ES2388521T3 - Taste receptors of the hetero-oligomeric sweet t1r2-t1r3, cell lines that express said receptors and use thereof for the identification of compounds with sweet taste - Google Patents

Abstract

A taste receptor comprising at least one T1R2 polypeptide and at least one T1R3 polypeptide, wherein said taste receptor specifically binds to and / or is activated by a sweet taste stimulus, in which T1R2 dichopolypeptide is selected from the group that it consists of: (a) a polypeptide having an amino acid sequence at least 85% identical to SEQ ID NO: 6, a fragment thereof having a length of at least 40 amino acids, or a fragment of an amino acid sequence that is at at least 90% identical to SEQ ID NO: 6 and having a length of at least 20 amino acids; (b) a polypeptide encoded by a polynucleotide that, under stringent hybridization conditions, hybridizes with the inverse complement of SEQ ID NO: 10; and (c) a polypeptide encoded by a polynucleotide that, under stringent hybridization conditions, hybridizes with the reverse complement of a polynucleotide encoding the polypeptide of SEQ ID NO: 6; and wherein said T1R3 polypeptide is selected from the group consisting of: (d) a polypeptide having an amino acid sequence at least 85% identical to SEQ ID NO: 7, a fragment thereof having a length of at least 40 amino acids, or a fragment of an amino acid sequence that is at least 90% identical to SEQ ID NO: 7 and having a length of at least 20 amino acids; (e) a polypeptide encoded by a polynucleotide that, under stringent hybridization conditions, hybridizes with the inverse complement of SEQ ID NO. : 9; and (f) a polypeptide encoded by a polynucleotide that, under stringent hybridization conditions, hybridizes with the reverse complement of a polynucleotide encoding the polypeptide of SEQ ID NO: 7.

Description

Taste receptors of the hetero-oligomeric sweet t1r2-t1r3, cell lines that express said receptors and use thereof for the identification of compounds with sweet taste.

Background of the invention

Field of the Invention

The present invention relates in part to the discovery that T1R receptors are assembled to form functional taste receptors. In particular, it has been found that the coexpression of T1R1 and T1R3 results in a taste receptor that responds to stimuli of the umami flavor, which includes monosodium glutamate. In addition, it has been found that coexpression of the T1R2 and T1R3 receptors results in a taste receptor that responds to sweet-tasting stimuli, including natural and artificial sweeteners. In addition, the present description relates to the use of hetero-oligomeric taste receptors comprising T1R1 / T1R3 and T1R2 / T1R3 in assays to identify compounds that respond respectively to Umami flavor stimuli and sweet taste stimuli.

In addition, the invention relates to the construction of cell lines that stably or transiently coexpress a combination of T1R2 and T1R3 under constitutive or inducible conditions.

The use of these cell lines is also provided in cell-based assays to identify umami and sweet taste modulating compounds, in particular high-performance screening assays that detect the activity of receptors through the use of fluorometric imaging.

Description of the related technique

WO 02/064631 A, citable under article 53 (3) EPC, describes T1R taste receptors and genes encoding them.

The taste system provides sensory information about the chemical composition of the outside world. Mammals are believed to have at least five basic taste modalities: sweet, bitter, acid, salty, and umami. See, for example, Kawamura et al., Introduction to Umami: A Basic Taste (1987); Kinnamon et al .., Ann. Rev. Physiol., 54: 715-731 (1992); Lindemann, Physiol. Rev., 76: 718-766 (1996); Stewart et al., Am. J. Physiol., 272: 1-26 (1997). It is believed that each taste modality is mediated by a receptor or receptors of differentiated proteins that are expressed in the taste receptor cells found on the surface of the tongue (Lindemann, Physiol. Rev., 76: 718-716 (1996)) . Taste receptors that recognize sour, sweet and umami taste stimuli belong to the G protein-coupled receptor superfamily (GPCR) (Hoon et al., Cell, 96: 451 (1999); Adler et al., Cell, 100 : 693 (2000)). (It is believed that other taste modalities are mediated by ion channels).

G protein-coupled receptors mediate many other physiological functions, such as endocrine function, exocrine function, heart rate, lipolysis, and carbohydrate metabolism. The biochemical analysis and molecular cloning of a series of said receptors has revealed many basic principles regarding the function of these receptors. For example, US Patent No. 5,691,188 describes how after binding of a ligand to a GPCR, the receptor undergoes a conformational change that leads to the activation of a heterotrimeric G protein promoting the displacement of GDP bound by GTP on the surface. of the Gay subunit the subsequent dissociation of the Gade subunit the G1 and Gy subunits. Galibres subunits and G1 complexes activate elements downstream of a variety of signal transduction pathways.

This invention relates to the T1R class of three GPCR members specific to taste. Previously, the hypothesis that T1R receptors acted as taste receptors for candy had been established (Hoon et al., Cell, 96: 541-551 (1999); Kitagawa et al., Biochem. Biophys. Res. Commun., 283 : 236-242 (2001); Max et al., Nat. Genet., 28: 58-63 (2001); Montmayeur et al., Nat. Neurosci., 4: 492-498 (2001); Sainz et al. , J. Neurochem., 77: 896-903 (2001)), and in recent dates, Nelson et al. (2001) have shown that rat T1R2 and rat T1R3 act in combination to recognize sweet taste stimuli. The present invention relates to the discovery that, as is the case with rat T1R2 / T1R3, human T1R2 and T1R3 act in combination to recognize sweet taste stimuli. In addition, the present description refers to the discovery that human T1R1 and T1R3 act in combination to recognize umami taste stimuli. Therefore, T1R2 / T1R3 is likely to act as a sweet taste receptor, and T1R1 / T1R3 is likely to act as a umami taste receptor in mammals. The probable explanation of the functional codependency of T1R1 and T1R3, and of the functional codependency of T1R2 and T1R3, is that, like the GABAB receptor, structurally related (Jones et al., Nature, 396: 5316 -5322 (1998); Kaupmann et al., Nature, 396: 683-687 (1998); White et al., Nature, 396: 679-682 (1998); Kuner et al., Science, 283: 74-77 (1999)), T1Rs act as heterodimeric complexes.

The identification of the characterization of taste receptors that act as sweet and umami receptors is significant, since it will facilitate the use of these receptors in assays to identify compounds that modulate (potentiate or block) the sweet and umami taste. These compounds would be useful for improving the taste and palatability of food, beverages, medicinal compounds for human or animal consumption. In particular, an assay using a functional sweet receptor would allow the identification of new sweeteners.

Summary of the invention

The present invention relates to the discovery that different combinations of T1R, when co-expressed, produce functional taste receptors that respond to taste stimuli. In particular, the present invention relates to the discovery that the coexpression of T1R2 and T1R3 produces a heterooligomeric taste receptor that responds to sweet taste stimuli.

The present invention also relates to cell lines that coexpress T1R2 and T1R3, preferably human. In preferred embodiments, these cell lines will express high amounts of the receptors, constitutively or inducibly. These cell lines include cells that express T1R2 and T1R3 in a transient or stable manner.

In addition, the present invention provides assays, preferably high performance screening assays, which utilize the taste receptor T1R2 / T1R3, preferably high yielding cell-based assays, to identify compounds that modulate the sweet or umami taste. The description also provides analyzes that include taste tests to confirm that these compounds modulate the sweet taste.

Objects

With this objective, it is an object to provide a family of G-protein-coupled mammalian receptors, referred to herein as T1R, that mediates the taste perception.

Another object is to provide fragments and variants of said T1Rs that retain the activity, for example, that are activated and / or linked to taste stimuli of sweet or umami.

Still another object is to provide nucleic acid sequences or molecules encoding said T1R, fragments or variants thereof.

Another object is to provide expression vectors that include nucleic acid sequences encoding said T1Rs, fragments or variants thereof, which are operatively linked to at least one regulatory sequence, such as a promoter, enhancer or other sequence involved in the transcription and / or translation of positive or negative genes and / or protein export.

A further object is to provide human or non-human cells, for example, mammalian, yeast, worm or insect cells, which functionally express at least one of said T1R, or fragments or variants thereof, and preferably a combination of the T1R or fragments or variants thereof.

Another object is to provide T1R polypeptides or fusion proteins that include at least one fragment of at least one of said T1R.

Another object is to provide an isolated nucleic acid molecule encoding a T1R polypeptide, comprising a nucleic acid sequence that is at least 50%, preferably 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence having one of the hT1R nucleic acid sequences identified below, and their conservatively modified variants.

Another additional object is to provide an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence that is at least 35% to 50%, preferably 60%, 75%, 85%, 90 %, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group of one of the T1R amino acid sequences identified below, and their conservatively modified variants, in which the fragment has a length of at least 20, preferably, 40, 60, 80, 100, 150, 200 or 250 amino acids. Optionally, the fragment can be an antigenic fragment that binds to an anti-T1R antibody.

Another object is still to provide an isolated polypeptide comprising a variant of said fragment, in which there is a maximum variation of 10, preferably 5, 4, 3, 2 or 1 amino acid residues.

Another object is to provide combinations of T1R1 / T1R3, in which T1R1 and / or T1R3 is a variant or fragment, and combinations of T1R2 / T1R3, in which T1R2 and / or T1R3 is a variant or fragment.

Another object is still to provide agonists or antagonists of said T1R, or its fragments or variants.

Another object is still to provide a peptide that interacts with the PDZ domain (referred to herein as PDZIP), which can facilitate the expression on the surface of integral plasma membrane proteins, specifically GPCRs, such as T1Rs. It is also an object to provide vectors that include PDZIP, host cells expressing said vectors, and methods for using PDZIP to facilitate surface expression.

A preferred object is to provide tests, especially high performance tests, to identify flavor modulating compounds, in particular sweet taste and umami modulating compounds. Preferably, said assays will use a combination of the T1Rs, or their fragments or variants, or genes encoding said T1Rs, or their fragments or variants, which are described herein. More preferably, said combinations will comprise hT1R1 / hT1R3 and hT1R2 / hT1R3.

A particularly preferred object is the identification of compounds that modulate the taste receptors T1R1 / T1R3 or T1R2 / T1R3, for example, that enhance the ability of these receptors to respond to taste stimuli. For example, as described below, it has been found that 5’-IMP or 5’-GMP enhances the sensitivity of umami (T1R1 / T1R3) to L-glutamate. These modulating compounds can enhance the activity of different taste stimuli of sweet or umami, and provide enhanced flavors, and / or that the same flavor is raised at a reduced concentration of the particular compound that arouses sweet or umami flavor, whose activity enhances a Taste modulator identified using the assays of the present invention.

Another object is still to provide preferred assays to evaluate one or more compounds for a flavor comprising: a step of contacting said one or more compounds with at least one of the T1Rs, their described fragmenes or variants, preferably combinations of human T1Rs.

A more specific object is to provide a method for selecting one or more compounds for their ability to enhance, mimic, block and / or modulate the perception of sweet taste in a mammal, preferably in a human being, comprising a step of contacting one or more compounds with a combination of hT1R2 and hT1R3, or a complex comprising a fragment, a chimera, or a variant of hT1R2 and / or hT1R3.

Another specific object is to provide a method for selecting one or more compounds for their ability to enhance, mimic, block and / or modulate the perception of taste, especially the perception of Umami flavor in a mammal, preferably a human being, comprising a step of contacting said one or more compounds with a combination of hT1R1 and hT1R3, or a complex comprising a fragment, a chimera, or a variant of hT1R1 and hT1R3.

Another specific object is to produce cells that coexpress hT1R2 and hT1R3, or their fragment, variant or chimera, for use to identify compounds that enhance, mimic, block and / or modulate the perception of taste, especially the perception of sweet taste.

Another specific object is to produce cells that coexpress hT1R1 and hT1R3, or their fragment, variant or chimera, for use in assays to identify compounds that enhance, mimic, block and / or modulate the perception of taste, especially the perception of Umami flavor .

Another object is to produce non-human animals that have been genetically modified to express or not express one or more T1R.

Another object is still to use a compound identified using an assay that uses T1R, or a combination thereof, as flavoring ingredients in food and beverage compositions. In particular, an object is to use a compound that interacts with hT1R2 and / or hT1R3 as a blocker, enhancer, modulator or imitator of the sweet taste, and a compound that interacts with hT1R1 and / or hT1R3 as a blocker, enhancer, modulator

or impersonator of the umami flavor, in food and beverage compositions.

Another object is to use T1R, in particular non-human T1R, to identify compounds that modulate the taste of animal feed formulations for use, for example, in fish farming.

A preferred object is to provide eukaryotic cell lines, preferably of mammalian or insect, that stably coexpress hT1R1 / hT1R3 or hT1R2 / hT1R3, preferably HEK-293 cell lines, which also express a G protein, for example, Ga15 or other protein G which, when expressed in association with T1R2 / T1R3 or T1R1 / T1R3, produces a functional gustatory receptor.

Another preferred object is to provide eukaryotic cell lines, preferably mammalian or insect, stably expressing T1R1 / T1R3 or T1R2 / T1R3, preferably hT1R1 / hT1R3 or hT1R2 / hT1R3. In a preferred embodiment, said cell lines will comprise HEK-293 cells that stably express Ga15 or other G protein that is associated with T1R1 / T1R3 or T1R2 / T1R3 to produce a sweet taste or functional umami receptor.

It is also an object to provide assays, preferably high performance assays, that employ HEK-293 or other cell lines that express stably or transiently T1R1 / T1R3 or T1R2 / T1R3, under constitutive or inducible conditions, to identify compounds that modulate taste sweet or umami.

Another specific object is to identify compounds that enhance, mimic, block and / or modulate the umami taste receptor T1R1 / T1R3, based on their ability to affect the binding of lactisol (a sweet taste inhibitor) or a related compound from the point of Structural view with the taste receptor T1R1 / T1R3 (umami).

