Functional Characterization of the Human Sweet Taste Receptor: High

Chapter 23

Functional Characterization of the Human Sweet Taste Receptor: High-Throughput Screening Assay Development and Structural Function Relation

Downloaded by EMORY UNIV on January 26, 2016 | http://pubs.acs.org Publication Date: March 4, 2008 | doi: 10.1021/bk-2008-0979.ch023

Xiaodong Li and Guy Servant Senomyx, Inc., 4767 Nexus Center Drive, San Diego, C A 92121

The human sweet taste receptor is a heteromeric complex of T1R2 and T1R3. We have developed a high throughput screening (HTS) assay for the receptor and demonstrated a tight correlation between the in vitro receptor activity and in vivo taste behavior. Furthermore, we developed a human-rat chimeric receptor system, and discovered different functional domains on both subunits of the receptor.

Introduction TIRs, a family o f class C G protein-coupled receptors (GPCRs), are selectively expressed in the taste buds (1-6). Functional expression of TIRs in HEK293 cells revealed that different combinations of TIRs respond to sweet and umami taste stimuli (6, 7). T1R2 and T1R3, when co-expressed in HEK293 cells, recognize a diverse set of natural and synthetic sweeteners. Similarly, T1R1 and T1R3, when coexpressed in HEK293 cells, respond to umami taste stimulus L-glutamate. This response is enhanced by 5'-ribonucleotides, a hallmark of umami taste. Recent experiments with knockout mice confirmed that TIRs indeed mediate mouse sweet and umami tastes (8, 9), 368

© 2008 American Chemical Society In Sweetness and Sweeteners; Weerasinghe, Deepthi K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

369 The class C GPCRs possess a large N-terminal extracellular domain, often referred to as the Venus flytrap domain (VFD) (10), and are known to function as either homodimers, in the cases of metabotropic glutamate receptors (mGluRs) and the calcium-sensing receptor (CaR), of heterodimers, in the case of the γ-aminobutyric acid type Β receptor ( G A B A R ) (10). The functional expression data suggest a heterodimer mechanism for TIRs: both T1R1 and T1R2 need to be coexpressed with T1R3 to be functional, which is supported by the overlapping expression patterns of TIRs in rodent tongue. Nonetheless, there has been no direct evidence that TIRs function as a heteromeric complex. It is possible that T1R3 is not a functional component of sweet and umami taste receptors, but merely a chaperone protein, which facilitates the proper folding or intracellular translocation of T1R1 and T1R2. The distinct ligand specificities of T1R1/T1R3 and T1R2/T1R3 receptors suggest that T1R1 and T1R2 play more important roles in ligand binding in sweet and umami taste receptors than T1R3. Support for this hypothesis was provided recently by results from mouse genetics where human T1R2 transgenic mice, generated on the T1R2 knockout background, displayed sweetener taste preferences similar to those of humans (9). However, the functional role of T1R3 and the overall structure/function relationship of T1R taste receptors remain largely unknown.


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Another intriguing observation of the T1R2/T1R3 receptor is the structural diversity of its ligands. This receptor is able to recognize every sweetener tested, including carbohydrates, sweet amino acids and derivatives, sweet proteins, and synthetic sweeteners (7). Additionally, the receptor exhibits stereo-selectivity for certain molecules. For example, it responds to D-tryptophan but not L tryptophan (7), which is in correlation with the sensory data. It is still a puzzle as to how this single receptor can recognize such a large collection of diverse chemical structures. In this study, we report the development of an HTS assay using the human sweet taste receptor. The in vitro receptor activity is highly correlated with human taste behavior. Furthermore, we utilized the species differences in T1R ligand specificity to demonstrate that the sweet taste receptor indeed functions as a heteromeric complex, and that there are likely more than one ligand binding sites on the receptor (11).

A High Throughput Screening Assay for the Human Sweet Receptor T1R2/T1R3 We have developed a very sensitive cell-based assay platform for the detection of human sweet receptor modulators. Our assay was developed using the phospholipase C (PLC) effector pathway, where the human sweet receptor dimmer T1R2/R3 couples to G a l 5 and P L C activation (7) causing a net increase

In Sweetness and Sweeteners; Weerasinghe, Deepthi K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

370 in calcium mobilization inside the cells. The sweetener-induced calcium mobilization can readily be monitored using calcium sensitive dyes such as Fluo3 and a Fluorometric Imaging Plate Reader (FLIPR) (7) and Figure 1.

