WO2013159227A1 - Bitter taste receptor constitutively active mutants - Google Patents

Abstract

The human bitter taste receptors (T2Rs) belong to the G-protein coupled receptor (GPCR) superfamily. T2Rs share little homology with the large subfamily of Class A GPCRs, and their mechanisms of activation are pooriy understood. Guided by biochemical and molecular approaches, we identified two conserved amino acids Gly28^1.46 and Ser285^7.47 present on transmembrane (TM) helices, TM1 and TM7, which might play important roles in T2R activation. We mutated Gly28^1.46 and Ser285747 in T2R4 to G28A, G28L, S285A, S285T and S285P, and carried out pharmacological characterization of the mutants. S285A mutant displayed agonist- independent activity (~ 3 fold over basal wild type T2R4 or S285T or S285P). Ser285^7.47 stabilizes the inactive state of T2R4 by a network of hydrogen-bonds connecting important residues on TM1-TM2-TM7. Thus far, S285A is the first constitutively active T2R mutant reported, and gives novel insights into T2R activation.

Description

BITTER TASTE RECEPTOR CONSTITUTIVELY ACTIVE MUTANTS

PRIOR APPLICATION INFORMATION

The instant application claims the benefit of US Provisional Patent Application 61/638,189, filed April 25, 2012.

BACKGROUND OF THE INVENTION

The human taste perception is one of the most important chemosensations. Humans can sense five basic tastes which are sweet, bitter, umami, salt and sour. Bitter taste which is sensed by bitter taste receptors (T2Rs) is the most complex and the least understood among the human taste sensations, and part of this complexity is due to the large number of receptors (25 T2Rs) that code for bitter taste sensation compared to only three receptors that code for both sweet and umami tastes. Recent studies have shown that T2Rs are expressed in many extra-oral tissues including the brain (Singh ei al. 2011 b), where their physiological function needs to be determined. The T2Rs are cell surface receptors and belong to the G-protein coupled receptor (GPCR) superfamily (Chandrashekar et al. 2000).

There are a very limited number of structure-function studies on T2Rs (Sakurai et al. 20 0, Brockhoff et al. 2010) and none have addressed the role of hydrogen- bond networks in T2R activation. Using amino acid sequence analysis and molecular modeling approaches, we identified two conserved residues present on TM1 and TM7 of T2Rs, Gly28¹-⁴⁶ and Ser285^{7 47} that might play important roles in inter- and intrahelical hydrogen-bonding and receptor activation. To elucidate the structural and functional role of these two conserved residues in T2Rs, we pursued mutagenesis using N-terminal FLAG epitope tagged T2R4 receptor. The amino acid numbering used in this manuscript incorporates the residue number from the receptor sequence (e.g. Gly28) and a residue number (e.g. 1.46) from a generic numbering system developed by Ballesteros and Weinstein (Ballesteros & Weinstein 1995). The two residues at positions 1.46 and 7.47 are 88% and 70% conserved in T2Rs (Singh ei al. 201 1a), however only glycine at position 1.46 is conserved in Class A GPCRs (Smith 2010, Singh et al. 201 1a). In rhodopsin, Gly51^1,46 occurs as naturally occurring polymorphic variants G51A, G51V, and G51 D which cause autosomal dominant retinitis pigmentosa (ADRP) (Sung et al. 1991 , Dryja et al. 1991 , Macke et al. 1993). Although these ADRP mutants fold properly, they displayed thermally destabilized structures and were severely defective in signal transduction (Bosch-Presegue et al. 2011 , Bosch et al. 2003).

SUMMARY OF THE INVENTION

According to an aspect of the invention, there is provided an isolated or purified constitutively active mutant (CAM) of bitter taste receptor (T2R) selected from the group consisting of:

a) a T2R CAM receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, T2R55 and T2R60, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to histidine 214 of the T2R4 wild type sequence;

b) a T2R CAM receptor selected from the group consisting of: T2R1 , T2R3,

T2R4, T2R5, T2R7, T2R8, T2R9, T2R1_.0, T2R13, T2R14, T2R16, T2R39, T2R40, T2R48, T2R49, T2R50 and T2R55, wherein said T2R CAM receptor further

comprises an alanine residue at the amino acid residue corresponding to serine 285 of the T2R4 wild type sequence;

c) a T2R CAM receptor selected from the group consisting of: T2R4,

T2R13 and T2R41.wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to glutamine 216 of the T2R4 wild type sequence;

d) a T2R CAM receptor selected from the group consisting of: T2R4, T2R7, T2R8 and T2R10, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to valine 234 of the T2R4 wild type sequence;

e) a T2R CAM receptor selected from the group consisting of: T2R4, T2R10 and T2R55, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to methionine 237 of the T2R4 wild type sequence;

f) a T2R CAM receptor selected from the group consisting of: T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, , T2R14, , T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50 and T2R55, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue

corresponding to isoleucine 55 of the T2R4 wild type sequence;

g) a T2R CAM receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R7, T2R8, T2R9, T2R16, T2R38, T2R40, T2R41 , T2R55 and T2R60, wherein said T2R CAM receptor further comprises a alanine residue at the amino acid residue corresponding to histidine 123 of the T2R4 wild type sequence;

h) a T2R4 CAM receptor, wherein said T2R4 mutant receptor further comprises an alanine residue at amino acid residue corresponding to asparagine 132 of the T2R4 wild type sequence.

According to a further aspect of the invention, there is provided an isolated or purified constitutively active mutant (CAM) of bitter taste receptor (T2R) selected from the group consisting of:

a) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:2; b) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:3; c) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:4; d) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:5; e) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:6; f) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:7; g) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:8; and

h) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:9. According to another aspect of the invention, there is provided a method of preparing a constitutively active mutant (CAM) bitter taste receptor (T2R) comprising: a) subjecting a T2R receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, T2R55 and T2R60, to site directed mutagenesis such that the histidine residue corresponding to histidine 214 is mutated to alanine; or

b) subjecting a T2R receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R39,

T2R40, T2R48, T2R49, T2R50 and T2R55 to site directed mutagenesis such that the serine residue corresponding to serine 285 of the T2R4 wild type sequence is mutated to alanine ;or

c) subjecting a T2R receptor selected from the group consisting of: T2R4, T2R13 and T2R41 to site directed mutagenesis such that the glutamine residue corresponding to glutamine 216 of the T2R4 wild type sequence is mutated to alanine; or

d) subjecting a T2R receptor selected from the group consisting of: T2R4, T2R7, T2R8 and T2R10 to site directed mutagenesis such that the valine residue corresponding to valine 234 of the T2R4 wild type sequence is mutated to alanine; or e) subjecting a T2R receptor selected from the group consisting of: T2R4, T2R10 and T2R55 to site directed mutagenesis such that the methionine residue corresponding to methionine 237 of the T2R4 wild type sequence is mutated to alanine; or

f) subjecting a T2R receptor selected from the group consisting of: T2R3,

T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, , T2R14, , T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50 and T2R55 to site directed mutagenesis such that the isoleucine residue corresponding to isoleucine 55 of the T2R4 wild type sequence is mutated to alanine; or

g) subjecting a T2R receptor selected from the group consisting of: T2R1 ,

T2R3, T2R4, T2R7, T2R8, T2R9, T2R16, T2R38, T2R40, T2R41 , T2R55 and T2R60 to site directed mutagenesis such that the histidine residue corresponding to histidine 123 of the T2R4 wild type sequence is mutated to alanine; or

h) subjecting a T2R4 receptor to site directed mutagenesis such that asparagine residue 132 is mutated to alanine; and recovering a nucleic acid molecule encoding the T2R4 CAM receptor.

According to yet another aspect of the invention, there is provided a method of determining if a compound of interest is a human bitter taste receptor blocker comprising:

adding a compound of interest to a suitable cell line expressing a constitutively active bitter taste receptor mutant as described above; and

measuring the activity of the constitutively active bitter taste receptor mutant, wherein the compound of interest is a bitter taste receptor antagonist or inverse agonist if said compound reduces the activity of the constitutively active bitter taste receptor mutant to approximately the same activity as a wild type bitter taste receptor.

According to a yet further aspect of the invention, there is provided a nucleic acid molecule encoding a bitter taste receptor (T2R) constitutively active mutant (CAM) comprising a nucleotide sequence encoding the amino acid sequence as set forth in any one of SEQ ID NOs: 2-9 or as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. A. Two-dimensional representation of the T2R4 amino acid sequence with the octapeptide FLAG-tag at the N-terminus. The coding region of T2R4 without the FLAG-tag is 299 amino acids. The receptor consists of seven transmembrane (TM) helices, a short N-terminus, three extracellular loops (ECLs) and three intracellular loops (ICLs), and a cytoplasmic tail which constitutes a short helix-8 that runs parallel to the membrane. The residues mutated in this study, Gly28 on TM1 and Ser285 on TM7 are shown. B. The sequence alignment of TM1 and TM7 of T2R1 (SEQ ID NOs: 10 and 11 ), T2R4 (SEQ ID NOs: 12 and 13) and opsin (SEQ ID NOs: 14 and 15) are shown. The conserved residue mutated in this study, residues 1.46 and 7.47 are also shown.

Figure 2. Characterization of Ga16/44 chimera-mediated signaling of the wild type T2R4 and mutants. A. Receptor activity was determined by measuring the agonist independent and dependent changes in intracellular calcium using HEK293T cells transiently co-transfected with WT-T2R4 or mutants and Ga16/44 (see methods. WT-T2R4 and the four mutants were stimuiated

concentrations of quinine and the amount of calcium mobilized (Δ RFU) was measured using Fluo-4NW dye. The results are expressed as a percentage of the WT-T2R4 activity and are from at least three independent experiments performed in 5 duplicate. B. Cell surface expression of wild type T2R4 and mutants. The bar plots represent the cell surface expression of the WT-T2R4 and mutants as determined by ELISA assay. Data was normalized to WT-T2R4.

