WO2017030862A1 - Method of modulating fat taste perception - Google Patents

Abstract

Methods for identifying a compound that mimics or modulates fat taste perception employ screening recombinant cell or cell line-based assays that monitor the effect of a test compound on the function, activity or pathway stimulation of a nucleic acid sequence encoding GPR113, optionally co-expressed with Gna14, with one or more T1R receptors, or with one or more additional G proteins, or with chimeric molecules containing at least a portion of Gna14. A test compound that mimics or modulates the effect of dietary fat causes a change in the physical or functional characteristic of the contacted cells or cell lines in comparison to a control cell. Other cell-free assays that measure the ability of a test compound to disrupt or displace the binding of the GPR113 protein with the Gna14 protein and optionally other proteins, e.g., TIRs, are also useful. Reagents and devices employing such recombinant cells are also disclosed.

Description

METHOD OF MODULATING FAT TASTE PERCEPTION

[001] CROSS-REFERENCE TO RELATED APPLICATIONS

[002] This application claims the benefit of the priority of United States patent application No. 62/207,793, filed August 20, 2015.

[003] SEQUENCE LISTING

[004] Applicant hereby incorporates by reference the Sequence Listing material filed in electronic form herewith. This file is labeled "MON23PCT_ST25.txt".

[005] BACKGROUND OF THE INVENTION

[006] The mammalian and animal taste systems detect the nutrient content of food to guide food consumption. The understanding of taste reception mechanisms has progressed tremendously in the past 20 years. Morphologically defined type II cells in taste buds are known receptor cells for sweet, umami, and bitter substances. Type 1 taste receptors (TIRs) form sweet and umami receptors and type 2 taste receptors (T2Rs) are bitter receptors. TIRs and T2Rs are not expressed in the same taste cells, and activation of TIR- and T2R-expressing cells elicits the preference and avoidance, respectively. Type III cells are sour taste cells, which together with T2R-expressing cells encode information of high salt. Among basic taste qualities, the receptors for sour substances and salts remain unknown. Animal taste systems use, for example, sweet and umami/amino acid taste quality recognition to recognize dietary carbohydrates and proteins, respectively.

[007] In addition to basic tastes, there are other compounds that contribute importantly to taste, but which do not fit into any of the accepted five categories of basic taste. Among these other taste qualities or taste modifiers is fat, which is highly preferred by humans and mice. Fat is one of the macronutrients, along with carbohydrates and proteins. However, the determination of the taste recognition of fat by animals has been debatable. Fat plays a prominent role in food palatability. It is an important factor for several aspects of oral perception of food, including texture and mouth feel. However, previously developed fat mimetics, which focused on matching fat's tactile properties, failed in the marketplace. The likely reason for this is that these fat mimetics fail to replicate effects of fat on the taste system. [008] Several fat reception mechanisms have been proposed, but none are agreed upon. Recent studies suggested that fat taste is mediated by a subset of type II taste bud cells, and that it involves a Trpm5 -dependent mechanism¹'². G protein coupled receptors Gpr40 and Gprl 20 were proposed to be fatty acid receptors³ , but later studies demonstrated that these GPCRs are not involved in oral sensing of fatty acids or fat⁴'⁵. Although earlier studies of Ci/3(5-knockout mice (lacking a gene that encodes the leukocyte differentiation antigen CD36, also known as fatty acid translocase, FAT) suggested that CD36 mediates gustatory component to fat preference ⁶'⁷, this was not confirmed in a recent study of Ci/3(5-knockout mice⁸. Furthermore, CD36 immunoreactivity was reported predominantly in gustducin (Gnat3)-expressing bitter taste bud cells of the mouse circumvallate papillae ⁷, which elicit aversive behavior, being inconsistent with the animal preference for fat. However, our gene expression analysis revealed that CD36 is not expressed in any taste cells (unpublished data). Therefore, none of these proteins (GPR40, GPR120 or CD36) has been unequivocally proven to function as a fat taste receptor.

[009] GPRl 13 (also referred to alternatively as ADGRF3), an "orphan" G protein-coupled receptor, was reported to be expressed in a subset of taste bud cells. See, US Patent No. 8,669,066, filed in 2011, which is a lengthy specification referring to naturally occurring taste- specific genes and gene products, including a 20 page listing of hundreds of such genes and gene products. Within this reference, cells that purportedly express GPRl 13 are referred to as possibly representing a novel taste cell population. However, this specification theorized in one portion of the specification, that GPRl 13 is involved in a new taste modality "such as fat" (col. 85, lines 55 et seq); and in another portion, GPRl 13 cells are noted as potentially representing salt sensing cells (col. 86, lines 58 et seq). No data provided in that specification confirmed or disputed either theory.

[0010] Another 2011 publication ¹⁰ stated that genetically engineered mutant mice with a disrupted Gprl 13 gene did not display altered behavioral and neural taste responses, and likely does not function as a receptor for taste ligands.

[0011] There remains a need in the art for compositions and methods for determining and analyzing fat taste perception.

[0012] SUMMARY OF THE INVENTION

[0013] In one aspect, cell -based methods are provided for identifying a compound that modulates fat taste perception. One method for identifying a compound that mimics or modulates fat taste perception comprises contacting a recombinant cell or cell line that expresses the G protein-coupled receptor GPRl 13 in vitro with a test compound; and assaying for a detectable change in the physical or functional characteristic of the contacted cells or cell lines in comparison to a reference cell or cell line control. The detectable change permits the determination that the test compound mimics the effect of dietary fat. In another embodiment, the cell or cell line also expresses the G protein Gnal4.

[0014] In another aspect, a recombinant cell or cell line useful in such methods comprises a nucleic acid sequence or molecule that coexpresses GPRl 13 and another G protein-coupled receptor or a G protein under the control of a suitable expression system, wherein said sequence or molecule is heterologous to the cell. In one embodiment, the G protein is Gnal4. In another embodiment, the receptor is a T1R. A recombinant cell or cell line comprises a nucleic acid sequence or molecules that encode for GPRl 13 and another G protein-coupled receptor under the control of a suitable expression system, wherein said reference nucleic acid sequences are heterologous to the cell. In some embodiments, the nucleic acid sequences encoding the proteins are homologs, or chimeric or modified versions of the naturally occurring Gprll3 and Gnal4 genes of a selected human or non-human mammal or animal.

[0015] In another aspect, a taste cell population or cell line is provided that comprises a nucleic acid sequence that encodes one or more of GPRl 13, T1R1, T1R2, T1R3, Gnal4, Gnal5, and Gnat3 under the control of an expression system heterologous to the nucleic acid sequence. In some embodiments, the nucleic acid sequences encoding the proteins are homologs, or chimeric or modified versions of the naturally occurring genes of a selected human or non-human mammal or animal.

[0016] In another aspect, a multi-well test plate device is provided that comprises or contains a recombinant cell or taste cell population as described herein.

[0017] In another aspect, a recombinant mammalian cell is provided that is ablated for its wild type Gprll3 and Gnal4 genes. In another aspect, there is provided a genetically engineered animal model that does not contain its wild type Gprll3 and/or Gnal4 genes, nor does it express the wild type GPRl 13 and Gnal4 proteins.

[0018] In another aspect, a humanized genetically engineered animal model that does not contain its wild type Gprll3 and/or Gnal4 genes but incorporates in its genome a construct comprising the human GPRl 13 and/or human Gnal4 encoding nucleic acid sequences. [0019] In another aspect, non-cell -based methods are provided for identifying a compound that mimics or modulates fat taste perception. One such method includes (a) introducing a test compound into a cell-free mixture of GPR113 receptor protein and one or more of the G proteins alpha, beta and gamma, where G protein alpha is Gnal4; and (b) measuring the displacement of GPR113 receptor protein from its association with the G proteins in the absence and presence of fat, wherein the ability of the test compound to induce dissociation of the GPR113-G proteins complex in the absence of fat indicates that the compound is able to mimic fat taste perception, and the ability of the test compound to modulate lipid-induced dissociation of the GPR113-G proteins complex indicates that the compound is able to modulate fat taste perception.

[0020] In another aspect, a non-cell-based method for identifying a compound that mimics or modulates fat taste perception comprises (a) introducing a test compound into a cell-free mixture of a GPR113 receptor protein and a second receptor protein with G proteins alpha, beta and gamma, where G protein alpha is Gnal4, Gnal5, Gnat3 or a chimeric G alpha protein; and (b) measuring the displacement of GPR113 and one or more of the second receptor proteins from their association with the G proteins in the absence and presence of fat, wherein the ability of the test compound to induce dissociation of the GPCRs-G proteins complex in the absence of fat indicates that the compound is able to mimic fat taste perception, and the ability of the test compound to modulate lipid-induced dissociation of the GPCRs-G proteins complex indicates that the compound is able to modulate fat taste perception.

[0021] Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows an electrophoretic gel (top panel) showing the results of PCR performed using mouse multiple tissue cDNA panels and RNA isolated from various mouse tissue. An amplified product of the expected size for Gprll3 (417 bp) was observed in cDNAs derived from the testis and vallate and foliate taste papillae, but not fungiform papillae or other tissues tested. NTC is the control and M the molecular marker. The bottom panel is a G3PDH control.

[0024] FIG. 2 are micrographs showing the spatial distribution oi Gprll3 and Gnal4 mRNAs in the oral cavity, in the circumvallate papillae (CvP), the palate, and the fungiform papillae (FuP) of wild-type B6 mice and <S¾??7a-knockout {Sim- la ^~) mice that lack type II (sweet, umami and/or bitter) taste bud cells (TBCs). Expression of Gprll3 and Gnal4 mRNAs in the oral cavity was confined to the taste buds distributed in the posterior gustatory papillae, i.e., the CvP of B6 mice. The lack of signal in CvP of <S¾??7a-knockout mice that lack type II TBCs demonstrates that Gprll3 mRNA is expressed in the type II (sweet/umami/bitter) TBCs.

[0025] FIGs. 3A-3G shows identification of TBCs expressing Gprll3 by dual in situ hybridization (dISH). FIG. 3A is a photograph showing the results of a dISH assay in cells using Gprll3 and Trpm5 (a marker of type II TBCs) probes. FIG. 3B is a photograph of a dISH assay in cells using Gprll3 and Pkd2ll (a marker of type III TBCs) probes. FIG. 3C is a photograph showing the results of a dISH assay in cells using Gprll3 and NTPDase2 (a marker of type I TBCs) probes. FIG. 3D is a photograph of a dISH assay in cells using Gprll3 and T1R3 (also referred to as Taslr3) probes. FIG. 3E is a photograph of a dISH assay in cells using Gprll3 and T2R5 (also referred to as Tas2r5) probes. FIG. 3F is a photograph showing the results of a dISH assay in cells using Gprll3 and Gnal4 (referred to as G14) probes. FIG. 3G is a photograph of a dISH assay in cells using Gprll3 and

Gustducin (also known as Gnat3 and shown as Ggust in figure) probes. Expression oi Gprll3 mRNA was confined to a subset of 7>p»25-expressing TBCs as expected from the results in Sim 1 o-knockout mice that lack type II TBCs (FIG. 2). Expression oi Gprll3 mRNA was also confined to a subset oi TlR3 (also 7os7r3)-expressing TBCs, and completely overlapped with Gnal4 expression.

[0026] FIG. 4A is a graph showing intake of fatty acid (linoleic acid) emulsions of twelve wild-type mice expressing Gprll3 (WT, Gprll3^+I+ , symbol♦) and fourteen genetically engineered knock out mice in which the Gprll3 gene was ablated {Gprll3 KO, GprllS^'1' symbol■) in two-bottle preference tests in which the taste emulsions included linoleic acid. The graph plotted volume intake over 24 hours over an average of 48 hours vs. concentration of fatty acid.

[0027] FIG. 4B is a graph showing similar intake of fatty acid (oleic acid) emulsions of the mice of FIG. 4A in which the taste emulsions included oleic acid. The graph plots the same parameters as FIG. 4A.

[0028] FIG. 4C is a graph showing intake of dietary oil (corn oil) emulsions of the mice of FIG. 4A in which taste emulsions included corn oil. The graph plots the same parameters as FIG. 4A. [0029] FIG. 4D is a graph showing similar intake of dietary oil (soybean oil) emulsions of the mice of FIG. 4A when the taste emulsions included soybean oil. The graph plots the same parameters as FIG. 4A.

[0030] FIG. 4E is a graph showing intake of fatty acid (linoleic acid) emulsion of ten wild- type mice expressing Gnal4 (WT, Gnal4^+I+ , symbol♦) and ten genetically engineered knock out mice in which the Gnal4 gene was ablated (Gnal4 KO, GnaH^'1' symbol■) in two-bottle preference tests in which the taste emulsions included linoleic acid. The graph plotted volume intake over 48 hours vs. concentration of fatty acid (nM). The graph plots the same parameters as FIG.4E.

[0031] FIG. 4F is a graph showing similar intake of fatty acid (oleic acid) emulsions of the mice of FIG. 4E, when the taste emulsions included oleic acid. The graph plots the same parameters as FIG.4E.