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§ 18000

20

40

60

80

Time (seconds) Figure 1. Aspartame induces an increase in intracellular calcium concentration within HEK293 cells stably expressing the human sweet receptor (T1R2/R3) and Gal5 on FLIPR™.

In Vitro Receptor Activity We undertook the characterization of 44 different molecules reported to taste sweet or modulate sweet taste in humans. We performed dose-response analysis of each individual molecule in our G a l 5 cell based FLIPR assay. A summary of the result can be found in Table I. Sweeteners fall into different classes based on potency and efficacy. The more potent sweetener class (with EC50s < Î mM) contains the commonly known artificial sweeteners such as neotame, aspartame, alitame, saccharin, cyclamate, the sweet proteins as well as the terpenoids, terpenoid glycosides and dihydrochalcone (NHDC) (representative dose-response curves of some of these members are shown in Figure 2). Only one of these molecules, 2, 4-dihydroxybenzoic acid (DHB), is a partial agonist on the sweet receptor, inducing 45% of maximal receptor activity. The remaining molecules are as efficacious as D-Fructose at activating the sweet receptor. The less potent sweeteners class (EC50s > 1 mM) contains natural


371 carbohydrate sweeteners as well as D - and L - amino acids known to taste sweet to humans (Table I). D - Alanine and Glycine behave as partial agonists on the sweet receptor (Figure 2, Table I). EC50s for maltose, D-glucose, D-sorbitol, D (+)-galactose, α-lactose and L-glucose could not be determined due to the low potency of these sweeteners in the assay. However, these molecules tested at a concentration of lOOmM consistently induced activation levels corresponding to ~20%-50% of maximal receptor activity.


Table I. Summary of potencies and efficacies of sweet receptor agonists Sweeteners Guanidinoacetic acid Neotame P-4000 Perillartine SC-45647 Super Aspartame Monellin Pine tree Rosin sweetener NC-002740-01 Thaumatin NHDC CC-00100 Mogroside V NC-00420 Dulcin Alitame Rebaudioside A NC-00576 Cyanosuosan Steviocide Sucralose Glycyrrhizic Acid CMB° 5-(3-hydroxyphenoxy)tetrazole Saccharin Aspartame AcesulfameK Cyciamate D-Tryptophan DHB*

ECSO(tJM) 0.11 ± 0 . 0 6 0.40 ± 0 . 2 1 0.83 ± 0.40 1.45 ± 1.03 1.62 ± 0 . 2 0 2.16 ± 0 . 2 6 3.37 ± 0.76 3.62 ± 1 . 4 1 3.94 ± 0 . 5 9 6.11 ± 3 . 0 6 10.2 ± 4 . 0 1 12.7 ± 1 . 4 9 13.4 ± 2 . 9 7 13.6 ± 1.65 13.8 ± 2 . 4 3 13.8 ± 0 . 9 2 14.4 ± 2.53 20.2 ± 1.48 21.7 ± 0 . 5 8 23.1 ± 4 . 2 9 26.0 ± 4.00 29.3 ± 7 . 1 5 29.3 ± 12.9 37.0 ± 3 . 0 0 43.3 ± 11.2 123 ± 33.7 125 ± 2 1 . 4 490 ± 1 3 3 523±115 957 ± 2 0 8

Emax 134 ± 7.00 106 ± 7 . 9 4 139 ± 4.00 104 ± 3 . 1 0 106 ± 1 . 5 3 106 ± 3 . 0 6 95.5 ± 1.64 125 ± 7 . 1 4 95.6 ± 1 . 5 2 101 ± 8.96 128 ± 3 . 6 1 97.7 ± 3 . 7 9 117 ± 6 . 9 3 99.7 ± 1 . 5 3 97.7 ± 3.79 99.7 ± 1.64 100 ± 3 . 4 9 103 ± 1.00 102 ± 2.08 105 ± 12.3 106 ± 3 . 4 6 97.7 ± 2 . 5 2 87.7 ± 4.73 128 ± 8 . 5 0 105 ± 4 . 0 8 117 ± 5 . 2 3 102 ± 2.00 118 ± 5 . 1 2 112 ± 8 . 9 6 45.0 ± 8 . 5 5

Ν 28 4 3 4 10 10 11 4 12 19 20 10 5 10 5 10 14 10 3 21 3 30 3 3 28 24 16 13 11 12

Continued on next page.


