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J. Biol. Chem., Vol. 279, Issue 43, 45068-45075, October 22, 2004
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From the
Department of Physiology and Biophysics and Howard Hughes Medical Institute, Mount Sinai School of Medicine, New York, New York 10029
Received for publication, June 17, 2004 , and in revised form, August 4, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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T1R2 and T1R3 are subclass 3 G-protein-coupled receptors (1–7). Other members of this subclass are metabotropic glutamate receptors (mGluRs),1 calcium-sensing receptors, pheromone receptors, and other taste/olfactory receptors (T1R1, 5.24 odor receptor) (8). Each member of this family has a large extracellular amino-terminal domain (ATD) followed by a cysteine-rich linker domain and a seven-transmembrane-spanning helical region.
The solved crystal structures of the ATD of homodimeric metabotropic glutamate type 1 receptor (mGluR1) show that the mGluR1 ligand-binding region consists of two amino-terminal protomers (9). Each protomer comprises LB1 and LB2 domains that form a clamshell-like structure with the ligand-binding domain lying between LB1 and LB2. The free-form I (open-open_R) is thought to be in the resting state, whereas the free-form II (closed-open_A) is thought to be the active state. Agonist binding stabilizes the active closed-open_A conformer and promotes a shift of equilibrium toward the active state. The role of the cysteine-rich region, which links the ATD to the transmembrane domain, is presently unknown.
Based on sequence homology and predicted secondary structural similarity to mGluR1, it seems likely that the T1R2 + T1R3 sweet receptor will also have open-open and open-closed forms and that small sweet compounds may stabilize the active form of T1R2 + T1R3 by binding within the cleft. However, unlike the mGluRs, the sweet receptor is heterodimeric with each monomer potentially playing a distinct role. The T1R2 protomer is the ligand-specifying component for many agonists (10). Although T1R3 is shared by both the sweet receptor (T1R2 + T1R3) (1, 2) and the amino acid receptor (T1R1 + T1R3) (1, 11), and is essential for both sweet and umami taste (10, 12), its physical role in taste signal detection/transmission is unknown.
Six sweet tasting proteins, ranging in size from 6 to 
22 kDa, have been discovered (13). The mass of these protein sweeteners makes them too large to fit within the presumptive small molecule-binding pocket, suggesting that they stabilize the active conformation of the sweet receptor in a different manner. Identifying the binding sites for protein sweeteners might provide insights into the molecular events leading to receptor activation. Monellin and brazzein are intensely sweet proteins the structures of which have been solved (14, 15); these two proteins are not structurally similar or similar to any of the other sweet proteins. We used the fact that the protein sweeteners (and also aspartame) are only perceived as sweet by Old World primates (16–18) to identify a region of the human sweet receptor that is necessary for it to respond to brazzein.
Heterologously expressed human T1R2 (hT1R2) + human T1R3 (hT1R3) responds to monellin and thaumatin, but mouse T1R2 (mT1R2) + mouse T1R3 (mT1R3) does not (1, 2). Using mixed pairs of human and mouse T1Rs and mouse/human chimeric receptors we have determined that the residues required for a human-like response to monellin lie within the ATD of T1R2. hT1R2 + hT1R3 responds to brazzein, but hT1R2 + mT1R3 does not, indicating that residues in hT1R3 are required for receptor activity toward brazzein. We have located these human-specific residues within a small area in the cysteine-rich region of T1R3 (amino acids 536–545); this is the first case in which the cysteine-rich region of a G-protein-coupled receptor has been implicated in receptor function. Replacement of the cysteine-rich region of mouse T1R3 with the corresponding human segment allows hT1R2 + humanized mT1R3 to respond to brazzein. This same segment of T1R3 influences receptor activity toward monellin as well.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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l6 chimeras were generated by polymerase chain reaction (PCR) with mutagenic primers and cloned into pCDNA3. The five-residue carboxyl-terminal tail of Gl6 was replaced by its counterpart from Ggust(DCGLF) or Gi3(ECGLY). Construction of human/mouse chimeras of T1Rs was performed by PCR using overlapping primers (19). The integrity of all DNA constructs was confirmed by automated DNA sequencing. Point mutations in genes were made using the same overlapping PCR strategy.