[0032] FIG. 4G is a graph showing intake of dietary oil (corn oil) emulsions of the mice of FIG. 4E, when the taste emulsions included corn oil. The graph plots the same parameters as FIG.4E.

[0033] FIG. 4H is a graph showing similar intake of dietary oil (soybean oil) emulsions of the mice of FIG. 4E, when the taste emulsions included soybean oil. The graph plots the same parameters as FIG.4E.

[0034] FIG. 5A is a graph showing preference scores to fatty acid (linoleic acid) emulsions of twelve wild type mice expressing Gprll3 (WT, Gprll3^+I+ , symbol♦) and fourteen genetically engineered knock out mice in which the Gprll3 gene was ablated (Gprll3 KO, Gprll3^~'^~ symbol■) in two-bottle preference tests in which taste emulsions included linoleic acid. The graph plotted preference in percentage vs. concentration of fatty acid.

[0035] FIG. 5B is a graph showing similar preference to fatty acid (oleic acid) emulsions of the mice of FIG. 5 A in which the taste emulsions included oleic acid. The graph plots the same parameters as FIG. 5A.

[0036] FIG. 5C is a graph showing preference to dietary oil (corn oil) emulsions of the mice of FIG. 5A in which taste emulsions included corn oil. The graph plots the same parameters as FIG. 5A. [0037] FIG. 5D is a graph showing similar preference to dietary oil (soybean oil) emulsions of the mice of FIG. 5 A when the taste emulsions included soybean oil. The graph plots the same parameters as FIG. 5A.

[0038] FIG. 5E is a graph showing preference to fatty acid (linoleic acid) emulsions often wild type mice expressing Gnal4 (WT, Gnal4^+I+ , symbol♦) and ten genetically engineered knock out mice in which the Gnal4 gene was ablated (Gnal4 KO, GnaH^'1' symbol■) in two- bottle preference tests in which taste emulsions included linoleic acid. The graph plotted preference in percentage vs. concentration of fatty acid (nM).

[0039] FIG. 5F is a graph showing similar preference to fatty acid (oleic acid) emulsions of the mice of FIG. 5E, when the taste emulsions included oleic acid. The graph plots the same parameters as FIG.5E.

[0040] FIG. 5G is a graph showing preference to dietary oil (corn oil) emulsions of the mice of FIG. 5E, when the taste emulsions included corn oil. The graph plots the same parameters as FIG.5E.

[0041] FIG. 5H is a graph showing similar preference to dietary oil (soybean oil) emulsions of the mice of FIG. 5E, when the taste emulsions included soybean oil. The graph plots the same parameters as FIG.5E.

[0042] FIG. 6A is a graph showing normalized licking rates of twelve wild type mice expressing Gprll3 (WT, Gprll3^+I+, symbol♦) and eleven to fourteen genetically engineered knock out mice in which the Gprll3 gene was ablated (Gprll3 KO, Gprlli^'1' symbol■) in brief-access gustometer assay in which taste emulsions included linoleic acid. The graph plotted lick rate vs. concentration of fatty acid.

[0043] FIG. 6B is a graph showing similar normalized licking rates of the mice of FIG. 6A in which the taste emulsions included oleic acid. The graph plots the same parameters as FIG. 6A.

[0044] FIG. 6C is a graph showing similar normalized licking rates of the mice of FIG. 6A in which taste emulsions included corn oil. The graph plots the same parameters as FIG. 6A.

[0045] FIG. 6D is a graph showing similar normalized licking rates of the mice of FIG. 6A when the taste emulsions included soybean oil. The graph plots the same parameters as FIG. 6A. [0046] FIG. 6E is a graph showing normalized licking rates often wild type mice expressing Gnal4 (WT, Gnal4^+I+, symbol♦) and ten genetically engineered knock out mice in which the Gnal4 gene was ablated (Gnal4 KO, Gnal4^~'^~ symbol■) in brief-access gustometer assay in which taste emulsions included linoleic acid. The graph plotted lick rate vs. concentration of fatty acid.

[0047] FIG. 6F is a graph showing similar normalized licking rates of the mice of FIG. 6E in which the taste emulsions included oleic acid. The graph plots the same parameters as FIG. 6E.

[0048] FIG. 6G is a graph showing similar normalized licking rates of the mice of FIG. 6E in which taste emulsions included corn oil. The graph plots the same parameters as FIG. 6E.

[0049] FIG. 6H is a graph showing similar normalized licking rates of the mice of FIG. 6E when the taste emulsions included soybean oil. The graph plots the same parameters as FIG. 6E.

[0050] FIG. 7 is the cDNA sequence oiMus musculus GprlB SEQ ID NO: 1, which is a novel variant of the sequence provided under NCBI Ref No. BC027015.1. Light gray highlighting on the sequence figure shows an extra CTG triplet at nt position 1237-45 of SEQ ID NO: 1, which is not a part of the NCBI Ref No. BC027015.1. The published sequence includes only two CTG triplets at that site (indicated by dark gray highlighting).

[0051] FIG. 8 is the protein sequence of Mus musculus GPRl 13 SEQ ID NO: 2, which is a novel variant of the sequence provided under NCBI Ref No.BC027015. Light gray highlighting shows a leucine (L) residue at each AA position 413, 414 and 415 in SEQ ID NO: 2. This sequence differs from the NCBI Ref No. BC027015 that includes only two L residues (indicated by dark gray highlighting) at that position.

[0052] FIG. 9 is an alignment of known GPRl 13 protein sequences from this indicated mouse SEQ ID NO: 2, human SEQ ID NO: 3, dog SEQ ID NO: 4, cat SEQ ID NO: 5 and rat SEQ ID NO: 6 sequences.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] The methods and compositions described herein are based upon the inventors' discovery that the genes Gprll3 (which encodes a G protein coupled receptor, GPCR) and Gnal4 (which encodes a G protein alpha subunit) are involved in fat taste reception and downstream signal transduction. With this determination of molecular mechanisms of fat taste transduction in taste bud cells, assays for screening compounds that mimic or modulate fat taste are available as described herein. These assays and components of the assays, such as cell lines and recombinant cells are provided for screening fat substitutes and modifiers (e.g., enhancers, suppressors, and inhibitors of fat taste).

[0055] Definitions

[0056] All scientific and technical terms used herein have their known and normal meaning to a person of skill in the fields of biology, biotechnology, molecular biology and molecular genetics and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. However, for clarity, the following terms are particularly defined as follows:

[0057] As used herein, italicizing shall indicate a gene, e.g., TRP8, in contrast to its encoded protein product, which is indicated by a non-italicized symbol, e.g., TRP8. This convention is followed for all genes and proteins identified throughout this specification.

[0058] Additionally, when a gene or its encoded protein product is identified by one symbol, it is understood that other symbols known to represent the same gene or protein are to be included. For example, T1R2 or Taslr2 represent the same gene; and T1R2 and TAS 1R2 represent the same protein; and GPR113 mdADGRF3 represent the same gene and GPR113 and ADGRF3 represent the same protein, etc.

[0059] By "providing a fat taste" is meant that the test molecule alone creates a fat taste, i.e., recognition of a taste as one similar to a fatty acid or oil, as detected by oral taste cells.

[0060] By "modulates fat taste" or "modulates fat taste perception" is meant that the test molecule can increase or decrease the sensitivity of the subject to fat taste as detected by oral taste cells and as further indicated by a result in a suitable fat taste assay as described herein. Because excess intake of fat is undesirable for some medical conditions, in one embodiment, it is desirable to increase the perception of fat taste, thus allowing one to formulate food products that would have lower actual fat content but retain the same taste level. In some embodiments, the level of inhibition or upregulation of the expression or biological activity of a molecule or pathway of interest refers to a decrease (inhibition or downregulation) or increase (upregulation) of greater than from about 1% to about 99%, and more specifically, about or at least 1%, 2%, 3%, 4%, 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 28%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. Numbers falling within the listed numbers are also included herein. The inhibition or upregulation may be direct, i.e., operate on the molecule or pathway of interest itself, or indirect, i.e., operate on a molecule or pathway that affects the molecule or pathway of interest. In another embodiment, a test compound "modulates fat taste" when a statistically significant difference between the test and control levels is identified.

[0061] The term "test compound" as used herein can refer to any known or novel molecule for testing as fat taste substitute or a modulator of fat taste perception. Such compounds include, without limitation, a purified molecule, substantially purified molecule, molecules that are one or more components of a mixture of compounds, or a mixture of a compound with any other material that can be analyzed using the methods of the present invention. Such molecules may typically be found in known libraries of molecules, including those that have been pre- screened e.g., for safe use in humans or other animals. Suitable test molecules may be found, for example, in commercially available compound libraries. Such libraries may be readily obtained from vendors such as Otava, TimTec, Inc., Chem Bridge Corp., etc.¹¹ Test compounds can be organic or inorganic chemicals, or biomolecules, and all fragments, analogs, homologs, conjugates, and derivatives thereof. Biomolecules include proteins, polypeptides, nucleic acids, lipids, monosaccharides, polysaccharides, and all fragments, analogs, homologs, conjugates, and derivatives thereof. Test compounds can be of natural or synthetic origin, and can be isolated or purified from their naturally occurring sources, or can be synthesized or produced recombinantly using techniques known in the art. The compounds discussed herein also encompass "metabolites" which are unique products formed by processing the compounds of the invention by the cell or subject. Desirably, metabolites are formed in vivo.

[0062] "Taste cell", as used herein, refers to a mammalian sensory cell found in taste buds in the oral cavity of mammals, including fungiform papillae, foliate papillae, circumvallate papillae, soft palate, nasoincisor duct and epiglottis. These cells can be identified by their typical appearance under light and electron microscopy. They can also be identified by oral location along with expression of taste specific genes (and/or proteins) in oropharyngeal epithelia such as T1R1, T1R2, T1R3, T2Rs, Trpm5, PKD2L1, Car 4, Ddc, Snap25, and NTPDase2. Methods for culturing mammalian taste cells are known in the art.¹²

[0063] By use of the term "native cell" is meant a mammalian cell or cell line that naturally or endogenously expresses the indicated nucleic acid sequence, molecule or gene. For example, such native cells include mammalian oral taste cells or oral taste cell lines that naturally express Gprll3 and/or Gnal4.

[0064] As used herein, a "heterologous cell or cell line" means a mammalian cell or cell line established from cells that are not oral taste cells, and/or that do not naturally express the desired nucleic acid sequence, molecule or gene, e.g., Gprll3 or Gnal4. For example, a heterologous cell that does not natively express the GPRl 13 or Gnal4 protein may be a human kidney cell (HEK) cell that is genetically engineered to express the desired GPRl 13 or Gnal4 protein or a mammalian endocrine cell or cell line that does not naturally express GPRl 13 or Gnal4. In one embodiment, a useful endocrine cell or cell line is an intestinal or gut endocrine cell or cell line. In another embodiment an endocrine cell or cell line is a pancreatic endocrine cell or cell line. Still others are known and useful as provided in the methods described below.

[0065] A "recombinant cell" or "transformed cell" as used herein refers to a cell or cell line that is genetically engineered to express a desired nucleic acid sequence, molecule or gene that it does not naturally express in the desired amounts. In one embodiment, the recombinant cell expresses the desired nucleic acid sequence, molecule or gene e.g., Gprll3 or Gnal4, as defined below. Particularly desirable cells or cell lines are selected from among bacterial cells, amphibian cells including amphibian oocytes, insect cells and mammalian cells.

Mammalian cells useful in the invention include, but are not limited to, MDCK, BHK, HEK293, HEK293T, COS 1, COS7, NIH3T3, Swiss3T3 and CHO cells. The nucleic acid sequence, molecule or gene, e.g., Gprll3 and/or Gnal4 or a gene encoding chimeric Gna protein, optionally with one or more of TlRl, T1R2, and T1R3 may be expressed under the control of a regulatable or constitutive promoter.

[0066] By "expression level" is meant the quantitative expression of the nucleotide sequence (e.g., mRNA) of a desired protein encoding sequence (e.g., Gprll3 and/or Gnal4) or the quantitative expression of the resulting expressed protein (e.g., GPRl 13 and/or Gnal4) itself.

[0067] By "G protein" is meant a guanine nucleotide binding protein. A G protein is heterotrimeric and is form of an α, β, and γ subunit. Generally, the a subunit of a G protein confers most of the specificity of interaction between its receptor and its effectors in the signal transduction process. The β and γ subunits appear to be shared among different G proteins. G proteins mediate signal transduction in olfactory, visual, hormonal and neurotransmitter systems by coupling cell surface receptors to cellular effector enzymes. G proteins transduce an extracellular signal into an intracellular second messenger (e.g., cAMP, cGMP, IP3).^40-42

[0068] By "G protein coupled receptor" as used herein is meant a transmembrane or cell- surface receptor involved in taste transduction, which interacts with a G-protein to mediate taste signal transduction. Exemplary GPCR are described in cited references⁴⁴' ⁴⁵ as well as in numerous publications known to the art.