372


Table I. Continued Sweeteners L-Hydroxyproline Sucrose D-Alanine D-fructose Glycine D(-)tagatose Xylitol

EC50 (mM) 9.60 ± 1 . 1 4 22.4 ± 5 . 3 6 37.6 ± 15.2 44.6 ± 10.8 59.9 ± 3 0 . 4 73.4 ± 3 3 . 3 81.4 ± 14.6

Emax 77.4 ± 15.7 93.4 ± 3.46 53.2 ± 8.30 100 33.0 ± 9 . 3 8 80.6 ± 2 1 . 6 111 ± 4 . 4 1

Ν 5 24 7 68 9 9 5

Sweeteners Maltose a-D-glucose a-lactose D(+)galactose D-sorbitol L-glucose

% activity 54.0 ± 1.73 40.4 ± 2 . 8 8 34.0 ± 2 . 3 5 27.3 ± 3 . 1 0 26.8 ± 2 . 9 5 20.4 ± 2 . 7 0

Emax — — —

Ν 5 5 5 4 5 5

—

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2-carboxy-4'-methoxybenzophenone * 2, 4-dihydroxybenzoic acid

'12(H Neotame Super Aspartame Thaumatin Dulcin Stevioside Acesulfame Κ D-Tryptophan D-faictose D-Alanine

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log Cone. (M) Figure 2. Dose-response analysis of different sweeteners in HEK293 cells stably expressing the human sweet receptor (T1R2/R3) and Gal5 on FLIPR™ (Manuscript in preparation).


373


Correlation with taste data A key question regarding the assay is whether the relative potencies of the different sweeteners characterized in the assay system correlates with their relative sweetness intensities in taste tests. The sweetness intensity of different sweeteners is often reported as sweetness relative to sucrose on a weight-byweight basis (Pw) (12). This method, although still popular in the sensory field, is flawed by a lack of consideration for the significant variation of molecular weight that can be observed between different sweeteners. To correct for this limitation, Morini et al. (12) transformed Pw values of several sweeteners into molar relative sweetness (MRS) values. Table II shows the M R S values for 10 sweeteners and their corresponding relative potency to sucrose in our assay, The order of potency is almost identical between the two sets of data, with the only difference being that saccharin and aspartame have roughly the same M R S values while saccharin is about three fold more potent than aspartame in our assay. The most noticeable difference between the M R S and relative potency values were observed with neotame, P-4000 and perillartine. Although the order of potency for these sweeteners is the same in the two 2 sets of data, their absolute relative potency to sucrose is 5 to 15 fold greater than their corresponding M R S values. For the remaining sweeteners (guanidinoacetic acid, alitame, sucralose, saccharin, aspartame, cyclamate and D-tryptophan) the M R S and the relative potency values in the assay vary only by -three fold at the most. The calculated correlation coefficient between the two data sets is 0.9625 (Figure 3).

Mapping of Functional Domains Having demonstrated that a number of agonists exhibit differential activities between the human and rodent sweet taste receptors, we initiated experiments to begin to map where these agonists bind. The human and rat sweet receptors expressed in HEK293 cells functionally couple to a variety of G proteins including G i i i , a chimera of G i with the C-terminal tail of G i (7). The human but not rat T1R2/T1R3 are selectively activated by a group of sweeteners, including aspartame, neotame, and cyclamate (7). These data are consistent to behavioral/sensory data with each of these sweeteners. To map the key domains, we generated chimeric TIRs between human and rat receptors. Each T1R chimera consists of two halves, an N-terminal extracellular domain and the C terminal transmembrane and intracellular domain from different species. For example, a chimeric T1R2, termed T1R2H-R, is composed of the N-terminus of human T1R2 linked to the rat T1R2 C-terminal sequence. Combinations of a

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Table II. Calculated Relative Potency of Different Sweeteners to Sucrose in the Assay and Their Relative Sweetness to Sucrose in Human Taste Tests