To construct pRluc-hT1R2 and pGFP-hT1R3, hT1R2 and hT1R3 were amplified by PCR using primers that removed the stop codons and introduced a novel NruI restriction site. The restriction fragments containing the hT1R2 or hT1R3 coding portions were inserted into the GFP- or Rluc-BRET plasmids between the EcoRI and EcoRV sites. This placed either GFP or Rluc at the COOH terminus of the designated T1R sequence. To construct GFP-tagged hT1R3 mutants, hT1R3 mutant plasmids were digested with EcoRI and KpnI then inserted into the hT1R3-GFP plasmids between the EcoRI and KpnI sites. This placed GFP at the COOH terminus of the designated mutant hT1R3 sequence.
Functional Expression—HEK293 EBNA (HEK293E) cells were cultured at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For calcium imaging experiments, cells were seeded onto 6-well plates, and plasmid DNAs were transiently cotransfected into HEK293E cells using Mirus TransIT-293 (Panvera, Madison, WI). T1R2 or T1R3 or their mutants or chimeras were transfected using 0.6 µg of plasmid, Gl6-i3 or G16-igust at 0.3 µg, and pDsRed-2 vector at 0.1 µg per well. We found that G16-i3 produced a larger signal than G16-igust, so it was used for all but the initial assays. The transfection efficiencies were estimated by visualizing DsRed fluorescence or by immunohistochemistry of T1R3 and were typically >20%. After 24 h, cells were trypsinized and replated onto polylysine-coated 24- or 96-well tissue culture plates (200,000 cells/well for a 24-well plate and 40,000/well for a 96-well plate) and cultured in low glucose Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal bovine serum (Invitrogen) and lx glutaMAX-1 (Invitrogen). After an additional 24 h, transfected cells were loaded with 200 µl for a 24-well plate or 50 µl for a 96-well plate of 3 µM fluo-4 acetoxymethyl ester (Molecular Probes) in Dulbecco's phosphate-buffered saline (DPBS) (Invitrogen) for 1.5 h at room temperature. Then the transfected cells were washed with DPBS once and incubated in 200 µl of DPBS/well for a 24-well plate and 50 µl for a 96-well plate. Stimulation was performed with 200 µl of DPBS supplemented with tastants. Calcium mobilization was recorded using an Olympus Fluoview confocal microscope. Acquisition and data analysis of time series images were performed using Fluoview 2.1 version software. Cells were scanned at 3.2 s/image, and at the third scan, tastant was added into the recorded wells; the response was recorded for an additional 27 scans, and the total recording time was 96 s. To quantify the calcium change, 
F/F was calculated as the change of fluorescence level of the whole image field (peak - base line) normalized to the base-line level. For those non-responding wells, F/F was calculated as the change of fluorescence between the fluorescence level of the frame at which fluorescence normally reached the peak after addition of tastants and the normalized base-line level.
Response Profiles—For each pair of T1R2 and T1R3, the calcium response to six sweeteners was recorded. The typical concentration of sweeteners was as follows: sucrose (75 mM), monellin (0.1%, 89 µM), brazzein (0.25%, 380 µM), D-tryptophan (10 mM), aspartame (2.5 mM), and N-saccharin (1 mM). These sweeteners were chosen for their structural diversity. Brazzein was a gift from Dr. Hellekant; monellin, D-tryptophan, and N-saccharin were purchased from Sigma, and aspartame was purchased from Lab Safety Chemical.
Immunostaining—HEK293E cells were transfected with T1R2 + T1R3 or T1R3 mutants, and G16-i3. After 24 h, transfected HEK293E cells were replated onto polylysine-coated coverslips. After an additional 24 h of growth in low glucose medium supplemented with glutaMAX-1 and dialyzed fetal bovine serum, cells were rinsed with 1x DPBS and then fixed for 5 min with 4% paraformaldehyde. After three washes with 1x phosphate-buffered saline, cells were blocked in 2% horse serum for 20 min and then incubated with anti-T1R3 polyclonal antibody (see below) for 30 min. Alexa fluro-488-conjugated anti-rabbit secondary antibodies were used to visualize the staining.
Polyclonal antibodies were made against epitopes within the extracellular domain of mT1R3 (HEGLVPQHDTSGQQLGK) and hT1R3 (EEAGLRSRTRPSSP) and affinity purified using peptide columns (12). No immunostaining was seen in mock transfected cells (supplemental Fig. 3). Using these antibodies we observed specific staining of mouse (12) and human taste receptor cells (data not shown). Images were taken using an Olympus Fluoview confocal laser scanning microscope.