[0069] By "modulates fat taste perception" is meant that the test compound alters the function or activity of the referenced G protein-coupled receptor, e.g., GPRl 13 or T1R, or G protein, e.g., Gnal4, in the cell, or otherwise affects the function or activity of another nucleic acid sequence, molecule or gene in the biochemical pathways in the cell from that of a control or reference cell. Assays using taste-related G proteins are also described in referenced publications ^40-42 and such assay formats may be useful in detecting compounds that modulate fat taste perception as described herein.

[0070] By "control or reference cell" is meant a cell that contains and normally expresses the referenced wild type G protein-coupled receptor, e.g., GPRl 13 and/or TlRs, and/or G protein, e.g., Gnal4, when not contacted with a test compound. The control cell is characterized by a certain function or activity of the GPRl 13 and Gnal4 proteins and their respective pathways. In one embodiment, the control cell is characterized by certain functions or activities of the referenced G protein-coupled receptor, e.g., GPRl 13 or T1R, or G protein, e.g., Gnal4 in the cell in the absence of any test compound. In another embodiment, the control cell has an altered function or activity of the referenced G protein-coupled receptor and/or G protein, when in the presence of a test compound or molecule, e.g., fatty acid or triglyceride, that has a known effect on enhancing fat taste perception in the mouth. Thus, the comparison of the effect of the test molecule may be against a positive or negative control cell and the method permits an identification of a result that is correlated with providing or modulating (e.g., increasing or decreasing) fat taste perception.

[0071] By "modulates expression" is meant that the test compound affects the quantitative expression of a desired nucleic acid sequence, molecule or gene or its encoded protein as compared to the normal level or a control or reference level. In one embodiment, modulation of the gene or its protein is an increase in expression or activity. In another embodiment, by modulates expression is meant a decrease in expression or activity of the gene or encoded protein. By "control level" is meant the level of expression of the gene encoding the reference protein in a cell in the absence of a test compound. In one embodiment, the control level is the level of expression of the gene obtained when the same cell or cell line is contacted by a control test compound or molecule, which has a known effect on expression of the gene or its encoded protein in the cells. Thus, the comparison of the effect of the test molecule in the method permits an identification of a result that is correlated with providing or modulating the production of proteins important in fat taste perception.

[0072] By "functional activity" is meant the expected normal activity of a certain expression product, e.g., mR A or protein, when expressed in a cell.

[0073] By "mammalian" is meant primarily a human, but also common laboratory mammals, such as primates, mice, rats, etc. as well as common household pets, e.g., dogs, cats, etc. having native oral cells or taste cells that are capable of discriminating fat taste from other tastes.

[0074] By "animal physiological or behavioral assays for fat taste" and "human

psychophysical assays for fat taste" is meant assays that are known or adapted to function as described herein and in the examples that rely on physiological or behavioral reactions in response to increased or decreased fat taste of a compound applied to the oral taste cells of a mammalian test animal or human. Examples of known assays in non-human animals include use of a taste preference tests, taste threshold tests, conditioned taste aversion generalization tests, taste discrimination tests gustatory nerve recording assays. Examples of known assays in humans include use of taste assays to measure taste quality perception, intensity, sensitivity, and hedonics¹⁴. In another embodiment, such assays include electrophysiological responses or calcium imaging of isolated taste cells, isolated taste buds, or taste slices¹⁵. Other suitable assays are known to those of skill in the art and may be applied in the methods described below.

[0075] By "reference nucleic acid sequence" or "reference protein" is meant as a simple reference for one or more of encoding nucleic acid sequences, or proteins of Gprl 13, Gnal4, T1R members, Gnat3, Gnal5 and chimeric versions, modified versions or other variants thereof. [0076] The terms "a" (or "an") refers to one or more, for example, "an assay" is understood to represent one or more assays. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. As used herein, the term "about" means a variability of up to 5% or up to 10 % from the reference given, unless otherwise specified. While various embodiments in the specification are presented using "comprising" language, which is inclusive of features in addition to the specifically recited features or steps; under other circumstances, a related embodiment is also intended to be interpreted and described using "consisting of or "consisting essentially of language.

[0077] The words "consist", "consisting", and its variants, are to be interpreted to exclude features in addition to those features specifically recited, or to include only additional features of minor significance.

[0078] Fat Taste Genes, Proteins and Pathways

[0079] The discovery of the GPCR (Gprl 13), which detects fat contacting taste bud cells, and the G protein alpha subunit (Gnal4), which is coupled with this GPCR and which upon its activation by dietary fat triggers intracellular signal transduction cascade that excites taste cells, allows the development of screening assays and assay components. As described in the examples below, the proteins GPR113 and Gnal4 are co-expressed in the same cells of the mouse circumvallate papillae (Cvp) that also express Trpm5 and T1R3 (i.e. , in a subset of type II cells). Knockout (KO) mice with deletions of the Gprl 13 or Gnal4 genes have reduced appetitive responses to fatty acids and triglycerides in behavioral taste tests compared with their wild type littermates, but they have unaltered responses to taste stimuli representing the five basic taste qualities (sweet, bitter, salty, sour and umami)¹⁰. These data suggest that these genes are expressed by a specialized type/set of taste receptor cells that use a dedicated G protein coupled transduction pathway to detect dietary fat, and that activation of these cells evokes a taste sensation that triggers appetitive behavior. These results demonstrate that both Gprl 13 and Gnal4 are involved in fat reception, which culminates in behavioral preference. Therefore, these genes and the proteins encoded thereby are useful as targets in assays for screening fat taste substitutes that do not require the presence of fat to activate fat-sensitive receptors and cells, and fat taste modulators that require the presence of fat to activate fat- sensitive receptors and cells.

[0080] Similarities across vertebrate species (including humans) in sequences of the Gprl 13 and Gnal4 genes, and GPR113 and Gnal4 proteins, and in their patterns of cellular expression, demonstrate that these genes and proteins are involved in fat taste recognition in different species. As also described in the methods herein, the following genes and encoded proteins are useful in these methods of screening for fat taste perception

[0081] As described herein and in Table 1 below, a variety of mammalian species have sequences useful in the methods and compositions described herein. In the experiments of the example, the murine Gprll3 nucleic acid sequence and encoded protein represented by the NCBI database accession numbers in the second row of Table 1 for "mouse" were employed. However, the inventors have also discovered another variant or modified sequence for mouse Gprll3 nucleic acid sequence and encoded protein, which appear in FIGs. 7 and 8 and are referenced as SEQ ID NO: 1 and 2, respectively. Other variants oi Gprlli nucleic acid sequence and encoded protein from a variety of species are further identified in Table 1 and can be useful in the compositions and methods of this invention. Still other naturally occurring splice variants, or modified versions of these sequences, or sequences from other mammalian species identified as encoding GPRl 13 proteins as well as modified or variants of GPRl 13 proteins may be used in the compositions and methods described herein.

[0082] Similarly in the experiments and examples below, the murine Gnal4 nucleic acid sequence and encoded protein sequence referenced by the NCBI database accession numbers of the first row of Table I in the second mouse entry were employed. Other variants of Gnal4 nucleic acid sequence and encoded protein from a variety of species are further identified in Table 1 and can be useful in the compositions and methods of this invention. Still other naturally occurring splice variants, or modified versions of these sequences, or sequences from other mammalian species identified as encoding Gnal4 proteins as well as modified or variants of Gnal4 proteins may be used in the compositions and methods described herein.

[0083] Other variants of Gprll3 and Gnal4 nucleic acid sequence and encoded protein from a variety of species are further identified in Table 1 and can be useful in the compositions and methods of this invention. 4] TABLE I

SEQ ID Nos and/or NCBI Accession Nos for indicated GPRl 13 and

Gnal4 cDNA and encoded proteins

Species GPR113 Gnal4

cDNA Protein cDNA Protein Mouse (Mus

SEQ ID NO: 1 SEQ ID NO: 2 BC027015 AAH27015 musculus)

Mouse (Mus

NM_001014394 NP 001014416

musculus)

Human (Homo

NM_001145169 NP 001138641 NM_004297 NP_004288 sapiens)

Human (Homo

AK293887 BAG57276 XM_011519224 XP 011517526 sapiens)

Human (Homo

AY140955 AAN46669

sapiens)

Human (Homo

BC152988 AAI52989

sapiens)

Human (Homo

NM_001145168 NP_001138640

sapiens)

Human (Homo

NM_153835 NP_722577

sapiens)

Dog Canis

XM_005630226 XP_005630283 XM_541272 XP_541272 familiaris

Cat (Felis

XM_006930448 XP 006930510 XM_003995421 XP_003995470 catus)

Rat (Rattus

NM_001106712 NP_001100182 NM_001013151 NP_001013169 norvegicus)

[0085] Similarly the nucleic acid and amino acid sequences of the Gnat3 nucleic acid sequences and Gnat3 proteins and Gnal5 nucleic acid sequences and Gnal5 proteins mentioned herein, and the various species of wild type genes, are known and publically available from the above-mentioned databases as well as other public sources. Table II lists the nucleic acid sequence and encoded protein represented by various NCBI database accession numbers for Gnat3 for several mammalian species. Other variants of Gnat3 nucleic acid sequence and encoded protein from a variety of species can be useful in the compositions and methods of this invention. Still other naturally occurring splice variants, or modified versions of these sequences, or sequences from other mammalian species identified as encoding Gnat3 proteins as well as modified or variants of Gnat3 proteins may be used in the compositions and methods described herein. [0086] Table II also lists the nucleic acid sequence and encoded protein represented by various NCBI database accession numbers for Gnal5 for several mammalian species. Other variants οΐ θηαΐδ nucleic acid sequence and encoded protein from a variety of species can be useful in the compositions and methods of this invention. Still other naturally occurring splice variants, or modified versions of these sequences, or sequences from other mammalian species identified as encoding Gnal 5 proteins as well as modified or variants of Gnal5proteins may be used in the compositions and methods described herein.

[0087] TABLE II

1.

[0088] Similarly the nucleic acid and amino acid sequences of the TIR family of nucleic acid sequences and TIR proteins mentioned herein, and the various species of wild type genes, are known and publically available from the above-mentioned databases as well as other public sources. Table III lists the nucleic acid sequence and encoded protein represented by various NCBI database, Ensemble database, or UniProt database accession numbers for T lRl, T 1R2 and T 1R3 for several mammalian species. Other variants οΐ ΤΙϋΙ, T1R2 and T1R3 nucleic acid sequence and encoded protein from a variety of species can be useful in the compositions and methods of this invention. Still other naturally occurring splice variants, or modified versions of these sequences, or sequences from other mammalian species identified as encoding TlRl, T 1R2 and/or T 1R3 proteins as well as modified or variants of TlRl, T 1R2 and/or T1R3 proteins may be used in the compositions and methods described herein.

[0089] TABLE III

[0090] For use in the methods described below, the genes and proteins mentioned herein can be readily selected from naturally occurring (wild type) genes of humans, mice, or other suitable animal or mammalian species. It is anticipated that modified versions of these genes and proteins may be used in place of the wild type genes and proteins in the methods described. When the term "gene" or "nucleic acid sequence or molecule" is used in the following descriptions, it is understood that the terms encompasses a targeted nucleic acid sequence, e.g., GPR113 and/or Gnal4, and any variants, orthologs, mutated, substituted, chimeric or otherwise modified versions described herein. It is also understood that these GPR113 and/or Gnal4 nucleic acid sequences may be used in conjunction or co-expression with the TIR family of nucleic acid sequences, the Gnat3 nucleic acid sequence or the Gnal5 nucleic acid sequence. Therefore, the terms also apply to the use of the TIR nucleic acid sequences when expressed with the GPR113 and/or Gnal4 nucleic acid sequence. These methods include contacting a recombinant cell that expresses nucleic acid sequence(s) with a test compound. In one embodiment, the nucleic acid sequence is an ortholog of GPR113 and/or Gnal4. In one embodiment, the nucleic acid sequence is a mammalian or human gene. The method further includes determining whether the test compound modulates expression of the referenced nucleic acid sequence.

[0091] By "modified" versions of the identified nucleic acid sequence, e.g., cDNA, for the components of this invention is meant to include chimeric sequences formed by the fusion of all or fragments of a referenced nucleic acid sequence, e.g., of Gprll3 with all or fragments of a referenced nucleic acid sequence from a second referenced nucleic acid sequence, Gnal4. Chimeric nucleic acid sequences can contain sequences fused from orthologous nucleic acid sequences or gene sequences from different species, from different genes, from naturally occurring nucleic acid sequences, from modified nucleic acid sequences, from codon optimized sequences, or from sequences modified by nucleic acid substitutes, e.g., replacement of one nucleic acid with a nucleic acid containing a different substituent or structure. Similarly such nucleic acid sequences may be modified by the addition of tags/labels.

[0092] Additionally, such "modified" versions of the identified nucleic acid sequences herein can include any transcript including splice variants generated from an orthologous gene (of the animals of interest) in a conserved syntenic block with the mouse genome where the reference gene is located. For example, other useful nucleic acid sequences for human Gprll3 are variants generated from the conserved syntenic block with the mouse genome where Gprl 13 is located are considered Gprl 13 orthologs.