Accordingly, the present invention provides a taste receptor consisting of at least one T1R2 polypeptide and at least one T1R3 polypeptide, wherein said taste receptor specifically binds and / or is activated by a sweet taste stimulus, in which said T1R2 polypeptide is selected from the group consisting of:

(to)

 a polypeptide having an amino acid sequence at least 85% identical to SEQ ID NO: 6, a fragment thereof having a length of at least 40 amino acids, or a fragment of an amino acid sequence that is at least 90% identical to SEQ ID NO: 6 and having a length of at least 20 amino acids;

(b)

 a polypeptide encoded by a polynucleotide that hybridizes under stringent hybridization conditions with the reverse complement of SEQ ID NO: 10; Y

(C)

a polypeptide encoded by a polynucleotide that hybridizes under stringent hybridization conditions with the reverse complement of a polynucleotide encoding the polypeptide of SEQ ID NO: 6;

and wherein said T1R3 polypeptide is selected from the group consisting of:

(d)

 a polypeptide having an amino acid sequence at least 85% identical to SEQ ID NO: 7, a fragment thereof having a length of at least 40 amino acids, or a fragment of an amino acid sequence that is at least 90% identical to SEQ ID NO: 7 and having a length of at least 20 amino acids;

(and)

 a polypeptide encoded by a polynucleotide that hybridizes under stringent hybridization conditions with the reverse complement of SEQ ID NO: 9; Y

(F)

a polypeptide encoded by a polynucleotide that hybridizes under stringent hybridization conditions with the reverse complement of a polynucleotide encoding the polypeptide of SEQ ID NO: 7.

In addition, the present invention provides a composition comprising the receptor described above, wherein at least one of the T1R polypeptides is coupled to amino acids that represent all or part of another receptor coupled to protein G. In addition, the present invention provides a host cell expressing the receptor. An expression vector is also provided comprising the nucleic acid sequence encoding the receptor.

In addition, the invention provides a method for identifying compounds that modulate the perception of taste by identifying compounds that bind, activate, inhibit and / or modulate the receptor described above.

Brief description of the figures

Figure 1 contains an alignment of human and rat T1R sequences, the human calcium detector receptor, and the rat metabotropic glutamate receptor.

Figure 2 contains the experimental results of an amplification by RT-PCR demonstrating that hT1R2 and hT1R3 are expressed in the gustatory tissue.

Figure 3a-3b contains functional data (intracellular calcium responses) produced by different sweet taste stimuli in HEK cells stably expressing Ga15 that are transiently transfected with human T1R2, T1R3, and T1R2 / T1R3 at various concentrations of sweet taste stimuli (figure 3a); dose responses of human T1R2 / T1R3 for various sweet taste stimuli (Figure 3b); the human T1R2 / T1R3 responses to sucrose in the presence of gurmarin, and the endogenous 12-adrenergic receptor responses to isoproterenol in the presence of gurmarin. Figure 3c contains the normalized response to different sweeteners.

Figure 4 contains the intracellular calcium responses in HEK cells stably expressing Ga15, transiently transfected with hT1R2 / hT1R3, rT1R2 / rT1R3, hT1R2 / rT1R3 and rT1R2 / hT1R3 in response to 350 mM sucrose, 25 mM tryptophan, 15 mM aspartame, and 0.05% monelin.

Figure 5 contains the results of an assay based on a fluorescence plate reactor, in which HEK cells stably expressing Ga15 were transiently transfected with hT1R2 and hT1R3, or only with hT1R3, and contacted Fluo-4 calcium dye and a sweet taste stimulus (12.5 mM cyclamate).

Figure 6 contains standardized dose response curves demonstrating that hT1R2 and hT1R3 act in combination as a human sweet receptor, based on their specific dose interaction with various sweet stimuli (trp, cyclamate, sucrose, neotame, asparamo, saccharin and Acek).

Figure 7 contains structural information related to mGluR1 and T1R1 demonstrating that key ligand binding moieties are observed in these molecules.

Figure 8a-8c contains functional data demonstrating that HEK cells stably expressing Ga15, which are transiently transfected with T1R1 / T1R3, respond to glutamate in an intracellular calcium based assay. Figure 8a demonstrates that intracellular calcium increases in response to increasing concentrations of glutamate; Figure 8b demonstrates that intracellular calcium responds to IMP (2 mM), glutamate (0.5 mM) and 0.2 mM IMP; and Figure 8c shows the responses of human T1R1 / hT1R3 to glutamate in the presence and absence of 0.2 mM IMP.

Figures 9a-9b, respectively, contain the results of an immunofluorescence staining assay using Myc-labeled hT1R2, and a FACS experiment demonstrating that the incorporation of the PDZIP peptide (SEQ ID NO: 6) enhances the expression of a T1R ( hT1R2) on the plasma membrane.

Figure 10a to 10b contains calcium imaging data demonstrating that hT1R2 / hT1R3 responds to different sweet stimuli.

Figure 11 shows the responses of cell lines stably expressing hT1R1 / hT1R3 by automatic fluorescence imaging, to umami flavor stimuli.

Figure 12 shows the responses of a cell line stably expressing hT1R2 / hT1R3 by automatic fluorescence imaging, to sweet taste stimuli.

Figure 13 shows the dose response curves determined using automatic fluorescence imaging, for a cell line that inductively expresses the human T1R1 / T1R3 taste receptor for L-glutamate in the presence and absence of IMP 0 , 2 mM.

Figures 14 and 15 show the response of a cell line that inductably expresses the human T1R1 / T1R3 taste receptor (clone I-17) to an L-amino acid panel. In Figure 14, different Caminoácidos were tested at 10 mM in the presence and absence of 1 mM IMP. In Figure 15, the dose responses for active amino acids in the presence of 0.2 mM IMP were determined.

Figure 16 demonstrates that lactisol inhibits the activities of human T1R2 / T1R3 and human T1R1 / T1R3 receptor.

Detailed description of the invention

Therefore, the invention provides functional taste receptors, preferably human taste receptors, which are produced by coexpression of a combination of different T1R, T1R2 / T1R3, and their corresponding isolated nucleic acid sequences comprised in expression vectors which, after coexpression , produce a functional gustatory receptor, that is, a sweet taste receptor (T1R2 / T1R3).

As indicated in the literature, members of the TCR family of GPCRs specific to taste cells are known, and are identified in Hoon et al., Cell, 96: 541-551 (1999), and in WO 00 documents / 06592, WO 00/06593, and US 2003008344 (serial number US 09 / 799,629).

More specifically, the invention relates to the coexpression of the GPCRs specific to taste cells T1R2 / T1R3. These nucleic acids and the receptors they encode are called members of the "T1R" family of taste cell-specific GPCRs. In specific embodiments of the invention, members of the T1R family that are co-expressed will include: rT1R2, rT1R3, mT1R2, mT1R3, hT1R2, and hT1R3. Although no limitation is intended by the theory, it is believed that these specific GPCRs of taste cells are components of the taste transduction pathway, and are involved in the detection of the taste of sweet flavor stimuli.

It is hereby established that members of the T1R family act in combination with other members of the T1R family to act as taste receptors for sweet and umami. As will be described in more detail below in the experimental examples, it has been shown that heterologous cells that coexpress hT1R2 and hT1R3 are selectively activated by sweet taste stimuli in a manner that reflects human sweet taste. For example, HEK-293-Ga15 cells that coexpress hT1R2 and hT1R3 respond specifically to cyclamate, sucrose, aspartame and saccharin, and dose responses for these compounds correlate with psychophysical taste thresholds. Therefore, the cells that coexpress hT1R2 and hT1R3 can be used in selections, preferably high yield selections, to identify compounds that mimic, modulate, block and / or enhance the sweet taste sensation.

In addition, as the data in the experimental examples support, it has been shown that cells that coexpress hT1R1 and hT1R3 are selectively activated by glutamate (monosodium glutamate) and by 5'-ribonucleotides in a way that reflects human umami taste. For example, HEK-293-Ga15 cells that coexpress hT1R1 and hT1R3 respond specifically to glutamate, and dose responses for this compound with umami flavor correlate with their psychophysical taste detection threshold. In addition, 5’-ribonucleotides, such as IMP, enhance the glutamate response of the T1R1 / T1R3 receptor, a characteristic synergy of the umami flavor. Thus, the cells that coexpress hT1R1 and hT1R3 can be used in selections, preferably high yield selections, to identify compounds that mimic, modulate, block and / or enhance the umami taste sensation.

In addition, as the experimental data show in the examples, it has been shown that cells that coexpress T1R1 / T1R3 stably and inducibly selectively respond to Umami taste stimuli Lglutamate and L-aspartate, and only weakly respond to other L-amino acids and at much higher concentrations, which provides further evidence that the T1R1 / T1R3 receptor can be used in assays to identify compounds that modulate (potentiate or block) umami flavor stimuli.

In addition, as experimental data support in the examples, it has been shown that cell lines that coexpress T1R1 / T1R3 or T1R2 / T1R3 respectively respond to stimuli of sweet or sweet taste and in a quantitative dose response manner, which further supports the conclusion that the T1R1 / T1R3 and T1R2 / T1R3 receptors can be used to identify agonists and receptor antagonists, for example, MSG substitutes, umami flavor blockers, new artificial and natural sweeteners, and sweet taste blockers.

In addition, as supported by the data in the experimental examples, it has been shown that the lactisol sweet taste blocker inhibits the sweet taste receptor T1R2 / T1R3 and the taste taste receptor of the Umami T1R1 / T1R3. This suggests that tests that select compounds that affect the binding of lactisol to T1R2 / T1R3 or T1R1 / T1R3 can be used to identify compounds that potentiate, mimic, modulate or block the sweet or umami taste. The fact that lactisol inhibits the T1R1 / T1R3 and T1R2 / T1R3 receptors suggests that these receptors may share a common subunit to which lactisol binds and potentially other taste modulators. Therefore, this suggests that some compounds that enhance, mimic, modulate or block the sweet taste may have a similar effect on the umami flavor or vice versa.

In addition, as the data in the experimental examples support, it has been shown that cell lines that stably coexpress T1R, that is, T1R1 / T1R3 or T1R2 / T1R3, when tested by formation of automatic fluorescence images respond very effectively to various stimuli of sweet taste and umami, that is, at magnitudes substantially greater than transiently transfected cells. Therefore, these cell lines are especially suitable for use in high performance screening assays to identify compounds that modulate, block, mimic or potentiate the sweet or umami flavor. However, the invention also includes assays using cells that transiently express a T1R2 / T1R3 receptor.

In addition, although the application contains data demonstrating that some T1Rs act in combination, in particular T1R1 / T1R3 and T1R2 / T1R3, and that these receptor combinations can be used in assays, preferably high performance assays, it should be noted that the present application also provides for tests that use T1R1, T1R2 and T1R3 individually or in combination with other proteins, for example, other GPCRs.

Compounds identified with T1R assays can be used to modulate the taste of food and beverages. Suitable assays described in more detail below include, as an example, whole cell assays and biochemical assays, including direct binding assays using one of a combination of different T1R receptors, their chimeras or fragments, especially fragments containing binding domains. to the N-terminal ligand. Examples of tests suitable for use in the invention are described in more detail below and are known in the field of GPCRs.

The assays can be designed to quantify the binding of different compounds or mixtures of compounds to T1R taste receptors or combinations of T1R taste receptors or T1R receptors expressed in combination with other heterologous proteins (other than T1R), for example, other GPCRs, or for quantify the activation of cells expressing T1R taste receptors. This can be done by stable expression.

or transient taste receptors in heterologous cells, such as HEK-293, CHO and COS cells.

The assays will preferably also use cells that also express (preferably stably) a G protein, such as Ga15 or Ga16 or other promiscuous G proteins or G protein variants, or an endogenous G protein. In addition they can also express G1 and Gy proteins.

The effect of a compound on sweet taste or umami using cells or compositions that express or contain the receptors or receptor combinations identified above can be determined by various means, including the use of calcium sensitive dyes, voltage sensitive dyes, assays of CAMP, direct binding assays using fluorescently labeled ligands or radioactive ligands, such as 3Hglutamate, or transcriptional assays (using a suitable indicator, such as luciferase or beta-lactamase).

Assays that can be used with one or more T1R according to the description include, as an example, assays that use a genetic selection for living cells; assays using whole cells or fragments of purified T1R membranes or proteins; assays using second messengers, such as cAMP and IP3, assays that detect the translocation of arrestin to the cell surface, assays that detect loss of receptor expression on the cell surface (internalization) by tested ligands, ligand binding assays direct, competitive binding assays with inhibitors, assays using a protein translated in vitro, assays that detect conformational changes after ligand binding (e.g., as evidenced by proteolysis, fluorescence, or NMR), behavioral tests that use transgenic non-human animals that express a T1R or a combination of T1R, such as flies, worms or mice, or assays using cells infected with recombinant viruses that contain T1R genes.

Structure-based analyzes are also considered, in which the X-ray crystal structure of a T1R or a T1R fragment (or a combination of T1R, or a combination of a T1R with another protein) is determined and used to predict, by means of molecular modeling techniques, the compounds that will bind and / or enhance, mimic, block or modulate the particular receptor or combination of T1R receptors. More specifically, the description provides for the determination of the crystalline structure of T1R1 / T1R3 (preferably hT1R1 / hT1R3) and / or T1R2 / T1R3 (preferably hT1R2 / hT1R3), and the use of said crystalline structures in design methods based on structure to identify molecules that modulate the activity of the T1R receptor.

The invention includes, in particular, biochemical assays performed using cells, for example, mammalian, yeast, insect or other heterologous cells that express the taste receptors of the present invention, preferably receptors comprising the N-terminal domains of T1R2 and / or T1R3. The effect of a compound in these assays can be determined using competitive binding assays, for example, using glutamate or radioactive IMP, fluorescence (eg, fluorescence polarization, FRET), or GTPy35S binding assays. As indicated, in a preferred embodiment, these assays will use cell lines that stably coexpress T1R2 / T1R3 and a suitable G protein, such as Ga15. Other suitable G proteins include the chimeric G proteins and variants described in US 2002/0143151 (US applications serial number 09 / 984,292 and 60 / 243,770).

