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Bitter taste receptor T2R1 is activated by dipeptides and tripeptides
Biochemical and Biophysical Research Communications 398 (2010) 331–335
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Bitter taste receptor T2R1 is activated by dipeptides and tripeptides Jasbir Upadhyaya a, Sai Prasad Pydi a, Nisha Singh a, Rotimi E. Aluko b, Prashen Chelikani a,* a b
Department of Oral Biology, University of Manitoba, Winnipeg, Canada MB R3E 0W4 Department of Human Nutritional Sciences and The Richardson Center for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, Canada MB R3T 6C5
a r t i c l e
i n f o
Article history: Received 20 June 2010 Available online 27 June 2010 Keywords: G-protein coupled receptors (GPCRs) Bitter taste receptors T2R1 Bitter peptides Molecular modeling
a b s t r a c t Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, the molecular targets or receptors in humans for these bitter peptides were poorly characterized and least understood. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. In addition, we tested the ability of peptide inhibitors of the blood pressure regulatory protein, angiotensin converting enzyme (ACE) to activate T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium was measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Among the peptides examined, the bitter tri-peptide Phe-Phe-Phe (FFF), is the most potent in activating T2R1 with an EC50 value in the micromolar range. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The molecular models showed that the bitter peptides bind within the same binding pocket on the receptor. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2 forming a cap like structure on the binding pocket. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Humans are capable of sensing five basic tastes which are, sweet, umami, bitter, sour and salt. While the signal transduction for sweet, umami and bitter tastes is through G-protein coupled receptors (GPCRs), the sour and salt tastes are sensed by ion channels. In humans more than 30 GPCRs are known to mediate taste perception, with 25 of these predicted to sense bitter tastes alone. The human genome encodes 25 bitter taste receptors (T2Rs) localized as clusters on chromosomes 5p15, 7q31 and 12p13 [1–3]. While sweet taste perception is well studied and the sweet taste receptors (T1Rs) well characterized, very little is known regarding the bitter and umami tastes sensed by GPCRs. The human T2Rs are between 290 and 333 amino acids, and have extracellular amino-termini that are 5–19 amino acids long, and intracellular carboxyl-termini that are 7–32 amino acids long. Amino acid sequence analysis of the T2Rs showed that the intracellular loops have a higher degree of sequence similarity and the most divergent parts in T2R sequences are the extracellular loop
* Corresponding author. Address: D319, Department of Oral Biology, 780 Bannatyne Avenue, University of Manitoba, Winnipeg, Canada MB R3E 0W4. Fax: +1 204 789 3913. E-mail address: [email protected] (P. Chelikani). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.097
regions. The signaling events in the perception of bitter taste start with the binding of specific water-soluble molecules (tastants or ligands) to the extracellular and/or transmembrane domains of T2Rs initiating an intracellular signaling cascade. Based on selectivity towards bitter tastants there are at least two different classes of T2Rs, those like T2R16 are broadly tuned and recognize a wide variety of bitter ligands [4], while others like T2R4 are very specific and recognize only selective ligands [5]. However, the mechanisms underlying these differences in specificities have so far not been elucidated. In addition, the ligand binding pocket of T2Rs, as well as the molecular changes that take place in T2Rs upon ligand binding is poorly understood. Recently, it was shown that the human T2Rs are activated by the bitter tasting extracts of the milk protein, casein [6]. It was the first report to show that bitter receptors can be activated by bitter peptides. However, in that study only two synthesized bitter di-peptides, Gly-Phe and Gly-Leu, were shown to activate the bitter receptor, T2R1. In addition, the ligand binding pocket on the T2Rs for these peptides was not elucidated. In this report, using a synthetic T2R1 gene and a heterologous expression system, we showed that various food protein derived and bitter tasting tri and di-peptides activate the human bitter receptor, T2R1. Furthermore to elucidate the potential ligand binding pocket of T2R1 for these tri- and di-peptides, we used homology modeling to construct molecular models of the T2R1 bound
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to different ligands. Results from our molecular modeling showed that the ligand binding pocket for T2R1 was located close to the extracellular surface. In addition, we showed that amino acid residues from TM1, TM2, TM3 and TM7 and residues from ECL-1 and ECL-2 contribute to ligand binding in T2R1.
2. Materials and methods 2.1. Materials The detergent n-dodecyl-b-D-maltoside (DM) was purchased from Anatrace (Maumee, OH, USA). The monoclonal antibody, rho1D4, was prepared by the Cell Culture Center (Minneapolis, MN, USA) from a cell line provided by R.S. Molday (University of British Columbia, Vancouver, Canada). The S-17 polyclonal antibody was from Abcam, MA, USA. Fetal bovine serum and DME High Glucose were purchased from Sigma and Invitrogen. Common chemicals and bitter compounds were purchased either from Fisher or Sigma. Fluo-4NW was purchased from Invitrogen (Carlsbad, CA, USA). Bitter peptides were synthesized commercially (Genescript, NJ, USA). 2.2. Molecular biology and cell culture The synthetic T2R1-rho1D4 gene consisted of 954 bp, of which the 897 bp encodes T2R1. The salient features of the synthetic T2R1 gene include, a Kozak consensus (GCCACCATGG) 50 to the ATG start codon [7], a bovine rhodopsin C8 peptide tag (ETSQVAPA) immediately 50 to the natural stop codon of T2R1, and restriction sites for EcoRI at the 50 end and NotI at the 30 end to facilitate cloning. The sequence encoding the human T2R1 gene was optimized for mammalian cell codon usage [8], and the gene was synthesized commercially (Genescript, NJ, USA). The synthetic T2R1 gene was then inserted into the EcoRI and NotI sites of the mammalian expression vector pcDNA3.1 (Invitrogen). The T2R1-Rho38 gene which contains the first 38 amino acids of mouse rhodopsin placed in frame with the 50 -end of T2R1 coding sequence was a gift from Dr. Kenji Maehashi (Tokyo University of Agriculture, Tokyo, Japan) [6]. The T2R1 genes in pcDNA3.1 were transiently expressed in C6glioma cells using lipofectamine 2000 (Invitrogen) and as described by the manufacturer.