[0093] By "modified" versions of the identified protein sequence for the components of this invention is meant to include chimeric sequences formed by the fusion of all or fragments of a referenced protein, e.g., of GPR113 with all or fragments of a referenced protein sequence from a second referenced protein sequence, e.g., Gnal4. Chimeric protein sequences can contain sequences fused from orthologous protein sequences or protein sequences from different species, from different proteins, from naturally occurring protein sequences, from modified protein sequences, from codon optimized sequences, or from sequences modified by amino acid substitutes, e.g., replacement of one amino acid with an amino acid containing a different substituent, charge, or structure or a conservative amino acid replacement. Similarly such protein or amino acid sequences may be modified by the addition of tags/labels. Other modified versions of the amino acid sequences or protein sequences for the reference proteins herein can include protein sequences or fragments having at least a 70%, 80%, 90% up to 99% or more sequence identity with a sequence identified in this disclosure.

[0094] The terms "percent (%) identity", "sequence identity", "percent sequence identity", or "percent identical" in the context of amino acid sequences refers to the residues in the two sequences which are the same when aligned for correspondence. Percent identity may be readily determined for amino acid sequences over the full-length of a protein, polypeptide, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequences. A suitable amino acid fragment may be at least about 8 amino acids in length, and may be up to about 700 amino acids. Generally, when referring to "identity", "homology", or "similarity" between two different sequences, "identity", "homology" or "similarity" is determined in reference to "aligned" sequences. "Aligned" sequences or "alignments" refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Sequence alignment programs are available for amino acid sequences, e.g., the "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match- Box" programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs.¹⁶

[0095] The term "amino acid substitution" and its synonyms described above are intended to encompass modification of an amino acid sequence by replacement of an amino acid with another, substituting, amino acid. The substitution may be a conservative substitution. It may also be a non-conservative substitution. The term conservative, in referring to two amino acids, is intended to mean that the amino acids share a common property recognized by one of skill in the art. For example, amino acids having hydrophobic nonacidic side chains, amino acids having hydrophobic acidic side chains, amino acids having hydrophilic nonacidic side chains, amino acids having hydrophilic acidic side chains, and amino acids having hydrophilic basic side chains. Common properties may also be amino acids having hydrophobic side chains, amino acids having aliphatic hydrophobic side chains, amino acids having aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with electrically charged side chains, amino acids with electrically charged acidic side chains, and amino acids with electrically charged basic side chains. Both naturally occurring and non- naturally occurring amino acids are known in the art and may be used as substituting amino acids in embodiments. Methods for replacing an amino acid are well known to the skilled in the art and include, but are not limited to, mutations of the nucleotide sequence encoding the amino acid sequence. Reference to "one or more" herein is intended to encompass the individual embodiments of, for example, 1, 2, 3, 4, 5, 6, or more.

[0096] In addition to the protein sequences referenced by public database accession numbers as provided herein, nucleic acid sequences encoding protein variants are provided. The coding sequences may be generated using site-directed mutagenesis of the wild-type nucleic acid sequence(s). Alternatively or additionally, web-based or commercially available computer programs, as well as service based companies may be used to back translate the amino acids sequences to nucleic acid coding sequences, including both RNA and/or cDNA.¹⁷ In one embodiment, the RNA and/or cDNA coding sequences are designed for optimal expression in human cells.

[0097] Codon-optimized coding regions can be designed by various different methods. This optimization may be performed using methods which are available on-line, published methods, or a company which provides codon optimizing services.

[0098] Methods, Assays and Assay Components

[0099] Several types of assays can be used to screen compounds that mimic or modulate fat taste. Modulation of the function or activity oi Gprlli and Gnal4 can be assessed using a variety of in vitro and in vivo assays, including cell-based models as described herein. Such assays can be used to test for inhibitors and activators of the protein or fragments thereof. Assays using cells expressing the subject fat-taste specific proteins, either recombinant or naturally occurring, can be performed using a variety of methods, in vitro, in vivo, and ex vivo, as known in the art and described herein. To identify molecules capable of modulating activity thereof, assays are performed to detect the effect of various candidate modulators on activity preferably expressed in a cell. Various methods for assessing the capability of a molecule to modulate the expression and/or activity of the subject proteins are known in the art and are useful herein. Methods which may be adopted for use in the screening assays discussed herein are known in the art and are described in the referenced publications ^18~29, each of which is incorporated herein by reference. In one embodiment, the method is performed with an additional animal physiological or behavioral assay for fat taste.

[00100] Various embodiments of the methods for screening fat taste perception compounds are described as follows. In one embodiment, a method for identifying a compound that mimics or modulates fat taste perception comprises contacting a recombinant cell or cell line that expresses the G protein-coupled receptor GPR113 in vitro with a test compound. After a sufficient period of contact, an assay is performed to identify a detectable change in the physical or functional characteristic of the contacted cells or cell lines in comparison to a reference cell or cell line control, thereby determining that the test compound mimics the effect of dietary fat. In one embodiment of this method the recombinant cell co-expresses another G protein-coupled receptor. In another embodiment of this method the recombinant cell co-expresses the G protein, Gnal4. In still another embodiment, the recombinant cell co- expresses a TIR receptor. As stated above, the TIR receptor used in the method can be TlRl, T1R2, T1R3, or combinations of TIRs.

[00101] The method can also employ a recombinant cell co-expressing Gnat3 receptor with GPR113 and/or Gnal4. Similarly the recombinant cell can co-expresses the G protein, Gnal5 with GPR113 and/or Gnal4, and optionally with one or more of the TIR family. In the method the G proteins or G protein-coupled receptor proteins are optionally chimeric proteins comprising all or a fragment of one G protein fused to all or a fragment of another G protein. For example, the chimeric proteins can comprise fused fragments or proteins of one or more of Gnal4, Gnal5, TlRl, T1R2, T1R3, Gnat3 and GPR113, in any combination. The chimeric proteins used in the methods may further comprise a detectable label.

[00102] In one embodiment of the method, the detectable change is the formation or change in the formation of a protein comprising all or a fragment of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3. In another embodiment, the detectable change is an increase in the signal transduction activity of a G protein-coupled receptor pathway. In still a further embodiment, the detectable change in the contacted cells or cell lines is a change in the expression of enzymes downstream in a G protein-coupled receptor pathway of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3. In yet a further embodiment, the detectable change is a change in the level of intracellular calcium or ion transport or a change of the cell membrane potential.

[00103] The method can also employ a detectable change in function or activity of other G proteins or proteins in their respective pathways. For example, the detectable change is a change in the interaction between GPR113 protein and its ligands. In another example, the detectable change is a change in the signaling, activity or activation of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3. [00104] Still other methods as described herein are high-throughput assays involving multiple cells, cell lines, test molecules and references. The assay techniques used in these methods include in one aspect an imaging assay. In one embodiment, the method uses G protein-coupled receptors or G proteins encoded by genes of human or non-human mammalian origin, or are encoded by modified nucleic acid sequences. In another embodiment, the methods employ genes modified by one or more replaced or inserted nucleotides or modified nucleotides that do not occur in the normal mammalian gene.

[00105] In certain methods, the G protein-coupled receptors or G proteins or the nucleic acid sequences encoding them are associated with detectable labels or reporter genes, which are alone or in concert with a label partner, capable of generating a detectable signal. The methods employ labels or reporter genes that generate a signal selected from a colorimetric, fluorescent, luminescent, chemiluminescent or electrochemical signal.

[00106] In one embodiment, the recombinant cells useful in these methods are taste cells. Alternatively, the recombinant cell is a cell that does not naturally comprise a nucleic acid sequence expressing one or more of GPRl 13, Gnal4, Gnal5, T1R1, T1R2, T1R3, and Gnat3.

[00107] Additionally in these methods one or more G protein-coupled receptors or G proteins or the nucleic acid sequences encoding them is associated with a sequence enabling transport of the product of the expressed protein to or through the cell membrane.

[00108] In one aspect, the Gprl 13 and Gnal4 proteins are useful in screening for compounds which alter fat taste perception. These compounds may be useful as enhancers of fat taste and would allow one to formulate food products that would have lower actual fat content but retain the same fat taste level.

[00109] In one embodiment, the method of identifying a compound that modulates fat taste perception includes identifying a compound that alters the function or activity of GPRl 13. In one embodiment, the method of identifying a compound that modulates fat taste perception includes identifying a compound that alters the function or activity of GPRl 13 with Gnal4. Such modulation includes modulation that increases or decreases responses to taste stimuli. In one embodiment, the test compound modulates or alters the function or activity of GPRl 13 with one or more of T1R1, T1R2 or T1R3. In one embodiment, the method includes contacting a recombinant cell that expresses a reference nucleic acid sequence GPRl 13 and optionally co-expresses one or more of Gnal4, T1R1, T1R2 or T1R3 with a test compound. In some embodiments of these assays, GPRl 13 protein is contacted with a test compound, and molecular interactions between the compound and the GPRl 13 receptor are detected. If a test compound binds to, or otherwise interacts with GPRl 13 or its encoding nucleic acid, it triggers a detectable event. If a test compound binds to or otherwise interacts with Gnal4, it triggers a detectable interaction or a change in the normal interaction between GPRl 13 and Gnal4. In certain embodiments the GPRl 13 protein is expressed in cultured cell lines by transient or stable transfection. Presence of endogenous intracellular transduction components triggers, upon activation of GPRl 13, detectable changes in the cells, such as increase in the intracellular calcium or change in the membrane potential.

[00110] Because in native taste cells GPRl 13 and Gnal4 proteins are co-expressed and likely coupled in a fat taste signal transduction pathway, in certain embodiments Gnal4 may be a more effective transduction component compared with endogenous transduction components present in cell lines used for heterologous expression of GPRl 13. Therefore, some assays will use cultured cell lines with heterologously co-expressed GPRl 13 and Gnal4 proteins. Furthermore, some assays will include one or more chimeric G proteins composed of fragments of Gna 14 and another G protein (for example Ga 15) ; these other G proteins and their fragments will be used to achieve both effective coupling of the chimeric G protein to GPRl 13 (due to the presence of Gnal4), and effective activation of endogenous downstream signaling pathway of cultured cell lines (due to the presence of Gal 5 or another G protein).

[00111] In other embodiments, because in native taste cells GPRl 13 is co-expressed with TIR receptors, and because TIRs and other GPCRs are known to form dimers, heterodimers, homooligomers and/or heterooligomers, it is likely that GPRl 13 forms such types of interactions with TIRs. Therefore, co-expression of GPR113 with T1R1, T1R2 and/or T1R3 may increase sensitivity of the assay. Therefore, some assays will use cultured cell lines with heterologously co-expressed GPRl 13 and TIR proteins.

[00112] In still other embodiments, assays employ cultured cell lines with heterologously co- expre ssed GPR113, TlR and Gna 14 (or chimeras of Gna 14 with Ga 15 or other G proteins) .

[00113] It may also be desirable to use assays that also employ detection of changes in expression of the reference gene or gene product. In general, to obtain high level expression of a cloned gene, such as those cDNAs encoding the GPRl 13 and/or Gnal4 genes, the gene in question is subcloned into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and, if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. These sequences can be produced in plasmid based systems or viral vector systems, of which many are commercially available. Suitable plasmid and viral vectors are well known to those of skill in the art and are not a limitation of the present invention. Briefly, the nucleic acid sequence encoding the gene GPR113 and/or Gnal4 is inserted into a vector or plasmid which contains other optional flanking sequences, a promoter, an mR A leader sequence, an initiation site and other regulatory sequences capable of directing the multiplication and expression of that sequence in vivo or in vitro. As used herein, a vector may include any genetic element including, without limitation, naked DNA, a phage, transposon, cosmid, episome, plasmid, bacteria, or a virus. As used herein, the term vector refers to a genetic element which expresses, or causes to be expressed, the desired construct that expresses the selected reference nucleic acid sequence or inhibits the expression of the reference nucleic acid sequence in the target cell in vitro or in vivo.

[00114] As well known in the art, a nucleotide sequence is inserted into an expression vector, transformed or transfected into an appropriate host cell and optionally cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).

[00115] Bacterial expression systems for expressing the fat-taste specific protein(s) are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al, Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits for such expression systems are

commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells as well as for the host cells mentioned explicitly herein are well known in the art and are also commercially available. For example, retroviral expression systems may be used in the present invention. As described herein, the subject putative fat taste specific genes are preferably expressed in human cells such as HEK-293 cells which are widely used for high throughput screening.

[00116] In one embodiment, the vector is a non-pathogenic virus. In another embodiment, the vector is a non-replicating virus. In one embodiment, a desirable viral vector may be a retroviral vector, such as a lentiviral vector. In another embodiment, a desirable vector is an adenoviral vector. In still another embodiment, a suitable vector is an adeno-associated viral vector. A variety of adenovirus, lentivirus and AAV strains are available from the American Type Culture Collection, Manassas, Virginia, or available by request from a variety of commercial and institutional sources. Further, the sequences of many such strains are available from a variety of databases including, e.g., PubMed and GenBank. One of skill in the art given the teachings provided herein can readily construct a suitable adenoviral vector to carry and express a nucleotide sequence as described herein, e.g., a nucleic acid construct that encodes a protein, e.g., Gprl 13 and/or Gnal4, by resort to well-known publications and patents directed to such viral vectors.