In addition, altered receptors having improved properties can be constructed and expressed, for example, enhanced surface expression or G-protein coupling. These T1R variants can be incorporated into cell-based and biochemical assays.

It is anticipated that the findings herein that are related to human T1R will be extended to other species, for example, to rodents, pigs, monkeys, dogs and cats, and perhaps other non-mammals, such as fish. In this regard, in the following example 1 several fish T1R fragments are identified. Therefore, the present invention has application in the selection of compounds for use in animal feed formulations.

The invention also includes the use of different allelic variants of T1R2 and T1R3, and combinations thereof, thereby allowing the identification of compounds that cause a specific taste sensation in individuals expressing these allelic variants, or compounds that cause specific taste sensations in all individuals These compounds can be used to make foods that are generally tastier.

Nucleic acids encoding T1R also provide valuable probes for the identification of taste cells, since nucleic acids are specifically expressed in taste cells. For example, probes for T1R polypeptides and proteins can be used to identify taste cells present in foliated, circumvalated and fungiform papillae, as well as taste cells present in the taste strip, the oral cavity, the gastrointestinal epithelium, and the epiglottis. In particular, methods for detecting T1R can be used to identify taste cells sensitive to sweet taste stimuli and / or umami or other flavor stimuli that represent other taste modalities. For example, from the work presented herein, it can be predicted that cells stably or transiently expressing T1R2 and / or T1R3 will respond to umami taste stimuli. Similarly, it can be predicted that cells expressing T1R1 and / or T1R3 will respond to umami taste stimuli. The nucleic acids encoding the T1R proteins and polypeptides of the invention can be isolated from a variety of sources, can be genetically modified, amplified, synthesized and / or expressed recombinantly according to the methods described in WO 00/035374 .

The examples provide a list of T1R2 and T1R3 that can be expressed according to the invention. However, it should be emphasized that the invention includes the expression and use of other specific T1R2 and T1R3 or of fragments, variants or chimeras constructed based on said T1R sequences and, in particular, the T1Rs of other species having the length and length Percent sequence identity or hybridization capacity required.

As described, an important aspect of the invention is the plurality of modulator selection methods, for example, activators, inhibitors, stimulants, enhancers, agonists and antagonists, of these GPCRs specific to taste cells. These taste transduction modulators are useful for modulating taste signaling pathways. These selection methods can be used to identify agonists and antagonists of high affinity of taste cell activity. These modulating compounds can then be used in the food industry to adapt the taste, for example, to modulate the sweet taste of food.

This invention rectifies the lack of prior understanding regarding sweet taste, since it identifies T1R receptors and specific T1R receptor combinations that mediate the sweet taste sensation. Therefore, in general, this application refers to the inventors' discoveries in relation to the T1R class of taste-specific G-protein coupled receptors and their specific function in the perception of taste and the relationship of these discoveries to a better understanding of the molecular basis of taste.

The molecular basis of sweet taste and umami taste (the taste of monosodium glutamate) is enigmatic. Recently, a class of three taste-specific G-protein coupled receptors, called T1R, has been identified. The overlapping of T1R expression patterns and the demonstration that the GABAB receptor, structurally related, is heterodimeric, suggest that T1Rs act as heterodimeric taste receptors. In the examples that follow, the inventors herein describe the functional coexpression of human T1R1, T1R2 and T1R3 in heterologous cells; the cells that coexpress T1R1 and T1R3 are activated by stimuli of umami flavor; and the cells that coexpress T1R2 and T1R3 are activated by sweet taste stimuli. The activity T1R1 / T1R3 and T1R2 / T1R3 correlates with the psychophysical detection thresholds. In addition, it has been discovered that IMP 5’-ribonucleotide enhances the response of T1R1 / T1R3 to glutamate, a characteristic synergy of umami flavor. These findings demonstrate that specific T1Rs and, in particular, different combinations of T1Rs act as taste receptors for sweet and umami.

It is believed that the human perception of bitter, sweet and umami taste is mediated by G-protein coupled receptors (Lindemann, B., Physiol. Rev., 76: 718-766 (1996)). In recent dates, the evaluation of the human genome revealed the T2R class of bitter taste taste receptors (Adler et al., Cell, 100: 613-702 (2000); Chandrasgekar et al., Cell, 100: 703-711 (2000 ); Matsunami et al., Nature, 404: 601-604 (2000)), but taste receptors for sweet and umami taste have not been identified. Recently, another class of candidate taste receptors, the T1Rs, has been identified. The T1Rs were first identified large-scale sequencing of a cDNA library derived from rat gustatory tissue, which identified T1R1, and subsequently by degenerate PCR based on T1R1, which led to the identification of T1R2 (Hoon et al., Cell, 96: 541-551 (1999)). Recently, the inventors of the present invention and others identified a third -and possibly final-member of the T1R family, T1R3, in the human genome database (Kitagawa et al., Biochem. Biophys. Res. Commun. , 283 (1): 236-242 (2001); Max et al., Nat. Genet., 28 (1): 58-63 (2001); Sainz et al., J. Neurochem., 77 (3): 896-903 (2001); Montmayeur et al., Nat. Neurosci., 4, 492-498 (2001)). Revealingly, rat T1R3 maps in a genomic range containing Sac, a locus that influences the sweet taste in the mouse (Fuller et al., J. Hered., 65: 33-36 (1974); Li et al., Mamm. Genome, 12: 13-16 (2001)). Therefore, it was predicted that T1R3 would act as a sweet taste receptor. Recent studies of high-resolution genetic mapping have reinforced the connection between mouse T1R3 and Sac (Fuller TC, J. Hered., 65 (1): 33-36 (1974); Li et al., Mamm. Genome, 12 ( 1): 13-16 (2001)).

Interestingly, all family C receptors that have been functionally expressed to date metabotropic glutamate receptors, the GABAB receptor, the calcium detector receptor (Conigrave, A.D., Quinn,

S.J. and Brown, E.M., Proc. Natl Acad. Sci. USA, 97, 4814-4819 (2000)), and an olfactory fish receptor (Speca, D.J. et al., Neuron, 23, 487-498 (1999)) - have been shown to be activated by amino acids. This common feature raises the possibility that T1R recognize amino acids, and that T1R may be involved in the detection of glutamate in addition to the sweet-tasting amino acids. Alternatively, it has been proposed that a transcriptional variant of the mGluR4 metabotropic glutamate receptor be the umami taste receptor due to its selective expression in rat taste tissue, and the similarity of the receptor activation threshold with the psychophysical detection threshold of the glutamate (Chaudhari et al., Nat. Neurosci., 3: 113-119 (2000)). This hypothesis is difficult to reconcile with the extremely low expression level of the mGluR4 variant in gustatory tissue, and the more or less unaltered glutamate taste of mice with inactivated mGluR4 (Chaudhari and Roper, Ann. NY Acad. Sci., 855: 398-406 (1998)). In addition, the gustatory variant is unlikely from the structural point of view, not only because it lacks most of the remains that form the glutamate binding pocket of the wild-type receptor, but also because it lacks approximately half of the domain of Globular N-terminal glutamate binding (Kunishima et al., Nature, 407: 971-977 (2000)).

A comparative analysis of the expression patterns of T1R in rodents has shown that each T1R2, and probably T1R1, is coexpressed with T1R3 (Hoon et al., Cell, 96: 541-551 (1999); Kitagawa et al., Biochem. Biophys. Res. Commun., 283: 236-242 (2001); Max et al., Nat. Genet., 28: 58-63 (2001); Montmayeur et al., Nat. Neurosci., 4: 492-498 (2001); Sainz et al., J. Neurochem., 77: 896-903 (2001)). In addition, dimerization is emerging as a common theme of family C receptors: the metabotropic glutamate receptor and the calcium detector receptor are homodimers (Romomano et al., J. Biol. Chem., 271: 28612-28616 ( 1996); Okamoto et al., J. Biol. Chem., 273: 13089-13096 (1998); Han et al., J. Biol. Chem., 274: 100008-100013 (1999); Bai et al., J. Biol. Chem., 273: 23605-23610 (1998)), and the structurally related GABAB receptor is heterodimeric (Jones et al., Nature, 396: 674-679 (1998); Kaupmann et al., Nature, 396: 683-687 (1998); White et al., Nature, 396: 679-682 (1998); Kuner et al., Science, 283: 74-77 (1999)). The inventors of the present invention have demonstrated, by functional coexpression of T1R in heterologous cells, that human T1R2 acts in combination with human T1R3 as a taste taste of sweet, and that human T1R1 acts in combination with human T1R3 as receptor Gustatory of Umami.

The findings analyzed herein are especially significant, since previously the development of improved artificial sweeteners has been hampered by the lack of sweet taste tests. Indeed, the five commercial artificial sweeteners that are most frequently used, all of which activate hT1R2 / hT1R3, were discovered by chance by chance. Similarly, there are no other tests, apart from sensory tests, which are a very laborious process, to identify compounds that modulate umami taste. These problems are now alleviated because, as established by the experimental results analyzed below, human sweet and umami taste receptors have been identified, and trials for these receptors have been developed, in particular assays that employ cells expressing stable form a functional T1R gustatory receptor, that is, the sweet or umami taste receptor.

Based on these, the invention provides assays for detecting and characterizing flavor modulating compounds, in which members of the T1R family act, as they do in the taste bud, as indicator molecules for the effect on sweet and umami taste. of flavor modulating compounds. In particular, tests to identify compounds that modulate, mimic, potentiate and / or block the sweet taste are provided and are within the invention. The methods for testing GPCR activity, and especially compounds that affect GPCR activity, are well known and are applicable to the member of the T1R family of the present invention and their functional combinations. Previously suitable trials have been identified.

In particular, the GPCRs of the present invention can be used in assays, for example to measure changes in ligand binding, ionic concentration, membrane potential, current flow, ion flow, transcription, interactions. of ligand-receptor, second messenger concentrations, in vitro and in vivo. In another embodiment, members of the T1R family can be expressed recombinantly in cells, and modulation of taste transduction through GPCR activity can be assayed by measuring changes in Ca2 + levels and other intracellular messengers, such as cAMP. , CGMP, or IP3.

In certain assays, a domain of a T1R polypeptide, for example, an extracellular, transmembrane or intracellular domain, is condensed with a heterologous polypeptide, thereby forming a chimeric polypeptide, for example, a chimeric protein with GPCR activity. In particular, the use of fragments of T1R1, T1R2 or T1R3 containing the N-terminal ligand binding domain is contemplated. These proteins are useful, for example, in assays to identify ligands, agonists, antagonists or other T1R receptor modulators. For example, a T1R polypeptide can be expressed in a eukaryotic cell as a chimeric receptor with a heterologous chaperone sequence that facilitates plasma membrane traffic, or maturation and directed transport through the secretory pathway. The optional heterologous sequence may be a peptide that interacts with the PDZ domain, such as a fragment of the C-terminal PDZIP (SEQ ID NO: 1). PDZIP is an ER export signal that, according to the present description, has been shown to facilitate surface expression of heterologous proteins, such as the T1R receptors described herein. More specifically, in one aspect of the description, PDZIP can be used to stimulate suitably targeted transport of problematic membrane proteins, such as olfactory receptors, T2R taste receptors, and T1R taste receptors described herein.

These chimeric T1R receptors can be expressed in any eukaryotic cell, such as HEK-293 cells. Preferably, cells containing a G protein, preferably a promiscuous G protein, such as Ga15

or Ga16 or other promiscuous G protein capable of connecting a wide range of GPCR to an intracellular signaling pathway or a signaling protein, such as phospholipase C. The activation of these chimeric receptors in these cells can be detected using any conventional method, such as by detecting changes in intracellular calcium by detecting FURA-2 dependent fluorescence in the cell. If the preferred host cells do not express an appropriate G protein, they can be transfected with a gene encoding a promiscuous G protein, such as those described in US 2002028121 (US Application Serial No. 09 / 984,297, filed October 29, 2001) and US 20020143151 (US application serial number 09 / 989.497, filed on November 21, 2001).

Other methods for testing taste transduction modulators include in vitro ligand binding assays that employ T1R polypeptides, portions thereof, that is, the extracellular domain, the transmembrane region or combinations thereof, or chimeric proteins comprising one or more. domains of a member of the T1R family; oocytes or tissue culture cells expressing T1R polypeptides, fragments, or fusion proteins; phosphorylation and dephosphorylation of members of the T1R family; G protein that binds to GPCR; ligand binding assays; changes in voltage, membrane potential and conductance; ion flow assays; changes in the second intracellular messengers, such as cGMP, cAMP and inositol triphosphate (IP3); and changes in intracellular calcium levels.

In addition, the description provides methods for detecting the expression of T1R proteins and nucleic acids, which allow investigation of the regulation of taste transduction and the specific identification of taste receptor cells. Members of the T1R family also provide nucleic acid probes useful for paternity and forensic investigations. The T1R genes are also useful as nucleic acid probes to identify taste receptor cells, such as foliate, fungiform, circumvallated taste receptor cells, the taste strip and the epiglottis. T1R receptors can also be used to generate monoclonal and polyclonal antibodies useful for identifying taste receptor cells.

From a functional point of view, T1R polypeptides comprise a family of related G 7-transmembrane protein coupled receptors, which are believed to be involved in taste transduction and that can interact with G protein to mediate the transduction of taste signals (see, for example, Fong, Cell Signal, 8: 217 (1996); Baldwin, Curr. Opin. Cell Biol., 6: 180 (1994)). From a structural point of view, the nucleotide sequences of the members of the T1R family encode related polypeptides comprising an extracellular domain, 7-transmembrane domains, and a cytoplasmic domain. The related T1R family genes of other species share at least about 50%, and optionally 60%, 70%, 80% or 90% match in the nucleotide sequence through a region of at least about 50 nucleotides in length , optionally 100, 200, 500 or more nucleotides in length, with the T1R nucleic acid sequences described in the examples herein, or their conservatively modified variants, or encode polypeptides that share at least about 35% at 50%, and optionally 60%, 70%, 80% or 90% coincidence in the amino acid sequence along a region of at least 25 amino acids in length, optionally 50 to 100 amino acids in length, with a sequence of T1R polypeptide described below in the examples, or its conservatively modified variants.