Figure 3. Pharmacological characterization of WT-T2R4 and mutants. Shown are the activity after stimulation (A, Top panel) with a single saturating concentration

10 of quinine to determine the maximal agonist-induced or intrinsic signal and agonist- independent or basal activity (B, Bottom panel). Calcium mobilized (ARFU) is normalized to WT-T2R4 cell surface expression as determined by ELISA. One way ANOVA analysis of WT-T2R4 and S285A mutant showed p<0.05 and p< 0.01 at high agonist concentration and in absence of agonist, respectively.

15 Figure 4. Effect of receptor density on basal Ca²⁺ mobilization. WT-T2R4

(circular dots) and S285A (square dots) constructs were transiently expressed in HEK293T cells at different receptor densities by varying amounts of DNA used in each transfection (3pg, 6 pg and 9 pg DNA per 10⁶ cells). Receptor expression levels were determined by cell surface ELISA and normalized to WT-T2R4 transfected with

20 3pg DNA per 10⁶ cells, which was taken as 100%. The slope of WT-T2R4 was 0.185 ±0.13 of calcium mobilized (ARFU), while that of the S285A was 1 .06 ±0.27, respectively. This means S285A displays 5-fold increase over basal WT-T2R4 activity.

Figure 5. Comparison of the hydrogen-bond network in the vicinity of residues 25 1 .46 and 7.47, in rhodopsin and T2R4. A, Rhodopsin_1 U19 represents the inactive structure of rhodopsin (Protein Data Bank code 1 U19) and CAM_Rhodopsin_2X72 represent the structure of the constitutively active mutant rhodopsin (Protein Data Bank code 2X72). B, T2R4_basal represents the inactive structure of wild type T2R4 built by homology modeling using the Rhodopsin_1 U19 template, and T2R4_S285A 30 mutant represents the constitutively active S285A mutant built by homology modeling using the CAM_Rhodopsin_2X72 template. In T2R4 basal model, an intrahelical hydrogen bond connects the side chains of S285^{7 47}, R63^{2 54} with Water 2017, and interhelical backbone contacts between N32¹'⁵⁰ and G28^{1 ,46} are observed. There is a rearrangement of this network in the constitutively active S285A mutant, due to loss of hydrogen bonding by the residue at position 7.47. In T2R4_S285A model, the side chain of R63^2,54 moves away from TM7 and is stabilized by waters 2013, 2014 and 2018. Furthermore, the intrahelical hydrogen bond between Ν32¹·⁵⁰ and G28¹⁴⁶ breaks off, and the side chain amine of N32^{1 50} hydrogen bonds with the backbone of R63²-⁵⁴. Broken lines represent hydrogen bonds, structural waters are shown as a cross.

Figure 6. Comparison of the hydrogen-bond network in the vicinity of residues 1.46 and 7.47, in a molecular model of S285P mutant. The S285P model was built by homology modeling using the rhodopsin_1 U19 as template. The interhelical H-bond interaction between the backbone N-atom of Pro285⁷'⁴⁷ with the side chain-NH2 of Arg63²⁵⁴ restrains basal activity of the receptor. This is similar to the H-bond interaction of Ser285^{7 47} with Arg63^{2 54} observed in the molecular model of wild type T2R4 that is proposed to stabilize the inactive state. Broken blue lines represent hydrogen bonds.

Figure 7. Amino acid sequence comparison of T2R4 (SEQ ID No. 1 ) and T2R4 S285A CAM (SEQ ID No. 2).

Figure 8. Two-dimensional representation of the T2R4 amino acid sequence with the FLAG-tag at the N-terminus. The receptor consists of seven transmembrane (TM) helices, a short N-terminus, three extracellular loops (ECLs) and three

intracellular loops (ICLs), and a cytoplasmic tail. The 23 ICL3 residues mutated to alanine in this study are displayed in broken rings. The constitutively active mutants identified in this study are represented in grey circles.

Figure 9. Representative calcium traces for HEK293T cells transiently transfected with T2R4 and select mutants. The mock transfected (pcDNA) control is shown. The cells are stimulated with 2.5 mM quinine (top panel) or assay buffer (lower panel). The calcium mobilized (ARFUs or Relative Fluorescence Units) was detected using the calcium sensitive dye Fluo 4NW (Invitrogen), and fluorescence measured using Flex Station III microplate reader.

Figure 10. A. Pharmacological characterization of the basal or agonist independent activity of WT-T2R4 and intracellular alanine mutants. Calcium mobilized (ARFU) is normalized to WT-T2R4 cell surface expression as determined by ELISA. The results were analyzed using one way ANOVA with Tukeys post hoc test, at significance level p < 0.05. B. Effect of receptor density on basal Ca²⁺ mobilization. The H214A, Q216A, V234A and M237A mutants displayed true constitutive activity. The slope values of the constitutively active mutants (CAMs) are shown next to the mutant. The H214A (2.35 ±1 .06) displayed the highest constitutive activity, with greater than 10-foid increase in basal activity over WT-T2R4 (0.19 ±0.05).

Figure 11. Homology models of the inactive (red) and constitutively active (yellow) WT-T2R4 built using the rhodopsin inactive (PDB: 1 U19) and CAM (PDB: 2X72) structures as templates. A. The left panel shows the intracellular view (from the cytoplasmic side) of the TM2-TM3-TM5-TM6 arrangement in both T2R4 structures. The intracellular loops (ICLs) are shown as threads, along with the location of the ICL3 CAMs. B. The right panel shows the membrane view of TM5-TM6, along with ICL3 CAMs and the packing interactions of the LxxSL motif on TM5. In the T2R4 CAM model, the cytoplasmic end of TM6 moves by around 2A towards the helical core.

Figure 12. Two-dimensional representation of the T2R4 amino acid sequence with the FLAG-tag at the N-terminus. The receptor consists of seven transmembrane (TM) helices, a short N-terminus, three extracellular loops (ECLs) and three

intracellular loops (ICLs), and a cytoplasmic tail. The intracellular residues mutated to alanine in this study, are displayed in broken rings, the constitutively active mutants are represented in grey circles, and the short helix in ICL2 Is shown.

Figure 13. A. Pharmacological characterization of the basal or agonist independent activity of WT-T2R4 and intracellular alanine mutants. Calcium mobilized (ARFU) is normalized to WT-T2R4 cell surface expression as determined by ELISA. The results were analyzed using one way ANOVA with Tukeys post hoc test, at significance level p < 0.05. B. Effect of receptor density on basal Ca^2÷ mobilization. Of the 83 alanine replacements, seven mutants displayed true constitutive activity. The slope values of the constitutively active mutants (CAMs) are shown next to the mutant. The H214A (2.35 ±1 .06) displayed the highest constitutive activity, with greater than 10-fold increase in basal activity overWT-T2R4 (0.19 ±0.05).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise,, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

Described herein are a plurality of expressed or isolated or purified T2R constitutively active mutants (CAM): I55A, H123A, N.132A, S285A, H214A, Q216A, V234A and M237A. It is of note that these residue locations are based on the wild- type T2R4 sequence as set forth in SEQ ID NO:1 .

As discussed above, there are at least 25 known T2R receptors. Accordingly and as discussed below, T2R receptors in which the above-referenced amino acids are conserved when compared to the T2R4 sequence can be used in the generation of corresponding CAMs. For example any T2R receptor having a serine residue at the residue corresponding to serine 285 in T2R4 can be mutated at that residue to alanine and a CAM T2R receptor will be obtained. Similarly, any T2R receptor having a histidine residue at the residue corresponding to histidine 214 in T2R4, or any T2R receptor having a glutamine residue corresponding to glutamine 216 in T2R4, or a valine residue at the residue corresponding to valine 234 in T2R4 or a methionine residue corresponding to methionine 237 or an isoleucine residue corresponding to isoleucine 55 in T2R4 or a histidine at the residue corresponding to histidine 123 in T2R4 or an aspargine residue corresponding to aspargine 132 in T2R4 respectively, can be mutated at that residue to alanine and a CAM T2R receptor will be obtained. The appropriate T2Rs for each of the 8 CAMs are listed below. In another embodiment of the invention, there is provided a purified or isolated

CAM T2R receptor comprising or having the amino acid sequence as set forth in any one of SEQ ID NOs: 2-9.

In another embodiment of the invention, there is provided a purified or isolated CAM T2R receptor comprising or having the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO:3.

As discussed below, the inventors mutated Gly28^{1 46} and Ser285⁷⁴⁷ in T2R4 to G28A, G28L, S285A, S285T and S285P, and carried out pharmacological

characterization of the mutants. Interestingly, S285A mutant displayed agonist- independent or constitutive activity, while the conservative replacement S285T displayed wild type basal activity. Ser285⁷⁴⁷ stabilizes the inactive state of T2R4 by forming a hydrogen bond with Arg63²⁵⁴. The constitutive activity of the S285A mutant is due to a loss in ability of the residue at position 7.47 in T2R4 to hydrogen bond. This leads to a re-arrangement of the hydrogen-bond network connecting TM1-TM2- TM7 causing the S285A mutant to adopt an active conformation. Surprisingly, no major changes in T2R function were observed upon mutation of the glycine at position 1.46. This hydrogen-bond network is compared and contrasted with that present in the dim light receptor, rhodopsin beiow. These interesting results, along with the inventors' previously reported findings that T2Rs lack important and conserved amino acid motifs present in Class A GPCRs (Singh et al. 2011a) add to the increasing body of evidence that T2Rs have different activation mechanisms compared to Class A GPCRs.

