY receptors, allow us to hypothesize, that it is the lack of the Asp that confers to the binding characteristics of the Y2. This is further supported by the fact that the only ligand affected by the mutation was the highly Y2-specific ligand hPYY(3–36). Possibly, this position has shifted in Y2 as compared to the ancestral receptor, to be a residue only important for the specificity of the Y2 receptor, i.e. interaction of truncated PYY. 4.3. Position 4.60 Two previous studies have investigated the hY1 mutant F4.60A. The first study reported a more than 130-fold decreased affinity

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Fig. 3. Receptor expression in HEK-293 cells. HEK-293 cells (A) were transfected with the wt hY2 (B); G2.68N (C); T3.40I (D); L4.60A (E) and Q6.55A (F) C-terminally fused to a GFP (shown in green). The nuclei are visualized by DAPI (shown in blue).

Fig. 4. Three dimensional model structure of the hY2 receptor based on the crystal structure of the human adenosine A2A receptor. The receptor model was based on homologies in the transmembrane regions of wt hY2 receptor with the hA2AR. The residues investigated by site-directed mutagenesis are shown as spheres. The model was made in PyMOL. (A) Side view of the receptor. (B) Extracellular view of the receptor.

of [125I]NPY and 40-fold decrease of [3H]BIBP3226 (Sautel et al., 1996). The second study added to these findings by reporting loss of affinity of [3H]J-104870 but retained binding for [125I]1229U91 and [125I]PYY (Kanno et al., 2001). Thus, the affinity of PYY and NPY were reported to be differentially affected by this mutation. This is notable since the two peptides are very similar in structure and thought to interact with the receptor in a similar manner. This position is a well conserved Leu in the majority of members in the Y2- and Y5-subfamiles, while a different non-polar amino acid (Phe) is occupying this position in the Y1 subfamily. Based on the assumption that a non-polar bulky amino acid is needed in position 4.60 of the Y receptors, we chose to change it to Ala with its small side chain. Our hY2 mutant L4.60A had a decreased affinity for hPYY(3–36) (p < 0.05) and BIIE0246 (p < 0.01). The fact that the latter showed a 46-fold reduction in affinity strongly indicates that this position is

important for antagonist binding. Since Ala has a much shorter side chain than Leu but retains the non-polar properties, it can be speculated that a supposed hydrophobic interaction between this position and the antagonist is weakened or even lost simply due to the increased distance. The reduced hPYY(3–36) affinity indicates that L4.60 is involved in interaction with this highly Y2 specific agonist, directly or indirectly. Thus, position 4.60 seems to be important in both hY2 and hY1 for subtype-selective non-peptide antagonist binding. 4.4. Position 6.55 Two articles have been published addressing the position N6.55 in hY1. An Ala substitution led to a greatly decreased affinity of [125I]NPY and [3H]BIBP3226 (Sautel et al., 1996). Moreover, [125I]PYY and the antagonist [3H]J-104870 also lost their affinity

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for this mutant (Kanno et al., 2001). However, the affinity of the antagonist [125I]1229U91 was unchanged (Kanno et al., 2001). This position shows a clear subtype-specific conservation being Asn in the Y1 subfamily, Gln in the Y2 subfamily, and His in Y5. Hence, in order to investigate if this position is important for Y2 specificity we mutated this residue to an Ala. In this study an increased affinity for the hY2 mutant Q6.55A receptor was observed for the peptide agonists [125I]pPYY (p < 0.05), pNPY and hPYY(3–36) (p < 0.01). The non-peptide antagonist BIIE0246 displayed a decreased affinity (p < 0.01). pNPY(13– 36) was the only ligand which retained wt affinity against this mutant. The exchange of a polar Gln to a non-polar Ala resulted in an increased affinity of the three longest peptides tested. The most likely explanation is that the exchange resulted in a conformational change of the receptor which indirectly enhances the ligand–receptor interaction. The same kind of exchange in hY1, a polar Asn to a non-polar Ala, resulted in a decreased affinity of NPY and a lost affinity of PYY. This indicates that this residue is of greater importance in the hY1 receptor than in hY2. 4.5. Position 3.40 From the sequence alignment it is clear that the majority of the receptors in the Y2 and Y5 subfamilies have a Thr at this position whereas the Y1 subfamily has an Ile in the majority of sequences. In order to investigate this difference between subfamilies we decided to make a hY2 mutant with a Thr to Ile exchange, i.e. T3.40I. The two peptide agonists pNPY and hPYY(3–36) both displayed a statistically significant increase in the affinity (p < 0.01) whereas the non-peptide antagonist BIIE0246 displayed a decreased affinity (p < 0.05) for T3.40I. The affinity of pNPY(13–36) was unchanged. NPY displays a somewhat higher affinity for Y1 than it does for Y2, 0.2 nM versus 0.7 nM (Michel et al., 1998). This is in concordance with our result where pNPY shows 0.10 ± 0.04 nM affinity for the Y1 imitating T3.40I mutant as compared to the 0.81 ± 0.16 nM affinity for the wt hY2. However, this mutation also resulted in an increased affinity of hPYY(3–36). Considering that this position is situated deep down inside the receptor, a plausible explanation is that the changes in affinities are due to an indirect effect. It has recently been shown that this position plays an important role in stabilizing the receptor conformations in the activation pathway of b2AR (Rasmussen et al., 2011). Thus the mutation of the polar Thr situated in a hydrophobic environment to a non-polar Ile mutation most probably affects the receptor conformation, possibly by stabilizing it, explaining the increased affinities. The decreased affinity of the Y2-selective antagonist BIIE0246 strengthens the probability of a different interaction pattern of this small non-peptide compared to the peptide ligands. We previously mutated a nearby position, Q3.37H which also resulted in a decreased affinity of BIIE0246 (Berglund et al., 2002). This entire region thus seems important for antagonist binding. 4.6. Interpretations and implications The 3D model of the hY2 receptor presented in Fig. 4 suggests that T3.40 is located at the part of TM3 facing TM6. According to this model, the substitution of the original Thr by a more hydrophobic Ile facilitates the hydrophobic contacts with F6.44, which has been recently shown to be an important switch in receptor activation according to the recent crystal structures of b2 receptors (Rasmussen et al., 2011). A higher stabilization of the active receptor conformation of the T3.40I mutant would explain the generally enhanced agonist binding affinities of this mutant, while being also in agreement with the observed decrease of antagonist binding.