Several domains or consensus amino acid sequences that are characteristic of members of the T1R family have also been identified. For example, members of the T1R family generally comprise a sequence that has at least about 50%, optionally 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95-99% or more coincidence with the consensus sequences of T1R 1 and 2 (SEQ ID NO: 2 and 3, respectively). Therefore, these conserved domains can be used to identify members of the T1R family, by coincidence, specific hybridization or amplification, or specific binding by antibodies generated against a domain. The T1R consensus sequences include, as an example, the following sequences:

Consensus sequence of family T1R 1: (SEQ ID NO: 2)

(TR) C (FL) (RQP) R (RT) (SPV) (VERKT) FL (AE) (WL) (RHG) E

Consensus sequence of the T1R 2 family: (SEQ ID NO: 3)

(LQ) P (EGT) (NRC) YN (RE) A (RK) (CGF) (VLI) T (FL) (AS) (ML)

These consensus sequences include those found in the T1R polypeptides described herein, but members of the T1R family from other organisms can be expected to comprise consensus sequences that have approximately 75% or more matches with the included consensus sequences. specifically described herein.

Specific regions of the nucleotide and amino acid sequences of T1R can be used to identify polymorphic variants, interspecific homologs, and alleles of members of the T1R family. This identification can be performed in vitro, for example, under stringent hybridization or PCR conditions (for example, using primers encoding the T1R consensus sequences identified above), or using the sequence information in a computer system for comparison with other sequences. of nucleotides The different alleles of the T1R genes within a single population of a species will also be useful to determine whether differences in allelic sequences control differences in taste perception among members of the population. PCR amplification and classical cloning techniques are useful for isolating new T1Rs, for example, when degenerate primers are sufficient to detect genes related between species.

Generally, the identification of polymorphic variants and alleles of the members of the T1R family can be made by comparing an amino acid sequence of approximately 25 amino acids or more, for example, 50-100 amino acids. An amino acid match of about at least 35% to 50%, and optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99% or more generally demonstrates that a protein is a polymorphic variant, an interspecific counterpart, or an allele of a member of the T1R family. A sequence comparison can be performed using any of the sequence comparison algorithms discussed below. Antibodies that specifically bind T1R polypeptides or conserved regions thereof can also be used to identify alleles, interspecific homologs and polymorphic variants.

Polymorphic variants, interspecific homologs and alleles of T1R genes can be confirmed by studying the specific expression of taste cells of the T1R gene or protein of assumptions. Generally, T1R polypeptides having an amino acid sequence described herein can be used as a positive control compared to the putative T1R polypeptide, to demonstrate the identification of a polymorphic variant or an allele of the member of the T1R family. Polymorphic variants, interspecific homologs and alleles are expected to retain the 7-transmembrane structure of a G-protein coupled receptor. For more details, see WO 00/06592, which describes related T1R family members, GPCR -B3, whose contents are incorporated herein by reference in a manner consistent with this description. Here, GPCR-B3 receptors are called rT1R1 and mT1R1. In addition, see WO 00/06593, which also describes members of the related T1R family, GPCR-B4. GPCR-B4 receptors are referred to herein as rT1R2 and mT1R2. As previously analyzed, the description also includes structure-based assays that use the X-ray crystal structure of a T1R or a combination of T1R, for example, hT1R2 / hT1R3 or hT1R1 / hT1R3, to identify molecules that modulate the activity of the T1R receptor and, therefore, that modulate the sweet and / or umami flavor.

The present invention also provides assays, preferably high performance assays, to identify molecules that potentiate, mimic, block and / or modulate T1R receptors. In some assays, a specific domain of a member of the T1R family is used in combination with a particular domain of another member of the T1R family, for example, an extracellular, transmembrane or intracellular region or domain. In other embodiments, an extracellular domain, a transmembrane region or a combination thereof can be bound to a solid substrate and used, for example, to isolate ligands, agonists, antagonists or any other molecule that can bind to a T1R polypeptide and / or Modulate your activity.

It is envisioned that various conservative mutations and substitutions are within the scope of the invention. For example, it is within the level of those skilled in the art to make amino acid substitutions using known protocols of recombinant gene technology that includes PCR, gene cloning, site-directed specific cDNA mutagenesis, host cell transfection, and in-transcription vitro. Then, the variants can be selected to determine their activity.

Definitions

As used herein, the following terms and expressions have the meanings attributed to them, unless otherwise indicated.

"Taste cells" include neuroepithelial cells that are organized into groups to form taste buds in the tongue, for example, foliated, fungiform and circumvalated cells (see, for example, Roper et al., Ann. Rev. Neurosci., 12: 329-353 (1989)). Taste cells are also found in the palate and other tissues, such as the esophagus and stomach.

"T1R" refers to one or more members of the family of G-protein coupled receptors that are expressed in gustatory cells, such as foliated, fungiform and circumvalated cells, as well as in palate and esophageal cells (see, for example, Hoon et al., Cell, 96: 541-551 (1999)). Family members are also referred to as GPCR-B3 and TR1 in WO 00/06592, as well as GPCR-B4 and TR2 in WO 00/06593. Taste receptor cells can also be identified based on morphology (see, for example, Roper, supra), or by the expression of proteins specifically expressed in taste cells. Members of the T1R family may have the ability to act as receptors for sweet taste transduction, or to distinguish between various other taste modalities. Representative T1R sequences, which include hT1R1, hT1R2 and hT1R3, are identified below in the examples.

"T1R" nucleic acids encode a GPCR family with 7-transmembrane regions that have a "G-protein coupled receptor activity," for example, can bind to G proteins in response to extracellular stimuli and stimulate second messenger production. , such as IP3, cAMP, cGMP and Ca2 + by stimulating enzymes, such as phospholipase C and adenylate cyclase (for a description of the structure and function of GPCRs see, for example, Fong, supra, and Baldwin, supra). A single taste cell can contain many differentiated T1R polypeptides.

The term "T1R family" therefore refers to polymorphic variants, alleles, mutants, and interspecific homologs that: (1) have at least about 35% to 50% coincidence in the amino acid sequence, optionally about 60%, 75% , 80%, 90%, 96%, 97%, 98% or 99% coincidence in the amino acid sequence, with a T1R polypeptide, preferably those identified in example 1, through a window of approximately 25 amino acids, optionally 50-100 amino acids; (2) specifically bind antibodies generated against an immunogen comprising an amino acid sequence preferably selected from the group consisting of the sequence of the T1R polypeptide described in Example 1 and its conservatively modified variants; (3) are encoded by a nucleic acid molecule that specifically hybridizes (with a size of at least about 100, optionally at least about 500-1000 nucleotides) under stringent hybridization conditions, with a sequence selected from the group consisting of T1R nucleic acid sequences contained in example 1, and their conservatively modified variants; or (4) comprise a sequence at least about 35% to 50% identical with an amino acid sequence selected from the group consisting of the amino acid sequence of T1R identified in example 1.

From the topological point of view, certain chemosensory GPCRs have an "N-terminal domain"; "Extracellular domains"; "Transmembrane domains" comprising 7-transmembrane regions, and corresponding cytoplasmic and extracellular loops; "Cytoplasmic domains", and a "C-terminal domain" (see, for example, Hoon et al., Cell, 96: 541-551 (1999); Buck and Axel, Cell, 65: 175-187 (1991)) . These domains can be structurally identified using methods known to those skilled in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains (see, for example, Stryer, Biochemistry (3rd ed., 1988); see also any of one series of sequence analysis programs based on the Internet, such as those found in dot.imgen.bcm.tmc.edu). These domains are useful for manufacturing chimeric proteins and for in vitro assays of the invention, for example, ligand binding assays.

Thus, "extracellular domains" refer to the domains of T1R polypeptides that protrude from the cell membrane and are exposed to the extracellular face of the cell. These domains generally include the "N-terminal domain" that is exposed to the extracellular face of the cell, and may optionally include portions of the extracellular loops of the transmembrane domain that are exposed to the extracellular face of the cell, that is, the loops between transmembrane regions 2 and 3, between transmembrane regions 4 and 5, and between transmembrane regions 6 and 7.

The "N-terminal domain" region begins at the N-terminal and extends to a region near the beginning of the first transmembrane domain. More specifically, in one embodiment of the invention, this domain begins at the N-terminal and ends approximately in the glutamic acid conserved at amino acid position 563 or so about 20 amino acids. These extracellular domains are useful for ligand binding assays in vitro, both in the soluble phase and in the solid phase. In addition, the transmembrane regions, described below, can also bind ligands in combination with the extracellular domain and, therefore, are also useful for in vitro ligand binding assays.

The "transmembrane domain," which comprises the seven "transmembrane regions," refers to the T1R polypeptide domain that extends across the plasma membrane, and may also include the corresponding cytoplasmic (intracellular) and extracellular loops. In one embodiment, this region corresponds to the domain of members of the T1R family that begins approximately in the glutamic acid conserved at amino acid position 536 plus or minus 20 amino acids, and ends approximately in the tyrosine moiety conserved at position 812 about 10 amino acids. The seven transmembrane regions and the extracellular and intracellular loops can be identified using conventional methods, as described in Kyte and Doolittle, J. Mol. Biol., 157: 105-132 (1982), or in Stryer, supra.

"Cytoplasmic domains" refer to domains of the T1R polypeptides that face the inner face of the cell, for example, the "C-terminal domain" and intracellular loops of the transmembrane domain, for example, the intracellular loop between the transmembrane regions 1 and 2, the intracellular loop between transmembrane regions 3 and 4, and the intracellular loop between transmembrane regions 5 and 6. The "C-terminal domain" refers to the region that encompasses the end of the last transmembrane domain and C -terminal protein, and that is normally located within the cytoplasm. In one embodiment, this region begins at the amino acid residue tyrosine conserved at position 812 or so about 10 amino acids and continues to the C-terminal of the polypeptide.

The term "ligand binding region" or "ligand binding domain" refers to sequences derived from a taste receptor, in particular a taste receptor that substantially incorporates at least the extracellular domain of the receptor. In one embodiment, the extracellular domain of the ligand binding region may include the N-terminal domain and, optionally, portions of the transmembrane domain, such as the extracellular loops of the transmembrane domain. The ligand binding region may be able to bind a ligand, and more specifically, a compound that enhances, mimics, blocks and / or modulates taste, for example, sweet taste or umami.

The term "heteromultimer" or the expression "heteromultimeric complex" in the context of the T1R receptors or T1R polypeptides of the invention, refers to a functional association of at least one T1R receptor and another receptor, generally another polypeptide of a T1R receptor. (or, alternatively, another entity that is not a T1R receptor polypeptide). To make it clearer, the functional codependency of the T1Rs is described in this application in a way that reflects their possible function as heterodimeric taste receptor complexes.

The term "functional effects" in the context of tests to test compounds that modulate the transduction of taste mediated by members of the T1R family includes the determination of any parameter that is directly or indirectly under the influence of the receptor, for example, functional effects , physicists and chemists. It includes ligand binding, changes in ion flow, membrane potential, current flow, transcription, G protein binding, phosphorylation or dephosphorylation of GPCR, assays based on conformational changes, signal transduction, receptor-ligand interactions, concentrations of second messengers (e.g. cAMP, cGMP, IP3, or intracellular Ca2 +), in vitro, in vivo and ex vivo, and also includes other physiological effects, such as increases or decreases in the release of neurotransmitters or hormones.

"Determination of the functional effect" in the context of the tests means a test for a compound that increases or decreases a parameter that is directly or indirectly under the influence of a member of the T1R family, for example, functional, physical and physical effects. Chemicals These functional effects can be measured by any means known to those skilled in the art, for example, changes in spectroscopic characteristics (eg, fluorescence, absorbance, refractory index), hydrodynamics (eg, shape), chromatographic, or properties of solubility, fixation of patches, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, expression of the T1R gene in oocytes; T1R expression in tissue culture cells; transcriptional activation of T1R genes; ligand binding assays; changes in voltage, membrane potential and conductance; ion flow assays; changes in the second intracellular messengers, such as cGMP, cAMP and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, conformational assays, and the like.

The "inhibitors", "activators" and "modulators" of T1R genes or proteins are used to refer to inhibitory, activating or modulating molecules identified using in vitro and in vivo assays for taste transduction, for example, ligands, agonists, antagonists, and their counterparts and mimetics.

Inhibitors are compounds that, for example, bind, partially or totally block stimulation, diminish, prevent, delay activation, inactivate, desensitize or under-regulate taste transduction, for example, antagonists. Activators are compounds that, for example, bind, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or overregulate the transduction of taste, for example, agonists. Modulators include compounds that, for example, alter the interaction of a receptor with extracellular proteins that bind activators or inhibitors (eg, ebnerin and other members of the family of hydrophobic carriers); G proteins; kinases (eg, rhodopsin kinase homologs and beta-adrenergic receptor kinases that are involved in the deactivation and desensitization of a receptor); and arrests, which also deactivate and desensitize receptors. Modulators may include genetically modified versions of members of the T1R family, for example, with altered activity, as well as natural and synthetic ligands, antagonists, agonists, small chemical molecules, and the like. These assays for inhibitors and activators include, for example, expressing members of the T1R family in cells or cell membranes, applying putative modulating compounds, in the presence or absence of taste stimulants, for example, sweet taste stimulants, and then determining the functional effects on taste transduction, as described above. Samples or assays comprising members of the T1R family that are treated with a potential activator, inhibitor or modulator are compared with control samples without the inhibitor, activator.

or modulator to study the degree of modulation. Positive control samples (for example, a sweet taste stimulant without added modulators) are assigned a relative T1R activity value of 100%.

Negative control samples (for example, buffer without an added gustatory stimulus) are assigned a relative T1R activity value of 0%. Inhibition of a T1R is achieved when a mixture of the positive control sample and a modulator produces a T1R activity value relative to the positive control of approximately 80%, optionally 50% or 25-0%. The activation of a T1R by a single modulator is achieved when the T1R activity value in relation to the negative control sample is 10%, 25%, 50%, 75%, optionally 100%, optionally 150%, optionally 200-500 %, or 1000-3000% higher.