Furthermore, the inventors recently identified a conserved LxxSL motif in TM5 of T2Rs. Mutational and molecular modeling analysis of this motif suggested that it performs a structural role by stabilizing the helical conformation of TM5 at the cytoplasmic end (Singh et al., JBC 286: 36032-36041 ). However, the LxxSL motif is very close to the third intracellular loop (ICL) and the role of this loop in T2R activation has not been studied to date. In different GPCR classes, 1CL3 was found to perform different roles. For example, in Ste2p a yeast GPCR, disulfide cross-linking

experiments show that the cytoplasmic ends of TM5 and TM6 that flank ICL3 undergo conformational changes upon ligand binding, whereas the center of the ICL3 loop does not (Umanah et al., Biochemistry 50: 6841 -6854). In neuropeptide Y1 receptor (NPY1), ICL3 constrains the inactive state of the receptor and mutations in ICL3 lead to agonist-independent or basal or constitutive activity. In contrast, a recent alanine scan mutagenesis of ICL3 in the melanocortin-3 receptor (MC3R) showed that ICL3 is important for ligand binding and signaling, but none of the mutants displayed constitutive activity.

As discussed below, the inventors have carried out alanine scan mutagenesis of the ICL3 and functionally characterized 23 alanine mutants of T2R4. The results based on site-directed mutagenesis, pharmacological characterization of the mutants, and molecular modeling analysis allowed the inventors to identify four constitutively active mutants (CAMs) in ICL3, with constitutive activity ranging from 2 to 10 fold over wild type T2R4. Taken together, the results show that the cytoplasmic ends of TM5 and TM6 play an important role in T2R activation, and ICL3 is involved in restraining T2Rs in an inactive conformation.

Humans can sense five basic tastes which are sweet, salt, sour, bitter and umami. Bitter taste which is sensed by bitter taste receptors (referred to as T2Rs), is the most complex and the least understood among the taste sensations. The molecules (i.e. tastants) that activate T2Rs, also known as T2R agonists, have diverse chemical structures and include plant derived compounds and natural alkaloids such as, quinine, caffeine, nicotine and morphine. Of nutritional and pharmaceutical or nutraceutical significance is the development of bitter blockers (antagonists and/or inverse agonists) that would improve the palatability of health promoting foods such as green vegetable extracts, as well as providing therapeutic benefits. However, the pharmacological tools necessary for the proper

characterization of these bitter blockers are lacking. Constitutively active mutants (CAM) of T2Rs are one such tool, and a proper experimental control for the

pharmacological characterization of a potential bitter blocker against a particular T2R. CAMs are mutations in the receptor that lock it in an active conformation and allow the receptor to signal, even in the absence of an agonist. Of the 25 T2Rs in humans, 17 have the amino acid serine in transmembrane helix 7 at position 7.47 (according to Ballesteros and Weinstein numbering) or corresponding to serine 285 in the T2R4 wild type sequence (SEQ ID NO:1 ). These serine containing bitter taste receptors are T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R39, T2R40, T2R48, T2R49, T2R50 and T2R55.

Replacement of the serine at position 7.47 in T2Rs with alanine results in a CAM phenotype, as observed in the case of serine to alanine mutation at 7.47 in T2R4. The CAMs of T2Rs can be used as pharmacological tools for the screening of potential bitter blockers.

Accordingly, in one aspect of the invention, there is provided a purified or isolated T2R CAM in which position 7.47 or amino acid 285 is an alanine wherein the corresponding wild type sequence has a serine at position 7.47 or amino acid 285 of the T2R4 wild type sequence.

Preferably, the wild type T2R having serine at amino acid 285 is selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R39, T2R40, T2R48, T2R49, T2R50 and T2R55.

Preferably, the T2R having a serine at amino acid position 285 of the wild type sequence is T2R4.

Of the 25 T2Rs in humans, 24 have the amino acid histidine at amino acid residue 214: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, T2R55 and T2R60.

Accordingly, in one aspect of the invention, there is provided a purified or isolated T2R CAM in which amino acid residue corresponding to 214 is an alanine wherein the corresponding wild type sequence has a histidine at position 214 of the wild type T2R4 sequence.

Preferably, the wild type T2R having histidine at amino acid 214 is selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2_.R47, T2R48, T2R49, T2R50, T2R55 and T2R60.

Preferably, the T2R having a histidine at amino acid position 2 4 of the wild type sequence is T2R4.

Of the 25 T2Rs in humans, 3 have the amino acid glutamine at position 216:

T2R4, T2R13 and T2R41 .

Accordingly, in one aspect of the invention, there is provided a purified or isolated T2R CAM in which amino acid 216 is an alanine wherein the corresponding wild type T2R4 sequence has a glutamine at this position.

Preferably, the wild type T2R having glutamine at amino acid 216 is selected from the group consisting of: T2R4, T2R13 and T2R41.

Preferably, the T2R having a glutamine at amino acid position 216 of the wild type sequence is T2R4.

Of the 25 T2Rs in humans, 4 have the amino acid valine at amino acid residue 234 of the wild type T2R4 sequence: T2R4, T2R7, T2R8 and T2R 0.

Accordingly, in one aspect of the invention, there is provided a purified or isolated T2R CAM in which amino acid residue corresponding to 234 is an alanine wherein the corresponding wild type T2R4 sequence has a valine at position 234.

Preferably, the wild type T2R having valine at amino acid 234 is selected from the group consisting of: T2R4, T2R7, T2R8 and T2R10.

Preferably, the T2R having a valine at amino acid position 234 of the wild type sequence is T2R4.

Of the 25 T2Rs in humans, 3have the amino acid methionine at amino acid residue 237: T2R4, T2R10 and T2R55.

Accordingly, in one aspect of the invention, there is provided a purified or isolated T2R CAM in which amino acid residue corresponding to 237 is an alanine wherein the corresponding wild type T2R4 sequence has a methionine at position 237.

Preferably, the wild type T2R having methionine at amino acid 237 is selected from the group consisting of: T2R4, T2R10 and T2R55. -Preferably, the T2R having a methionine at amino acid position 237 of the wild type sequence is T2R4.

Of the 25 T2Rs in humans, 21 have the amino acid isoleucine at amino acid residue 55: T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R14, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50 and T2R55.

Accordingly, in one aspect of the invention, there is provided a purified or isolated T2R CAM in which amino acid residue corresponding to position 55 is an alanine wherein the corresponding wild type T2R4 sequence has an isoleucine at position 55.

Preferably, the wild type T2R having isoleucine at amino acid 55 is selected from the group consisting of: T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, , T2R14, , T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50 and T2R55.

Preferably, the T2R having a methionine at amino acid position 237 of the wild type sequence is T2R4.

Of the 25 T2Rs in humans, 12 have the amino acid histidine at amino acid residue 123:T2R1 , T2R3, T2R4, T2R7, T2R8, T2R9, T2R16, T2R38, T2R40, T2R41 , T2R55 and T2R60.

Accordingly, in one aspect of the invention, there is provided a purified or isolated T2R CAM in which amino acid residue corresponding to 123 is an alanine wherein the corresponding wild type T2R4 sequence has a histidine at position 123.

Preferably, the wild type T2R having histidine at amino acid 123 is selected from the group consisting of: T2R1 , T2R3, T2R4, T2R7, T2R8, T2R9, T2R16, T2R38, T2R40, T2R41 , T2R55 and T2R60.

Preferably, the T2R having a methionine at amino acid position 123 of the wild type sequence is T2R4.

Of the 25 T2Rs in humans, 1 has the amino acid asparagine at amino acid residue 132: T2R4.

Accordingly, in one aspect of the invention, there is provided a purified or isolated T2R CAM in which amino acid residue corresponding to 132 is an alanine wherein the corresponding wild type T2R4 sequence has an asparagine at position 132.

Preferably, the T2R having an asparagine at amino acid position 132 of the wild type sequence is T2R4.

The sequence of the human T2R4 wild type receptor is provided below.

Human T2R4 wild type sequence (SEQ ID No. 1 )

MLRLFYFSAIIASVILNFVGIiMNLFITWNCKTWVKSHRISSSDRILFSLGITRFLMLGLF LVNTIYFVSSNTERSVYLSAFFVLCFMFLDSSSVWFVTLLNILYCVKITNFQHSVFLLL KRNISPKIPRLLLACVLISAFTTCLYITLSQASPFPELVTTRNNTSFNISEGILSLWSLVL SSSLQFIINVTSASLUHSLRRHIQKMQKNATGFWNPQTEAHVGAMKLMVYFLILYIPY SVATLVQYLPFYAGMDMGTKSICLIFATLYSPGHSVLIIITHPKLKTTAKK!LCFKK

The sequence of the T2R4 S285A CAM is provided below.

T2R4 S285A CAM Sequence (SEQ ID No. 2)

MLRLFYFSAIIASVILNFVGIIMNLFITWNCKTWVKSHRISSSDRILFSLGITRFLMLGLF LVNTIYFVSSNTERSVYLSAFFVLCFMFLDSSSVWFVTLLNILYCVKITNFQHSVFLLL KRNISPKIPRLLLACVLISAFTTCLYITLSQASPFPELVTTRNNTSFNISEGILSLWSLVL SSSLQFIINVTSASLLIHSLRRHIQKMQKNATGFWNPQTEAHVGAMKLMVYFLILYIPY SVATLVQYLPFYAGMDMGTKSiCLIFATLYSPGHAVLIIITHPKLKTTAKKILCFKK

The sequence of the human T2R4 H214A CAM is provided below.

T2R4 H214A CAM sequence (SEQ ID No. 3)

MLRLFYFSAIIASVILNFVGIIMNLFITWNCKTWVKSHRISSSDRILFSLGITRFLMLGLF LVNTIYFVSSNTERSVYLSAFFVLCFMFLDSSSVWFVTLLNILYCVKiTNFQHSVFLLL KRNISPKIPRLLLACVLISAFTTCLYITLSQASPFPELVTTRNNTSFNISEGILSLWSLVL SSSLQFIINVTSASLLIHSLRRAIQKMQKNATGFWNPQTEAHVGAMKLMVYFLILYIPY SVATLVQYLPFYAGMDMGTKSICLIFATLYSPGHSVLIIITHPKLKTTAKKILCFKK

The sequence of the T2R4 Q216A CAM receptor is provided below.