Sweetener

Potency in the assay relative to sucrose (A)

MRS values (B)

Log A

Log Β

Guanidinoacetic acid

199815

168000

5.301

5.225

Neotame

55602

11057

4.745

4.044

P-4000

26856

2293

4.429

3.360

Perillartine

15434

996

4.188

2.998

Alitame

1622

1937

3.210

3.287

Sucralose

861

755

2.935

2.878

Saccharin

517

161

2.713

2.207

Aspartame

182

172

2.259

2.236

Cyclamate

46

26

1.659

1.415

D-Tryptophan

43

21

1.631

1.322

Sucrose

1

1

0.000

0.000

receptors were expressed in HEK293 cells with G i i i and the cells were examined for their responses to aspartame, neotame and cyclamate (Fig. 4). a

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Aspartame and Neotame The chimera in which the N-terminal domain of human T1R2 was replaced by the homologous rat domain failed to respond to aspartame or neotame, when co-expressed with human T1R3. This result was consistent with the model in which these sweeteners required the extracellular domain of human T1R2. The reverse chimera, in which the rat N-terminal domain of T1R2 was replaced with the human and co-expressed with rat T1R3, exhibited a gain of function and now responded effectively to aspartame and neotame (Fig. 4B). These data suggests that the same domain of human T1R2 is also sufficient (in the context of sweet taste receptors) to enable activation by those two sweeteners. Further support for this model comes from experiments in which a mouse strain engineered to expressed human T1R2 responded to aspartame (9).


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Relative Potency to Sucrose in the HTS Assay (Log) Figure 3. Correlation analysis between the relative potency of different sweeteners to sucrose in the HTS assay and their relative sweetness to sucrose in human taste tests (Manuscript in preparation).

Cyclamate Cyclamate is another sweetener that is sweet to humans but not to rodents. Consistent with these findings, only the human sweet receptor responds to cyclamate in vitro. In contrast to the aspartame results, chimeric receptors composed of either the N - or C-terminal portion of human T1R2 and coexpressed with rat T1R3 failed to respond to cyclamate. Surprisingly, a chimeric receptor composed of the N-terminal domain of rat T1R3 and the C-terminal domain of human T1R3 when co-expressed with rat T1R2 was activated by cyclamate (Fig. 4C). It has been previously known that binding sites for certain positive and negative allosteric modulators of other family C GPCRs has been mapped to their transmembrane domains (13). Although the response to cyclamate was not tested in the mice engineered by Zhao et al, based on our data h T l R 3 but not h T l R 2 would be expected to convey cyclamate preference to mouse taste.



Figure 4. Sweeteners map to different domains/subunits of the human sweet taste receptor. (A) Differences between the activities of human and rat sweet taste receptors. (B) Aspartame and neotame were mapped to N-terminal extracellular domain of human T1R2. (C) Cyclamate was mapped to the C-terminal transmembrane domain of human T1R3. (D) Lactisole was mapped to the transmembrane domain of human T1R3. (Reproducedfrom reference 11. Copyright 2004 The National Academy of Sciences of the USA.)


377


Lactisole Lactisole, an aralkyl carboyxlic acid, specifically inhibits the activation of the human taste receptor by sweeteners in vitro and blocks sweet taste in humans. It has no effect on the rodent sweet receptor or sweet taste response. The effects of lactisole on the human and rat receptors in our assay system are shown in Fig. 4A. Using T1R chimeras we conducted mapping experiments and demonstrated that lactisole acts similar to cyclamate through the human T1R3 C-terminal domain to inhibit the receptor's response to sucrose and acesulfame Κ (Fig. 4D). This result further demonstrates the importance of T1R3 C-terminal domain in the sweet taste receptor function. To completely probe this response, we examined all 16 possible chimera combinations with lactisole. The results from are consistent with our model. Our results were later confirmed by other research groups (14-16).