The terms and the term "purified", "substantially purified" and "isolated", as used herein, refer to the state of being exempt from other different compounds with which the compound of the invention is normally associated in its natural state, so that the subject "purified", "substantially purified" and "isolated" comprises at least 0.5%, 1%, 5%, 10% or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample. In a preferred embodiment, these terms and expression refer to the compound of the invention comprising at least 95% of the mass, by weight, of a given sample. As used herein, the terms and the term "purified", "substantially purified" and "isolated", when referring to a nucleic acid or a protein, also refer to a state of purification or different concentration than which occurs naturally in the body of a mammal, especially a human being. Any degree of purification or concentration greater than that which occurs naturally in the body of a mammal, especially a human being, including (1) purification of other structures or associated compounds, or (2) association with structures or compounds with which it is not normally associated in the body of a mammal, especially a human being, is within the meaning of "isolated." The nucleic acid or protein, or the classes of nucleic acids or proteins described herein, may be isolated, or otherwise associated with structures or compounds with which they are not normally associated in nature, according to a variety of methods and processes known to those skilled in the art.

The terms "nucleic acid" or "nucleic acid sequence" refer to an oligonucleotide of deoxyribonucleotides or ribonucleotides in single or double stranded form. Expressions include nucleic acids, that is, oligonucleotides, which contain known analogs of natural nucleotides. Expressions also include nucleic acid-like structures with synthetic skeletons (see, for example, Oligonucleotides and Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the

N.Y. Academy of Sciences, vol. 660, eds. Baserga et al. (NYAS, 1992); Milligan, J. Med. Chem., 36: 1923-1937 (1993); Antisense Research and Applications (1993, CRC Press), WO 97/03211; WO 96/39154; Kill, Toxicol. Appl. Pharmacol., 144: 189-197 (1997); Strauss-Soukup, Biochemistry, 36: 8692-8698 (1997); Samstag, Antisense Nucleic Acid Drug Dev., 6: 153-156 (1996)).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating, for example, sequences in which the third position of one or more selected codons is substituted with residues of mixed bases and / or deoxyinosine (Batzer et al., Nucleic Acids Res. , 19: 5018 (1991); Ohtsuka et al., J. Biol. Chem., 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8: 9198 (1994)). The term "nucleic acid" is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical mimetic of a corresponding natural amino acid, as well as natural amino acid polymers and non-natural amino acid polymers.

The term "plasma membrane translocation domain" or simply "translocation domain" means a polypeptide domain that when incorporated into a coding sequence of a polypeptide can "chaperonar" or "translocate" more efficiently the hybrid protein ("fusion" ") Towards the cellular plasma membrane without the present domain. For example, a "translocation domain" can be derived from the amino terminal of the bovine rhodopsin receptor polypeptide, a 7-transmembrane receptor. However, rhodopsin from any mammal can be used, as well as other sequences that facilitate translocation. Therefore, the translocation domain is particularly effective for translocating 7-transmembrane fusion proteins to the plasma membrane, and a protein (for example, a taste receptor polypeptide) comprising an amino-terminal translocation domain will be transported to the membrane. plasma more effectively than without the domain present. However, if the N-terminal domain of the polypeptide is active after binding, as is the case with the T1R receptors of the present invention, the use of other translocation domains may be preferred. For example, an interaction peptide with the PDZ domain can be employed, as described herein.

The "translocation domain", the "ligand binding domain", and the chimeric receptor compositions described herein also include "analogs", or "conservative variants" and "mimetics" ("peptidomimetics") with structures and an activity that substantially correspond to the example sequences. Thus, the term "conservative variant" or the terms "analogous" or "mimetic" refers to a polypeptide having a modified amino acid sequence, such that the change or changes do not substantially alter the structure and / or activity of the polypeptide. (the conservative variant), as defined herein. These include conservatively modified variations of an amino acid sequence, that is, amino acid substitutions, additions or deletions of residues that are not critical to protein activity, or amino acid substitution with residues that have similar properties (by for example, acids, basic, positively or negatively charged, polar or non-polar, etc.), so that even critical amino acid substitutions do not substantially alter the structure and / or activity.

More specifically, the term "conservatively modified variants" applies to amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to nucleic acids encoding identical or fundamentally identical amino acid sequences, or when the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.

For example, all codons GCA, GCC, GCG and GCU encode the amino acid alanine. Thus, at each position where a codon specifies an alanine, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.

These variations in nucleic acids are "silent variations," which are a type of conservatively modified variation. Each nucleic acid sequence herein, which encodes a polypeptide, also describes any possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be modified to produce a functionally identical molecule. Accordingly, each silent variation of a nucleic acid, which encodes a polypeptide, is implicit in each sequence described.

Conservative substitution tables that provide functionally similar amino acids are well known in the art. For example, an example of a guide for selecting conservative substitutions includes (the original remainder followed by the substitution example): ala / gly or ser; arg / lys; asn / gln or his; asp / glu; cys / ser; gln / asn; gly / asp; gly / ala or pro; his / asn or gln; ile / leu or val; leu / ile or val; lys / arg or gln or glu; met / leu or tyr or ile; phe / met or leu or tyr; be / thr; thr / ser; trp / tyr; tyr / trp or phe; val / ile or leu. Another example guide uses the following six groups, each containing amino acids that are conservative substitutions with each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (I); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W) (see also for example, Creighton, Proteins, WH Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Verlag (1979) ). Those skilled in the art will appreciate that the substitutions identified above are not the only possible conservative substitutions. For example, for some purposes all charged amino acids can be considered as conservative substitutions, whether they are positive or negative. In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered "conservatively modified variations."

The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound having substantially the same structural and / or functional characteristics of the polypeptides, for example, translocation domains, ligand binding domains, or chimeric receptors of the invention. . The mimetic may be totally composed of synthetic, non-natural amino acid analogs, or it may be a chimeric molecule with part of natural peptide amino acids and with part of non-natural amino acid analogs. The mimetic can also incorporate any amount of conservative substitutions of natural amino acids, provided that such substitutions do not substantially alter the structure and / or activity of the mimetic.

As with the polypeptides of the invention that are conservative variants, routine experimentation will determine if a mimetic is within the scope of the description, that is, that its structure and / or its function are not substantially altered. Polypeptide mimetic compositions may contain any combination of unnatural structural components, which generally belong to three structural groups: a) linkage groups of residues other than natural amide bonds ("peptide bond"); b) unnatural residues instead of natural amino acid residues; or c) residues that induce a secondary structural imitation, that is, to induce or stabilize a secondary structure, for example, a conformation in beta spin, gamma spin, beta sheet, alpha helix, and the like. A polypeptide can be characterized as a mimetic when all or part of its moieties are linked by chemical means other than natural peptide bonds. The remains of the individual peptidomimetics may be linked by peptide bonds, other chemical bonds or coupling means such as, for example, glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N, N'-dicyclohexylcarbodiimide (DCC) or N, N '-diisopropylcarbodiimide (DIC). Connector groups that may be an alternative to the traditional amide bond ("peptide bond") include, for example, ketomethylene (eg, C (= O) -CH2-for -C (= O) -NH-), aminomethylene (CH2-NH), ethylene, olefin (CH = CH), ether (CH2-O), thioether (CH2-S), tetrazol (CN4), thiazole, retroamide, thioamide, or ester (see, for example, Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, vol. 7, pp. 267-357, "Peptide Backbone Modifications", Marcell Dekker, NY (1983)). A polypeptide can also be characterized as a mimetic because it contains all or some unnatural residues instead of natural amino acid residues; Unnatural remains are well described in the scientific and patent literature.

A "marker" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful markers include 32P, fluorescent dyes, electrodense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin,

or haptens and proteins that can be made detectable, for example, by incorporating a radioactive label into the peptide, or used to detect antibodies specifically reactive with the peptide.

An "oligonucleotide or labeled nucleic acid probe" is one that is covalently linked, through a chemical linker or link, or non-covalently, through ionic bonds, van der Waals, electrostatic or hydrogen , with a marker, so that the presence of the probe can be detected by detecting the presence of the marker attached to the probe.

As used herein, an "oligonucleotide or nucleic acid probe" is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through mating of complementary bases, usually through the formation of hydrogen bonds. As used herein, a probe may include natural (i.e., A, G, C or T) or modified bases (7-desazaguanosine, inosine, etc.). In addition, the bases in a probe can be linked by a link other than a phosphodiester bond, with the proviso that it does not interfere with hybridization. Thus, for example, the probes can be peptide nucleic acids in which the constituent bases are linked by peptide bonds instead of phosphodiester bonds. Those skilled in the art will understand that the probes can bind to target sequences that lack complete complementarity with the probe sequence depending on the stringency of the hybridization conditions. Optionally, the probes are directly labeled, for example, with isotopes, chromophores, light bulbs, chromogens, or indirectly labeled, for example, with biotin, to which a streptavidin complex can then be attached. The presence or absence of the selected sequence or sub-sequence can be detected by testing the presence or absence of the probe.

The term "heterologous", when used in reference to portions of a nucleic acid, indicates that the nucleic acid comprises two or more sub-sequences that are not in the same relationship with each other in nature. For example, nucleic acid is generally produced recombinantly, and has two or more sequences from unrelated genes arranged to make a new functional nucleic acid, for example, a promoter from one source and a coding region from another source. . Similarly, a heterologous protein indicates that the protein comprises two or more sub-sequences that are not in the same relationship with each other in nature (eg, a fusion protein).

A "promoter" is defined as a series of nucleic acid sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes the necessary nucleic acid sequences near the transcription initiation site, such as a polymerase II promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as far as several thousand base pairs from the transcription initiation site. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions.

An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional link between a nucleic acid expression control sequence (such as a promoter, or a series of transcription factor binding sites) and a second nucleic acid sequence, in which the expression control sequence directs the transcription of the corresponding nucleic acid to the second sequence.

As used herein, "recombinant" refers to a polynucleotide synthesized or otherwise manipulated in vitro (eg, a "recombinant polynucleotide"), to methods for using recombinant polynucleotides to produce gene products in cells or cells. other biological systems, or to a polypeptide (a "recombinant protein") encoded by a recombinant polynucleotide. A "recombinant medium" also includes the coupling of nucleic acids having various coding regions or promoter domains or sequences from different sources in an expression vector or module for expression, for example, inducible or constitutive expression of a fusion protein. which comprises a translocation domain of the description and an amplified nucleic acid sequence using a primer of the description.

As used herein, a "stable cell line" refers to a cell line that stably expresses, that is, over a prolonged period, a heterologous nucleic acid sequence, that is, a T1R or protein G. In preferred embodiments, these stable cell lines will be produced by transfecting appropriate cells, generally mammalian cells, for example, HEK-293 cells, with a linearized vector containing an expression construct of T1R, ie, T1R1, T1R2 and / or T1R3. Most preferably, these stable cell lines will be produced by co-transfecting two linearized plasmids expressing hT1R1 and hT1R3, or hT1R2 and hT1R3, and an appropriate selection procedure to generate cell lines that have these genes stably integrated into them. Most preferably, the cell line also stably expresses a G protein, such as Ga15.

The term "selectively hybridizes (or specifically) with" refers to the binding, duplex formation or hybridization of a molecule only with a specific nucleotide sequence under stringent hybridization conditions when this sequence is present in a complex mixture (by example, total or library cell DNA or RNA).

The term "stringent hybridization conditions" refers to conditions under which a probe will hybridize with its target sub-sequence, generally in a complex mixture of nucleic acids, but with no other sequence. Rigorous conditions depend on the sequence and will be different in different circumstances. Longer sequences that specifically hybridize at higher temperatures. A comprehensive guide to nucleic acid hybridization is found in Tijssen, Techniques in Biochemistry and Molecular Biology -Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). In general, the stringent conditions are selected to be approximately 5-10 ° C lower than the thermal melting point (Tf) for the specific sequence at a defined pH and ionic strength. Tf is the temperature (under an ionic strength, pH and concentration of defined nucleic acids) at which 50% of the complementary probes to the target hybridize with the target sequence in equilibrium (since the target sequences are present in excess , in Tf, 50% of the probes are occupied in the balance). The stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, generally about 0.01 to 1.0 M sodium ion concentration (or other salts), at pH 7, 0 to 8.3, and the temperature is at least about 30 ° C for short probes (eg, 10 to 50 nucleotides) and at least about 60 ° C for long probes (for example, greater than 50 nucleotides). Rigorous conditions can also be achieved with the addition of destabilizing agents, such as formamide. For selective or specific hybridization, a positive signal is at least twice the background, optionally 10 times the background hybridization. Examples of stringent hybridization conditions may be the following: 50% formamide, 5x SSC, and 1% SDS, incubation at 42 ° C, or 5x SSC, 1% SDS, incubation at 65 ° C, washed at 0, 2x SSC, and 0.1% SDS at 65 ° C. These hybridization and washing steps can be performed for, for example, 1, 2, 5, 10, 15, 30, 60 or more minutes.

Nucleic acids that do not hybridize with each other under stringent conditions remain substantially related if the encoding polypeptides are substantially related. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code. In these cases, nucleic acids generally hybridize under moderately rigorous hybridization conditions. Examples of "moderately stringent hybridization conditions" include hybridization in a 40% formamide buffer, 1M NaCl, 1% SDS at 37 ° C, and a 1x SSC wash at 45 ° C. These hybridization and washing steps can be performed for, for example, 1, 2, 5, 10, 15, 30, 60 or more minutes. A positive hybridization is at least twice the background. Those skilled in the art will readily recognize that other hybridization and washing conditions can be used to provide conditions of similar stringency.

An "antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene, or its fragments, that specifically binds and recognizes an antigen. Recognized immunoglobulin genes include genes from the constant regions kappa, lambda, alpha, gamma, delta, epsilon, and mu, as well as myriad genes from the variable region of immunoglobulins. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define the classes of immunoglobulins, IgG, IgM, IgA, IgD and IgE, respectively.