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50,000) were transferred into triplicate wells of a 96-well microplate (white Optiwell plates from Packard). To start the assay, 10 µl of Deep BlueC coelenterazine was added to each well to a final concentration of 5 µM. Bioluminescence resonance energy transfer (BRET) readings were performed using a FUSION plate reader (Packard). The BRET ratio was defined as ((emission at 500–530) - (emission 370–450) x Cf)/(emission at 370–450), where Cf corresponds to (emission at 500–530)/(emission at 370–450) for the receptor-Rluc construct expressed alone in the same experiment. |
RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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16-i3. Consistent with earlier characterizations of the sweet receptor, HEK293E cells expressing hT1R2 + hT1R3 responded to all tested sweet compounds, including D-tryptophan, N-saccharin, sucrose, aspartame, and monellin (Fig. 1A). As expected, HEK293E cells expressing hT1R2 + hT1R3 responded to brazzein with a strong calcium response (Fig. 1, A and B).
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Human Residues in the ATD of T1R2 Are Required for Responses to Monellin—To determine whether the extracellular or transmembrane/intracellular portions of hT1R2 were required for the response of the sweet receptor to monellin, we made a human/mouse chimera joining the extracellular domain of hT1R2 (residues 1–564) to the mouse transmembrane/intracellular domain of mT1R2 (mouse residues 569–843, corresponding to human 565–839). Chimera designations follow a scheme where the residues from hT1R2 or hT1R3 are specified in the name of the construct and the mouse portion of T1R3 indicated at the 5' or 3' end. (In this case, h.1–564.mT1R2 contains hT1R2 residues 1–564 and the remaining 3' end residues come from mT1R2.) The human/mouse chimera (h.1–564.mT1R2) was cotransfected with mT1R3 and G-protein and tested for responses to the panel of sweeteners (Fig. 1D). The response profile of mT1R3 + T1R2 chimera was indistinguishable from that of mT1R3 + fully human hT1R2, indicating that the human-specific residues of hT1R2 required for the sweet receptor to respond to monellin all reside in the extracellular portion.
Human Residues in the Cysteine-rich Region of T1R3 Are Required for Responses to Brazzein—That hT1R2 + mT1R3 did not respond to brazzein suggests that human-specific residues in hT1R3 are required for this response. This contrasts with the other human-specific sweeteners that activated hT1R2 + mT1R3 (1, 2, 10). We reasoned that the human-specific residues in T1R3 required for responsiveness to brazzein would reside in the ATD. To map the portion of hT1R3 essential for brazzein-induced activity, we generated several human/mouse chimeras encompassing the entire extracellular domain containing the presumptive ligand-binding domain and the cysteine-rich region (Fig. 2, A and B). The T1R3 chimeras, in combination with hT1R2, were tested for responses to brazzein and the panel of sweeteners. Only two chimeras, h.1–567.mT1R3 and h.1–545.mT1R3, responded to brazzein (Fig. 2B). h.1–567.mT1R3 displayed enhanced responses to brazzein, monellin, and aspartame, whereas h.1–545.mT1R3 had diminished responses to all sweeteners. The other constructs with less extensive 5' hT1R3 sequences (e.g. h.1–535.mT1R3) did not respond to brazzein but did respond to all other sweeteners in the panel and in some cases showed large increases in responsiveness to the small molecule sweeteners. These results indicate that human-specific residues of hT1R3 between 536 and 545 are required for responses to brazzein.
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Ala-537 and Phe-540 Are Important for hT1R3 Responses to Brazzein—Within region 536–545 of hT1R3 there are five amino acids that differ from mT1R3 (Fig. 3A). We mutated singly each of these five amino acids of hT1R3 to their mouse counterpart and then tested for "loss of function" (Fig. 3B). Significantly diminished responses to brazzein were seen with the A537T and F540P mutants. hT1R3(A537T) lost all responsiveness to brazzein but retained responses to the small molecule sweeteners and to monellin. hT1R3(F540P) had greatly diminished responses to brazzein, normal responses to monellin, and greatly elevated responses to the small molecule sweeteners (Fig. 3B). The selective and severe loss of responsiveness to brazzein by hT1R3(A537T) demonstrates that Ala-537 is an essential determinant of the response of hT1R3 to brazzein.