cells washed once with PBS and incubated with the calcium sensitive dye Fluo-4NW (Invitrogen) for 1 h. Receptor activation was determined by measuring changes in intracellular calcium after application of different concentrations of ligands (peptides) using Flexstation-3 fluorescence plate reader (Molecular Devices, CA, USA) at 525 nm following excitation at 494 nm. The calcium efflux was calculated using the following formula
½Ca2þ ¼ K d ðF F min Þ=ðF max FÞ where F is the experimentally measured fluorescence intensity; Fmin is the measured fluorescence intensity in the absence of Ca2+; Fmax is the measured fluorescent intensity of the Ca2+-saturated dye; and Kd is the dissociation constant of the Ca2+ indicator. Kd value of Fluo 4 calcium indicator dye is 345 nM [9]. Dose–response curves were generated and EC50 values calculated by nonlinear regression analysis using PRISM software version 4.03 (GraphPad Software Inc, San Diego, CA) after subtracting the responses of mock-transfected cells. 2.5. Molecular modeling of the T2R1 receptor The model of T2R1 receptor was built by homology modeling using the crystal structure of the active opsin (PDB ID: 3DQB) as template. The transmembrane regions of T2R1 were modeled using the program MODELLER 9V7 [10]. Loop regions of the receptor were modeled using ModLoop server [11]. Side chains of the amino acids were refined with SCWRL4 [12]. Then the whole molecule was energy minimized by 1000 steps of steepest descent (SD) and 1000 steps of conjugate gradients using SPDBV 4.0.1. Molecular dynamics simulations for 10 ps were performed for the active model using openMM Zephyr [13]. The quality of the model was verified by using the program PROCHEK [14] and 98.2% of the residues were in allowed regions of the Ramachandran Plot. The ligands were docked to the receptor using the program AutoDock Vina [15]. The binding site for the ligand on T2R1 was defined by forming a cube with the dimensions 60 80 70 around the protein with a grid point spacing 0.375 Å and center grid boxes 51.807, 12.467 and 38.921 in X, Y and Z dimensions, respectively. We performed 50 genetic algorithms runs for each ligand. In each run the best pose was saved. Finally, all poses were superimposed and the most frequent orientation of the ligand was taken as final pose. The receptor–ligand complexes generated as above were used for further analysis.
2.3. RT-PCR analysis 3. Results and discussion Genomic analysis of DNA extracted from C6-glioma cells by reverse transcriptase-PCR was done using a MJ Mini thermal cycler (Biorad laboratories, USA). The following primers were used, GAP DH (amplicon size 105 bp) Forward-ATGACTCTACCCACGGCAAG, Reverse-GATCTCGCTCCTGGAAGATG; T2R1-rho38 (amplicon size 129 bp) Forward-CCGTCACCCACTCTTCATCT, Reverse-GGGACCATA AACCCTGCATA; T2R1-rho1D4 (amplicon size 118 bp), Forward-A GAATGCCACCATCCAGAAG, Reverse-AGGCTGAAGATCAGGAGCAG; T2R1 (amplicon size 80 bp), Forward-CTCATTGTGGTTGTCCATGC, Reverse-GCCAGGCAAAATAGAAGCAG. GAPDH was used as an internal control; a control with reverse transcriptase omitted from the reaction was run to rule out the presence of contaminating genomic DNA, along with a control with no template present. The transcribed cDNA was used as a negative control. 2.4. Calcium assays Around 1.0–1.5 105 C6-glioma cells were plated into each well of a 96-well tissue culture treated BD-falcon biolux plates and plasmid DNA (200 ng/well) was transfected using liopfectamine 2000. Following 24 h transfection, the media was removed,
3.1. Expression of T2R1 Similar to most other GPCRs, the native T2R receptors are poorly expressed. There are various methods to improve the expression of GPCRs, the two frequently used methods are; codon-optimization of the genes to suit the expression system [16], and addition of the first 20–38 amino acids of rhodopsin to the N-terminal sequences of the poorly expressed GPCRs [5]. The two T2R1 constructs used in this study, T2R1-rho1D4 and T2R1-rho38 genes carry the above features and their construction is described in Section 2.2. The expression of these two genes was analyzed by PCR. Reverse transcriptase-PCR analysis confirmed the expression of the transiently-transfected T2R1-rho1D4 and T2R1-rho38 genes in glial cells. In addition, both the T2R1 genes were expressed at similar levels as observed on a 2% agarose gel (Fig. 1). DNA from untransfected glial cells was used to analyse the expression of endogenous T2R1 receptor, and no transcript was observed which shows that glial cells do not express any native T2R1 receptor (Fig. 1). The localization of the expressed T2R1-rho1D4 protein at the plasma membrane of glial cells was confirmed by immunofluorescence (Fig. 4, Supporting information).