T2R4 Q216A CAM sequence (SEQ ID No. 4)

MLRLFYFSAMASVILNFVGIIMNLFITWNCKTWVKSHRISSSDRILFSLGITRFLMLGLF LVNTIYFVSSNTERSVYLSAFFVLCFMFLDSSSVWFVTLLNILYCVKITNFQHSVFLLL KRNISPKIPRLLLACVLISAFTTCLYITLSQASPFPELVTTRNNTSFN!SEGILSLWSLVL SSSLQFIINVTSASLLIHSLRRHIAKMQKNATGFWNPQTEAHVGAMKLMVYFLILYIPY SVATLVQYLPFYAGMDMGTKSICLIFATLYSPGHSVLIIITHPKLKTTAKKILCFKK

The sequence of the T2R4 V234A CAM receptor is provided below.

T2R4 V234A CAM sequence (SEQ ID No. 5)

MLRLFYFSAIIASVILNFVGIIMNLFITWNCKTWVKSHRISSSDRILFSLGITRFLMLGLF LVNTIYFVSSNTERSVYLSAFFVLCFMFLDSSSVWFVTLLNILYCVKITNFQHSVFLLL KRNlSPKIPRLLLACVLISAFTTCLYITLSQASPFPELVTTRNNTSFNISEGiLSLWSLVL SSSLQFIINVTSASLLIHSLRRHIQKMQKNATGFWNPQTEAHAGAMKLMVYFLILYIPY SVATLVQYLPFYAGMDMGTKSICLIFATLYSPGHSVLMITHPKLKTTAKKILCFKK

The sequence of the T2R4 M237A CAM receptor is provided below.

T2R4 M237A CAM sequence (SEQ ID No. 6)

MLRLFYFSAIIASVILNFVGIIMNLFITWNCKTWVKSHRISSSDRILFSLGITRFLMLGLF LVNTIYFVSSNTERSVYLSAFFVLCFMFLDSSSVWFVTLLNILYCVKITNFQHSVFLLL KRNISPKIPRLLLACVLISAFTTCLYITLSQASPFPELVTTRNNTSFNISEGILSLWSLVL SSSLQFIINVTSASLLIHSLRRHIAKMQKNATGFWNPQTEAHVGAAKLMVYFLILYIPY SVATLVQYLPFYAGMDMGTKSICLIFATLYSPGHSVLIIITHPKLKTTAKKILCFKK

The sequence of the human T2R4 I55A CAM receptor is provided below. T2R4 I55A CAM sequence (SEQ ID No. 7)

MLRLFYFSAilASVILNFVGIlMNLFITWNCKTWVKSHRISSSDRALFSLGITRFLMLGL FLVNTIYFVSSNTERSWLSAFFVLCFMFLDSSSVWFVTLLN!LYCVKITNFQHSVFLLL KRNISPKIPRLLLACVLISAFTTCLYITLSQASPFPELVTTRNNTSFNISEGILSLWSLVL SSSLQFIINVTSASLLIHSLRRHIQKMQKNATGFWNPQTEAHVGAMKLMVYFLILYIPY SVATLVQYLPFYAGMDMGTKSICLIFATLYSPGHSVLlllTHPKLKTTAKKiLCFKK

The sequence of the T2R4 H123A CAM receptor is provided below.

T2R4 H123A CAM sequence (SEQ ID No. 8)

MLRLFYFSAIIASVILNFVGIIMNLFITWNCKTWVKSHRISSSDRILFSLGITRFLMLGLF LVNTIYFVSSNTERSVYLSAFFVLCFMFLDSSSVWFVTLLNILYCVKITNFQASVFLLL KRAISPKIPRLLLACVLISAFTTCLYITLSQASPFPELVTTRNNTSFNISEGILSLWSLVL SSSLQFIINVTSASLLIHSLRRHiQKMQKNATGFWNPQTEAHVGAMKLMVYFLILYIPY SVATLVQYLPFYAGMDMGTKSICLIFATLYSPGHSVLIIITHPKLKTTAKKILCFKK The sequence of the T2R4 N132A CAM receptor is provided below.

T2R4 N132A CAM sequence (SEQ ID No. 9)

MLRLFYFSAIIASVILNFVGIIMNLFITWNCKTWVKSHRISSSDRILFSLGITRFLMLGLF LVNTIYFVSSNTERSVYLSAFFVLCFMFLDSSSVWFVTLLNILYCVKITNFQHSVFLLL KRAISPKIPRLLLACVLISAFTTCLYITLSQASPFPELVTTRNNTSFNISEGILSLWSLVL SSSLQFIINVTSASLLIHSLRRHIQKMQKNATGFWNPQTEAHVGAMKLMVYFLILYIPY SVATLVQYLPFYAGMDMGTKSICLIFATLYSPGHSVLIIITHPKLKTTAKKILCFKK

Accordingly, in an embodiment of the invention, there is provided an isolated or purified constitutively active mutant (CAM) of bitter taste receptor (T2R) mutant selected from the group consisting of:

a) a T2R CAM receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, T2R55 and T2R60, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to histidine 214 of the T2R4 wild type sequence;

b) a T2R CAM receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R39, T2R40, T2R48, T2R49, T2R50 and T2R55, wherein said T2R CAM receptor further

comprises an alanine residue at the amino acid residue corresponding to serine 285 of the T2R4 wild type sequence;

c) a T2R CAM receptor selected from the group consisting of: T2R4, T2R13 and T2R41.wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to glutamine 216 of the T2R4 wild type sequence;

d) a T2R CAM receptor selected from the group consisting of: T2R4, T2R7, T2R8 and T2R10, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to valine 234 of the T2R4 wild type sequence; e) a T2R CAM receptor selected from the group consisting of: T2R4, T2R10 and T2R55, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to methionine 237 of the T2R4 wild type sequence;

f) a T2R CAM receptor selected from the group consisting of: T2R3, T2R4,

T2R5, T2R7, T2R8, T2R9, T2R10, , T2R14, , T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50 and T2R55, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue

corresponding to isoleucine 55 of the T2R4 wild type sequence;

g) a T2R CAM receptor selected from the group consisting of: T2R1 , T2R3,

T2R4, T2R7, T2R8, T2R9, T2R16, T2R38, T2R40, T2R41 , T2R55 and T2R60, wherein said T2R CAM receptor further comprises a alanine residue at the amino acid residue corresponding to histidine 123 of the T2R4 wild type sequence;

h) a T2R4 CAM receptor, wherein said T2R4 mutant receptor further comprises an alanine residue at amino acid residue corresponding to aspargine 132 of the T2R4 wild type sequence.

As will be appreciated by one of skill in the art, in the above method, a nucleic acid molecule encoding the selected T2R is mutated through site directed

mutagenesis means and the resulting mutated nucleic acid molecule is recovered and/or purified and/or isolated. The nucleic acid molecule encoding the CAM can then be engineered for expression in a suitable cell line, either for expression of the CAM for recovery or in a cell line for testing and/or screening, as discussed herein.

Accordingly, in another aspect of the invention, there is provided a method of generating or producing such a T2R CAM by following the steps as outlined above, specifically, the mutation of the selected wild type T2R nucleotide sequence at the selected amino acid residue to alanine, thereby producing the CAM.

According to another aspect of the invention, there is provided a cell or cell line engineered to express the constitutively active bitter taste receptor mutant(s) described herein. As will be appreciated by one of skill in the art, there are a wide variety of suitable cells or cell lines for in vitro expression of the T2R CAMs which will be readily apparent to one of skill in the art. Such cells or cell lines can then be used to screen for bitter blockers as antagonists or inverse agonists, as discussed herein.

In another aspect of the invention, there is provided a T2R CAM comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 2-9. In another aspect of the invention, there is provided a nucleic acid molecule encoding an amino acid sequence that is a peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 2-9.

As will be appreciated by one of skill in the art, modifications made to the T2R4 CAM peptide sequence that do not significantly affect the constitutive activity of this peptide are within the scope of the invention. For example, conservative changes within highly non-conserved amino acids are likely to be tolerated. It is of note that such conserved amino acids can be readily determined by comparison of two or more of the T2R sequences. Furthermore, the 3D structures of these receptors have been modeled and accordingly it is well within routine skill in the art to determine what amino acid locations would tolerate modification (and what modifications would be tolerated). Other such possible substitutions will be readily apparent to one of skill in the art and/or through routine experimentation.

As will be appreciated by one of skill in the art, taste and flavour companies interested in discovering new bitter blockers can use these T2R CAMs as

experimental controls. A bitter blocker (antagonist and/or inverse agonist) is expected to decrease the constitutive activity of these CAMs, as discussed below.

The current technique(s) for elucidating putative bitter blockers is to use competition and/or inhibition assays where premixing of the agonist and putative antagonist is performed and the cellular response of this mixture is then compared to those of agonist responses.

The CAMs in T2Rs are more accurate in testing the efficacy of the putative bitter blockers. In an experimental technique known as reversal of basal activity, putative bitter blockers should be able to reverse the basal activity of CAMs, depending on their potency. Using CAMs allows for the pharmacological classification of the putative bitter blockers as antagonists or inverse agonists, depending on their ability to attenuate the basal signal to either wild type or lower than wild type levels.

As will be appreciated by one of skill in the art, as used in this context,

"isolated" or "purified" does not require absolute purity but rather that the receptor is greatly enriched or overexpressed compared to its natural environment.