Mutants with Ligand-selective Effect To more precisely identify the amino acids essential for recognition of aspartame, neotame and cyclamate we generated receptor variants containing site-specific amino acid substitutions. Our hypothesis was that substitutions in the N-terminal domain of T1R2 that affected aspartame and neotame would not affect activation by cyclamate and substitutions in the T1R3 transmembrane domain that eliminated activation by cyclamate would not affect aspartame or neotame. Critical amino acid residues in the T1R2 N-terminal domain were selected by aligning the sequences of T1R2 with mGluRl (Fig. 5A). Among the eight residues that are crucial in ligand binding in mGluRl (17), three are conserved in human T1R2 (S144, Y218, and E302). Each of the three residues was changed to alanine and the resulting receptors were tested for their response to different sweeteners in HEK293 cells. Substitution of Y218 to A abolished the responses to all sweeteners tested including cyclamate (data not shown). Y218 might be important for the overall conformation of the sweet taste receptor. However, it is also possible that that Y218A failed to express or target to the cell surface, considering that equivalent substitutions in mGluRl (18) and mGluR8 (19) led to partially functional receptors. In contrast, the variants containing S144A and E302A selectively reduced the response to aspartame and neotame but were still activated by cyclamate. Cell lines stably expressing the S144A and E302A h T l R 2 variants and coexpressed with wild type h T l R 3 and G did not respond to aspartame or neotame at the physiologically relevant concentrations, but did respond to cyclamate (Fig. 5B). Cells stably expressing receptors are generally more sensitive than those produce by transient expression. However, in this case, the stable cells still failed to respond to aspartame or neotame. u l 5



378 The cyclamate-binding site was further dissected by creating substitutions within the three extracellular loops o f the T1R3 C-terminal domain. Alignment of human and rodent TlR3s revealed multiple amino acid differences in the three extracellular loops. Replacing the human T1R3 second (loop-2) or third (loop-3) extracellular loops with the homologous rat sequences abolished the cyclamate response without affecting the sucrose or aspartame responses. In contrast, replacing the first extracellular (loop 1) had no obvious effect on the response to cyclamate, suggesting important roles for extracellular loop-2 and loop-3 in recognizing cyclamate. Interestingly, none of these loop replacements affected ability of lactisole to inhibit the receptor, suggesting a different binding mechanism. In summary, amino acid substitutions in T1R2 or T1R3 result in selective interference of activities induced by different sweeteners, consistent with the chimeric receptor results. Taken together, the above results demonstrate that human sweet taste receptor functions as a heteromeric complex of T1R2 and T1R3. Both subunits are required for recognizing different sweeteners, and our data indicate the existence of multiple binding pockets on the receptor for different classes of agonists. The presence of multiple ligand-binding sites provides a possible explanation for the structural diversity of sweeteners. G protein Interaction Site Studies in HEK293 cells identified a different in G protein-coupling efficiency between the human and rat sweet taste receptors. For example, both human and rat receptors respond to sweeteners when co-expressed with G i / n but only the human receptor efficiently responds when co-expressed with G | (7) (Fig. 6A). This differential response allowed us to map the receptor-G protein interactions using the chimeric receptors described above. a

5

a

5

Replacing the C-terminus of human T1R2 with the corresponding rat sequence abolished coupling. In contrast, substitution of the rat T1R2 C-terminal half with human sequence enabled coupling to G and respond to sucrose and acesulfame Κ (Fig. 6). These results suggest residues in T1R2 but not T1R3 are critical for G -coupling. Substituting the T1R3 C-terminal domain had no effect on G ir-mediated coupling (Fig. 6B). This observation demonstrates the important role of T1R2 in G protein-coupling in our functional expression system. Gustducin (20) has been proposed to be an endogenous G protein for the sweet taste receptor, and we speculate that T1R2 should be the subunit responsible for coupling in taste cells. G A B A R is the other example of heteromeric family C GPCR, whereas one subunit ( G A B A R 1 ) is responsible for ligand-binding, and the other ( G A B A R 2 ) for G protein coupling (21-24). The sweet taste receptor is different from G A B A R in that the same subunit is required for both ligand recognition and G-protein coupling. a{5

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B

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Figure 5. Mutants with sweetener-selective effect (A) Two point mutations in the human T1R2 N-terminal extracellular domain abolish response to aspartame and neotame without affecting cyclamate. (B) Mutations in the extracellular loop ofhTlR3 abolish response to cyclamate without affecting aspartame. (Reproducedfrom reference II. Copyright 2004 The National Academy of Sciences of the USA.)



