Valine 738 and lysine 735 in the fifth transmembrane domain of rTas1r3 mediate insensitivity towards lactisole of the rat sweet taste receptor
© Winnig et al; licensee BioMed Central Ltd. 2005
Received: 03 February 2005
Accepted: 07 April 2005
Published: 07 April 2005
The sweet taste inhibitor lactisole acts on the human sweet taste receptor heteromer TAS1R2-TAS1R3 but not on its rodent counterpart. Recently, it was shown that the lactisole sensitivity of the human sweet taste receptor involves the part of TAS1R3 encompassing the seven transmembrane regions but not the huge N-terminal domain. Using mutational analysis we investigated which amino acid residues distinguish lactisole insensitive rat from sensitive human T1R3 receptors.
The functional analysis of specific receptor mutants in HEK293T cells revealed that the exchange of valine 738 in the fifth transmembrane domain of rTas1r3 by an alanine is sufficient to confer lactisole sensitivity to the rat sweet taste receptor. The sensitivity of this receptor mutant is ~2 fold lower than the sensitivity of the human sweet taste receptor. Additional substitution of lysine 735 by phenylalanine in rTas1r3 results in a rat sweet taste receptor that is as sensitive to lactisole as its human counterpart. The exchange of valine 738 to alanine was accompanied by a ~50% reduction in receptor efficacy. This effect was seen with all six different sweet compounds examined.
The lactisole insensitivity of rat sweet taste receptor is caused by only two amino acids in transmembrane region five, which is critical for the interaction of lactisole with the sweet taste receptor. The observation that the mutant receptor simultaneously displays a generally reduced sensitivity towards all agonists suggests that the lactisole insensitivity of the rodent receptor might be more likely caused by the inaccessibility of the lactisole binding site rather then by its direct disruption.
Sweet and umami taste involve TAS1R receptors, which belong to the subclass 3 of the GPCR super family and are distantly related to the V2R pheromone-, metabotropic glutamate- and calcium sensing receptors [1–3]. They have a large N-terminal extracellular domain that is linked by a cysteine-rich domain to the seven transmembrane domains . Heterologous expression and functional analysis showed that cells co-transfected with Tas1R2-TAS1R3 responded to a variety of natural and artificial sweeteners, while the combination TAS1R1-TAS1R3 responded to L-amino acids [3–5]. In line with observed differences in the perception of these compounds between humans and rodents [6–8], the artificial sweeteners aspartame and cyclamate, and the sweet proteins brazzein, monellin, and thaumatin only activate the human but not the rodent TAS1R2-TAS1R3 heteromer [2, 3]. Further studies of mouse-human interspecies chimeras and point-mutated receptors revealed that cyclamate interacts with the part of hTAS1R3 encompassing the seven transmembrane regions, whereas aspartame interacts with the N-terminal domain of hTAS1R2 . Moreover, the cysteine-rich motif in the N-terminal domain of hTAS1R3 is required for the activation of the sweet receptor by brazzein . These results and modeling studies on the basis of the solved X-ray structure of the mGLUR1 ligand binding domain strongly suggest that multiple sites in both receptor subunits are involved in the activation of the sweet receptor by its ligands [9–12]. Interestingly, differences in taste perception are not limited to sweet compounds but also have been observed for 2-(4-methoxphenoxy) propanoic acid sodium salt (lactisole), which suppresses the sweet taste of various compounds in humans but not in rats [3, 13–17]. Functional studies of the human and rat sweet taste receptor recently showed that lactisole inhibits the human TAS1R2-TAS1R3 heteromer but not that of rats . Moreover it could be shown that that the lactisole sensitivity of the human sweet taste receptor involves the TAS1R3 subunit . Thus, the inhibition of the sweet response by lactisole in humans is due to structural differences between the rat and human sweet taste receptor. Using mutational analysis we investigated the molecular basis for these differences.