In another aspect of the invention, there is provided a method of determining if a compound of interest is a human bitter taste receptor blocker comprising:

adding a compound of interest to a suitable cell line expressing a constitutively active bitter taste receptor mutant; and

measuring the activity of the constitutively active bitter taste receptor mutant, wherein the compound of interest is a bitter taste receptor antagonist or inverse agonist if said compound reduces the activity of the constitutively active bitter taste receptor mutant to approximately the same activity as a wild type bitter taste receptor.

As will be appreciated by one of skill in the art, the activity of the T2R receptor may be determined by any means known in the art, for example, by measuring intracellular Ca²⁺ levels.

In some embodiments of the invention, the activity of the constitutively active bitter taste receptor mutant is compared to a control. The control may be a wild type bitter taste receptor. Aiternatively, the control could be a CAM T2R mutant exposed to a known negative compound (one that has no effect on CAM T2R activity) or a mock treated control. As will be appreciated by one of skill in the art, the control does not necessarily need to be repeated every time. Specifically, it is believed that one of skill in the art will be able to identify whether or not a specific compound of interest without necessarily repeating controls every time.

In this manner, bitter taste receptor agonists and reverse agonists can be identified.

According to another aspect of the invention, there is provided a bitter taste receptor agonist or reverse-agonist as identified by the above-described method.

According to a further aspect of the invention, there is provided a nucleic acid molecule encoding a human bitter taste receptor (T2R) having a serine at amino acid position 285 of the wild type sequence mutated to a non-polar amino acid.

According to a still further aspect of the invention, there is provided a nucleic acid molecule encoding a purified or isolated human bitter taste receptor (T2R) having a serine at position 7.47 of transmembrane helix 7 in the wild type receptor mutated to a non-poiar amino acid.

According to a still further aspect of the invention, there is provided a nucleic acid molecule encoding an amino acid sequence as set forth in any one of SEQ ID NOs: 2-9.

According to a still further aspect of the invention, there is provided a nucleic acid molecule encoding an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

As will be appreciated by one of skill in the art, such nucleic acid molecules may be inserted into a suitable expression vector and transfected into a suitable cell line so that the cell line expresses CAM T2R mutants. Such a cell line can be used to screen for bitter taste receptor agonists and reverse agonists as discussed herein.

Structure-function studies on T2Rs are very limited and none have reported a CAM, previously. This is the first report and successful characterization of a CAM in T2Rs.

As will be appreciated by one of skill in the art, bitter blockers that target T2R4 and other T2Rs that have a serine at a position corresponding to amino acid 285 of T2R4 {SEQ ID NO: 1) can be characterized using the S285A T2R4 mutant as set forth in SEQ ID NO: 2.

As will be appreciated by one of skill in the art, bitter blockers that target T2R4 and other T2Rs that have a histidine at a position corresponding to amino acid 214 of T2R4 (SEQ ID NO: 1 ) can be characterized using the H214A T2R4 mutant as set forth in SEQ ID NO: 3.

As will be appreciated by one of skill in the art, bitter blockers that target T2R4 and other T2Rs that have a glutamine at a position corresponding to amino acid 216 of T2R4 (SEQ ID NO: 1 ) can be characterized using the Q216A T2R4 mutant as set forth in SEQ ID NO: 4. As will be appreciated by one of skill in the art, bitter blockers that target T2R4 and other T2Rs that have a valine at a position corresponding to amino acid 234 of T2R4 (SEQ ID NO: 1 ) can be characterized using the V234A T2R4 mutant as set forth in SEQ ID NO: 5.

As will be appreciated by one of skill in the art, bitter blockers that target T2R4 and other T2Rs that have a methionine at a position corresponding to amino acid 237 of T2R4 can be characterized using the M237A T2R4 mutant as set forth in SEQ ID NO: 6.

As will be appreciated by one of skill in the art, bitter blockers that target T2R4 and other T2Rs that have an isoleucine at a position corresponding to amino acid 55 of T2R4 can be characterized using the I55A T2R4 mutant as set forth in SEQ ID NO: 7.

As will be appreciated by one of skill in the art, bitter blockers that target T2R4 and other T2Rs that have a histidine at a position corresponding to amino acid 123 of T2R4 can be characterized using the H123A T2R4 mutant as set forth in SEQ ID NO: 8.

As will be appreciated by one of skill in the art, bitter blockers that target T2R4 and other T2Rs that have a aspargine at a position corresponding to amino acid 132 of T2R4 can be characterized using the N132A T2R4 mutant as set forth in SEQ ID NO: 9.

Bitter blockers have immense nutraceutical potential. A large number of naturally occurring or plant derived compounds can be screened in a semi- or high- throughput format using these CAMs. This would allow classification of some of these putative bitter blockers into T2R antagonists and/or inverse agonists depending on their ability to attenuate the signal to varying percentages. For example, these compounds could then be screened against the US Food and Drug Administration (FDA) list of 3000 compounds in the SCOGS database classified as Generally Recognized as Safe (GRAS) for human consumption.

CAMs at other positions in T2Rs can be characterized using the technique described. As discussed above, an accurate estimate of basal activity is necessary for classifying a mutant as CAM although this does not necessarily need to be repeated every time. Basal activity can be calculated from slope of expression vs. basai activity for the mutants compared to same value for the wild type receptor (Hwa et al. 1997, Chakraborty et al. 2012).

To characterize the basal activity of T2R4 and mutants, intracellular Ca²⁺ assays were carried out with the receptors stimulated with buffer alone to determine the basal or agonist-independent signal. Basal Ca²⁺ levels of the WT-T2R4 and mutants corrected for cell surface expression levels were measured to assess constitutive activity. Among the mutants analyzed in this experiment, only the S285A mutant displayed a more than 3-fold increase in agonist-independent activity (Figure 3). To characterize the constitutive activity of the S285A mutant in more detail, we assayed the effect of receptor density on basal Ca²⁺ mobilization (Figure 4). WT- T2R4 (circular dots) and S285A (square dots) constructs were expressed in HEK293T cells at different receptor densities by varying amounts of DNA used in each

transfection (3pg to 9 pg DNA per 10⁶ cells). Receptor expression levels were determined by cell surface ELISA and normalized to WT-T2R4. The slope of WT- T2R4 was 0.185 ± 0.13 of calcium mobilized (ARFU), while that of the S285A was 1.06 ± 0.27, respectively (Figure 4). These results show that S285A mutant exhibits constitutive activity that is 3-5 fold higher than wild type T2R4 basal activity.

The inventors targeted two conserved residues present on TM1 and TM7 of T2R4 for structure-function analysis. This elucidated the role of a crucial amino acid Ser285⁷'⁴⁷ that is involved in locking T2R4 in the inactive state by interhelical hydrogen bonds. The S285A mutant in T2R4, which does not have the ability to hydrogen bond, showed constitutive activity, while the conservative substitution

S285T which retains the ability to hydrogen bond displayed normal wild type like basal activity. Previous studies on rhodopsin and other Class A GPCRs have shown that receptor activation involves breaking of hydrogen bonds and formation of new bonds (Hubbell et al. 2003, Sakmar et al. 2002). The recent elucidation of the crystal structures of a number of Class A GPCRs, including some active and G-protein bound structures, gives structural and

mechanistic insights into the activation and signal transduction by these receptors (Rasmussen et al, 2011 , Standfuss et al. 2011 ). Interestingly, these crystal structures show how water- mediated hydrogen-bond networks between the ligand binding pocket and the conserved Class A GPCR sequence motifs play important structural and functional roles. Indeed, the key role played by these structural waters in mediating interhelical interactions in GPCRs has only now begun to be appreciated (Angel et al. 2009, Nygaard et al. 2010). Recently, the inventors proposed that the intramolecular structural waters in combination with the group-conserved residues in β2-AR create an interface that facilitates the rotation of transmembrane (TM) helix, TM6 relative to TM7 during activation of P2-AR (Arakawa et al. 201 1). However, most of the conserved amino acid motifs that are present in Class A GPCRs are absent in T2Rs {Singh et al. 201 1a). In the absence of the crystal structure of a taste receptor and the very low sequence homology with Class A GPCRs, the role of hydrogen-bond networks including those mediated by structural waters, in taste receptor function was not studied thus far.

Similar to T2Rs, the residue at position 1 .46 is conserved as a glycine in a majority of Class A GPCRs, including rhodopsin. However, in rhodopsin, the naturally occurring variants of Gly51^{1 46} cause ADRP, and like most other ADRP mutations appear to cause destabilization of the opsin structure. It was hypothesized that the instability of Gly51^1.46 ADRP mutations can be due to a steric hindrance with the residue 7.47, which is Va!300 (Bosch et al. 2003). Interestingly, while the residue at position 1.46 is a glycine in T2Rs, the amino acid at 7.47 is a serine in 70%, and a proline in 18% of the T2Rs (Singh et al. 201 1a).

Thus the inventors have shown that Ser285⁷⁴⁷ stabilizes the inactive state of T2R4, and replacement of this residue with a non-polar amino acid, as in the case of S285A mutant, results in the receptor displaying a 3-5 fold increase in basal activity over wild type. While not wishing to be bound to a particular hypothesis, we propose based on molecular modeling analysis which included structural waters that in the absence of restraining interaction between Ser285⁷⁴⁷and Arg63²⁵⁴, the side chain of Arg63^2,54 moves away from TM7 and forms a continuous hydrogen bond network connecting Asn32¹⁵⁰ with structural waters. This results in the T2R4 adopting an active state conformation, with less number of intra- and interhelical contacts to that observed in the structure of the constitutively active rhodopsin mutant {Figure 5).