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Table 1 Summary of the biochemical and sensory properties of synthetic peptide ligands and their ability to activate the human bitter receptor, T2R1. Peptide
Bitterness thresholda (mM)
Rcafb
ACEinhibitory activityc
EC50d (mM)
GF FL IF FFF GLL IQW LKP Dextromethorphan*
1.2 1.5 1.5 0.2 1.5 – – –
0.83 0.67 0.67 5.0 0.67 – – –
No No Yes No No Yes No No
7.3 ± 1.1 7.2 ± 0.7 7.4 ± 0.9 0.37 ± 0.11 4.9 ± 0.7 6.1 ± 2 7.6 ± 1.2 0.031
a
Fig. 1. Reverse Transcriptase (RT)-PCR analysis of T2R1 expression in C6 glial cells. The PCR amplified DNA were resolved on a 2% agarose gel stained with ethidium bromide and visualized under UV light. RT-PCR analysis confirmed the expression of the transiently-transfected T2R1-rho38 and T2R1-rho1D4 genes in glial cells. Glial cells do not express any endogenous T2R1 gene. GAPDH was used as an internal control. M indicates low molecular weight DNA marker and size in base pairs is indicated next to the gel.
However, in our experiments the polyclonal antibody against the T2R1-rho38 did not bind to the receptor and the T2R1-rho38 protein could not be visualized either by immunofluorescence or by immunoblot analysis. 3.2. Food protein derived bitter peptides A wide variety of peptides derived from proteins have been found in various foods and fermentated products, such as miso (salted and fermented soybean paste), soy sauce, cheese, and fish
Published values from sensory test [20]. Ratio of bitterness to caffeine [20]. Published literature values [19,21]. d Values determined by calcium dose–response functional assays (from this study). * Not a peptide ligand (bitter compound). b
c
sauce. Hydrolysis of proteins by proteolytic enzymes during fermentation is usually accompanied by the formation of a bitter taste, which is considered to be due to the presence of peptides. Soybean-derived peptides can play an important role in physiology, particularly in the prevention of chronic diseases [17,18]. The presence of bioactive peptides in protein hydrolysates and fermented products, and especially the ACE-inhibitory peptides have been a major focus of research in recent years [18,19]. In this study, we have used bitter peptides synthesized commercially (Genescript, NJ, USA). These peptides were previously isolated from food
Fig. 2. Dose-dependent changes in intracellular calcium [Ca2+] induced by bitter ligands in C6 glial cells. (A) Dose–response curves of T2R1 stimulated with increasing concentrations (lM) of the tri-peptide FFF and the synthetic bitter compound dextromethorphan (DXM). (B) Dose–response curves of T2R1 stimulated with increasing concentrations (mM) of different bitter peptides. Bitter peptides are shown in the inset. (C) Relative activation rates of T2R1 in response to FFF and DXM calculated based on their EC50 values. Values were normalized to the EC50 value of DXM. (D) Relative activation rates of T2R1 in response to different peptides calculated based on their EC50 values. All values were normalized to the EC50 value of FFF.
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proteins and their sensory properties characterized by different groups [19–21] (Table 1). 3.3. Activation of T2R1 by tri and di-peptides To elucidate, if the addition of peptides induce a functional response in T2R1-expressing glial cells, we stimulated both T2R1rho1D4 and T2R1-rho38 expressing glial cells with different bitter peptides. Our initial results showed that both T2R1 constructs elicited a rapid increase in intracellular calcium and showed similar functional responses to the ligands tested (data not shown). However, the ease of detection of T2R1-rho1D4 using the monoclonal antibody rho1D4 helped us decide in favour of using T2R1-rho1D4 construct for subsequent experiments. The ligands that activate T2R1 were classified into two groups based on their potency. The first group comprises of ligands which are highly potent and have EC50 values for T2R1 in the micromolar range and include, the bitter peptide Phe-Phe-Phe (FFF) and bitter synthetic compound dextromethorphan (Table 1). The tri-peptide FFF consists of hydrophobic amino acids with phenyl groups which are considered important for bitter taste sensation [22]. L-Phenylalanine exhibits a slightly bitter taste with a threshold value of 20 mM, while D-phenylalanine exhibits a sweet taste with a threshold value of 2.2 mM. However, a 100 times greater bitterness than that of phenylalanine has been observed in the taste of tri-phenylalanine (Table 1). The bitter taste of any peptide is more apparent when the hydrophobic amino acid is located at the C-terminus [20]. For the tri-peptides, the middle amino acid residue is considered more important than both the C- and N-terminal amino acids [19]. Among all the tested peptides, FFF activated T2R1-expressing cells at concentrations of 0.125–1 mM that humans also perceive as bitter, with an EC50 value of 370 lM (Table 1 and Fig. 2). The bitter synthetic compound dextromethorphan also falls in the group of high potency bitter ligands that activate T2R1. Pre-