[00117] In yet another embodiment, the vector used herein is a bacterial vector. In one embodiment, the bacterial vector is Listeria monocytogenes. In another embodiment, the bacterial vector is live-attenuated or photochemically inactivated. The heterologous gene of interest, can be expressed recombinantly by the bacteria, e.g., via a plasmid introduced into the bacteria, or integrated into the bacterial genome, i.e., via homologous recombination.

[00118] Generally, each of these vectors also comprises a minigene. By "minigene" is meant the combination of a selected nucleotide sequence (e.g., an R A/DNA sequence that expresses or encodes a protein, e.g., GPR113 and/or Gnal4 as described herein) and the operably linked regulatory elements necessary to drive transcription, translation and/or expression of the gene product in the host cell in vivo or in vitro. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. These vectors also include conventional control elements that permits transcription, translation and/or expression of the nucleic acid construct in a cell transfected with the plasmid vector or infected with the viral vector. A great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue- specific, are known in the art and may be utilized. Suitable eukaryotic and prokaryotic promoters are well known in the art and can be selected by one of skill in the art. In one embodiment, the promoter is a constitutive promoter. In another embodiment, the promoter is an inducible promoter. In one embodiment, the promoter is selected based on the chosen vector or upon the cell into which the vector will be transduced, e.g., a promoter operable in a taste cell. Still other conventional expression control sequences include selectable markers or reporter genes, which may include sequences encoding geneticin, hygromicin, ampicillin or purimycin resistance, among others. Other components of the vector may include an origin of replication. Selection of these and other promoters and vector elements are conventional and many such sequences are available.³⁰

[00119] These vectors are generated using the techniques and sequences provided herein and publicly available, in conjunction with techniques known to those of skill in the art. Such techniques include conventional cloning techniques of gene and cDNA such as those described in texts³⁰, use of overlapping oligonucleotide sequences, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.

[00120] Thus, in one embodiment, using the information taught herein and publically available and known vector construction components and techniques, one of skill in the art can construct a viral vector (or plasmid) that expresses the desired construct, e.g., a nucleic acid sequence that encodes and thereby can express GPR113 and/or Gnal4, as well as one of the T1R or Gnat3 or Gnal5 or chimeric versions described herein. In one embodiment, the method includes comparing the function or activity (or possibly expression) of the reference encoded protein in the recombinant cell contacted with the test compound with a control cell. In one embodiment, the control cell is not contacted with a test molecule or is contacted with a molecule known not to affect fat taste perception. Thus, the comparison of the effect of the test molecule in the method permits an identification of a result that is correlated with providing or modulating fat taste.

[00121] An increase in mR A or protein function or activity, e.g., an increase in functional activity of the expression product by the cell or cell line contacted with the test molecule over that of a negative control cell or cell line identifies a test molecule that provides or modulates a fat taste. That is, the effect of the test molecule to alter the normal function or activity of the reference nucleic acid sequence, GPR113 and/or Gnal4, is related to its impact on providing or modulating a fat taste. Where the effect is that the test molecule allows maintenance of normal function or increases normal function of the reference protein, e.g., GPR113 and/or Gnal4, above those of the controls, that test molecule is indicated to be useful as a potential novel fat taste providing compound or a compound useful in modulating the fat taste perception of other components of an end composition, e.g., foodstuff, medicine, etc. Where the effect is that the test molecule decreases function or activity of the reference protein, the test molecule is indicated to have a potential inhibitory effect on fat taste when present in a composition.

[00122] In one embodiment, a method of identifying a molecule that modulates fat taste perception involves contacting a test molecule with a recombinant cell or cell line that expresses the reference protein(s) under in vitro culture conditions. Depending upon the specific assay and endpoint desired, suitable culture conditions include a temperature of about 37^°C; or a range of from about 32 to 40^°C. This temperature is maintained for about 10 minutes to 24 hours. In some embodiments, the time period is at least about 20 minutes, 30 minutes, 45, minutes, 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours or more.

[00123] Desirably, the cell or cell line is a recombinant cell or cell line which has been transformed to express the reference nucleic acid sequence. In one embodiment, the recombinant cell is selected from a bacterial cell, a yeast cell, an amphibian cell, an insect cell and a mammalian cell. In one embodiment, the mammalian cell is selected from MDCK, BHK, HEK293, HEK293T, COS1, COS7, NIH3T3, Swiss3T3, STC-1 and CHO cells. In another embodiment, the cell or cell line used in the method is a native oral taste cell or established oral taste cell line that endogenously expresses the fat taste gene(s). In another embodiment, the cell line or cell culture may be a heterologous (non-oral, non-taste) cell or cell line which expresses the encoded protein. In another embodiment the cell line or cell culture may be established from fat taste gene knockout mice and other knock-in/knock-out mice.

[00124] The effect of the test molecule on the functional activity of the expressed protein (or potentially on the level of expression) is assessed and quantified by any suitable means known in the art or described herein. If desired, the expression level of the gene product may be measured using conventional means including by measurement of protein or nucleic acid.

[00125] In one embodiment, the recombinant cell expresses a detectable reporter gene in operable association with the nucleic acid encoding the reference protein, which, upon expression, produces a detectable signal. In one embodiment, the reporter gene generates a signal selected from a colorimetric, fluorescent, luminescent, chemiluminescent or electrochemical signal. Suitable reporter genes are well known in the art. Such reporter sequences include, without limitation, DNA sequences encoding β-galactosidase (LacZ), alkaline phosphatase, green fluorescent protein (GFP), red fluorescent protein (RFP), their variants, such as EGFP, EYFP, Venus, etc. In one embodiment, a fluorescent plate reader is employed. These coding sequences, when associated with regulatory elements which drive their expression (i.e., "in operable association"), provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunocytochemistry. For example, where the reporter is green fluorescent protein variant or luciferase, the function, activity or expression may be measured visually by fluorescence or light production in a luminometer. [00126] In another embodiment, the function, activity or expression of the referenced protein is determined using a ligand that binds to the protein. In one embodiment, the ligand is an antibody. Such antibodies may be presently extant in the art or presently used commercially or may be developed by techniques now common in the field of immunology. Similarly, the ligands may be tagged or labeled with reagents capable of providing a detectable signal, depending upon the assay format employed. Such labels are capable, alone or in concert with other compositions or compounds, of providing a detectable signal. Where more than one ligand is employed in a screening method, e.g., such as in a sandwich ELISA, the labels are desirably interactive to produce a detectable signal. Most desirably, the label is detectable visually, e.g. colorimetrically. Other label systems that may be utilized in the methods of this invention are detectable by other means, e.g., colored latex microparticles (Bangs

Laboratories, Indiana) in which a dye is embedded, may be used in place of enzymes to provide a visual signal indicative of the presence of the resulting protein-ligand complex in applicable assays. Still other labels include fluorescent compounds, radioactive compounds or elements.

[00127] Measurement of the function, activity or level of the nucleic acid molecules or reference proteins according to these methods may employ conventional techniques known in the art. Such methods include methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, proteomics-based methods or

immunochemistry techniques. The most commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization; R ase protection assays; and PCR-based methods, such as reverse

transcription, real time polymerase chain reaction (RT-PCR) or qPCR. Alternatively, antibodies may be employed that can recognize specific DNA -protein duplexes. The methods described herein are not limited by the particular techniques selected to perform them.

[00128] Any of the methods described herein may employ a high throughput screening assay used to identify test molecules that provide or modulate fat taste in the mouth. In one embodiment, such an assay involves contacting in each individual well of a multi-well plate a different selected test molecule (e.g., nucleotide sequence, amino acid sequence, small molecules, etc.) with a cell or cell line that expresses the proteins. In one embodiment, the cell is transfected with an expression system (promoter, protein, marker gene) that expresses luciferase (or another marker gene) only when said cell expresses the protein. After the compound has been exposed to the expressing cell under appropriate culture conditions, the level of the marker gene (or luminescence) is conventionally measured. A change in the level of expression of the reference nucleic acid sequence-encoded protein expressed by the cell with the test molecule as compared to a control is correlated with the expression or lack of expression of the marker in each well.

[00129] Methods and systems for expressing heterologous nucleic acid sequences including genes encoding fat taste transduction proteins are well known in the art.³¹'³²

[00130] In another embodiment, the methods provided herein identify test compound(s) having the ability to modulate fat taste based on the activity of the expression product (i.e., protein). In various embodiments, the test compound is contacted directly with the expression product of the reference nucleic acid sequence or with a cell expressing the reference nucleic acid sequence. In one embodiment, the method includes contacting a cell that expresses reference nucleic acid sequences with a test compound. In another embodiment, the method includes contacting the expression product of a reference nucleic acid sequence, e.g., GPR113 and/or Gnal4, with the test compound.

[00131] The method further includes assaying the cell culture for a change in activity of the expression product of the referenced nucleic acid sequence or a pathway in which it participates verses a control level. The "activity" being assayed is any biologically relevant activity that is measurable using conventional means. In one embodiment, the activity is assayed using methods known in the art. In one embodiment, the activity is ion conductance. In another embodiment, the activity is receptor activity. In another embodiment, the activity is ion transport. The method further includes identifying the test compound as a potential fat taste modulator based on whether it modulates the activity of the expression product of the reference nucleic acid sequence, e.g., the proteins GPR113 and/or Gnal4.

[00132] In one embodiment, the test compound is a fat taste enhancer when it increases ion conductance, the activity of the receptor, or ion transport. The test compound is a fat taste inhibitor when it decreases ion conductance, the activity of the receptor, or ion transport. For example, a decrease or inhibition in the electrophysiological or functional activity of the expression product of the reference nucleic acid sequence, e.g GPR113 and/or Gnal4 (or a cell or cell line expressing the same) contacted with the test molecule compared to that of a negative control identifies a test molecule that evokes or modulates a fat taste. That is, the effect of the test molecule to alter the membrane potential or ion currents, or normal hyperpolarizing or depolarizing efflux of ions across the cell membrane, is related to providing or modulating a fat taste. In one embodiment, altering membrane potential is the

depolarization by influx of cations and/or efflux of anions. In another embodiment, altering membrane potential is the hyperpolarization by prolonged/continuous influx of cations and/or efflux of anions.

[00133] In one embodiment, the electrophysiological or functional activity of the expression product of the reference nucleic acid sequence GPR113 and/or Gnal4 is measured by electrodes. In one embodiment, the electrophysiological activity is a change in certain calcium ion indicators, sodium and potassium cations, or chloride anion fluxes. In one embodiment, the electrophysiological activity is the generation of action potential or depolarization of the cell or cell line.

[00134] In another embodiment, the functional assay for measuring the activity of the protein encoded by the reference nucleic acid sequence is an electrophysiological assay which uses an ion sensitive dye or ion sensitive protein. In one embodiment, the assay is performed using mammalian cells. In another embodiment, the assay is performed in frog oocytes. In one embodiment, the ion sensitive dye is a sodium or calcium sensitive dye. In one embodiment, the assay is a calcium imaging assay. Calcium imaging assays are described in the art ^33~36.

[00135] In one embodiment, the ion sensitive dye is sodium green tetraacetate (Molecular Probes), Na-sensitive Dye Kit (Molecular Devices), Fluor-4 or Fura Red. In one embodiment, the ion sensitive protein is chameleon or GCaMP variants.

[00136] In another embodiment, the functional assay is an electrophysiological assay which uses a voltage-sensitive (membrane potential-sensitive) dye. In one embodiment, the voltage- sensitive dye is selected from Molecular Devices Membrane Potential Kit (Cat#R8034), Di-4- ANEPPS (pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalen-yl)ethenyl)- 1 -(3- sulfopropyl)hydroxid- e, inner salt, DiSBACC4(2)(bis-(l,2-dibabituric acid)-triethine oxanol), Cc-2-DMPE (Pacific Blue l,2-dietradecanoyl-sn-glycerol-3phosphoethanolamine, triethylammonium salt) and SBFI-AM (1,3-benzenedicrboxylic acid, 4,4-[l,4, 10-trioxa-7,13- diazacylopentadecane-7,13-diylbis(5-methoxy-6,l,2- -benzofurandiyl)}bis-tetrakis

{(acetyloxy)methyl}ester (Molecular Probes). In another embodiment, electrophysiological activity is measured using a two electrode voltage clamping assay. In one embodiment, the assay is a patch clamp assay. Suitable patch clamp assays are well known in the art ³¹. See, also Example 5 below. [00137] In another embodiment, electrophysiological activity is measured using an ion flux assay. In one embodiment, atomic absorption spectroscopy is used to detect ion flux.³⁸ In one embodiment of any of the above methods, a fluorescent plate reader is employed.

[00138] In another aspect, potential modulators of fat taste are detected based on binding with the expression product of a gene, e.g., GPR113 and/or Gnal4. In one embodiment, the method includes contacting a test compound with the expression product or a fragment thereof. The method further includes determining whether the test compound binds the expression product (or fragment) of the reference nucleic acid sequence.