The hT1R3(G542N) mutation had no effect on T1R3 responses to brazzein or other sweeteners (Fig. 3B). The hT1R3(E545Q) mutant had decreased responsiveness to brazzein, and similarly decreased responses to the other sweeteners (Fig. 3B) that may be due to reduced efficacy of the receptor (surface expression and BRET activity for E545Q was normal (supplemental Figs. 1A and 2)). The responses of hT1R3(I536F) to all sweeteners were reduced in comparison with those of wild-type hT1R3, with a relative increase in responses to monellin over brazzein and to the protein sweeteners over the small molecule sweeteners (Fig. 3B). Thus, it appears that only A537T and F540P differentiate hT1R3 from mT1R3 in determining responsiveness to brazzein.
To determine whether substitution of any of these amino acids conferred brazzein responsiveness on mT1R3 we mutated singly each of these five amino acids of mT1R3 to the human counterpart and then tested for "gain of function" (Fig. 3C). Only the T542A mutation conferred brazzein responsiveness upon mT1R3, confirming the importance of Ala-537 for the responses of hT1R3 to brazzein. The other mouse to human mutations in this segment did not rescue brazzein activity but did have effects on the relative responses to other sweeteners in the test panel, implicating this region in determining, at least in part, responses to monellin and the small molecule sweeteners (see "Discussion").
T542A and P545F Confer Brazzein Responsiveness on mT1R3—In comparison with hT1R3, the mT1R3(T542A) mutant displayed reduced responsiveness to brazzein, suggesting that other residues might also contribute to the ability of hT1R3 to respond to brazzein. (Fig. 4A). Double and triple mutations were made by replacing mouse residues in mT1R3 with their corresponding human residues. The mutants tested were mT1R3(AF) (substituted by T542A and P545F), mT1R3(IA) (substituted by F541I and T542A), mT1R3(IF) (substituted by F541I and P545F), and mT1R3(IAF) (substituted by F541I, T542A, and P545F) (Fig. 4, A and B). As expected, only those mutants containing the T542A substitution responded to brazzein, but those that also contained the P545F substitution showed a greater response than did mT1R3(IA) or mT1R3(T542A) (Fig. 4, B and C).
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to 1.11 x 10-3). Although mT1R3 paired with hT1R2 responds to monellin (EC50 of 6.90 x 10-5), it is with much lower apparent affinity than that shown by hT1R3 + hT1R2 (EC50 of 1.13 x 10-5) (Fig. 4D). Responses to monellin were also altered by mutations in this region. The dose-response curves (Fig. 4D) showed that several of these mutants left-shifted the responses to monellin to varying degrees; mT1R3(P545F) (EC50 of 9.39 x 10-6) was comparable with hT1R3, mT1R3(AF) (EC50 of 4.06 x 10-6) was more sensitive than hT1R3, and mT1R3(IAFGE) (EC50 of 2.05 x 10-5) was slightly less sensitive than hT1R3.
Ala-537 and Phe-540 Substitutions Affect T1R3 Responses to Many Sweeteners—To investigate the physicochemical effects of residues Ala-537 and Phe-540 of hT1R3 on receptor activity toward brazzein, monellin, and small molecule sweeteners, we made various substitutions at these two positions (Fig. 5, A and C). As with the A537T mutant, substitution of Ala-537 by serine, glutamine, or valine abolished responses to brazzein without affecting the responses to monellin or the other sweeteners (Fig. 5B). hT1R3(A537G) had a markedly diminished response to brazzein and diminished responses to monellin and the other sweeteners (Fig. 5B). hT1R3(A537P) lost responsiveness to all of the sweeteners in our test panel. The surface expression of these mutants was comparable with that of hT1R3, and the BRET activities of hT1R2 + mutants were indistinguishable from those of hT1R2 + hT1R3 (supplemental Figs. 1B and 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Effects of Substitutions at Ala-537 and Phe-540 of hT1R3 on Receptor Responses—Substitution of hT1R3 Ala-537 with threonine, serine, glutamine, or valine abolished responses to brazzein. Only the A537G mutant retained partial activity toward brazzein; however, this mutation also suppressed responses to the other sweeteners tested, suggesting that sweetener signal transmission in general may be altered in this mutant. The inability of valine, a small hydrophobic residue, to substitute for A537, and the partial activity seen with A537G suggest that it is the small size of the side chain of alanine that is important at this position. Presumably, steric hindrance by the larger side chains of threonine, serine, glutamine, or valine led to the lack of responses shown by these mutants. Because the majority of Ala-537 substitutions selectively affected responses to brazzein, it seems likely that this is steric interference acting on the binding site of brazzein, either directly or indirectly.