An example of an immunoglobulin structural unit (antibody) comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The N-terminal of each chain defines a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer to light and heavy chains, respectively.

A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is bound to a region constant of a different or altered class, effector function and / or species, or a completely different molecule that confers new properties to the chimeric antibody, for example, an enzyme, toxin, hormone, growth factor, drug, etc .; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region that has a different or altered antigen specificity.

An "anti-T1R" antibody is an antibody or an antibody fragment that specifically binds to a polypeptide encoded by a T1R gene, cDNA or a sub-sequence thereof.

The term "immunoassay" is an assay that uses an antibody to specifically bind an antigen. An immunoassay is characterized by the use of the specific binding properties of a particular antibody to isolate, target and / or quantify an antigen.

The expression "specifically binds (or selectively)" with an antibody, or "is specifically (or selectively) immunoreactive with", when referring to a protein or a peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biological compounds. Thus, under certain immunoassay conditions, the specified antibodies bind to a specific protein at least twice the background, and do not bind substantially in a significant amount to other proteins present in the sample. Specific binding to an antibody under these conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies generated against a member of the T1R family can be selected from a specific species, such as rat, mouse or human, to obtain only polyclonal antibodies that are specifically immunoreactive with the T1R polypeptide or an immunogenic portion of this, and not with other proteins, except for orthologs or polymorphic variants and alleles of the T1R polypeptide. This selection can be achieved by extracting the antibodies that cross-react with T1R molecules from other species or other T1R molecules. Antibodies that recognize only members of the T1R family of GPCR but not GPCR of other families can also be selected.

A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid phase ELISA immunoassays can be used to select antibodies specifically immunoreactive with a protein (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Generally, a specific or selective reaction will be at least twice the signal or background noise, and more generally more than 10 to 100 times the background.

The term "selectively associated with" refers to the ability of a nucleic acid to "selectively hybridize" with another as defined above, or the ability of an antibody to "selectively (or specifically) bind" to a protein, such as It was defined above.

The term "expression vector" refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast cells. , fungal, vegetable, insect or mammalian. The expression includes linear or circular expression systems. Expression includes expression systems that remain episomic or integrate into the genome of the host cell. Expression systems may have the ability to self-replicate or not, that is, to direct only one transient expression in a cell. The expression includes "modules" of recombinant expression, which contain only the minimum elements necessary for transcription of the recombinant nucleic acid.

A "host cell" means a cell that contains an expression vector and that supports replication or expression vector expression. The host cells can be prokaryotic cells, such as E. coli, or eukaryotic cells, such as yeast, insect, amphibious, worm or mammalian cells, such as CHO, Hela, HEK-293 cells and the like, for example, cultured cells , explants, and cells in vivo.

Isolation and expression of T1R polypeptides

The isolation and expression of the T1R polypeptides of the invention can be performed as described below. PCR primers can be used for nucleic acid amplification encoding ligand binding regions of taste receptors, and libraries of these nucleic acids can optionally be generated. Individual expression vectors or expression vector libraries can then be used to infect or transfect host cells for the functional expression of these nucleic acids or libraries. These genes and vectors can be manufactured and expressed in vitro or in vivo. Those skilled in the art will recognize that the desired phenotypes can be obtained to alter and control the expression of nucleic acids by modulating the expression or activity of the genes and nucleic acids (eg, promoters, enhancers and the like) within the vectors of the invention. Any of the known methods can be used to increase or decrease expression or activity. The invention can be practiced together with any method or protocol known in the art, which are well described in the scientific and patent literature.

The nucleic acid sequences of the invention and other nucleic acids used to practice this invention, whether they are RNA, cDNA, genomic DNA, vectors, viruses or their hybrids, can be isolated from a variety of sources, genetically modified, amplified and / or expressed recombinantly. Any recombinant expression system that includes, in addition to mammalian cells, for example, bacterial, yeast, insect or plant systems can be employed.

Alternatively, these nucleic acids can be synthesized in vitro by well known chemical synthesis techniques, as described, for example, in Carruthers, Cold Spring Harbor Symp. Quant. Biol., 47: 411-418 (1982); Adams, Am. Chem. Soc., 105: 661 (1983); Belousov, Nucleic Acids Res., 25: 3440-3444 (1997); Frenkel, Free Radic. Biol. Med., 19: 373-380 (1995); Biommers, Biochemistry, 33: 7886-7896 (1994); Narang, Meth. Enzymol., 68:90 (1979); Brown, Meth. Enzymol., 68: 109 (1979); Beaucage, Tetra. Lett., 22: 1859 (1981); U.S. Patent No.

4,458,066. Double stranded DNA fragments can then be obtained by synthesizing the complementary strand and re-associating the strands with each other under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Nucleic acid manipulation techniques such as, for example, to generate mutations in sequences, subcloning, probe labeling, sequencing, hybridization and the like, are well described in the scientific and patent literature. See, for example, Sambrook, ed., Molecular Cloning: a Laboratory Manual (2nd ed.), Vol. 1-3, Cold Spring Harbor Laboratory (1989); Current Protocols in Molecular Biology, Ausubel, ed., John Wiley & Sons, Inc., New York (1997); Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, part I, Theory and Nucleic Acid Preparation, Tijssen, ed., Elsevier, N.Y. (1993).

Nucleic acids, vectors, capsids, polypeptides and the like can be analyzed and quantified by any of a number of general means well known to those skilled in the art. These include, for example, analytical biochemical methods, such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, by for example, fluid or gel precipitin reactions, immunodiffusion, immunoelectrophoresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), immunofluorescent assays, Southern blot analysis, Northern blot analysis, dot transfer analysis, gel electrophoresis (for example, SDS-PAGE), RT-PCR, quantitative PCR, other methods of signal amplification, targets or nucleic acids, radiolabeling, scintillation counting, and affinity chromatography.

Oligonucleotide probes can be used to amplify nucleic acid fragments encoding ligand binding regions of taste receptors. The nucleic acids described herein can also be cloned or quantitatively measured using amplification techniques. Amplification methods are also well known in the art and include, for example, polymerase chain reaction, PCR (PCR Protocols, a Guide to Methods and Applications, ed. Innis, Academic Press, NY (1990), and PCR Strategies, ed. Innis, Academic Press, Inc., NY (1995)), ligase chain reaction (LCR) (see, for example, Wu, Genomics, 4: 560 (1989); Landegren, Science, 241: 1077 (1988); Barringer, Gene, 89: 117 (1990)); transcription amplification (see, for example, Kwoh, Proc. Natl. Acad. Sci. USA, 86: 1173 (1989)); and self-sustained sequence replication (see, for example, Guatelli, Proc. Natl. Acad. Sci. USA, 87: 1874 (1990)); amplification of Q-beta replicase (see, for example, Smith, J. Clin. Microbiol., 35: 1477-1491 (1997)); automatic Q-beta replicase amplification assay (see, for example, Burg, Mol. Cell. Probes, 10: 257-271 (1996)); and other RNA polymerase mediated techniques (for example, NASBA, Cangene, Mississauga, Ontario); see also Berger, Methods Enzymol., 152: 307-316 (1987); Sambrook; Ausubel; U.S. Patent Nos. 4,683,195 and 4,683,202; Sooknanan, Biotechnology, 13: 563-564 (1995). Primers can be designed to retain the original "donor" 7-transmembrane receptor sequence. Alternatively, the primers can encode amino acid residues that are conservative substitutions (for example, a hydrophobic moiety with a hydrophobic moiety, see the above analysis) or functionally benign substitutions (for example, that do not prevent insertion into the plasma membrane, nor cause the rupture by peptidases, nor cause the anomalous folding of the receptor and the like). After being amplified, the nucleic acids, individually or in the form of a library, can be cloned according to methods known in the art, if desired, in any of a variety of vectors using standard molecular biology methods; The methods for cloning amplified nucleic acids in vitro are described, for example, in US Patent No. 5,426,039.

Primer pairs can be designed to selectively amplify ligand binding regions of members of the T1R family. These regions may vary for different ligands or taste stimulants. Thus, what could be a minimum binding region for a taste stimulant may be too limiting for a second taste stimulant. Accordingly, ligand binding regions of different sizes comprising different extracellular domain structures can be amplified.

The paradigms for designing degenerate primer pairs are well known in the art. For example, the computer program can be accessed with a consensus degenerate hybrid oligonucleotide primer ("COnsensus-DEgenerate Hybrid Oligonucleotide Primer", CODEHOP) at http://blocks.fhcrc.org/codehop.html, and is directly connected to the site of multiple BlockMaker sequence alignment for prediction of hybrid primers and starting with a set of related protein sequences, such as taste receptor ligand binding regions (see, for example, Rose, Nucleic Acids Res., 26: 1628 -1635 (1998); Singh, Biotechniques, 24: 318-319 (1998)).

Means for synthesizing pairs of oligonucleotide primers are well known in the art. "Natural" base pairs or synthetic base pairs can be used. For example, the use of artificial nucleobases offers a versatile strategy to manipulate primer sequences and generates a more complex mixture of amplification products. Various families of artificial nucleobases are capable of assuming multiple hydrogen bond orientations through internal bond rotations to provide a means for degenerate molecular recognition. The incorporation of these analogs to a single position of a PCR primer allows the generation of a complex library of amplification products. See, for example, Hoops, Nucleic Acids Res., 25: 4866-4871 (1997). Non-polar molecules can also be used to mimic the shape of natural DNA bases. An imitator analog of the hydrogen-free form for adenine can be replicated efficiently and selectively against an imitator analog of the non-polar form for thymine (see, for example, Morales, Nat. Struct. Biol., 5: 950-954 (1998)). For example, two degenerate bases may be the pyrimidine base 6H, 8H-3,4-dihydropyrimido [4,5-c] [1,2] oxazin-7-one, or the purine base N6-methoxy-2, 6-diaminopurine (see, for example, Hill, Proc. Natl. Acad. Sci. USA, 95: 4258-4263 (1998)). Examples of degenerate primers of the invention incorporate the nucleobase analog 5'-dimethoxytrityl-N-benzoyl-2'-deoxycytidine, 3 '- [(2cyanoethyl) - (N, N-diisopropyl)] phosphoramidite (the term "P" in the sequences, see above). This pyrimidine analog is linked by hydrogen bonds to purines, including residues A and G.

Polymorphic variants, alleles, and interspecific homologs that are substantially identical to a taste receptor described herein can be isolated using the nucleic acid probes described above. Alternatively, expression libraries can be used to clone T1R polypeptides and their polymorphic variants, alleles, and interspecific homologs, by detecting homologs expressed immunologically with antiserum or purified antibodies generated against a T1R polypeptide, which also recognize and selectively bind to the T1R counterpart.

Nucleic acids encoding ligand binding regions of gustatory receptors can be generated by amplification (eg, PCR) of appropriate nucleic acid sequences using pairs of degenerate primers. The amplified nucleic acid can be genomic DNA from any cell or tissue, or mRNA or DNA derived from cells expressing taste receptors.

In one embodiment, sequences encoding hybrid proteins can be constructed comprising nucleic acids encoding T1R fused to translocation sequences. Hybrid T1Rs comprising translocation motifs and taste-stimulating domains of other families of chemosensory receptors, in particular taste receptors, are also provided. These nucleic acid sequences may be operably linked to transcriptional or translational control elements, for example, transcription and translation initiation sequences, promoters and enhancers, transcription and translation terminators, polyadenylation sequences, and others. Useful sequences to transcribe DNA in RNA. In the constitution of recombinant, vector and transgenic expression modules, a promoter fragment can be employed to direct the expression of the desired nucleic acid in all desired cells or tissues.

In another embodiment, the fusion proteins may include C-terminal or N-terminal translocation sequences. In addition, fusion proteins may comprise other elements, for example, for detection, protein purification or other applications. Domains that facilitate detection and purification include, for example, metal chelating peptides, such as polyhistidine extensions, histidine-tryptophan modules, or other domains that allow purification on immobilized metals; maltose binding protein; protein A domains that allow purification on immobilized immunoglobulins; or the domain used in the FLAGS affinity / extension purification system (Immunex Corp., Seattle, WA).

The inclusion of cleavable linker sequences, such as factor Xa (see, for example, Ottavi, Biochimie, 80: 289-293 (1998)), motive for recognition of subtilisin proteases (see, for example, Polyak, Protein Eng., 10: 615-619 (1997)); enterokinase (Invitrogen, San Diego, CA), and the like, between the translocation domain (for effective plasma membrane expression) and the rest of the newly translated polypeptide, may be useful to facilitate purification. For example, a construct may include a polypeptide encoding a nucleic acid sequence linked to six histidine residues, followed by a thioredoxin, an enterokinase disruption site (see, for example, Williams, Biochemistry, 34: 1787-1797 (1995 )), and a C-terminal translocation domain. The histidine residues facilitate detection and purification, while the enterokinase rupture site provides a means to purify the desired protein or proteins from the rest of the fusion protein. The technology relating to vectors encoding fusion proteins and the application of fusion proteins are well described in the scientific and patent literature (see, for example, Kroll, DNA Cell. Biol., 12: 441-453 (1993 )).

Expression vectors, both as individual expression vectors and in the form of expression vector libraries, comprising sequences encoding a ligand binding domain, can be introduced into a genome or into the cytoplasm or nucleus of a cell, and expressed by a variety of conventional techniques, well described in the scientific and patent literature (see, for example, Roberts, Nature,

328: 731 (1987); Berger, supra; Schneider, Protein Expr. Purif., 6435: 10 (1995); Sambrook; Tijssen; Ausubel The product information provided by the manufacturers of biological reagents and experimental equipment also provides information regarding known biological methods. Vectors can be isolated from natural sources, obtained from sources such as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods.