vious functional studies showed that T2R1-expressing HEK293 cells were activated by dextromethorphan at a minimal or threshold concentration of 10 lM [23]. However, EC50 values were not determined in that study owing to the high signal noise at higher concentrations of dextromethorphan in HEK293 cells. Glial cells have comparatively lower basal calcium noise, and thus we were able to do a dose–response analysis for T2R1 activation by dextromethorphan, which resulted in an EC50 of 31 lM (Fig. 2). The second group encompasses peptides of medium potency and have EC50 values for T2R1 in the millimolar range, it includes di-peptides Gly-Phe (GF), Phe-Leu (FL), Iso-Phe (IF) and tri-peptides Gly-Leu-Leu (GLL), Leu-Arg-Pro (LKP) and Iso-Glu-Trp (IQW). In our study, tri-peptides have been found to be more potent than the di-peptides in activating T2R1, the rationale being that bitterness of peptides is intensified as the number of amino acids in the peptide chain increases. The tri-peptides LKP and IQW show saturated responses at concentrations of 10–15 mM, whereas signal saturation with other di- and tri-peptides was observed with 15–20 mM. At concentrations of LKP and IQW above 15 mM, a decrease in the signal was noticed due to a nonspecific inhibitory effect of the peptides in the assay. Alternatively, this might be due to desensitization of the receptor with higher concentrations of the peptides. Concentration ranges that span two orders of magnitude are often observed for T2Rs and their agonists. This was also seen with the peptides used in this study, as T2R1 was half maximally activated by micromolar concentrations of high potency ligands and by millimolar concentrations of medium potency peptides. 3.4. T2R1 ligand binding pocket The structure of T2R1 receptor was built by homology modeling using opsin as a template and as described in Section 2.5. The
Fig. 3. (A) Side view of T2R1 receptor showing the ligand binding pocket. Transmembrane helices (TM) of the T2R1 are shown along with the ligand binding pocket present towards the extracellular surface represented as a mesh. (B) Extracellular view of the ligand binding pocket of T2R1. The seven TM helices along with the three extracellular loops (ECL1–3) are shown in this enlarged view of the binding pocket. The amino acid residues Asn66 on TM2, Glu74 on ECL1, Asn89, Glu90 on TM3 and Asn163 on ECL2 involved in binding to the ligand, are also shown. The different ligands bind within the same binding pocket on T2R1 and are represented as ball and sticks. The color coding for the ligands are as follows, the tri-peptides FFF and IQW are represented in blue and green, the di-peptides GF and IF are represented in dark pink and yellow and the bitter compound dextromethorphan is represented in cyan. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
J. Upadhyaya et al. / Biochemical and Biophysical Research Communications 398 (2010) 331–335 Table 2 The amino acid residues lining the ligand binding pocket of T2R1. Ligand
FFFb
IQWb
Dextromethorphan GFb IFb
Amino acids in the 4 Å binding pocket of T2R1a Residues in the transmembrane helices
Residues in extracellular loop region
Iso62, Asn66, Val69, Iso70, Leu86, Asn89, Glu90, His240, Leu260, Phe261, Phe262, Iso263 Leu19, Thr23, Iso62, Phe63, Asn66, Val69, Iso70, Leu85, Leu86, Asn89, Glu90, Phe261, Iso263, Leu264, Glu267 Phe6, Iso62, Asn66, Asn163, Phe257, Leu260, Phe261, Iso263 Phe17, Leu19, Asn66, Val69, Iso70, Leu86, Phe257, Phe261 Phe17, Leu19, Phe22, Thr23, Iso26, Iso62, Phe63, Asn66, Iso70, Phe261, Iso263, Leu264
Glu74, Asn163
Glu74
335
Acknowledgments This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) individual Discovery grants to PC and RA. SP is supported by a University of Manitoba Graduate Fellowship (UMGF). RA is also a recipient of an operating grant from the Advanced Foods and Materials Network (AFMNet) of Centre of Excellence. PC holds a New Investigator Salary Award from Heart and Stroke Foundation of Canada (HSFC). Appendix A. Supplementary data
Glu74
a
Amino acid residues involved in hydrogen bond formation with the ligand are shown in bold. b Peptide ligands.
final model was docked with five different ligands using AutoDock Vina. Unlike sweet taste receptors where the ligand binding pocket is located in the N-terminal domain, our docking studies showed that the ligand binding pocket for T2R1 was located close to the extracellular surface (Fig. 3A). For all the ligands docked, amino acid residues from TM1, TM2, TM3 and TM7 and residues from ECL-1 and ECL-2 contributed to ligand binding (Fig. 3B). Results from our docking studies showed that the ligands are bound in different orientations within the same binding pocket; this could be due to the differences in their chemical structures which influence their interactions with the amino acids in the binding pocket. Table 2 lists the amino acids lining the binding pocket, though only a few are found to interact with most of the ligands docked. Except for minor variations in the orientation of the bound ligands (Fig. 3B), no major differences were observed between the mode of binding of the high and low potency bitter ligands to T2R1. In our docking studies, the only amino acid residue that is found to be involved in hydrogen bonding with all the ligands is Asn66 present on TM2 (Fig. 3B and Table 2). The other important amino acids in the ligand binding pocket of T2R1 that are involved in interactions with the ligands are Asn89, Asn163, Glu90 and Glu74.
4. Conclusion In this study we characterized in vitro a receptor present in the human oral cavity that is activated by some of the food protein derived bitter tasting di- and tri-peptides. We showed that bitter peptides activate the human bitter receptor T2R1, at concentration ranges that humans also perceive as bitter. Some of the peptides with ACE-inhibitory activity were also able to activate the T2R1 receptor. Among the peptides tested, the tri-peptide FFF showed high efficacy with an EC50 in the micromolar range. Results from the molecular modeling studies of the receptor-ligand complexes show that T2R1 receptor has only one predominant binding site for the agonist ligands. We identify few amino acid residues on T2R1 that might be important for ligand binding, and are potential targets for future structure–functions studies on T2R1.