[00139] The binding assay can be performed using either a cell-based or cell-free method.³⁹ With respect to cell-free binding assays, test compounds can be synthesized or otherwise affixed to a solid substrate, such as plastic pins, glass slides, plastic wells, and the like. In one embodiment, the test compounds are immobilized, e.g., utilizing conjugation of biotin and streptavidin by techniques well known in the art. The test compounds are contacted with the expression product of the gene, e.g., GPR113 and/or Gnal4, or functional fragment thereof, and washed. Bound polypeptide can be detected using standard techniques in the art (e.g., by radioactive or fluorescence labeling of the polypeptide or functional fragment, by ELISA methods, and the like). Alternatively, the expression product can be immobilized to a solid substrate and the test compounds contacted with the bound polypeptide or functional fragment thereof. In another embodiment, antibodies reactive with the expression product or functional fragment can be bound to the wells of a plate, and the polypeptide trapped in the wells by antibody conjugation. Test compounds can be incubated in with the expression product prior to, concurrent with or after the addition of antibody, and amount of polypeptide-antibody complex can be quantitated.

[00140] In another embodiment, a fusion protein can be provided which includes a domain that facilitates binding of the polypeptide to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates, which are then combined with cell lysates (e.g., ³⁵S-labeled) and the test compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized. The radiolabel is detected directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of expression product or functional fragment thereof found in the bead fraction quantitated from the gel using standard electrophoretic techniques.

[00141] With respect to cell-based assays, any suitable cell can be used, including bacteria, yeast, insect cells (e.g., with a baculovirus expression system), frog oocytes, avian cells, mammalian cells or any other cell described herein. In one embodiment, the assay is carried out in a cell line that naturally expresses the gene(s) GPR113 and/or Gnal4, e.g., oral taste cell line. Further, in other embodiments, it is desirable to use non-transformed cells (e.g., primary cells) as transformation may alter the characteristics of the cells.

[00142] The screening assay can be used to detect compounds that bind to or modulate the activity of the expression product of GPR113 and/or Gnal4. In one embodiment, a cell is transiently or stably transformed with a polynucleotide encoding a gene of GPR113 and/or Gnal4 or functional fragment, and can be stably transformed, for example, by stable integration into the genome of the organism or by expression from a stably maintained episome (e.g., Epstein Barr Virus derived episomes), according to techniques described herein and those well known in the art.

[00143] In a cell-based assay, the compound to be screened can interact directly with the expression product or functional fragment thereof (i.e., bind to it) and generate or modulate the activity thereof. Alternatively, the compound can be one that modulates polypeptide activity (or the activity of a functional fragment) at the nucleic acid level. For example, the compound can modulate transcription of the gene of GPR113 and/or Gnal4, modulate the accumulation of mR A (e.g., by affecting the rate of transcription and/or turnover of the mR A), and/or modulate the rate and/or amount of translation of the mRNA transcript.

[00144] In one embodiment, the method further includes recombinantly expressing the reference nucleic acid sequence prior to assaying for binding of the expression product and test compound.

[00145] In another embodiment, in vivo assays may be employed by generating and using genetically engineered animal models, such as knockout mice, genetically ablated for Gprll3 and/or Gnal4 expression are used for screening compounds in vivo. These mice retain ability to detect non-gustatory or sensory properties of fat, and to display responses to fat in behavioral tests. However, they have deficient gustatory responsiveness to fat. Therefore, compounds that are found to activate GPR113 using in vitro assays will not evoke gustatory sensation in KO mice. Consequently, KO mice have diminished behavioral taste responses to such compounds. In particular, modifiers of fat taste are not effective in these mice.

[00146] In one specific assay format, a method for identifying a compound that mimics or modulates fat taste perception comprises (a) introducing a test compound into a cell-free mixture of GPR113 receptor protein and at least one of G proteins alpha, beta and gamma, where G protein alpha is Gnal4; and (b) measuring the displacement of GPR113 receptor protein from its association with the G protein in the absence and presence of fat, wherein the ability of the test compound induce dissociation of the GPR113-G proteins complex in the absence of fat indicates that the compound is able to mimic fat taste perception, and the ability of the test compound to modulate lipid-induced dissociation of the GPR113-G protein complex indicates that the compound is able to modulate fat taste perception. In one embodiment, the G protein is a Gnal4 G protein. In another embodiment the G protein is a chimeric protein encoded by the fusion of one or more G proteins to another protein or protein fragment.

[00147] In another format, the method for identifying a compound that mimics or modulates fat taste perception comprises (a) introducing a test compound into a cell-free mixture of a GPR113 receptor protein and a second receptor protein with a G proteins alpha, beta and gamma, where G protein alpha is Gnal4, Gnal5, Gnat3 or a chimeric G alpha protein; and (b) measuring the displacement of GPR113 and one or more of the second receptor proteins from their association with the G protein in the absence and presence of fat, wherein the ability of the test compound to induce dissociation of the GPCRs-G proteins complex in the absence of fat indicates that the compound is able to mimic fat taste perception, and the ability of the test compound to modulate lipid-induced dissociation of the GPCRs-G proteins complex indicates that the compound is able to modulate fat taste perception. In one embodiment, the G protein is a Gnal4 G protein. In another embodiment, the G protein is a chimeric protein encoded by the fusion of one or more G proteins to another protein or protein fragment. In still another embodiment, the second receptor protein is a TIR protein.

[00148] Human and mouse taste receptors are orthologs, but they are known to differ in responsiveness to some taste compounds. To create an in vivo assay better at predicting human sensory perception of fat, humanized mice are generated. Gprl 13-KO mice are used to incorporate in their genome a construct with the human GPR113 gene. In another assay, Gprl 13- and Gnal4-double-KO mice are used to incorporate in their genome constructs with human GPR113 and GNA14 genes. These mice are responsive to compounds that are found to interact with GPR113 using in vitro assays. In particular, modifiers of fat taste are effective in these mice. Still other known assays and methods may be developed or used to identify fat taste modulators.

[00149] A variety of known assay formats are disclosed in publications relating to taste sensation and modulation of GPCRs involved in such taste sensations. The assay formats described therein^45"48 are incorporated by reference herein.

[00150] Compositions and Reagents for Use in Methods

[00151] Provided herein are compositions for use in screening for modulators of fat taste. In one embodiment, the composition includes a recombinant cell which expresses a reference nucleic acid sequence selected from described above. In one embodiment, the reference nucleic acid sequence is GPR113. In another embodiment, the reference nucleic acid sequence is Gnal4. In another embodiment, the reference nucleic acid sequence includes the genes encoding T1R1, 2 or 3 co-expressed with is GPR113 and/or Gnal4. In one

embodiment, the reference nucleic acid sequence is a mammalian or human reference nucleic acid sequence.

[00152] Any of the embodiments used in the methods above are also useful. Specific embodiments include a recombinant cell or cell line that comprises a nucleic acid sequence that coexpresses GPR113 and another G protein-coupled receptor under the control of a suitable expression system, wherein said sequence is heterologous to the cell. Another embodiment is the same cell or cell line that also co-expresses Gnal4. In another embodiment, the recombinant cell or cell line additionally co-expresses a T1R receptor. Other recombinant cell or cell lines coexpress one or more of T1R1, T1R2, T1R3, or combinations of TIRs. Other recombinant cell or cell lines additionally coexpress Gnat3 and/or Gnal5. The G protein-coupled receptors or G proteins are chimeric proteins comprising a fragment of one G protein fused to all or a fragment of another G protein or a fragment of one G protein fused to all or a fragment of a G protein-coupled receptor or a fragment of one G protein-coupled receptor fused to all or a fragment of another G protein-coupled receptor. The chimeric protein comprises fused fragments or proteins of one or more of Gnal4, Gnal5, T1R1, T1R2, T1R3, Gnat3 and GPR113. The recombinant cells use sequences encoding the G protein-coupled receptors or G proteins or chimeric proteins which are of human, mouse or other mammalian or animal origin. [00153] Many types of cells and cell lines are known in the art which are useful in the invention as previously disclosed above. The selected reference nucleic acid sequence(s) may be expressed in an oral taste cell or oral taste cell line, a heterologous cell, a transformed cell or endocrine cell, or any cell line that natively or recombinantly expresses the desired reference gene(s). In one embodiment, the cell is an amphibian oocyte. In another embodiment, the cell is a mammalian cell. In one embodiment, the mammalian cell is selected from HEK293, HEK293T, STC-1, Swiss3T3, CHO, BHK, NIH3T3, COS1, COS7 cell, monkey L cell, African green monkey kidney cell and Ltk-cell. In one embodiment, the cell is a recombinant cell which stably expresses the reference nucleic acid sequence(s), i.e., the reference nucleic acid sequence has been integrated into the genome of the host cell. In another embodiment, the cell transiently expresses the reference nucleic acid sequence(s). The recombinant cell also be an insect cell, a mammalian taste cell or a mammalian non-taste cell.

[00138] A taste cell population or cell line as used and developed herein can contain a nucleic acid sequence that encodes one or more of GPR113, Gnal4, Gnal5, T1R1, T1R2, T1R3, and Gnat3 under the control of an expression system heterologous to the nucleic acid sequence. The taste cell population or cell line can contain a chimeric reference nucleic acid sequence. The taste cell or cell line is an oral taste cell or oral taste cell line of human or non- human mammalian lineage.

[00139] As stated above, other useful compositions are assay reagents, such as a multi-well test plate device comprising a recombinant cell or taste cell population as described above. Still other useful cells are a recombinant mammalian cell that is ablated for its wild type Gprll3 and Gnal4 genes. A genetically engineered animal model that does not express the wild type Gprll3 and/or Gnal4 genes is also useful.

[00140] In still another embodiment, a component of the assays and methods described herein is a knockout or double knock-out animal model, which has been ablated in GPR113 and/or Gnal4, and optionally ablated in one or more of the T1R genes discussed herein. Generation of the KO models may employ any techniques known in the art.

[00141 ] Specific Embodiments of this Disclosure

[00142] In one embodiment, a method for identifying a compound that mimics or modulates fat taste perception comprises: contacting a recombinant cell or cell line that expresses the G protein-coupled receptor GPR113 in vitro with a test compound; andassaying for a detectable change in the physical or functional characteristic of the contacted cells or cell lines in comparison to a reference cell or cell line control, thereby determining that the test compound mimics or modulates the effect of dietary fat. In another embodiment, the recombinant cell co- expresses another G protein-coupled receptor. In another embodiment, the recombinant cell of these methods co-expresses the G protein, Gnal4. In another the recombinant cell used in these methods co-expresses a T1R receptor. In another embodiment of any of these methods the T1R receptor is TlRl, T1R2, T1R3, or combinations of TIRs. In other embodiments of these methods, the recombinant cell co-expresses the G protein, Gnat3. In embodiments of any of these methods, the recombinant cell co-expresses the G protein, Gnal5. In other embodiments of any of these methods, the G proteins or G protein-coupled receptor proteins are chimeric proteins comprising all or a fragment of one G protein fused to all or a fragment of another G protein. In other embodiments of any of these methods, chimeric proteins comprise fused fragments or proteins of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3. In other embodiments of any of these methods, chimeric proteins further comprise a detectable label. In other embodiments of any of these methods, the proteins or nucleic acid sequences encoding them are of human, mouse or other mammalian or animal origin.

[00143] In other embodiments of any of these methods, the detectable change is the formation or change in the formation of a protein comprising all or a fragment of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3. In other embodiments of any of these methods, the detectable change is an increase in the signal transduction activity of a G protein-coupled receptor pathway. In other embodiments of any of these methods, the detectable change in the contacted cells or cell lines is a change in the expression of enzymes downstream in a G protein-coupled receptor pathway of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3. In other embodiments of any of these methods, the detectable change is a change in the level of intracellular calcium or ion transport. In other embodiments of any of these methods, the detectable change is a change of the cell membrane potential. In other embodiments of any of these methods, the detectable change is a change in expression, function or activity of other G proteins or proteins in their respective pathways. In other embodiments of any of these methods, the detectable change is a change in the interaction between GPR113 protein and its ligands. In other embodiments of any of these methods, the detectable change is a change in the signaling, activity or activation of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3. [00144] In other embodiments of any of these methods, the format used is a high- throughput method comprising multiple cells, cell lines, test molecules and references. In other embodiments of any of these methods, the assaying step comprises an imaging assay.

[00145] In other embodiments of any of these methods, the G protein-coupled receptors or G proteins are encoded by genes of human or non-human mammalian origin, or are encoded by modified nucleic acid sequences. In other embodiments of any of these methods, the genes are modified by one or more replaced or inserted nucleotides or modified nucleotides that do not occur in the normal mammalian gene. In other embodiments of any of these methods, the G protein-coupled receptors or G proteins or the nucleic acid sequences encoding them are associated with detectable labels or reporter genes, which are alone or in concert with a label partner, capable of generating a detectable signal.