Nucleic acids can be expressed using expression modules, vectors or viruses, which are expressed stably or transiently in cells (eg, episomic expression systems). Selection markers can be incorporated into expression modules and vectors to confer a selectable phenotype to transformed sequences and cells. For example, selection markers can encode for episomic maintenance and replication, so that introduction into the host genome is not necessary. For example, the marker can encode antibiotic resistance (e.g., chloramphenicol, kanamycin, G418, blasticidine, hygromycin), or herbicide resistance (e.g., chlorosulfurone or Basta) to allow selection of transformed cells with the sequences of desired DNA (see, for example, Blondelet-Rouault, Gene, 190: 315-317 (1997); Aubrecht, J. Pharmacol. Exp. Ther., 281: 992-997 (1997)). Because selectable marker genes that confer resistance to substrates, such as neomycin or hygromycin, can only be used in tissue cultures, chemoresistant genes are also used as selectable markers in vitro and in vivo.

A chimeric nucleic acid sequence can encode a T1R ligand binding domain within any 7-transmembrane polypeptide. Because 7-transmembrane receptor polypeptides have similar primary and secondary tertiary sequences and structures, structural domains (e.g., extracellular domain, TM domains, cytoplasmic domain, etc.) can be easily identified by sequence analysis. For example, homology model formation, Fourier analysis, and helical periodicity detection can identify and characterize the seven domains in a 7transmembrane receptor sequence. Fast Fourier transform (FFT) algorithms can be used to evaluate the dominant periods that characterize the hydrophobicity and variability profiles of the sequences analyzed. The enhancement of the periodicity detection and the periodicity index of alpha-helices can be performed, for example, as in Donnelly, Protein Sci., 2: 55-70 (1993). Other alignment and model formation algorithms are well known in the art; see, for example, Peitsch, Receptors Channels, 4: 161-164 (1996); Kyte and Doolittle, J. Med. Bio., 157: 105-132 (1982); Cronet, Protein Eng., 6: 59-64 (1993).

The present invention also includes not only DNA and proteins having the specified amino acid and nucleic acid sequences, but also DNA fragments, in particular fragments of, for example, 60, 80, 100, 150, 200 or 250 nucleotides or more, as well as protein fragments of, for example, 20, 30, 50, 70, 100 or 150 amino acids or more, with the proviso that said fragments have the percentage of sequence matching required. Optionally, the nucleic acid fragments can encode an antigenic polypeptide, which is capable of binding to an antibody generated against a member of the T1R family. In addition, a protein fragment of the invention may optionally be an antigenic fragment, which is capable of binding to an antibody generated against a member of the T1R family.

Chimeric proteins are also contemplated, comprising at least 10, 20, 30, 50, 70, 100 or 150 amino acids.

or more, of one of at least one of the T1R polypeptides described herein, coupled to additional amino acids that represent all or part of another GPCR, preferably a member of the 7-transmembrane superfamily. These chimeras can be formed from the receptors of the present invention and from another GPCR, or they can be formed by combining two or more of the T1R receptors of the present invention. In one embodiment, a chimera bearing corresponds to or derives from the extracellular domain of a T1R polypeptide of the invention. In another embodiment, a part of the chimera corresponds to or derives from the extracellular domain and one

or more of the transmembrane domains of a T1R polypeptide described in the present invention, and the rest of the part or parts may be derived from another GPCR. Chimeric receptors are well known in the art, and the techniques for creating them and the selection and boundaries of domains or fragments of G-protein coupled receptors for incorporation therein are also well known. Thus, this knowledge of those skilled in the art can be easily used to create such chimeric receptors. The use of these chimeric receptors can provide, for example, a taste selectivity characteristic of one of the receptors specifically described in the present invention, coupled with the signal transduction characteristics of another receiver, such as a known receiver used in the systems. of prior art test.

As indicated above, these chimeras, analogous to the native T1R receptor, or the association or combination of native T1R receptors, will bind and / or be activated by molecules that normally affect sweet taste or umami taste. Receptors or combinations of functional chimeric T1R receptors are molecules that, when expressed alone or in combination with other T1R or other GPCRs (which in turn can be chimeric), bind or are activated by taste stimuli, in particular sweet taste stimuli (T1R2 / 3) or umami (T1R / 3). The molecules that cause the sweet taste include natural and artificial sweeteners, such as sucrose, aspartame, xylitol, cyclamate, etc. Molecules that cause umami taste include glutamate and glutamate analogs, and other compounds that bind to native T1R1 and / or T1R3, such as 5’-nucleotides.

For example, a domain, such as a ligand binding domain, an extracellular domain, a transmembrane domain, a cytoplasmic domain, an N-terminal domain, a C-terminal domain, or any combination thereof, can be covalently linked to a heterologous protein For example, an extracellular domain of T1R can bind to a heterologous GPCR transmembrane domain, or a heterologous GPCR extracellular domain can bind to a T1R transmembrane domain. Other heterologous proteins can be chosen, for example, the green fluorescent protein.

Host cells for expressing the T1R polypeptides of the invention are also within the scope of the invention. To obtain high levels of expression of a cloned nucleic acid or gene, such as cDNAs encoding T1Rs, fragments or variants of the invention, those skilled in the art generally subclone the nucleic acid sequence into an expression vector that It contains a strong promoter to direct transcription, a transcription / translation terminator, and for a nucleic acid encoding a protein, a ribosome binding site for initiation of translation. Suitable bacterial promoters are well known in the art and are described, for example, in Sambrook et al. However, eukaryotic or bacterial expression systems can be used.

Any of the known methods can be employed to introduce foreign nucleotide sequences into host cells. These include the use of transfection with calcium phosphate, polybrene, fusion of protoplasts, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well-known methods for introducing genomic DNA, cDNA, and synthetic DNA cloned or other material genetic stranger in a host cell (see, for example, Sambrook et al.). It is only necessary that the particular genetic modification procedure used be able to successfully introduce at least one nucleic acid molecule into the host cell that is capable of expressing the T1R fragment or variant of interest.

After introducing the expression vector into the cells, the transfected cells are cultured under conditions that favor the expression of the receptor, fragment, or variant of interest, which is then recovered from the culture using conventional techniques. Examples of such techniques are well known in the art (see, for example, WO 00/06593).

T1R polypeptide detection

In addition to the detection of T1R genes and gene expression using nucleic acid hybridization technology, immunoassays can also be used to detect T1R, for example, to identify taste receptor cells, and variants of members of the T1R family. Immunoassays can be used to analyze T1R qualitatively or quantitatively. An overview of the applicable technology can be found in Harlow and Lane, Antibodies: A Laboratory Manual (1988).

1. Antibodies against family members T1R

Methods for producing monoclonal and polyclonal antibodies that react specifically with a member of the T1R family are known to those skilled in the art (see, for example, Coligan, Current Protocols in Immunology (1991); Harlow and Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2nd ed., 1986); and Kohler and Milstein, Nature, 256: 495-497 (1975)). These techniques include the preparation of antibodies by the selection of antibodies from recombinant antibody libraries in phage or similar vectors, as well as the preparation of polyclonal and monoclonal antibodies by immunization of rabbits.

or mice (see, for example, Huse et al., Science, 246: 1275-1281 (1989); Ward et al., Nature, 341: 544-546 (1989)).

A series of immunogens comprising T1R can be used to produce antibodies specifically reactive with a member of the T1R family. For example, a recombinant T1R polypeptide, or an antigenic fragment thereof, can be isolated as described herein. Suitable antigenic regions include, for example, the consensus sequences that are used to identify members of the T1R family. Recombinant proteins can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above. A recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Alternatively, a synthetic peptide derived from the sequences described herein and conjugated to a carrier protein can be used as an immunogen. The natural protein can also be used, in pure or impure form. The product is then injected into an animal capable of producing antibodies. Monoclonal or polyclonal antibodies can be generated, for later use in immunoassays to measure the protein.

The methods for producing polyclonal antibodies are well known to those skilled in the art. For example, an inbred breed of mice (for example, BALB / C mice) or rabbits can be immunized with the protein using a conventional adjuvant, such as Freund's adjuvant, and a conventional immunization protocol.

The animal's immunological response to the preparation of the immunogen is controlled by performing test bleeds and determining the titre of reactivity against T1R. When duly high titers of the antibody against the immunogen are obtained, blood is collected from the animal and the antiserum is prepared. If desired, subsequent fractionation of the serum can be performed to enrich protein reactive antibodies (see, Harlow and Lane, supra).

Monoclonal antibodies can be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells of an animal immunized with a desired antigen can be immortalized, generally by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol., 6: 511-519 (1976)). Alternative immortalization methods include transformation with Epstein-Barr virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies that arise from individual immortalized cells are selected for the production of antibodies with the specificity and affinity desired by the antigen, and the yield of the monoclonal antibodies produced by these cells can be enhanced by various techniques, including injection into the peritoneal cavity. of a vertebrate host. Alternatively, DNA sequences encoding a monoclonal antibody, or a binding fragment thereof, can be isolated by selecting a library of DNA from human B cells according to the general protocol outlined by Huse et al., Science, 246 : 1275-1281 (1989).

Monoclonal antibodies and polyclonal serum are collected and titrated against the immunogenic protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Generally, polyclonal antisera with a titer of 104 or greater are selected, and tested for cross-reactivity with other polypeptides other than T1R, or even other members of the T1R family or other related proteins from other organisms, using a competitive binding immunoassay. Polyclonal antisera and specific monoclonal antibodies will normally bind with a Kd of at least about 0.1 mM, more usually at least about 1 pM, optionally at least about 0.1 pM or better, and optionally 0.01 pM or better.

When specific antibodies of members of the T1R family are available, individual T1R proteins and T1R protein fragments can be detected by a variety of immunoassay methods. For an analysis of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites and Terr, eds., 7th ed., 1991). In addition, the immunoassays of the present invention can be performed in any of several configurations, which are thoroughly analyzed in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow and Lane, supra.

2. Immunological binding assays

T1R proteins, fragments and variants can be detected and / or quantified using any of a series of well-recognized immunological binding assays (see, for example, U.S. Patents 4,366,241, 5,376,110, 4,517,288, and 4,837,168). For an analysis of general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed., 1993); Basic and Clinical Immunology (Stites and Terr, eds., 7th ed., 1991). Immunological binding assays (or immunoassays) generally employ an antibody that specifically binds to a chosen protein or antigen (in this case, a member of the T1R family, or an antigenic sub sequence thereof). The antibody (for example, anti-T1R) can be produced by any of a number of means well known to those skilled in the art and as described above.

Immunoassays often employ a marker agent to specifically bind and label the complex formed by the antibody and the antigen. The labeling agent may itself be one of the moieties comprising the antibody / antigen complex. Thus, the labeling agent can be a labeled T1R polypeptide or a labeled anti-T1R antibody. Alternatively, the labeling agent may be a third moiety, such as a secondary antibody, that specifically binds to the antibody / T1R complex (a secondary antibody is generally antibody specific to the species from which the first antibody is derived). Other proteins capable of specifically binding to constant regions of immunoglobulins, such as protein A or protein G, can also be used as a marker agent. These proteins show a strong non-immunogenic reactivity with the constant regions of immunoglobulins from a variety of species (see, for example, Kronval et al., J. Immunol., 111: 1401-1406 (1973); Akerstrom et al., J. Immunol., 135: 2589-2542 (1985)). The labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule, such as streptavidin, can specifically bind. A variety of detectable moieties are well known to those skilled in the art.

Incubation and / or washing steps may be necessary throughout the tests after each reagent combination. The incubation steps may vary from about 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the incubation time will depend on the assay format, the antigen, the volume of solution, the concentrations and the like. Typically, the tests will be performed at room temperature, although they can be performed over a range of temperatures, such as from 10 ° C to 40 ° C.

A. Non-competitive test formats

Immunoassays to detect a T1R polypeptide in a sample can be competitive or non-competitive. Non-competitive immunoassays are assays in which the amount of antigen is measured directly. In a preferred "sandwich" assay, for example, anti-T1R antibodies can be directly bound to a solid substrate on which they are immobilized. These immobilized antibodies then capture the T1R polypeptide present in the test sample. To the T1R polypeptide thus immobilized is then bound a labeling agent, such as a second T1R antibody carrying a label. Alternatively, the second antibody may lack a label but, in turn, may be bound by a third specific labeled antibody to the antibodies of the species from which the second antibody is derived. The second or third antibody is generally modified with a detectable moiety, such as biotin, to which another molecule, for example streptavidin, specifically binds to provide a detectable moiety.

B. Competitive test formats

In competitive assays, the amount of T1R polypeptide present in the sample is measured indirectly by measuring the amount of a known (exogenous) added T1R polypeptide displaced (by competition) from an anti-T1R antibody by an unknown T1R polypeptide present in a sample. In a competitive assay, a known amount of T1R polypeptide is added to a sample, and the sample is then contacted with an antibody that specifically binds to T1R. The amount of exogenous T1R polypeptide bound to the antibody is inversely proportional to the concentration of the T1R polypeptide present in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of T1R polypeptide bound to the antibody can be determined by measuring the amount of T1R polypeptide present in the T1R / antibody complex or, alternatively, by measuring the amount of remaining uncomplexed protein. The amount of T1R polypeptide can be detected by providing a labeled T1R molecule.

A hapten inhibition assay is another preferred competitive assay. In this assay, the known T1R polypeptide is immobilized on a solid substrate. A known amount of anti-T1R antibody is added to the sample, and the sample is then contacted with the immobilized T1R. The amount of anti-T1R antibody bound to the known immobilized T1R polypeptide is inversely proportional to the amount of T1R polypeptide present in the sample. Again, the amount of immobilized antibody can be detected by detecting the immobilized fraction of the antibody or the fraction of the antibody that remains in solution. Detection can be direct, in which the antibody is labeled, or indirectly, by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.

C. Cross-reactivity determinations

Immunoassays in competitive binding format can also be used for cross-reactivity determinations. For example, a protein at least partially encoded by the nucleic acid sequences described herein can be immobilized on a solid support. Proteins (for example, T1R polypeptides or homologs) are added to the assay that compete for the binding of the antiserum to the immobilized antigen. The ability of the added proteins to compete for the binding of the antiserum to the immobilized protein is compared with the ability of the T1R polypeptide encoded by the nucleic acid sequences described herein to compete with itself. The percentage of cross reactivity for the above proteins is calculated, using conventional calculations. Antisera with less than 10% cross reactivity with each of the added proteins listed above are selected and pooled. Cross-reactivity antibodies are optionally removed from the collected antisera by immunoabsorption with the proteins considered added, for example, homologues with a distant relationship. In addition, in the cross-reactivity determinations peptides comprising amino acid sequences representing conserved motifs that are used to identify members of the T1R family can be employed.