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Bitter taste receptor T2R1 is activated by dipeptides and tripeptides Jasbir Upadhyaya a, Sai Prasad Pydi a, Nisha Singh a, Rotimi E. Aluko b, Prashen Chelikani a,* a b
Department of Oral Biology, University of Manitoba, Winnipeg, Canada MB R3E 0W4 Department of Human Nutritional Sciences and The Richardson Center for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, Canada MB R3T 6C5
a r t i c l e
i n f o
Article history: Received 20 June 2010 Available online 27 June 2010 Keywords: G-protein coupled receptors (GPCRs) Bitter taste receptors T2R1 Bitter peptides Molecular modeling
a b s t r a c t Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, the molecular targets or receptors in humans for these bitter peptides were poorly characterized and least understood. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. In addition, we tested the ability of peptide inhibitors of the blood pressure regulatory protein, angiotensin converting enzyme (ACE) to activate T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium was measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Among the peptides examined, the bitter tri-peptide Phe-Phe-Phe (FFF), is the most potent in activating T2R1 with an EC50 value in the micromolar range. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The molecular models showed that the bitter peptides bind within the same binding pocket on the receptor. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2 forming a cap like structure on the binding pocket. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Humans are capable of sensing five basic tastes which are, sweet, umami, bitter, sour and salt. While the signal transduction for sweet, umami and bitter tastes is through G-protein coupled receptors (GPCRs), the sour and salt tastes are sensed by ion channels. In humans more than 30 GPCRs are known to mediate taste perception, with 25 of these predicted to sense bitter tastes alone. The human genome encodes 25 bitter taste receptors (T2Rs) localized as clusters on chromosomes 5p15, 7q31 and 12p13 [1–3]. While sweet taste perception is well studied and the sweet taste receptors (T1Rs) well characterized, very little is known regarding the bitter and umami tastes sensed by GPCRs. The human T2Rs are between 290 and 333 amino acids, and have extracellular amino-termini that are 5–19 amino acids long, and intracellular carboxyl-termini that are 7–32 amino acids long. Amino acid sequence analysis of the T2Rs showed that the intracellular loops have a higher degree of sequence similarity and the most divergent parts in T2R sequences are the extracellular loop
* Corresponding author. Address: D319, Department of Oral Biology, 780 Bannatyne Avenue, University of Manitoba, Winnipeg, Canada MB R3E 0W4. Fax: +1 204 789 3913. E-mail address: [email protected] (P. Chelikani). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.097
regions. The signaling events in the perception of bitter taste start with the binding of specific water-soluble molecules (tastants or ligands) to the extracellular and/or transmembrane domains of T2Rs initiating an intracellular signaling cascade. Based on selectivity towards bitter tastants there are at least two different classes of T2Rs, those like T2R16 are broadly tuned and recognize a wide variety of bitter ligands [4], while others like T2R4 are very specific and recognize only selective ligands [5]. However, the mechanisms underlying these differences in specificities have so far not been elucidated. In addition, the ligand binding pocket of T2Rs, as well as the molecular changes that take place in T2Rs upon ligand binding is poorly understood. Recently, it was shown that the human T2Rs are activated by the bitter tasting extracts of the milk protein, casein [6]. It was the first report to show that bitter receptors can be activated by bitter peptides. However, in that study only two synthesized bitter di-peptides, Gly-Phe and Gly-Leu, were shown to activate the bitter receptor, T2R1. In addition, the ligand binding pocket on the T2Rs for these peptides was not elucidated. In this report, using a synthetic T2R1 gene and a heterologous expression system, we showed that various food protein derived and bitter tasting tri and di-peptides activate the human bitter receptor, T2R1. Furthermore to elucidate the potential ligand binding pocket of T2R1 for these tri- and di-peptides, we used homology modeling to construct molecular models of the T2R1 bound
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to different ligands. Results from our molecular modeling showed that the ligand binding pocket for T2R1 was located close to the extracellular surface. In addition, we showed that amino acid residues from TM1, TM2, TM3 and TM7 and residues from ECL-1 and ECL-2 contribute to ligand binding in T2R1.
2. Materials and methods 2.1. Materials The detergent n-dodecyl-b-D-maltoside (DM) was purchased from Anatrace (Maumee, OH, USA). The monoclonal antibody, rho1D4, was prepared by the Cell Culture Center (Minneapolis, MN, USA) from a cell line provided by R.S. Molday (University of British Columbia, Vancouver, Canada). The S-17 polyclonal antibody was from Abcam, MA, USA. Fetal bovine serum and DME High Glucose were purchased from Sigma and Invitrogen. Common chemicals and bitter compounds were purchased either from Fisher or Sigma. Fluo-4NW was purchased from Invitrogen (Carlsbad, CA, USA). Bitter peptides were synthesized commercially (Genescript, NJ, USA). 2.2. Molecular biology and cell culture The synthetic T2R1-rho1D4 gene consisted of 954 bp, of which the 897 bp encodes T2R1. The salient features of the synthetic T2R1 gene include, a Kozak consensus (GCCACCATGG) 50 to the ATG start codon [7], a bovine rhodopsin C8 peptide tag (ETSQVAPA) immediately 50 to the natural stop codon of T2R1, and restriction sites for EcoRI at the 50 end and NotI at the 30 end to facilitate cloning. The sequence encoding the human T2R1 gene was optimized for mammalian cell codon usage [8], and the gene was synthesized commercially (Genescript, NJ, USA). The synthetic T2R1 gene was then inserted into the EcoRI and NotI sites of the mammalian expression vector pcDNA3.1 (Invitrogen). The T2R1-Rho38 gene which contains the first 38 amino acids of mouse rhodopsin placed in frame with the 50 -end of T2R1 coding sequence was a gift from Dr. Kenji Maehashi (Tokyo University of Agriculture, Tokyo, Japan) [6]. The T2R1 genes in pcDNA3.1 were transiently expressed in C6glioma cells using lipofectamine 2000 (Invitrogen) and as described by the manufacturer.