Furthermore, recent molecular modeling studies using BiHelix and

SuperBi Helix Monte Carlo methods on T2R38 predicted the hydrogen bond

interactions between TM3 and TM6 or between TM5 and TM6 to play a role in receptor activation (Jan et al., Journal of Chemical Information and Modeling 52:

1875-1885). Results from these studies suggest the cytoplasmic end of TM6 moves 2A, while no major changes were observed in the movement of TM5. Previously, the inventors have reported in T2R1 that the highly conserved LxxSL motif performs an important structural role by stabilizing the helical conformation of TM5 (Singh et al., 2011 ). Results from this study suggest that the residues of the LxxSL motif also perform a functional role by forming a network of hydrogen-bond interactions with residues present in ICL3 and TM6, including the highly conserved His214 (96% conserved in T2Rs). Therefore, the conserved LxxSL motif at the cytoplasmic end of TM5 plays both a structural and functional role in T2Rs.

In this study, there were a number of mutants that showed no detectable signal or the signal was not saturated even with 5 mM quinine. While not wishing to be bound to a particular theory or hypothesis, the inventors speculate that the

conformational changes in the receptor brought about by these mutations might have altered the ligand binding pocket thereby affecting quinine binding. It is possible that the signal can be rescued when induced with a different ligand. However, in the absence of other well-defined agonists and antagonists for T2R4, this aspect could not be pursued further. Similarly, in the absence of well characterized T2R4 ligands, for the mutants that showed high EC₅₀ mutant/wild type ratio, whether this loss in function results because of altered receptor activation and/or defective ligand binding could not be characterized. The results show that the network of interactions, involving conserved residues at the cytoplasmic ends of TM5 and TM6, play an important role stabilizing the inactive state of T2Rs. Changes in this network brought about by mutations such as H214A cause the receptor to adopt an active conformation, and it involves the movement of TM6. The H214A mutant showed constitutive activity of up to 10-fold over WT-T2R4, one of the highest reported for a GPCR mutant. This study opens up new areas of research on bitter taste signaling, development of assays based on the CAMs identified in this study would allow pharmacological characterization of novel bitter taste blockers into antagonists and inverse agonists.

The intracellular region of T2R4 consists of 87 amino acids, including 4 alanines. An N-terminal FLAG tagged T2R4 (WT-T2R4) was used as the base receptor and the entire intracellular region that includes 16 amino acids in ICL1 , 28 amino acids in ICL2, 23 amino acids in ICL3 and 16 amino acids in the C-terminus of T2R4 were replaced with alanines and the mutants pharmacologically characterized (Fig. 11 ).

The intensely bitter tasting natural alkaloid, quinine, activates T2R4 in a concentration dependent manner. The 83 alanine mutants of T2R4 displayed varied levels of cell surface expression, and calcium signaling upon stimulation with quinine (Table 2). Majority of the 16 alanine mutants in ICL1 showed no significant changes in either cell surface expression and/or quinine induced changes in calcium mobilization. Six ICL1 mutants showed defective agonist induced signaling. The S51A, S52A, D53A and L56A mutants showed no detectable increase in intracellular calcium mobilization upon stimulation with quinine, while the F57A mutant displayed no concentration dependent increase in signal, and the signal was not saturated even at the highest quinine concentration. Cell surface ELISA revealed that all these mutants where properly expressed on the surface with expression levels ranging from 60% to 130% of WT-T2R4. Surprisingly, two mutants located in the middle of ICL1 displayed contrasting signaling efficacies, with the R48A mutant showing a 50% decrease, while the H47A mutant showed more than a 2-fold increase in EC₅₀ mutant/wild type ratio (Table 2). While the GPCR natural variant database does not list a T2R4 variant in this loop, this region has naturally occurring variants in T2R38, T2R43 and T2R46,

Interestingly, the ICL2 in T2R4 has two triads of leucines, one in the center of the loop and the other at the end of the loop, at the ICL2-TM4 interface (Fig. 12). Among the ICL2 alanine mutants, only 15 of the 28 alanine replacements showed significant agonist induced signaling, with 7 of the mutants displaying defective ligand binding, as shown by more than a 1 .5-fold increase in EC50 mutant/wild type ratio (Table 2). Of the 83 alanine mutants, the C1 15A mutant in ICL2 showed the highest EC50 mutant/wild type ratio of 2.5 (Table 2). The mutants L140A and L141A part of the second leucine triad in ICL2 were able to signal in response to quinine treatment, but not in a concentration dependent manner. Interestingly a majority of the mutants that were unable to signal were properly expressed at the cell surface. Compared to ICL2 mutants, the ICL3 mutants showed better agonist induced signaling with 14 of the 23 mutants displaying quinine induced signaling. Further, all of the mutants were targeted to the cell surface. Only three ICL3 mutants 1215A, F225A and P228A showed more than a 2-fold increase in EC₅₀ mutant/wild type ratio.

All the C-terminal alanine mutants showed quinine dependent increase in signal, with most of them showing increased sensitivity towards the ligand (Table 2). Except for the C304A mutant, the EC50 mutant/wild type ratio of majority of the C- terminal alanine mutants was less than 1.0. The P293A mutant showed EC50 mutant/wild type ratio of 0.30, the lowest among the 83 alanine mutants.

To obtain insights into the role of intracellular residues in T2R activation, the agonist-independent or basal activity of WT-T2R4 and all the 83 alanine mutants was characterized. Basal Ca²⁺ levels of the WT-T2R4 and mutants corrected for cell surface expression were measured to assess agonist-independent activity (Fig. 13). Interestingly, the mutation of residues present towards the C-terminal end of ICL1 showed high basal Ca²⁺ levels, similarly few mutants present in the ICL2 and ICL3 showed statistically significant values, while none of the amino acid replacements in the C-terminus showed increased basal signaling (Fig. 13A). To obtain a more accurate estimate of basal activity, the effect of receptor density on Ca²⁺ mobilization was assayed. Since not all activities are linear relative to receptor expression, basal activity was calculated from slope of expression vs. basal activity for critical mutants compared to same value for the WT-T2R4. Therefore, all the 1 1 intracellular mutants that showed statistically significant increase in basal activity were expressed in HEK293T cells at different receptor densities, by varying the amount of DNA used in each transfection. The results show that only I55A, H123A, N132A, H214A, Q216A, V234A and M237A mutants exhibit a true CAM phenotype, with constitutive activity ranging from 2 to 15 fold over WT-T2R4 (Fig. 12B). The H214A mutant present at the TM5-ICL3 interface displayed the highest constitutive activity, interestingly, this histidine is 96% conserved among the T2Rs.

RESULTS

Design and analysis of mutations

Guided by amino acid sequence analysis of 188 T2Rs and molecular modeling, the inventors identified five conserved residues Gly^{1 46} and Asn^{1 50} on TM1 , Arg²'⁵⁴ on TM2, His⁷⁴⁶ and Ser^{7 47}on TM7 that might play important roles in receptor activation by mediating an intricate network of hydrogen bonds connecting TM1-TM2-TM7 in T2Rs. The roles of three of these residues Asn^{1 ,50}, Arg^{2 54} and His^7,46 were recently elucidated by structure-function analysis (Singh et al. 2011 a). Molecular modeling analysis (with no structural waters introduced in the models) showed that the highly conserved residue, Asn^{1 50} establishes backbone contacts with the conserved Gly^{1 46} and He^{1 53} on TM1 , while its side chain functional group is hydrogen-bonded to the side chain functional groups of Arg²⁵⁴ on TM2 and Ser⁷⁴⁷ on TM7 respectively. This intricate network is absent or significantly modified in the inactive and hyperactive mutants of T2R1 (Singh et al. 2011 a). To further elucidate the interplay between TM1- TM2-TM7 in T2Rs, the two remaining key players in this hydrogen-bond network, Gly^{1 ,46} and Ser^7-47 in T2R4 were targeted for structure-function analysis.

Figure 1 shows a secondary structure representation of T2R4 amino acid sequence, and the amino acid sequence alignment of TM1 and TM7 of T2R1 , T2R4 and opsin. Based on the analysis of 188 T2R sequences, T2R4 is found to be missing a residue at position 7.48 (Singh et al. 2011 a). Furthermore, while the residue at position 1.46 is a glycine in T2Rs, the amino acid at 7.47 is conserved as a serine in 70.2% or as proline in 18.1 % of the 188 T2Rs (Singh et al. 201 1a). in the remaining 11 % of the T2Rs, no amino acid conservation was observed at position 7.47. The highly conserved residues in the two helices (Ballesteros and Weinstein residue) in both Class A GPCRs and T2Rs are also highlighted.

An N-terminal FLAG tagged-T2R4 heterologously expressed in HEK293T cells was used in this study. The inventors tried heterologous expression of T2R1 in HEK293T cells; unfortunately it showed high basal calcium signal (noise) under the assay conditions, which precluded using T2R1 in the current study. Two types of mutations were made in T2R4 at each position. First, mutations were made to a smaller amino acid such as alanine; with the expectation that this substitution will have a minimal effect on receptor folding and ligand binding. Second, guided by a molecular model of T2R4, mutations were made with either a non-conserved substitution such as glycine to leucine or with conserved substitution such as serine to threonine. Furthermore, at position 7.47 an additional S285P mutation was made to mimic T2Rs that contain proline at this position. Functional characterization

The natural alkaloid quinine and the synthetic bitter compound denatonium benzoate, are agonists for T2R4. Both of these compounds are capable of stimulating T2R4 and cause a concentration-dependent increase in intracellular calcium in cells expressing T2R4 (Singh et al. 2011 b). Taste sensory analysis of both the bitter compounds using the analytical instrument E-Tongue™ from Alpha MOS (Toulouse, France) showed that quinine has the most intense bitterness. Further, quinine, an important secondary metabolite, has higher efficacy for T2R4 and was thus selected for use as the agonist in the present study.