The A537P substitution of hT1R3 resulted in a receptor that was unresponsive to all sweeteners tested. Ala-537 is in a segment predicted to have a 
strand conformation (20). The neighboring C538 residue is predicted to be buried and may be in a disulfide bond with another conserved cysteine in this region (20). A change in backbone flexibility (either more flexible (A537G) or less flexible and kinked (A537P)) might alter the formation of the predicted strand and thereby alter the conformation of this region in a way that makes it less able to transmit the signal through the receptor. This suggests that the cysteine-rich region may couple ligand binding effects in the ATD to receptor output in the transmembrane/cytoplasmic domain to modulate the strength of coupling. It is particularly surprising to find such an effect for the cysteine-rich region of T1R3 in light of T1R2 appearing to be the ligand-binding monomer for most ligands (1, 2, 10). Thus, mutations at Ala-537 (and at F540, see below) may exert two separate effects: 1) altered access to the brazzein binding site and 2) altered signal transmission for all sweeteners in our test panel.
Specific mutations at position Phe-540 reduced the response to brazzein (F540Y, F540P), to monellin (F540L), or to brazzein and monellin (F540A and F540H). In addition, hT1R3 (F540P) also enhanced responses to the small molecule sweeteners. Substitution of hT1R3 by proline, native to mT1R3 at this position, makes the response profile of hT1R2 + hT1R3 (F540P) nearly identical to that of hT1R2 + mT1R3, indicating that this substitution plays a key role in differentiating responses of the wild-type human receptor (hT1R2 + hT1R3) from those of the mixed species receptor (hT1R2 + mT1R3) (see Fig. 1B). Brazzein-induced activity of T1R3 benefits from large nonpolar side groups at position 540, although there is not a strict requirement for a hydrophobic residue here, because tyrosine at this position supported near normal activity. Monellin-induced activity was best supported by Phe-540 itself or F540Y, suggesting that an aromatic residue is important for this response.
The selective alteration of responses to brazzein by some T1R3 mutants at Ala-537 and Phe-540 argues that at least some of the essential interactions of brazzein with the receptor occur at a site distinct from the small molecule binding site, i.e. the brazzein and small molecule binding site (presumed to be in the cleft of T1R2) may not overlap in part or in entirety.
The Effect of T1R2 on Brazzein-induced Activity—It is tempting to assume that brazzein, unlike the other human-specific sweet ligands, interacts primarily with T1R3 because it is the species of T1R3 monomer that determines responsiveness to brazzein. In preliminary studies, we have determined that hT1R2 is better able than mT1R2, when paired with the humanized mT1R3, to support brazzein activity, suggesting that there are human T1R2 residues that interact favorably with brazzein (data not shown). This suggests that brazzein may interact with both T1R2 and T1R3. We and others have modeled the structure of brazzein docked to hT1R3 (21) or hT1R2 (data not shown); such models may prove useful for generating testable hypotheses regarding the structure of receptor-sweetener complexes.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains three supplemental figures.
¶ Associate Investigator of Howard Hughes Medical Institute.
|| To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Mount Sinai School of Medicine, Box 1677, 1425 Madison Ave., New York, NY 10029. Tel.: 212-659-8612; Fax: 212-849-2599; E-mail: max{at}inka.mssm.edu.
1 The abbreviations used are: mGluR, metabotropic glutamate receptor; BRET, bioluminescence resonance energy transfer; DPBS, Dulbecco's phosphate-buffered saline; ATD, amino-terminal domain; GFP, green fluorescent protein.
2 Q. Ji., P. Jiang, Z. Liu, L. A. Snyder, L. M. J. Benard, R. F. Margolskee, and M. Max, manuscript in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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T. Masuda, |