cells washed once with PBS and incubated with the calcium sensitive dye Fluo-4NW (Invitrogen) for 1 h. Receptor activation was determined by measuring changes in intracellular calcium after application of different concentrations of ligands (peptides) using Flexstation-3 fluorescence plate reader (Molecular Devices, CA, USA) at 525 nm following excitation at 494 nm. The calcium efflux was calculated using the following formula
½Ca2þ ¼ K d ðF F min Þ=ðF max FÞ where F is the experimentally measured fluorescence intensity; Fmin is the measured fluorescence intensity in the absence of Ca2+; Fmax is the measured fluorescent intensity of the Ca2+-saturated dye; and Kd is the dissociation constant of the Ca2+ indicator. Kd value of Fluo 4 calcium indicator dye is 345 nM [9]. Dose–response curves were generated and EC50 values calculated by nonlinear regression analysis using PRISM software version 4.03 (GraphPad Software Inc, San Diego, CA) after subtracting the responses of mock-transfected cells. 2.5. Molecular modeling of the T2R1 receptor The model of T2R1 receptor was built by homology modeling using the crystal structure of the active opsin (PDB ID: 3DQB) as template. The transmembrane regions of T2R1 were modeled using the program MODELLER 9V7 [10]. Loop regions of the receptor were modeled using ModLoop server [11]. Side chains of the amino acids were refined with SCWRL4 [12]. Then the whole molecule was energy minimized by 1000 steps of steepest descent (SD) and 1000 steps of conjugate gradients using SPDBV 4.0.1. Molecular dynamics simulations for 10 ps were performed for the active model using openMM Zephyr [13]. The quality of the model was verified by using the program PROCHEK [14] and 98.2% of the residues were in allowed regions of the Ramachandran Plot. The ligands were docked to the receptor using the program AutoDock Vina [15]. The binding site for the ligand on T2R1 was defined by forming a cube with the dimensions 60 80 70 around the protein with a grid point spacing 0.375 Å and center grid boxes 51.807, 12.467 and 38.921 in X, Y and Z dimensions, respectively. We performed 50 genetic algorithms runs for each ligand. In each run the best pose was saved. Finally, all poses were superimposed and the most frequent orientation of the ligand was taken as final pose. The receptor–ligand complexes generated as above were used for further analysis.
2.3. RT-PCR analysis 3. Results and discussion Genomic analysis of DNA extracted from C6-glioma cells by reverse transcriptase-PCR was done using a MJ Mini thermal cycler (Biorad laboratories, USA). The following primers were used, GAP DH (amplicon size 105 bp) Forward-ATGACTCTACCCACGGCAAG, Reverse-GATCTCGCTCCTGGAAGATG; T2R1-rho38 (amplicon size 129 bp) Forward-CCGTCACCCACTCTTCATCT, Reverse-GGGACCATA AACCCTGCATA; T2R1-rho1D4 (amplicon size 118 bp), Forward-A GAATGCCACCATCCAGAAG, Reverse-AGGCTGAAGATCAGGAGCAG; T2R1 (amplicon size 80 bp), Forward-CTCATTGTGGTTGTCCATGC, Reverse-GCCAGGCAAAATAGAAGCAG. GAPDH was used as an internal control; a control with reverse transcriptase omitted from the reaction was run to rule out the presence of contaminating genomic DNA, along with a control with no template present. The transcribed cDNA was used as a negative control. 2.4. Calcium assays Around 1.0–1.5 105 C6-glioma cells were plated into each well of a 96-well tissue culture treated BD-falcon biolux plates and plasmid DNA (200 ng/well) was transfected using liopfectamine 2000. Following 24 h transfection, the media was removed,
3.1. Expression of T2R1 Similar to most other GPCRs, the native T2R receptors are poorly expressed. There are various methods to improve the expression of GPCRs, the two frequently used methods are; codon-optimization of the genes to suit the expression system [16], and addition of the first 20–38 amino acids of rhodopsin to the N-terminal sequences of the poorly expressed GPCRs [5]. The two T2R1 constructs used in this study, T2R1-rho1D4 and T2R1-rho38 genes carry the above features and their construction is described in Section 2.2. The expression of these two genes was analyzed by PCR. Reverse transcriptase-PCR analysis confirmed the expression of the transiently-transfected T2R1-rho1D4 and T2R1-rho38 genes in glial cells. In addition, both the T2R1 genes were expressed at similar levels as observed on a 2% agarose gel (Fig. 1). DNA from untransfected glial cells was used to analyse the expression of endogenous T2R1 receptor, and no transcript was observed which shows that glial cells do not express any native T2R1 receptor (Fig. 1). The localization of the expressed T2R1-rho1D4 protein at the plasma membrane of glial cells was confirmed by immunofluorescence (Fig. 4, Supporting information).