Results and discussion
Lactisole inhibited the responses of cells transfected with the cDNAs for the human receptor to all tested sweeteners (Fig. 1a, 1b) while it did not inhibit the responses of cells transfected with the rat sweet receptor (Fig. 1a, 1c). This is well in line with previous studies of the receptor function [3, 9]. Based on the inhibitory effect of lactisole on the human but not on the rat receptor heteromer, we next asked which amino acids cause the functional difference of the receptors. We therefore analyzed cells co-transfected with cDNAs of the interspecies sweet taste receptor combinations, i.e. hTAS1R3-rTas1r2 or rTas1r3-hTAS1R2. The combination hTAS1R2-rTas1r3 responded to aspartame, saccharin, sucrose, and stevioside but not to cyclamate and neohesperidine dihydrochalcone (Fig. 1d). The responses induced by all four agonists were lactisole insensitive (Fig. 1d). The combination rTas1r2-hTAS1R3 clearly responded to the sweeteners cyclamate and neohesperidine dihydrochalcone, although with markedly reduced amplitudes. Aspartame, saccharin, sucrose, and stevioside failed to activate this receptor combination (Fig. 1e). This indicates that the hTAS1R3 subunit is involved in the response of the human sweet taste receptor to cyclamate and neohesperidine dihydrochalcone. Notably, the responses were lactisole sensitive (Fig. 1e). In line with previous observations  we therefore conclude that the lactisole sensitivity is solely mediated by the hTAS1R3 subunit.
To test if rTas1r3-V738A displays the same degree of lactisole sensitivity as the human receptor, we determined the half maximal inhibitory concentration (IC50) of lactisole on rTas1r3-V738A and compared it with the IC50 values of the human receptor and the mutants m6, m6/1, m6/2, and m6/3. Although all variants except the lactisole insensitive mutant m6/2 were inhibited by lactisole, we observed subtle differences (Fig. 3d). The IC50 values were 0.05 ± 0.01 for the human receptor, 0.06 ± 0.01 for m6, 0.11 ± 0.01 m6/1, 0.05 ± 0.02 for m6/3 and 0.16 ± 0.03 for rTas1r3-V738A (Fig. 3d). Thus, the human receptor and the mutants m6 and m6/3 behave identical while the mutants m6/1 and rTas1r3-V738A are ~2 fold less lactisole sensitive. This result shows that the replacement of lysine 735 by phenylalanine is required for full lactisole sensitivity of the mutant rat receptor.
The lactisole insensitivity of rat sweet taste receptor is caused by valine 738 in transmembrane region five. The additional replacement of a lysine residue by a phenylalanine at amino acid position 735 again in transmembrane region five is sufficient to increases the lactisole sensitivity of the mutant rat receptors to the level of the human counterpart. Thus, transmembrane region five is critical for the interaction of lactisole with the sweet receptor. The observation that the mutant receptors displays a generally reduced sensitivity towards all agonists suggests that the lactisole insensitivity of the rodent receptor might be more likely caused by an inaccessibility of the lactisole binding site rather then by its direct disruption. Instead the lactisole insensitivity of the rodent receptor more is likely a side effect that is caused by a steric alteration in the fifth transmembrane region of the rat TAS1R3 subunit induced by the valine at amino acid 738. Recently functional studies of the mouse and human sweet taste receptor in combination with mutational analysis led to a model of the lactisole binding site in human TAS1R3 . This model predicts that the alanine 733 in human TAS1R3 which corresponds to valine 738 of rat TAS1R3 is not directly involved in lactisole binding although it lies in close proximity to the lactisole binding pocket. The proximity of this amino acid to the predicted lactisole binding pocket further supports our conclusion that valine 738 in rat sweet taste receptor prevents the access of lactisole to its binding site.
Cloning of the TAS1R cDNAs
hTAS1R2 was constructed via amplification of all exons from genomic DNA by recombinant polymerase chain reactions (PCR) . hTAS1R3 was cloned from testis cDNA. rTas1r2 was cloned from rat vallate papillae cDNA. PCR products were cloned into a pcDNA5/FRT expression vector (Invitrogen), fused with a C-terminal FLAG-epitope for immuncytochemical detection, sequenced, and checked for comparable protein expression by immuncytochemistry of transfected HEK293T cells.