[00146] In other embodiments of any of these methods, the recombinant cell is a taste cell. In other embodiments of any of these methods, the recombinant cell is a cell that does not naturally comprise a nucleic acid sequence expressing one or more of GPR113, Gnal4, Gnal5, T1R1, T1R2, T1R3, and Gnat3. In other embodiments of any of these methods, the label or reporter gene generates a signal selected from a colorimetric, fluorescent, luminescent, chemiluminescent or electrochemical signal. In other embodiments of any of these methods, one or more G protein-coupled receptors or G proteins or the nucleic acid sequences encoding them is associated with a sequence enabling transport of the product of the expressed protein to or through the cell membrane. In other embodiments of any of these methods, the expression of said G protein-coupled receptors or G proteins or the nucleic acid sequences encoding them is determined using a ligand that binds to the protein.

[00147] In another embodiment, a recombinant cell or cell line comprises one or more nucleic acid sequences that coexpress GPR113 and another G protein-coupled receptor under the control of a suitable expression system, wherein the sequence is heterologous to the cell. In another embodiment, the recombinant cell or cell line co-expresses Gnal4. In another embodiment of these cells, the recombinant cell or cell line co-expresses a T1R receptor. In other embodiments these recombinant cells or cell lines express a T1R receptor which is T1R1, T1R2, T1R3, or combinations of TIRs. In other embodiments of these cell or cell lines the cell co-expresses Gnat3 receptor. In other embodiments these recombinant cells or cell lines the recombinant cell co-expresses Gnal5. In other embodiments these recombinant cells or cell lines the recombinant cell or cell line express G protein-coupled receptors or G proteins which are chimeric proteins comprising a fragment of one G protein fused to all or a fragment of another G protein or a fragment of one G protein fused to all or a fragment of a G protein- coupled receptor or a fragment of one G protein-coupled receptor fused to all or a fragment of another G protein-coupled receptor. In other embodiments these recombinant cells or cell lines express a chimeric protein that comprises fused fragments or proteins of one or more of GPR113, Gnal4, Gnal5, T1R1, T1R2, T1R3, and Gnat3.

[00148] In other embodiments these recombinant cells or cell lines contain a nucleic acid sequences or chimeric nucleic acid sequences that further comprise a detectable label. In other embodiments these recombinant cells or cell lines contain nucleic acid sequences encoding the G protein-coupled receptors or G proteins or chimeric proteins are of human, mouse or other mammalian or animal origin. In other embodiments the recombinant cell or cell line described herein is a bacterial cell, an amphibian cell, an insect cell, a mammalian taste cell or a mammalian non-taste cell. In one embodiment, the recombinant mammalian cell is MDCK, BHK, HEK293, HEK293T, COS, NIH3T3, Swiss3T3 or CHO.

[00149] In another embodiment, a taste cell population or cell line comprises a nucleic acid sequence that encodes one or more of GPR113, Gnal4, Gnal5, T1R1, T1R2, T1R3, and Gnat3. In one embodiment of these taste cell populations or cell lines, one or more of the nucleic acid sequences is a chimeric sequence. In one embodiment of these taste cell populations or cell lines, the cell or cell line is an oral taste cell or oral taste cell line of human or non-human mammalian lineage.

[00150] In one embodiment a multi-well test plate device comprises a recombinant cell or taste cell population of any of those described herein.

[00151] In one embodiment a recombinant mammalian cell is ablated for its wild type Gprll3 and Gnal4 genes.

[00152] In one embodiment, a genetically engineered animal model is provided that does not express the wild type Gprll3 and/or Gnal4 genes. In one embodiment, a humanized genetically engineered animal model is provided that does not express its wild type Gprll3 and/or Gnal4 genes but incorporates in its genome a construct comprising the human GPR113 and/or human GNA14 encoding nucleic acid sequences.

In another embodiment, a method for identifying a compound that modulates fat taste perception comprises: introducing a test compound into a cell-free mixture of GPR113 receptor protein and G proteins alpha, beta and gamma; and measuring the displacement of GPR113 receptor protein from its association with the G protein in the absence and presence of fat, wherein the ability of the test compound induce dissociation of the GPR113-G proteins complex in the absence of fat indicates that the compound is able to mimic fat taste perception, and the ability of the test compound to modulate lipid-induced dissociation of the GPR113-G protein complex indicates that the compound is able to modulate fat taste perception. In one embodiment of this method, the G protein is Gnal4. In another embodiment of these methods, the G protein is a chimeric protein encoded by the fusion of one or more G proteins to another protein or protein fragment.

[00153] In one embodiment, a method for identifying a compound that modulates fat taste perception comprises: introducing a test compound into a cell-free mixture of a GPR113 receptor protein and a second receptor protein with G proteins alpha, beta and gamma; and measuring the displacement of GPR113 and one or more of the second receptor proteins from their association with the G protein in the absence and presence of fat, wherein the ability of the test compound to induce dissociation of the GPCRs-G proteins complex in the absence of fat indicates that the compound is able to mimic fat taste perception, and the ability of the test compound to modulate lipid-induced dissociation of the GPCRs-G proteins complex indicates that the compound is able to modulate fat taste perception. In another embodiment of this method, the G protein is Gnal4. In another embodiment of these methods, the G protein is a chimeric protein encoded by the fusion of one or more G proteins to another protein or protein fragment. In another embodiment of this method, the second receptor protein is a TIR protein. In another embodiment of these methods, the TIR receptor is T1R1, T1R2, T1R3, or combinations of TIRs.

[00154] The following examples are illustrative only and are not intended to be a limitation on the present invention.

[00155] EXAMPLES

[00156] The following examples are provided to illustrate certain aspects of the claimed invention. The invention is not limited to these examples. These examples demonstrate that Gprll3 is expressed in most TlR3-expressing cells in taste buds of circumvallate, and probably foliate, papillae of mice. Gprll3 knockout mice exhibited reduced preference for both fatty acids and dietary oils, while their wild type littermates preferred them. These results demonstrate that Gprll3 is involved in fat reception, which culminate in behavioral preference.

[00157] EXAMPLE 1 [00158] Taste-related behaviors are intimately associated with which type of cell is activated. Thus, it is very important to determine the specific cells expressing Gprl 13 and evaluate if the Gprl 13 receptor is associated with taste cells linked to preference behavior or to possible novel taste cell subsets that elicit preference. Our preliminary data suggest that Gprl 13 expression is confined to a subset of TlR3-expressing cells in circumvallate taste papillae.

[00159] Polymerase chain reactions (PCRs) were performed using mouse multiple tissue cDNA, including heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis, and embryonic tissue (7 day, 11 day, 15 day, and 17 day). Real time PCR (RT-PCR) was performed on RNA isolated from mouse circumvallate papillae, fungiform papillae and foliate papillae. Non-template controls (NTC) were negative controls. A molecular marker (M) was indicated in the gels, as shown in FIG.1.

[00160] The top panel of FIG. 1 shows the gel, which revealed an amplified product of the expected size for Gprl 13 (417bp) in the testis, circumvallate papillae and foliate taste papillae, but not the fungiform papillae or other tissues tested. The bottom panel was the G3PDH control.

[00161] EXAMPLE 2

[00162] In situ hydridization (ISH) was performed on cryosections from fresh-frozen gustatory papillae of wild type B6 mice and Skn la-knockout mice that lack type II

(sweet/umami/bitter) taste receptor cells. The probes used were an approximately 150 base- long antisense digoxigenin (DIG)-RNA fragment from 2.3 kb in vitro transcription (IVT) product for Gprl 13 (NCBI Ref: NM_001014394) and an approximately 150 base-long antisense DIG-RNA fragment from the 2.5 kb IVT product of Gnal 4 (NCBI Ref: BC027015) The hybridization conditions were 58°C, for about 48 hours, followed by two washings at 58 °C for 30 minutes per washing, in 0.2xSSC. Signal development used nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, toluidine-salt. The cells were kept at room temperature overnight.

[00163] FIG. 2 shows the results of this assay and the spatial distribution of Gprl 13 and Gnal 4 mRNAs in the oral cavity of wild type B6 mice and Skn-la KO mice that lack type II (sweet/umami/bitter) taste receptor cells. Expression of Gprl 13 and Gnal4 mRNAs in the oral cavity was confined to the taste buds distributed in the posterior gustatory papillae, i.e., the circumvallate papillae (abbreviated as CvP) of B6 mice. The lack of signal in the Skn-la KO mice revealed that Gprl 13 mRNA is expressed only in the type II TBCs. [00164] EXAMPLE 3

[00165] To further confirm which taste cell types express Gprl 13, we performed double- labeling in situ hybridization (dISH) analyses. dISH was employed to identify TBCs expressing Gprl 13 mRNA. Cryosections from fresh-frozen gustatory papillae of wild type B6 mice were hybridized with probes generated by antisense DIG-RNA and FITC-RNA fragmented to approximately 150 base-long sequences from

2.3 kb of Gprl 13 (NCBI Ref: NM_001014394); 3.2 kb of TRPM5 (NCBI Ref: AF228681);

3.4 kb (1.3 kb + 2.1 kb) of PKD2L1 (NCBI Ref: NM_181422); 1.8 kb of NTPDase2 (NCBI Ref: NM_009849);

2.2 kb of T1R3 (NCBI Ref: AF337039); 1 kb of T2R5 (NCBI Ref: AF227147); and

2.5 kb of Gnal4 (NCBI Ref: BC027015); and

1 kb of Gnat3 (NCBI Ref: BC 147839).

[00166] Hybridization was performed at 58°C, for about 40 hours. Washing was performed at 58 °C, 30 min x2, in 0.2xSSC. Signal development employed Alexa488 label using ABC- HRP (Vector Labs) and TSA (Perkin Elmer) for detection of the FITC-RNA probes and using HNPP (Roche Diagnostics) for detection of the DIG-RNA probes.

[00167] The results are shown in FIGs. 3A to 3G. Expression of Gprl 13 mRNA was confined to a subset of 7>p»25-expressing TBCs as expected by the results in the Skn-la KO mice of FIG. 2. Expression of Gprl 13 mRNA was also confined to a subset of T1R3- expressing TBCs, and completely overlapped with Gnal4 expression. Expression of Gprl 13 mRNA was confined to a subset of 7>p»25-expressing TBCs as expected by the results in the Skn-la KO mice (Fig. 2). Expression of Gprl 13 mRNA was also confined to a subset of 77R3-expressing TBCs, and completely overlapped with Gnal4 expression.

[00168] EXAMPLE 4

[00169] To determine if GPR113 expressed in taste cells is involved in fat perception, two- bottle preference tests with taste solutions including fatty acids and dietary oils were conducted, which provide functional evidence that GPR113 deficiency in taste cells alters fat taste perception.

[00170] The results of these assays are shown in FIGs. 4A-4D shows fatty acids and oils intake by twelve wild type mice expressing GPR113 (WT, GPR 113^+/+, symbol♦) and fourteen genetically engineered knock out mice in which the Gprll3 gene was ablated (Gprll3KO, Gprll3^~'^~ symbol■) in two-bottle preference tests. The fatty acids were linoleic acid (FIG. 4A) and oleic acid (FIG. 4B). The oils were corn oil (FIG.4C) and soybean oil (FIG. 4D). The results, which plotted volume intake over 24 hours over an average of 48 hours vs. concentration of fatty acid/oil, showed that the KO mice had lower fat intakes for all.

[00171] Feeding behavior in Gprll3 knockout mice indicates that they are indifferent to dietary oils and fatty acids, while their wild type littermates preferred these compounds. These observations indicate that GPR113 is involved in fat taste perception. Because GPR113 is expressed in taste bud cells, fat taste information mediated by GPR113 must be conveyed to the brain via gustatory nerves to influence taste-related behavior.

[00172] The results of similar assays are shown in FIGs. 4E-4H, which shows fatty acids and oils intake by ten wild type mice expressing Gnal4 (WT, Gnal4^+/+ , symbol♦) and ten genetically engineered knock out mice in which the Gnal4 gene was ablated GnaH d, Gnal4^~'^~ symbol■) in two-bottle preference tests. The fatty acids were linoleic acid (FIG. 4E) and oleic acid (FIG. 4F). The oils were corn oil (FIG.4G) and soybean oil (FIG. 4H). The results, which plotted volume intake over 48 hours vs. concentration of fatty acid/oil, showed that the KO mice had lower fat intakes for all.

[00173] The data in FIGs. 5A-5D were shown as preference vs. concentration of fatty acid/oil. These results also showed that Gprll3 KO mice have lower fat preferences. The data in FIGs. 5E-5H were shown the data of preference vs. concentration of fatty acid/oil. These results also showed that Gnal4 KO mice have lower fat preferences.