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Table 1 Summary of the biochemical and sensory properties of synthetic peptide ligands and their ability to activate the human bitter receptor, T2R1. Peptide
Bitterness thresholda (mM)
Rcafb
ACEinhibitory activityc
EC50d (mM)
GF FL IF FFF GLL IQW LKP Dextromethorphan*
1.2 1.5 1.5 0.2 1.5 – – –
0.83 0.67 0.67 5.0 0.67 – – –
No No Yes No No Yes No No
7.3 ± 1.1 7.2 ± 0.7 7.4 ± 0.9 0.37 ± 0.11 4.9 ± 0.7 6.1 ± 2 7.6 ± 1.2 0.031
a
Fig. 1. Reverse Transcriptase (RT)-PCR analysis of T2R1 expression in C6 glial cells. The PCR amplified DNA were resolved on a 2% agarose gel stained with ethidium bromide and visualized under UV light. RT-PCR analysis confirmed the expression of the transiently-transfected T2R1-rho38 and T2R1-rho1D4 genes in glial cells. Glial cells do not express any endogenous T2R1 gene. GAPDH was used as an internal control. M indicates low molecular weight DNA marker and size in base pairs is indicated next to the gel.
However, in our experiments the polyclonal antibody against the T2R1-rho38 did not bind to the receptor and the T2R1-rho38 protein could not be visualized either by immunofluorescence or by immunoblot analysis. 3.2. Food protein derived bitter peptides A wide variety of peptides derived from proteins have been found in various foods and fermentated products, such as miso (salted and fermented soybean paste), soy sauce, cheese, and fish
Published values from sensory test [20]. Ratio of bitterness to caffeine [20]. Published literature values [19,21]. d Values determined by calcium dose–response functional assays (from this study). * Not a peptide ligand (bitter compound). b
c
sauce. Hydrolysis of proteins by proteolytic enzymes during fermentation is usually accompanied by the formation of a bitter taste, which is considered to be due to the presence of peptides. Soybean-derived peptides can play an important role in physiology, particularly in the prevention of chronic diseases [17,18]. The presence of bioactive peptides in protein hydrolysates and fermented products, and especially the ACE-inhibitory peptides have been a major focus of research in recent years [18,19]. In this study, we have used bitter peptides synthesized commercially (Genescript, NJ, USA). These peptides were previously isolated from food
Fig. 2. Dose-dependent changes in intracellular calcium [Ca2+] induced by bitter ligands in C6 glial cells. (A) Dose–response curves of T2R1 stimulated with increasing concentrations (lM) of the tri-peptide FFF and the synthetic bitter compound dextromethorphan (DXM). (B) Dose–response curves of T2R1 stimulated with increasing concentrations (mM) of different bitter peptides. Bitter peptides are shown in the inset. (C) Relative activation rates of T2R1 in response to FFF and DXM calculated based on their EC50 values. Values were normalized to the EC50 value of DXM. (D) Relative activation rates of T2R1 in response to different peptides calculated based on their EC50 values. All values were normalized to the EC50 value of FFF.
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proteins and their sensory properties characterized by different groups [19–21] (Table 1). 3.3. Activation of T2R1 by tri and di-peptides To elucidate, if the addition of peptides induce a functional response in T2R1-expressing glial cells, we stimulated both T2R1rho1D4 and T2R1-rho38 expressing glial cells with different bitter peptides. Our initial results showed that both T2R1 constructs elicited a rapid increase in intracellular calcium and showed similar functional responses to the ligands tested (data not shown). However, the ease of detection of T2R1-rho1D4 using the monoclonal antibody rho1D4 helped us decide in favour of using T2R1-rho1D4 construct for subsequent experiments. The ligands that activate T2R1 were classified into two groups based on their potency. The first group comprises of ligands which are highly potent and have EC50 values for T2R1 in the micromolar range and include, the bitter peptide Phe-Phe-Phe (FFF) and bitter synthetic compound dextromethorphan (Table 1). The tri-peptide FFF consists of hydrophobic amino acids with phenyl groups which are considered important for bitter taste sensation [22]. L-Phenylalanine exhibits a slightly bitter taste with a threshold value of 20 mM, while D-phenylalanine exhibits a sweet taste with a threshold value of 2.2 mM. However, a 100 times greater bitterness than that of phenylalanine has been observed in the taste of tri-phenylalanine (Table 1). The bitter taste of any peptide is more apparent when the hydrophobic amino acid is located at the C-terminus [20]. For the tri-peptides, the middle amino acid residue is considered more important than both the C- and N-terminal amino acids [19]. Among all the tested peptides, FFF activated T2R1-expressing cells at concentrations of 0.125–1 mM that humans also perceive as bitter, with an EC50 value of 370 lM (Table 1 and Fig. 2). The bitter synthetic compound dextromethorphan also falls in the group of high potency bitter ligands that activate T2R1. Pre-