Construction of chimeric receptor cDNAs
The receptor chimeras of Tas1r3 were constructed using recombinant PCR. In the first reactions the N-terminal and the C-terminal part containing the seven transmembrane domains of hTAS1R3 and rTas1r3 were amplified from plasmid DNA using Ultra Pfu Polymerase (Stratagene). For the hTAS1R3 and rTas1r3 N-termini the forward vector-anchored primer (CMV: CGCAAATGGGCGGTAGGCGTG) and a reverse gene specific primer (CACAGCCGGCTCGCCC) were used. The hTAS1R3 and rTas1r3 C-terminal part were amplified with forward gene specific primer (GGGCGAGCCGGCTGTG) and a reverse vector-anchored primer (TAGAAGGCACAGTCGAGG). PCR conditions were: 5 min, 94°C, 24 cycles 1 min 94°C, 0.5 min 64°C, 2 min 72°C; 5 min 72°C. The amplicons were purified using the Highpure PCR Product Purification Kit (Roche) and used as template for the second PCR. To create the chimeric receptor tas1r3-hN-rTM, equimolar amounts (~50 ng) of the hTAS1R3 N-terminus and rTas1r3 transmembrane domains were combined without additional oligonucleotides. PCR conditions were: 5 min 94°C; 15 cycles of 2 min 64°C, 2 min 72°C and 0.5 min 94°C; followed by 5 min 64°C and 10 min 72°C. The identical protocol and combining the rTas1r3 N-terminus with the human transmembrane regions resulted in receptor chimera tas1r3-rN-hTM. These chimeras were cloned into pcDNA5/FRT.
Construction of Tas1r3 mutants
Receptor mutants were generated by site directed mutagenesis according to the QuikChange protocol (Stratagene). The forward and reverse complement primers contained the desired mutations and annealed to the same sequence on opposite strands of the plasmids. The following rTas1r3 receptor variants were generated: m1: S576L; m2: R632Q, S635P, S637Q; m3: M644L A645S; m4: A702T; m5: Q717H, V718M, V723A, E725V; m6: L735F, V738A, I740A. m6/1: V738A, I740A, m6/2: L735F, I740A, m6/3: L735F, V738A. m7: A793V, Y794L, Q795R. Subsequently a rTas1r3-V738A variant was generated. All constructs were checked by sequencing.
The cDNAs were transiently transfected into HEK293T cells stably expressing the chimeric G-protein subunit Gα16gust44  using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. 3–4 hours after transfection, DMEM was replaced by low-glucose DMEM supplemented with GlutaMAX and 10% dialyzed FBS (Invitrogen). 22–44 hours later, cells were loaded for 1 hour with the calcium sensitive dye Fluo4-AM (2 μg/ml in DMEM, Molecular Probes). Cells were washed 3x in solution C1 (130 mM NaCl, 5 mM KCl, 10 mM Hepes, 2 mM CaCl2, and 5 mM Glucose, pH 7,4). Calcium mobilization was monitored by an automated fluorometric imaging plate reader (FLIPR, Molecular Devices). Ligands (Sigma-Aldrich, Merck) were dissolved in C1 solution. All data were collected from at least two independent experiments carried out in triplicate. The obtained calcium signals were corrected for the response of mock transfected cells and normalized to the fluorescence of cells prior to the stimulus using ΔF/F=(F-F0)/F0. Concentration-response curves and EC50 and IC50 values were calculated in SigmaPlot by nonlinear regression using the function f = ((a-d)/(1+(x/EC50)nH)+d) and f = (a-b)/ [1+(x/IC50)nH]+b respectively.
HEK-293T/Gα16gust44 cells were seeded on coverslips coated with 10 μg/ml poly-D-lysine and transfected with the respective cDNAs. 48 h after transfection cells were washed with PBS and fixed and permeabilized for 5 min in acetone:methanol (1:1). Non-specific binding was reduced by incubating the cells in 5% goat serum for 1 h. To detect the receptors, antiserum against the FLAG-epitope (anti-FLAG M2 (Sigma), 1:2000 in 3% goat serum) was added to the cells for 1 h at room temperature (RT). After washing the cells three times with PBS we added Alexa488-conjugated goat antiserum against mouse IgG ((Molecular Probes), 1:1000 in 3% goat serum) for 1 h at RT. The cells were embedded in Fluorescent Mounting Medium (Dako) and analyzed using a fluorescence microscope (Zeiss Axioplan, Jena) and a camera (RT Slide, Visitron Systems, Munich).
List of abbreviations
G-protein coupled receptor
- HEK293T/ Gα16gust44:
Human embryonic kidney cells stably expressing the
- large T:
antigen and the G protein chimera Gα16gust44
half maximal effective concentration
half maximal inhibitory concentration
fluorometric imaging plate reader
We thank Jay Slack (Cincinnati) for the HEK293T Gα 16gust44 cell line, Hartwig Schmale and Nicole Burhenne (Hamburg) for the rTas1r3 cDNA, and Konrad Hoppe for providing stevioside, cyclamate, and aspartame. This project was supported through DFG-grants ME 1024 / 1–2 and ME 1024 / 2-1.
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