Functional analysis of wild type and mutant T2R4 receptors were determined by measuring changes in intracellular calcium of HEK293T cells transiently expressing these receptors after application of different concentrations of the T2R4 agonist quinine. All four mutants displayed varied levels of signaling (Figure 2). The G28L and S285T mutants showed increased potency towards the agonist quinine, displayed as a left shift in dose-response. Among the five mutants, S285T displayed the highest potency with an EC50 value of 0.60 ± 0.38 mM compared to 1.00 ± 0.38 mM for WT-T2R4. The signals from TM7 mutants S285A and S285P were not saturated at the normal concentration range used for the other mutants, hence an additional higher concentration of 5mM quinine was used (Figure 2A). Cell surface ELISA assay showed that all the five mutants were properly folded and expressed on the cell surface at similar levels (Figure 2B).

Characterization of intrinsic and basal signaling

To characterize the intrinsic and basal activity of T2R4 and mutants, intracellular Ca^2÷ assays were carried out with the receptors stimulated with buffer alone to determine the basal or agonist-independent signal, and with a single saturating concentration of quinine to determine the maximum agonist-induced or intrinsic signal (Figure 3). To measure intrinsic activity, the highest concentration as shown in figure 2, was used for WT-T2R4 and the mutants. The WT-T2R4, G28A, G28L and S285T receptors showed similar intensity in signal at saturating

concentrations of either 2.5 mM or 5 mM quinine, while in the case of S285A and S285P mutants a significant difference in signal intensity was observed hence the highest concentration of 5mM quinine was used. The intrinsic signal of the G28A, G28L and S285P mutants was in the range of WT-T2R4, while S285T showed slightly reduced signal. Interestingly, the S285A mutant showed a 2-fold increase in intrinsic signal compared to WT-T2R4 (Figure 3A). Basal Ca²⁺ levels of the WT-T2R4 and mutants corrected for cell surface expression levels were measured to assess constitutive activity. Among the mutants analyzed, only the S285A mutant displayed a 3-fold increase in agonist-independent activity (Figure 3B).

To characterize the constitutive activity of the S285A mutant in more detail, we assayed the effect of receptor density on basal Ca²⁺ mobilization (Figure 4). WT-T2R4 (circular dots) and S285A (square dots) constructs were expressed in HEK293T cells at different receptor densities by varying the amounts of DNA used in each

transfection (3 pg to 9 pg DNA per 10⁶ cells). Receptor expression levels were determined by cell surface ELISA and normalized to WT-T2R4. The slope of WT- T2R4 was 0.185 ± 0.13 of calcium mobilized (ARFU), while that of the S285A was 1.06 ± 0.27, respectively (Figure 4). These results show that the S285A mutant exhibits constitutive activity that is 3-5 fold higher than wild type T2R4 basal activity. As pursued with other constitutively active GPCR mutants (Chakraborty ef al. 2012), we could not assess reversal of basal activation with the S285A mutant, because of the lack of an inverse agonist or antagonist for T2R4.

Molecular modeling analysis

We built homology models of the inactive WT-T2R4 and the constitutively active S285A mutant using the inactive and constitutively active mutant (CAM) rhodopsin crystal structures as templates (Figure 5). We also analyzed the water molecules in the immediate vicinity of Gly28¹'⁴⁶, Asn32^{1 50}, Arg63^{2 M} and Ser285^{7 47}. In rhodopsin, residues at positions 7.46 and 7.47 are Ala299 and Val300, while in T2R4 they are His284 and Ser285 respectively. Interestingly, in both the inactive and CAM rhodopsin structures the side chain of Val300^{7 47}packs against Gly51^{1 46}. In the inactive rhodopsin structure (PDB ID 1 U19), Asp83^2,54 is hydrogen bonded to the water network through water 2017, and a separate network of interactions between TM1 and TM7 are observed. In the CAM rhodopsin structure (PDB ID 2X72) there is a continuous hydrogen bond network connecting residues Gly51^1'46, Asn55^1,50,

Asp83^{2 54} and Aia277⁷⁴⁷ with structural waters (water 2016 in 2X72 corresponds to water 2017 in IU19). In the inactive (basal) models of WT-T2R4 and S285P mutant (Fig. 6),an interhelical hydrogen bond contact was observed between residues 7.47 and 2.54 (Figure 5). The molecular model of the constitutively active S285A mutant suggests that the break of the hydrogen bond between Ala285^{7 47} and Arg63^{2 54} enables the side chain of Arg63^{2 54} to establish a continuous network of hydrogen bonds connecting the highly conserved Asn32^1,50 with structural waters, similar to that observed in the CAM rhodopsin structure. However, this is where the similarity ends, as no interhelicai contacts were observed between Ala at 7.47 in the T2R4_S285A model, with any of the other residues at positions 2.54, 1.50 and 1.46. Further, intrahelical contacts between residues 1.47 and 1 .50 were also lost in the

T2R4_S285A model (Figure 5).

Structure-function analysis on T2Rs revealed unique signature residues in TM helices that are distinct from Class A GPCRs (Singh et al., 2011 ; Pydi et al., 2012, J Neurochemistry 286: 36032-36041 ). Previous structure-function studies on T2R4 targeted the N-terminus and TM regions that play a role in receptor expression and activation (Pydi et al., 2012; Pydi et al., 2013, Biochem Biophys Res Comm 430: 179- 182). The intracellular region of T2R4 consists of 87 amino acids, including 4 alanines. Using an N-terminal FLAG tagged T2R4 (WT-T2R4) as the base receptor, the entire ICL3 region that includes 23 amino acids in ICL3 of T2R4 were replaced with alanines and the mutants characterized biochemically (Fig. 1 ).

Functional characterization

The natural alkaloid, quinine, acts as an agonist and activates T2R4 in a concentration dependent manner. The 23 alanine mutants in ICL3 of T2R4 displayed varied levels of calcium mobilization upon stimulation with quinine (Table 1 ). Only 14 of the 23 ICL3 alanine mutants displayed quinine induced signaling in a concentration dependent manner (Table 1 ). Three 1CL3 mutants, Q216A, T230A and V234A showed an increase in agonist induced response; however their response was not saturated even at the highest concentration of 5 mM quinine (Table 1 ). Going higher than a 5 mM quinine concentration results in a significant increase in non-specific calcium mobilization in HEK293T cells expressing WT-T2R4 or mutants. The R213A, Q219A, K220A, Q229A, E231A and H233A mutants showed no detectable or statistically significant increase in intracellular calcium mobilization above mock transfected cells, upon stimulation with quinine. In figure 9, representative calcium traces for select mutants, stimulated with a saturating concentration of 2.5 mM quinine are shown, along with the mock transfected cells and buffer alone controls. Interestingly, three mutants 1215A, F225A and P228A display altered receptor activation and/or defective ligand binding, as shown by a 2-fold increase in EC₅o mutant/wild type ratio (Table 1 ). Ceil surface ELISA revealed that all the ICL3 mutants where expressed on the surface with expression levels ranging from 80% to 140% of WT-T2R4.

Identification and characterization of CAMs in T2R4-ICL3

To obtain insights into the role of intracellular residues in T2R activation, The agonist-independent activity of WT-T2R4 and ail the 23 alanine mutants was characterized. Basal Ca²⁺ levels of the WT-T2R4 and mutants corrected for cell surface expression were measured to assess agonist-independent activity (Fig. 10). Interestingly, R213A, H214A, Q216A, N227A, V234A and M237A mutants showed statistically significant increased basal signaling (Fig. 10A). The effect of receptor density on Ca²⁺ mobilization was calculated from slope of expression vs. basal activity for these six mutants, and compared to the WT-T2R4. The results based on the slope values show that only H214A, Q216A, V234A and M237A mutants exhibit a true CAM phenotype, with constitutive activity ranging from 2 to 10 fold over WT-T2R4 (Fig. 10B). The H214A mutant present at the TM5-ICL3 interface displayed the highest constitutive activity. Interestingly, His214 is present in 24 of the 25 human T2Rs (96% sequence conserved).

Analysis of molecular models

To interpret the effect of the ICL3 CAMs on T2R4 structure and function, homology models of the inactive WT-T2R4, a constitutively active WT-T2R4 and CAMs were built using rhodopsin inactive (PDB: 1 U19) and CAM (PDB: 2X72) crystal structures as templates. The four CAMs in ICL3 identified in this study are present at the amino and carboxyl-terminus of T2R4-ICL3. Through an intricate network that involves side-chain and backbone interactions, His214, Gln216, Val234 and Met237 interact with the highly conserved LxxSL motif on TM5 (Fig. 11). Analysis of the molecular models suggests a major rearrangement at the cytoplasmic ends of TM5 and TM6 (Fig. 11 B). In the inactive T2R4, Leu207 and Ser210 of the LxxSL interact with Met237 and His214, respectively. In the T2R4 CAM model, the cytoplasmic end of TM6 moves by around 2A towards the helical core (Fig. 11 A). This results in change of interactions around the LxxSL motif. His214 now interacts with side chain of Ser210 and the backbone of Leu211 , while the side chain of Met237 moves away from Lue207 to interact with Leu211. These TM5-ICL3-TM6 interactions restrain the movement of the helices, and changes in this network, as in the case of H214A, Q216A, V234A and M237A mutants causes the receptor to adopt an active

conformation.

METHODS

Materials. Fetal Bovine Serum and DMEM High Glucose were purchased from Sigma and Invitrogen (Carlsbad, CA, USA). Common chemicals and bitter compounds were purchased either from Fisher or Sigma. Fluo-4NW™ and quinine hydrochloride, were purchased from Invitrogen and MP Biomedicals (Solon, OH, USA). All chemicals were of analytical grade and used without further purification.

Molecular Biology and Cell culture. Amino acid substitutions were introduced into the synthetic FLAG-T2R4 gene carried by the expression vector pcDNA3.1 (invitrogen) as described previously (Singh et al. 201 1 a). The wild type and mutant T2R4 genes in pcDNA3.1 were transiently expressed in HEK293T cells using lipofectamine 2000 (Invitrogen) according to previously published methods

(Upadhyaya er a/. 2010).