vious functional studies showed that T2R1-expressing HEK293 cells were activated by dextromethorphan at a minimal or threshold concentration of 10 lM [23]. However, EC50 values were not determined in that study owing to the high signal noise at higher concentrations of dextromethorphan in HEK293 cells. Glial cells have comparatively lower basal calcium noise, and thus we were able to do a dose–response analysis for T2R1 activation by dextromethorphan, which resulted in an EC50 of 31 lM (Fig. 2). The second group encompasses peptides of medium potency and have EC50 values for T2R1 in the millimolar range, it includes di-peptides Gly-Phe (GF), Phe-Leu (FL), Iso-Phe (IF) and tri-peptides Gly-Leu-Leu (GLL), Leu-Arg-Pro (LKP) and Iso-Glu-Trp (IQW). In our study, tri-peptides have been found to be more potent than the di-peptides in activating T2R1, the rationale being that bitterness of peptides is intensified as the number of amino acids in the peptide chain increases. The tri-peptides LKP and IQW show saturated responses at concentrations of 10–15 mM, whereas signal saturation with other di- and tri-peptides was observed with 15–20 mM. At concentrations of LKP and IQW above 15 mM, a decrease in the signal was noticed due to a nonspecific inhibitory effect of the peptides in the assay. Alternatively, this might be due to desensitization of the receptor with higher concentrations of the peptides. Concentration ranges that span two orders of magnitude are often observed for T2Rs and their agonists. This was also seen with the peptides used in this study, as T2R1 was half maximally activated by micromolar concentrations of high potency ligands and by millimolar concentrations of medium potency peptides. 3.4. T2R1 ligand binding pocket The structure of T2R1 receptor was built by homology modeling using opsin as a template and as described in Section 2.5. The
Fig. 3. (A) Side view of T2R1 receptor showing the ligand binding pocket. Transmembrane helices (TM) of the T2R1 are shown along with the ligand binding pocket present towards the extracellular surface represented as a mesh. (B) Extracellular view of the ligand binding pocket of T2R1. The seven TM helices along with the three extracellular loops (ECL1–3) are shown in this enlarged view of the binding pocket. The amino acid residues Asn66 on TM2, Glu74 on ECL1, Asn89, Glu90 on TM3 and Asn163 on ECL2 involved in binding to the ligand, are also shown. The different ligands bind within the same binding pocket on T2R1 and are represented as ball and sticks. The color coding for the ligands are as follows, the tri-peptides FFF and IQW are represented in blue and green, the di-peptides GF and IF are represented in dark pink and yellow and the bitter compound dextromethorphan is represented in cyan. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
J. Upadhyaya et al. / Biochemical and Biophysical Research Communications 398 (2010) 331–335 Table 2 The amino acid residues lining the ligand binding pocket of T2R1. Ligand
FFFb
IQWb
Dextromethorphan GFb IFb
Amino acids in the 4 Å binding pocket of T2R1a Residues in the transmembrane helices
Residues in extracellular loop region
Iso62, Asn66, Val69, Iso70, Leu86, Asn89, Glu90, His240, Leu260, Phe261, Phe262, Iso263 Leu19, Thr23, Iso62, Phe63, Asn66, Val69, Iso70, Leu85, Leu86, Asn89, Glu90, Phe261, Iso263, Leu264, Glu267 Phe6, Iso62, Asn66, Asn163, Phe257, Leu260, Phe261, Iso263 Phe17, Leu19, Asn66, Val69, Iso70, Leu86, Phe257, Phe261 Phe17, Leu19, Phe22, Thr23, Iso26, Iso62, Phe63, Asn66, Iso70, Phe261, Iso263, Leu264
Glu74, Asn163
Glu74
335
Acknowledgments This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) individual Discovery grants to PC and RA. SP is supported by a University of Manitoba Graduate Fellowship (UMGF). RA is also a recipient of an operating grant from the Advanced Foods and Materials Network (AFMNet) of Centre of Excellence. PC holds a New Investigator Salary Award from Heart and Stroke Foundation of Canada (HSFC). Appendix A. Supplementary data
Glu74
a
Amino acid residues involved in hydrogen bond formation with the ligand are shown in bold. b Peptide ligands.
final model was docked with five different ligands using AutoDock Vina. Unlike sweet taste receptors where the ligand binding pocket is located in the N-terminal domain, our docking studies showed that the ligand binding pocket for T2R1 was located close to the extracellular surface (Fig. 3A). For all the ligands docked, amino acid residues from TM1, TM2, TM3 and TM7 and residues from ECL-1 and ECL-2 contributed to ligand binding (Fig. 3B). Results from our docking studies showed that the ligands are bound in different orientations within the same binding pocket; this could be due to the differences in their chemical structures which influence their interactions with the amino acids in the binding pocket. Table 2 lists the amino acids lining the binding pocket, though only a few are found to interact with most of the ligands docked. Except for minor variations in the orientation of the bound ligands (Fig. 3B), no major differences were observed between the mode of binding of the high and low potency bitter ligands to T2R1. In our docking studies, the only amino acid residue that is found to be involved in hydrogen bonding with all the ligands is Asn66 present on TM2 (Fig. 3B and Table 2). The other important amino acids in the ligand binding pocket of T2R1 that are involved in interactions with the ligands are Asn89, Asn163, Glu90 and Glu74.
4. Conclusion In this study we characterized in vitro a receptor present in the human oral cavity that is activated by some of the food protein derived bitter tasting di- and tri-peptides. We showed that bitter peptides activate the human bitter receptor T2R1, at concentration ranges that humans also perceive as bitter. Some of the peptides with ACE-inhibitory activity were also able to activate the T2R1 receptor. Among the peptides tested, the tri-peptide FFF showed high efficacy with an EC50 in the micromolar range. Results from the molecular modeling studies of the receptor-ligand complexes show that T2R1 receptor has only one predominant binding site for the agonist ligands. We identify few amino acid residues on T2R1 that might be important for ligand binding, and are potential targets for future structure–functions studies on T2R1.
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