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As for the other three positions, the structural interpretation is more complex. G2.68 is located at the boundary between TM2 and the first extracellular loop, and an effect in the loop conformation or flexibility is expected when this residue is substituted by a polar and bulky Asn. However, the lack of a significant effect in the binding of all ligands studied, with the only exception of the selective agonist hPYY(3–36), seems to suggest no major direct ligand interactions in this region. Interestingly, hPYY(3–36) also displays a moderately diminished affinity for the L4.60A mutant, supporting the idea that the original Leu at this position might contribute with additional hydrophobic interactions to the binding of this ligand. The unexpected increase in affinity of the Q6.55A mutant is quite intriguing. Previous mutagenesis data suggest an importance in agonist binding of the corresponding Asn in position 6.55 of the Y1 receptor (Kanno et al., 2001; Sautel et al., 1996). Moreover, crystal structures and mutagenesis data on other GPCRs, namely hA2AAR (Jaakola et al., 2008; Kim et al., 1995) and the hb2R (Rasmussen et al., 2011; Wieland et al., 1996), demonstrate a role of the corresponding Asn in ligand binding of the corresponding receptors. As described above, this position is conserved among species for each Y receptor subtype but differs between subtypes. This fact, together with the observation that it is invariably occupied by a bulky polar residue (Asn/Gln/His) and oriented towards the binding cavity in our 3D model, led to the hypothesis that it might be interacting with the peptidic ligand. However, the results from the current study point to a more complex model, in which removal of this polar, conserved position results in a significant increase in affinity for the most potent Y2 agonists pPYY, pNPY and hPYY(3–36). An alternative structural hypotheses would be that side chains at this position may interact with and stabilize the conformation of the long extracellular loop EL2, i.e. an intra-receptor interaction, and that the absence of such side chains may result in improved binding of long peptide ligands to the receptor. The Y2 receptor is known as the only NPY receptor to bind truncated fragments of the NPY and PYY peptides with high affinity. Our results show that the affinity of hPYY(3–36) was affected for all four receptor mutants whereas pNPY(13–36) retained wt affinity for all four mutants. This indicates that these mutant positions might explain part of the ability of the receptor to bind hPYY(3–36) but not pNPY(13–36), implying that even though they are both truncated they interact differently with the receptor. The effects of the mutated positions on the binding of the antagonist ligand BIIE0246 are clearly different from those on agonist binding (see Table 2). Here, removing the bulky Leu at position 4.60 results in a reduced affinity, reinforcing the concept that this residue has a direct hydrophobic interaction with the antagonist. However, also Q6.55 and T3.40 are important for the binding of this ligand. Interestingly, T3.40, L4.60 and Q6.55 are oriented towards the cavity defined between TM3-TM4-TM5-TM6, which has been previously proposed by other authors to accommodate antagonist ligands in the Y1 receptor (Kanno et al., 2001; Sautel et al., 1996). An interesting development of our findings will be to investigate G-protein interaction and signal transduction in the mutants that resulted in an increased affinity of agonists. Moreover, if any of these mutants will be found to be naturally occurring, it would be very interesting to investigate if it has any physiological relevance. Since all human NPY receptors are involved in appetite regulation, major focus is on the development of new anti-obesity drugs, i.e. antagonist for the Y1 and Y5 receptors and agonists for the Y2 and Y4 receptors. To date there are several NPY receptor selective ligands whose anti-obesity efficacy and side effects have been examined in preclinical and clinical studies (Walther et al., 2011). For a long time PYY(3–36) was the only peptide used for pharmacological characterization of the Y2 receptor but now other selective Y2 peptide agonists are available (Sato et al., 2009). However, small non-peptide agonists are still missing. The develop-

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