Functional assay. HEK293T cells were cultured in 6-well tissue culture dishes at 37°C in DMEM-F12 and 10% FBS. Cells that are 70-80% confluent were co- transfected at 1 :1 ratio with T2R4 or mutants, and Ga 16/44 chimera (Ueda et ai.

2003) (a gift from Dr. Takashi Ueda, Nagoya City University, Japan). After 6-8 hours of transfection, 1 X10⁵ cells per well were plated in 96 well tissue culture treated BD- falcon biolux microplates along with DMEM-F12 and 5% FBS. After 14-16 hours of incubation at 37°C, the media was removed from wells and 100 μΙ of 1X concentration Fluo-4 NW dye was added to each well. Then the cells were incubated at 37°C for 45 min followed by 45 min at room temperature. Receptor activation was determined by measuring changes in intracellular calcium (Δ Relative Fluorescence Units) after application of different concentrations of quinine or buffer alone (for measuring basal activity) using Flexstation-3™ fluorescence plate reader (Molecular Devices, CA, USA) at 525 nm following excitation at 494 nm. Calcium mobilized was expressed as ARFU after subtracting the responses of cells transfected with piasmid carrying the Ga16/44 construct, and in certain experiments was normalized to wild type cell surface expression as determined by ELISA. The data presented was from two to five independent transfections in triplicate. Dose-response curves were generated and ECso values calculated by non-linear regression analysis using PRISM™ software version 4.03 (GraphPad Software Inc, San Diego, CA).

Cell surface ELISA. Cell surface expression of FLAG-T2R4 and the four mutants was detected by ELISA, as described below. HEK293T cells were

transfected with wild type or mutants using Lipofectamine 2000. After 8 hrs of transfection, 1.5 - 2X10⁵ cells per well were plated in poly-L-lysine coated plates. Following 18 - 20 hrs of plating, cells were washed 3 times with 1X PBS. The cells were then fixed in 4% paraformaldehyde for 12 min at room temperature, followed by - three 10 min washes with 1 X PBS. After blocking the cells with 2% BSA for 1 hour at room temperature, they were incubated with 1 g/ml (1 :1000) M1 anti-FLAG primary antibody over night at 4° C. Next day, the cells were washed with 1 % BSA dissolved in 1X PBS, 4 times (10-15 min each wash). The cells were then incubated with a concentration of 1 :5000 goat anti-mouse conjugated with horseradish peroxidase for 1 hour at room temperature, followed by washes as above. Empty cells were used as negative control. Color was developed by adding 200μΙ of SIGMAFAST™ OPD to each well by incubation in dark for 30 min, and absorbance was measured at 450 nm using Flexstation-3 fluorescence plate reader.

Statistics. To check the significance level of the calcium mobilized at basal (agonist-independent activity) and at the highest concentration of the agonist (for measuring intrinsic activity),statistical analysis were performed using one way ANOVA followed by "Tukey's Multiple Comparison Test" , P<0.05 considered to be statistically significant.

Molecular modeling. This was as described before (Singh et al. 2011a) with the following modifications. The T2R4 amino acid sequence without the FLAG tag was used for model building, inactive and constitutively active T2R4 models were built using rhodopsin crystal structures, PDB ID: 11)19 and PDB ID: 2X72 respectively. Structural waters were introduced into these models using PyMol. Molecular dynamics simulations (10ns) were performed on all of the receptor models using SYBYL X1 .3 molecular modeling suite (Tripos Inc, USA).

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

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Bitter taste receptor T2R1 is activated by dipeptides and tripeptides. Biochemical and biophysical research communications, 398, 331-335. Table 1. Pharmacological characterization of the T2R4 alanine mutants. Functional characterization of the mutants was pursued by measuring intracellular calcium mobilized after stimulating with different concentrations of agonist, quinine. Cell surface expression was determined by ELISA*.

* Intracellular loop 3 (ICL3). ND- not detected, no significant calcium mobilization detected; NS-not saturated, quinine concentration dependent increase in calcium mobilization not observed. Table 2. Pharmacological characterization of the T2R4 alanine mutants. Functional characterization of the mutants was pursued by measuring intracellular calcium mobilized after stimulating with different concentrations of a onist uinine. Cell surface ex re i *

Claims

1 . An isolated or purified constitutively active mutant (CAM) of bitter taste receptor (T2R) selected from the group consisting of:

a) a T2R CAM receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, T2R55 and T2R60, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to histidine 214 of the T2R4 wild type sequence;

b) a T2R CAM receptor selected from the group consisting of: T2R1 , T2R3,

T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R39, T2R40, T2R48, T2R49, T2R50 and T2R55, wherein said T2R CAM receptor further

comprises an alanine residue at the amino acid residue corresponding to serine 285 of the T2R4 wild type sequence;

c) a T2R CAM receptor selected from the group consisting of: T2R4,

T2R13 and T2R41 .wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to glutamine 216 of the T2R4 wild type sequence;

d) a T2R CAM receptor selected from the group consisting of: T2R4, T2R7, T2R8 and T2R10, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to valine 234 of the T2R4 wild type sequence;

e) a T2R CAM receptor selected from the group consisting of: T2R4, T2R10 and T2R55, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to methionine 237 of the T2R4 wild type sequence;

f) a T2R CAM receptor selected from the group consisting of: T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, , T2R14, , T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50 and T2R55, wherein said T2R CAM receptor further comprises an alanine residue at the amino acid residue corresponding to isoleucine 55 of the T2R4 wild type sequence;

g) a T2R CAM receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R7, T2R8, T2R9, T2R16, T2R38, T2R40, T2R41 , T2R55 and T2R60, wherein said T2R CAM receptor further comprises a alanine residue at the amino acid residue corresponding to histidine 123 of the T2R4 wild type sequence;

h) a T2R4 CAM receptor, wherein said T2R4 mutant receptor further comprises an alanine residue at amino acid residue corresponding to asparagine 132 of the T2R4 wild type sequence.

2. An isolated or purified constitutively active mutant (CAM) bitter taste receptor (T2R) selected from the group consisting of:

a) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:2; b) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:3; c) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:4; d) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:5; e) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:6; f) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:7; g) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:8; and

h) a peptide comprising the amino acid sequence as set forth in SEQ ID NO:9.

3. A method of preparing a constitutively active mutant (CAM) of bitter taste receptor (T2R) comprising:

a) subjecting a T2R receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R38, T2R39, T2R40, T2R41 , T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, T2R55 and T2R60, to site directed mutagenesis such that the histidine residue corresponding to histidine 214 is mutated to alanine; or

b) subjecting a T2R receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R39, T2R40, T2R48, T2R49, T2R50 and T2R55 to site directed mutagenesis such that the serine residue corresponding to serine 285 of the T2R4 wild type sequence is mutated to alanine ;or

c) subjecting a T2R receptor selected from the group consisting of: T2R4, T2R13 and T2R41 to site directed mutagenesis such that the glutamine residue corresponding to glutamine 216 of the T2R4 wild type sequence is mutated to alanine; or

d) subjecting a T2R receptor selected from the group consisting of: T2R4, T2R7, T2R8 and T2R10 to site directed mutagenesis such that the valine residue corresponding to valine 234 of the T2R4 wild type sequence is mutated to alanine; or e) subjecting a T2R receptor selected from the group consisting of: T2R4,

T2R10 and T2R55 to site directed mutagenesis such that the methionine residue corresponding to methionine 237 of the T2R4 wild type sequence is mutated to alanine; or

f) subjecting a T2R receptor selected from the group consisting of: T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, , T2R14, , T2R39, T2R40, T2R41 , T2R43,

T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50 and T2R55 to site directed mutagenesis such that the isoleucine residue corresponding to isoleucine 55 of the T2R4 wild type sequence is mutated to alanine; or

g) subjecting a T2R receptor selected from the group consisting of: T2R1 , T2R3, T2R4, T2R7, T2R8, T2R9, T2R16, T2R38, T2R40, T2R41 , T2R55 and T2R60 to site directed mutagenesis such that the histidine residue corresponding to histidine 123 of the T2R4 wild type sequence is mutated to alanine; or

h) subjecting a T2R4 receptor to site directed mutagenesis such that aspargine residue 132 is mutated to alanine; and

recovering a nucleic acid molecule encoding the T2R4 CAM receptor.

4. A method of determining if a compound of interest is a human bitter taste receptor blocker comprising:

adding a compound of interest to a suitable cell line expressing a constitutively active bitter taste receptor mutant according to claim 1 or 2; and

measuring the activity of the constitutively active bitter taste receptor mutant, wherein the compound of interest is a bitter taste receptor antagonist or inverse agonist if said compound reduces the activity of the constitutively active bitter taste receptor mutant to approximately the same activity as a wild type bitter taste receptor.

5. The method according to claim 4 wherein the activity is determined by measuring intracellular Ca²⁺ levels.

6. The method according to claim 4 wherein the activity of the constitutively active bitter taste receptor mutant is compared to a control.

7. The method according to claim 6 wherein the control is a wild type bitter taste receptor.

8. A nucleic acid molecule encoding a bitter taste receptor (T2R) constitutively active mutant (CAM) comprising a nucleotide sequence encoding the amino acid sequence as set forth in any one of SEQ ID NOs: 2-9.

9. A nucleic acid molecule encoding a bitter taste receptor (T2R) constitutively active mutant (CAM) comprising a nucleotide sequence encoding an amino acid sequence according to claim 1.

10. A nucleic acid molecule encoding a bitter taste receptor (T2R) constitutively active mutant (CAM) comprising a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 2.

1 1 . A cell or cell line engineered to express the constitutively active bitter taste receptor mutant according to claim 1 or 2.