[00174] Brief access gustometer assays were performed on the same mice to measure licking rates to the same fatty acids and oils. These results are shown in FIGs.6A to 6H. FIGs. 6A- 6D shows the results plotted as normalized licking rate vs. concentration of the same fatty acids (in mM) and oils (in %) by twelve wild type mice expressing GPR113 (WT, Gprll3^+I+ , symbol♦) and eleven to fourteen genetically engineered knock out mice in which the Gprll3 gene was ablated (Gprll3 KO, Gprll3^~'^~ symbol■). The fatty acids were linoleic acid (FIG. 5 A) and oleic acid (FIG. 5B). The oils were corn oil (FIG.5C) and soybean oil (FIG. 5D). The results showed that the KO mice had robustly decreased licking rates for the fats. FIGs. 6E-6H shows the results plotted as normalized licking rate vs. concentration of the same fatty acids (in mM) and oils (in %) by ten wild type mice expressing Gnal4 (WT, Gnal4^+I+ , symbol♦) and ten genetically engineered knock out mice in which the Gnal4 gene was ablated (Gnal4 KO, Gnal4^~'^~ symbol■). The fatty acids were linoleic acid (FIG. 5E) and oleic acid (FIG. 5F). The oils were corn oil (FIG.5G) and soybean oil (FIG. 5H). The results showed that the KO mice had robustly decreased licking rates for the fats.

[00175] All publications cited in this specification and in the reference section below and priority US provisional application No. 62/207,793, filed August 20, 2015, are incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

[00176] REFERENCES

1. Sclafani, A., et al., 2007 Fat and carbohydrate preferences in mice: the contribution of alpha-gustducin and Trpm5 taste-signaling proteins. Am J Physiol Regul Integr Comp Physiol, 293(4): p. R1504-13

2. Liu, P., et al, 2011 Transient receptor potential channel type M5 is essential for fat taste. J Neurosci, 31(23): p. 8634-42

3. Cartoni, C, et al., 2010 Taste preference for fatty acids is mediated by GPR40 and GPR120. J Neurosci, 30(25): p. 8376-82

4. Sclafani, A., S. Zukerman, and K. Ackroff, 2013 GPR40 and GPR120 fatty acid sensors are critical for postoral but not oral mediation of fat preferences in the mouse. Am J Physiol Regul Integr Comp Physiol, 305(12): p. R1490-7

5. A. Sclafani, P. Marambaud, K. Ackroff. 2015 Marked fat preference deficits in P2X2/P2X3 and Calhml but not CD36 and GPR40/120 knockout mice. Abstract of the AChemS 37th Annual Meeting, Chem. Senses, doi: 10.1093/chemse/bjv029, p. 72-73)

6. Sclafani, A., K. Ackroff, and N.A. Abumrad, 2007 CD36 gene deletion reduces fat preference and intake but not post-oral fat conditioning in mice. Am J Physiol Regul Integr Comp Physiol, 293(5): p. Rl 823-32 7. Laugerette F, Passilly-Degrace P, Patris B, Niot I, Febbraio M, Montmayeur JP, Besnard P. 2005 CD36 involvement in orosensory detection of dietary lipids, spontaneous fat preference, and digestive secretions. J Clin Invest 115 : 3177-3184

8. A. Sclafani, P. Marambaud, K. Ackroff. 2015 Marked fat preference deficits in P2X2/P2X3 and Calhml but not CD36 and GPR40/120 knockout mice. Abstract of the AChemS 37th Annual Meeting, Chem. Senses, doi: 10.1093/chemse/bjv029, p. 72-73

9. US Patent No. 8,669,066

10. Nelson et al, 2011 Abstracts from the 33^rd Annual Meeting of the Association for Chemoreception Sciences, Chem. Senses 31 :A28-29

1 1. Bhal et al, 2007 Mol. Pharmaceutics, 4(4):556-560.

12. Ozdener MH, Rawson NE. 2013 "Primary culture of mammalian taste epithelium." Methods Mol Biol. 2013; 945 :95- 107

13. Danilova, V. and G. Hellekant, 2003 Comparison of the responses of the chorda tympani and glossopharyngeal nerves to taste stimuli in C57BL/6J mice. BMC Neurosci, 2003. 4(1): p. 5

14. Frank, M.E., et al., 2003 Contemporary measurement of human gustatory function, in Handbook of Olfaction and Gustation, R.L. Doty, Editor. 2003, Marcel Dekker: New York. p. 783-804.

15. Tomchik, S.M., et al., 2007 Breadth of tuning and taste coding in mammalian taste buds. J Neurosci, 2007. 27(40): p. 10840-8.

16. J. D. Thomson et al, 1999 Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690

17. backtranseq by EMBOSS, http://www.ebi.ac.uk/Tools/st/ ; Gene Infinity

(http://www.geneinfinity.org/sms-/sms_backtranslation.html); ExPasy

(ht p://^'www. expasy.org/tools/)

18. International patent publication No. WO 2009/094610

19. US patent application publication No. 2008/0153120

20. International patent publication No. WO 2006/0821 10 21. International patent publication No. WO 2007/092185

22. US patent application publication No. 2009/0181404

23. US patent application publication No. 2005/0059094

24. US patent application publication No. 2009/0123942

25. US patent application publication No. 2004/0072254

26. US patent application publication No. 2009/013969

27. International patent publication No. WO 2005/014848

28. US patent application publication No. 2006/0223117

29. US Patent No. 5,693,756

30. Sambrook et al. 1989 "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY", its more recent updates and references cited therein.

31. International patent publication No. WO 2009/008950

32. US Patent No. 8,669,066

33. Chandrashekar, 2000 Cell, 100, 703-711

34. Nelson 2001 Cell 106, 381-390

35. Nelson, 2002 Nature 416, 199-202

36. Li, 2002 Proc. Natl. Acad. Sci. USA, 99, 4692-4696.

37. Ishimaru, PNAS, 103(33): 12571-7

38. Gill et al, 2003 Oct., Assay Drug Dev Technol. 1(5):709- 17

39. US patent publication No. 20120115795

40. US patent publication No.200300131 19

41. US patent publication No.20030166103

42. US Patent No. 7,314,716 43. Ming D. et al, 1998 Jul, "Characterization and solubilizaiton of bitter-responsive receptors that couple to gustducin", Proc. Natl. Acad. Sci. USA, 95:8933-38

44. Sainz, E et al, 2007 Functional characterization of human bitter taste receptors", Biochem J., 403:537-543

45. US patent publication No.20090221709

46. US Patent No. 7,803,982

47. US patent publication No. 20080305500

48. US patent publication No.20050084932

Claims

WHAT IS CLAIMED IS:

1. A method for identifying a compound that mimics or modulates fat taste perception comprising:

contacting a recombinant cell or cell line that expresses the G protein-coupled receptor GPR113 in vitro with a test compound; and

assaying for a detectable change in the physical or functional characteristic of the contacted cells or cell lines in comparison to a reference cell or cell line control, thereby determining that the test compound mimics or modulates the effect of dietary fat.

2. The method according to claim 1, wherein said recombinant cell co-expresses one or more of:

a. another G protein-coupled receptor or co-expresses the G protein, Gnal4, or b. a T1R receptor or

c. the TlR receptors TlRl, T1R2, T1R3, or combinations of TIRs; or d. the G protein, Gnat3, or

e. the G protein, Gnal5, or

f. G proteins or G protein-coupled receptor proteins which are chimeric proteins comprising all or a fragment of one G protein fused to all or a fragment of another G protein; or

g. chimeric proteins that comprise fused fragments or proteins of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3; or

h. chimeric proteins that further comprise a detectable label; or

i. proteins or nucleic acid sequences encoding them that are of human, mouse or other mammalian or animal origin.

3. The method according to any one of claims 1 or 2, wherein the detectable change is one or more of:

a. the formation or change in the formation of a protein comprising all or a fragment of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3; or b. an increase in the signal transduction activity of a G protein-coupled receptor pathway;

c. a change in the expression of enzymes downstream in a G protein- coupled receptor pathway of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3; or

d. a change in the level of intracellular calcium or ion transport; or e. a change of the cell membrane potential; or

f. a change in expression, function or activity of other G proteins or proteins in their respective pathways; or

g. a change in the interaction between GPR113 protein and its ligands; or h. a change in the signaling, activity or activation of one or more of GPR113, Gnal4, Gnal5, TlRl, T1R2, T1R3, and Gnat3.

4. The method according to claim 1, which is a high-throughput method comprising multiple cells, cell lines, test molecules and references.

5. The method according to claim 1, wherein the assaying step comprises an imaging assay.

6. The method according to claim 2, wherein:

a. the G protein-coupled receptors or G proteins are encoded by genes of human or non-human mammalian origin, or are encoded by modified nucleic acid sequences; or b. the genes are modified by one or more replaced or inserted nucleotides or modified nucleotides that do not occur in the normal mammalian gene; or

c. the G protein-coupled receptors or G proteins or the nucleic acid sequences encoding them are associated with detectable labels or reporter genes, which are alone or in concert with a label partner, capable of generating a detectable signal; or

d. the G protein-coupled receptors or G proteins or the nucleic acid sequences encoding them are associated with detectable labels or reporter genes, which are alone or in concert with a label partner and said label or reporter gene generates a signal selected from a colorimetric, fluorescent, luminescent, chemiluminescent or electrochemical signal; or

e. one or more G protein-coupled receptors or G proteins or the nucleic acid sequences encoding them is associated with a sequence enabling transport of the product of the expressed protein to or through the cell membrane; or

f. the expression of said G protein-coupled receptors or G proteins or the nucleic acid sequences encoding them is determined using a ligand that binds to the protein.

7. The method according claim 1, wherein the recombinant cell is a taste cell or wherein the recombinant cell is a cell that does not naturally comprise a nucleic acid sequence expressing one or more of GPR113, Gnal4, Gnal5, T1R1, T1R2, T1R3, and Gnat3.

8. A recombinant cell or cell line that comprises one or more nucleic acid sequences that coexpress GPR113 and another G protein-coupled receptor under the control of a suitable expression system, wherein said sequence is heterologous to the cell.

9. The recombinant cell or cell line according to claim 8, wherein recombinant cell co- expresses one or more of Gnal4, or a T1R receptor, or the T1R receptor T1R1, T1R2, T1R3, or combinations of TIRs, or Gnat3, or Gnal5.

10. The recombinant cell or cell line according to claim 8, wherein the G protein-coupled receptors or G proteins are chimeric proteins comprising a fragment of one G protein fused to all or a fragment of another G protein or a fragment of one G protein fused to all or a fragment of a G protein-coupled receptor or a fragment of one G protein-coupled receptor fused to all or a fragment of another G protein-coupled receptor or wherein said chimeric protein comprises fused fragments or proteins of one or more of GPR113, Gnal4, Gnal5, T1R1, T1R2, T1R3, and Gnat3 or wherein said nucleic acid sequences or chimeric nucleic acid sequences further comprise a detectable label, or wherein the nucleic acid sequences encoding the G protein- coupled receptors or G proteins or chimeric proteins are of human, mouse or other mammalian or animal origin.

11. The recombinant cell according to claim 8, which is a bacterial cell, an amphibian cell, an insect cell, a mammalian taste cell or a mammalian non-taste cell, or is a mammalian cell selected from MDCK, BHK, HEK293, HEK293T, COS, NIH3T3, Swiss3T3 or CHO.

12. A taste cell population or cell line that comprises a nucleic acid sequence that encodes one or more of GPR113, Gnal4, Gnal5, T1R1, T1R2, T1R3, and Gnat3, optionally wherein one or more of the nucleic acid sequences is a chimeric sequence and optionally wherein the cell or cell line is an oral taste cell or oral taste cell line of human or non-human mammalian lineage.

13. A multi-well test plate device comprising a recombinant cell or taste cell population of any one of claims 8 to 12.

14. A recombinant mammalian cell that is ablated for its wild type Gprll3 and Gnal4 genes.

15. A genetically engineered animal model that (a) does not express the wild type Gprll3 and/or Gnal4 genes; or (b) that is humanized and does not express its wild type Gprll3 and/or Gnal4 genes but incorporates in its genome a construct comprising the human GPR113 and/or human GNA14 encoding nucleic acid sequences.

16. A method for identifying a compound that modulates fat taste perception comprising: introducing a test compound into a cell-free mixture of GPR113 receptor protein and G proteins alpha, beta and gamma; and measuring the displacement of GPR113 receptor protein from its association with the G protein in the absence and presence of fat, wherein the ability of the test compound induce dissociation of the GPR113-G proteins complex in the absence of fat indicates that the compound is able to mimic fat taste perception, and the ability of the test compound to modulate lipid-induced dissociation of the GPR113-G protein complex indicates that the compound is able to modulate fat taste perception; or

introducing a test compound into a cell-free mixture of a GPR113 receptor protein and a second receptor protein with G proteins alpha, beta and gamma; and measuring the displacement of GPR113 and one or more of the second receptor proteins from their association with the G protein in the absence and presence of fat, wherein the ability of the test compound to induce dissociation of the GPCRs-G proteins complex in the absence of fat indicates that the compound is able to mimic fat taste perception, and the ability of the test compound to modulate lipid-induced dissociation of the GPCRs-G proteins complex indicates that the compound is able to modulate fat taste perception.

17. The method according to claim 16, wherein the G protein is Gnal4, or wherein the G protein is a chimeric protein encoded by the fusion of one or more G proteins to another protein or protein fragment, or wherein the second receptor protein is a TIR protein, or wherein said TIR receptor is T1R1, T1R2, T1R3, or combinations of TIRs.