Figure 6. G protein is coupled to sweet receptor through the T1R2 TMdomain. (A) Responses of human, rat and chimeric sweet taste receptors to sucrose andAceK. (B) G protein coupling is mapped to human T1R2. (Reproducedfrom reference 11. Copyright 2004 The National Academy of Sciences of the USA.)


381


Cyclamate Effect on Umami Receptor Umami is characterized as the savory taste of monosodium glutamate (MSG). The umami receptor was shown to be composed of T1R1 and T1R3, thus sharing a subunit in common with the sweet receptor (7, 25). Following our finding that cyclamate likely binds to T1R3, we predicted that cyclamate would also modulate the activity of the human umami receptor. Initial experiments showed that cyclamate was not an agonist of the human umami receptor. However, cyclamate enhanced the response of the umami receptor to L glutamate (Fig. 7A). This was demonstrated by a left shift in the dose response to glutamate (Fig. 7B). The effect of cyclamate was shown to require the human umami receptor since cyclamate had no effect on the carbachol response of the endogenous muscarinic acetylcholine receptor (Fig. 7A). It is noteworthy that cyclamate has comparable E C s for the sweet taste receptor (Fig. 7A) and umami taste receptor. Cyclamate shifted the dose-response curves for L glutamate by ~2 fold either in the presence or absence of the glutamate enhancer inosine monophosphate (IMP). These results suggest IMP and cyclamate are acting via different binding sites. We speculate that IMP binds to T1R1, since it has no effect on the sweet taste receptor (7). Other sweeteners, including sucrose, aspartame, saccharin, and D-tryptophan, had no effect on the human T1R1/T1R3 activities (not shown). Due to the intense sweet taste of cyclamate, the effect of cyclamate on M S G taste is difficult to determine. 50

Conclusion In summary, we developed a sweet receptor-based HTS assay and a series of human-rat chimeric receptors to probe the response of the sweet receptor to a variety of sweet taste modulators. Our work showed that the response of the receptor in the in vitro assay correlates with human sweet taste. Through functional mapping, we showed that 1) both T1R2 and T1R3 are required for function, 2) aspartame and neotame require the N-terminal extracellular domain of T1R2, 3) G protein coupling requires the C-terminal half of T1R2, and 4) cyclamate and lactisole require the transmembrane domain of T1R3. These findings demonstrated for the first time the different functional roles of T1R subunits in a heteromeric complex and the presence of multiple sweetener interaction sites on the sweet taste receptor. Because T1R3 is the common subunit in the sweet and the umami taste receptors, we predicted and confirmed the effect of cyclamate and lactisole on the umami taste receptor. Based on these results, we proposed a model (Fig. 8) for the interaction of ligands with the sweet and umami taste receptors. Although the rat and human receptors both respond to carbohydrate sweeteners, we speculated that carbohydrate sweeteners, including sucrose and fructose, bind to the N-terminal



Figure 7. Effect of cyclamate on umami receptor. (A) The responses of human T1R1/T1R3 stable cell line to threshold level of L-glutamate and endogenous M2 receptor agonist carbachol in the absence and presence of various concentrations of cyclamate. (B) Dose-responses of the human T1R1/T1R3 stable cell line with or without 0.2 mMIMP in the absence and presence of cyclamate. (Reproducedfrom reference 11. Copyright 2004 The National Academy of Sciences of the USA.)



Figure 8. Differentfunctional domains of the sweet taste receptor. (Reproducedfrom reference 11. Copyright 2004 The National Academy of Sciences of the USA.)


06

384


domain of T1R2. In addition to the N-terminal domain of T1R2 and the transmembrane domain of T1R3, other regions of the receptor, such as the transmembrane domain of T1R2, may also participate in ligand binding. The umami taste receptor is most likely to function in a similar fashion. Since neither glutamate nor IMP affects the sweet taste receptor we speculate that these umami modulators bind to T1R1. Furthermore, the coupling of the umami receptor with the G protein is mediated via the transmembrane domain of T1R1. A more detailed understanding of these receptors will emerge via additional mutagenesis, homology modeling, and x-ray crystallography experiments.

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Functional Characterization of the Human Sweet Taste Receptor: High

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