The immunoabsorbed and assembled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, of which it is believed that it is perhaps an allele or a polymorphic variant of a member of the T1R family, with the protein of the immunogen (ie, a T1R polypeptide encoded by the nucleic acid sequences described herein). To make this comparison, the two proteins are each tested at a wide range of concentrations, and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required to inhibit 50% of the binding is less than 10 times the amount of the protein encoded by the nucleic acid sequences described herein required to inhibit 50% of the binding, then it is said that The second protein specifically binds to polyclonal antibodies generated against a T1R immunogen.

Antibodies generated against conserved T1R motifs can also be used to prepare antibodies that specifically bind only GPCR of the T1R family, but not GPCR of other families.

Polyclonal antibodies that specifically bind a particular member of the T1R family can be prepared by subtracting the cross-reactivity antibodies using other members of the T1R family. Species-specific polyclonal antibodies can be prepared in a similar manner. For example, antibodies specific to human T1R1 can be prepared by subtracting antibodies that have cross-reactivity with orthologous sequences, for example, rat T1R1 or mouse T1R1.

D. Other test formats

A Western blot analysis (immunoblot) is used to detect and quantify the presence of the T1R polypeptide in the sample. The technique in general comprises separating the proteins from the sample by gel electrophoresis based on molecular weight, transferring the separated proteins to a suitable solid support (such as a nitrocellulose filter, a nylon filter, or a derivatized nylon filter) , and incubate the sample with antibodies that specifically bind to the T1R polypeptide. T1R antipolypeptide antibodies specifically bind to the T1R polypeptide on the solid support. These antibodies can be directly labeled or, alternatively, they can be subsequently detected using labeled antibodies (eg, labeled sheep anti-mouse antibodies) that specifically bind to the anti-T1R antibodies.

Other assay formats include liposome immunoassays (LIA), which employ liposomes designed to bind to specific molecules (eg, antibodies) and release encapsulated markers or reagents. The chemical compounds released are then detected according to conventional techniques (see, Monroe et al., Amer. Clin. Prod. Rev., 5: 34-41 (1986)).

E. Reduction of non-specific binding

Those skilled in the art will appreciate that it is often desirable to minimize non-specific binding in immunoassays. In particular, when the assay involves an antigen or an antibody immobilized on a solid substrate, it is desirable to minimize the amount of non-specific binding to the substrate. The means for reducing said non-specific binding are well known to those skilled in the art. Generally, this technique involves coating the surface substrate with a protein composition. In particular, protein compositions, such as bovine serum albumin (BSA), skimmed milk powder, and gelatin, are widely used, with milk powder being the most preferred.

F. Markers

The specific label or detectable group used in the assay is not a critical aspect of the invention, with the proviso that it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material that has a detectable physical or chemical property. These detectable markers have had extensive development in the field of immunoassays and, in general, almost any marker useful in such methods can be applied to the present invention. Thus, a marker is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. The markers useful in the present invention include magnetic spheres (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 125I, 14C, 35S) , enzymes (for example, horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric markers, such as colloidal gold or colored glass or plastic spheres (for example, polystyrene, polypropylene, latex, etc. ).

The label can be directly or indirectly coupled to the desired test component according to methods well known in the art. As indicated above, a wide variety of markers can be used, depending on the choice of the marker of the required sensitivity, the ease of conjugation with the compound, stability requirements, available instruments, and disposal forecasts.

Non-radioactive markers are often joined by indirect means. In general, a ligand molecule (eg, biotin) binds covalently to the molecule. The ligand then binds to another molecule (for example, streptavidin), which is inherently detectable or that is covalently bound to a signaling system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. The ligands and their targets can be used in any suitable combination with antibodies that recognize a T1R polypeptide, or secondary antibodies that recognize anti-T1R.

The molecules can also be conjugated directly with signal generating compounds, for example, by conjugation with an enzyme or a fluorophore. The enzymes of interest as markers are mainly hydrolases, in particular phosphatases, esterases and glycosidases, or oxidotases, in particular peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydroftalazindiones, for example, luminol. For an analysis of various signal or marker producing systems that can be used see US Patent No. 4,391,904.

Means for detecting markers are well known to those skilled in the art. Thus, for example, when the marker is a radioactive marker, the means for detection include a scintillation counter or a photographic film, as in an autoradiography. When the marker is a fluorescent marker, it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. Fluorescence can be detected by visual means, by means of a photographic film, by the use of electronic detectors, such as load-coupled devices (CCD) or photomultipliers, and the like. Similarly, enzyme markers can be detected by providing appropriate substrates for the enzyme and detecting the product of the resulting reaction. Finally, colorimetric markers can be detected simply by observing the color associated with the marker. Thus, in various trials with sticks, conjugated gold often appears as pink, while in various conjugated spheres the color of the sphere appears.

Some test formats do not require the use of marked components. For example, agglutination assays can be used to detect the presence of the target antibodies. In this case, the antigen-coated particles are bound by samples comprising the target antibodies. In this format, it is not necessary for any of the components to be labeled, and the presence of the target antibody is detected by a simple visual inspection.

Modulator Detection

Compositions and methods for determining whether a test compound specifically binds to a T1R receptor of the invention, in vitro and in vivo, are described below. Many aspects of cell physiology can be controlled to evaluate the effect of ligand binding to a T1R polypeptide of the invention. These assays can be performed on intact cells that express a chemosensory receptor, on permeabilized cells, or on membrane fractions produced by conventional methods or de novo synthesized proteins in vitro.

In vivo, taste receptors bind to taste stimulants and initiate the transduction of chemical stimuli into electrical signals. An activated or inhibited G protein, in turn, will alter the properties of target enzymes, channels and other effector proteins. Some examples are the activation of cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by stimulatory G protein, phospholipase C by Gq and other cognated G proteins, and modulation of various channels by Gi and other proteins G. The downstream consequences, such as the generation of diacylglycerol and IP3 by phospholipase C and, in turn, the mobilization of calcium by IP3, can also be studied.

The T1R proteins or polypeptides of the assay will preferably be selected from a polypeptide having the sequence of the T1R polypeptide selected from those described in Example 1, or its fragments or variants modified conservatively. Optionally, the fragments and variants may be fragments and antigenic variants that bind to an anti-T1R antibody. Optionally, the fragments and variants can be joined or activated by sweeteners or stimulants of umami taste.

Alternatively, the T1R proteins or polypeptides of the assay may be derived from a eukaryotic host cell, and may include an amino acid sequence that has an amino acid sequence match with the T1R polypeptides described in Example 1, or their fragments or variants modified conservatively. In general, the coincidence of the amino acid sequence will be at least 35% to 50%, or optionally 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. Optionally, the T1R proteins or polypeptides of the assays may comprise a domain of a T1R protein, such as an extracellular domain, a transmembrane domain, a cytoplasmic domain, a ligand binding domain and the like. In addition, as described above, the T1R protein, or a domain thereof, can covalently bind to a heterologous protein to create a chimeric protein used in the assays described herein.

Modulators of T1R receptor activity are assayed using T1R proteins or polypeptides as described above, both recombinant and natural. T1R proteins or polypeptides can be isolated, co-expressed in a cell, co-expressed in a membrane derived from a cell, co-expressed in a tissue or animal, both recombinant and natural. For example, tongue cuts, dissociated cells of a tongue, transformed cells, or membranes can be employed. Modulation can be tested using one of the in vitro or in vivo assays described herein.

For example, as described in the following experimental examples, it has been found that certain 51nucleotides, for example, 51-IMP or 51-GMP, potentiate the activity of L-glutamate to activate the umami taste receptor, or block the activation of Gustatory umami receptor by umami flavor stimuli, such as Lglutamate and L-aspartate.

1. In vitro binding assays

Taste transduction can also be studied in vitro with solid or soluble state reactions, using the T1R polypeptides of the invention. In a specific embodiment, the T1R ligand binding domains can be used in vitro in solid or soluble state reactions to assay for ligand binding.

For example, it is predicted that the N-terminal domain of T1R will be involved in ligand binding. More specifically, the T1Rs belong to a GPCR subfamily characterized by large extracellular N-terminal segments of approximately 600 amino acids. It is believed that these N-terminal segments form ligand binding domains and, therefore, are useful in biochemical assays to identify agonists and antagonists of T1R. It is possible that the ligand binding domain may be formed by additional portions of the extracellular domain, such as the extracellular loops of the transmembrane domain.

In vitro binding assays have been employed with other GPCRs that are related to T1Rs, such as metabotropic glutamate receptors (see, for example, Han and Hampson, J. Biol. Chem., 274: 10008-10013 (1999) ). These assays may involve displacing a radioactively or fluorescently labeled ligand, measuring changes in intrinsic fluorescence or changes in proteolytic susceptibility, etc.

The ligand binding to a heteromultimeric complex of T1R polypeptides of the invention can be tested in solution, in a bilayer membrane, optionally bound to a solid phase, in a lipid monolayer, or in vesicles. The binding of a modulator can be tested using, for example, changes in spectroscopic characteristics (for example, fluorescence, absorbance, refractory index), hydrodynamics (eg, shape), chromatographic, or solubility properties.

In another embodiment of the invention, a GTPy35S assay can be used. As described above, after activation of a GPCR, the Ga subunit of the G protein complex is stimulated to exchange the bound GDP for GTP. Ligand-mediated stimulation of the exchange activity of the G protein can be measured in a biochemical assay that measures the binding of radiolabeled GTPy35S added to the G protein in the presence of a putative ligand. Generally, the membranes containing the chemosensory receptor of interest are mixed with a complex of G proteins. Potential inhibitors and / or activators and GTPy35S are added to the assay, and the binding of GTPy35S to the G protein is measured. The binding can be measured by a liquid scintillation count or by any other means known in the art, including scintillation proximity assays (SPA). In other assay formats, fluorescently labeled GTPyS can be used.

2. Fluorescence polarization assays

In another embodiment, fluorescence polarization ("FP") based assays can be employed to detect and control ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous because they do not require a separation stage, such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These tests are carried out in real time, directly in solution, and do not require an immobilized phase.

The polarization values can be measured repeatedly and after the addition of reagents, since the polarization measurement is fast and does not destroy the sample. In general, this technique can be used to measure fluorophores polarization values from picomolar to micromolar levels. This section describes how fluorescence polarization can be used in a simple and quantitative manner to measure ligand binding to the T1R polypeptides of the invention.

When a fluorescently labeled molecule is excited with a polarized light in a plane, it emits light that has a degree of polarization that is inversely proportional to its molecular rotation. Large fluorescently labeled molecules remain relatively stationary during the excited state (4 nanoseconds in the case of fluorescein), and the polarization of light remains relatively constant between excitation and emission. Small fluorescently labeled molecules rotate rapidly during the excited state, and polarization changes significantly between excitation and emission. Therefore, small molecules have low polarization values, and large molecules have high polarization values. For example, a fluorescein-labeled single stranded oligonucleotide has a relatively low polarization value, but when hybridized with a complementary strand it has a higher polarization value. When FP is used to detect and control the binding of the taste stimulant, which can activate or inhibit the chemosensory receptors of the invention, fluorescently labeled taste stimulants or autofluorescent taste stimulants can be employed.

Fluorescence polarization (P) is defined as:

P = Int-IntU

n Intn + IntU

where n is the intensity of the light emission parallel to the excitation light plane, and IntU is the intensity of the light emission perpendicular to the excitation light plane. Being P, a proportion of light intensities, a dimensionless number. For example, together with these tests, the Beacon® and Beacon 2000T system can be used. These systems generally express polarization in millipolar units (1 polarization unit = 100 mP units).

The relationship between molecular rotation and size is described by the Perrin equation, and the reader is referred to the quotation of Jolley, M.E. (1991), in Journal of Analytical Toxicology, p. 236-240, which offers a thorough explanation of this equation. In summary, Perrin's equation states that polarization is directly proportional to the time of rotational relaxation, the time it takes for a molecule to rotate through an angle of approximately 68.5 °. The rotational relaxation time is related to viscosity (n), absolute temperature (T), molecular volume (V), and gas constant (R) by the following equation:

Rotational relaxation time = 3nV RT

The rotational relaxation time is small ('1 nanosecond) for small molecules (for example, fluorescein) and large (' 100 nanoseconds) for large molecules (for example, immunoglobulins). If the viscosity and temperature are kept constant, the rotational relaxation time and, therefore, the polarization, are directly related to the molecular volume. Changes in molecular volume may be due to interactions with other molecules, dissociation, polymerization, degradation, hybridization, or conformational changes of the fluorescently labeled molecule. For example, fluorescence polarization has been used to measure the enzymatic breakdown of large fluorescein-labeled polymers by proteases, DNases, and RNases. It has also been used to measure the equilibrium binding of protein / protein interactions, antibody / antigen binding, and protein / DNA binding.

A. High performance tests in solid and soluble state

In another embodiment, the invention provides soluble assays that employ a hetero-oligomeric T1R polypeptide complex; or a cell or tissue that coexpresses T1R polypeptides. Preferably, the cell will comprise a cell line that stably coexpresses a functional T1R2 / T1R3 (sweet) taste receptor. In another embodiment, the invention provides in vitro assays based on a solid phase in a high performance format, in which the T1R polypeptides, or a cell or tissue expressing the T1R polypeptides, binds to a phase substrate. solid or a taste stimulating compound, and is contacted with a T1R receptor, and binding is detected using an appropriate label or an antibody generated against the T1R receptor.

In the high performance assays of the invention, it is possible to select up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a different test against a potential modulator selected or, if the effects of the concentration or incubation time are to be observed, every 5-10 wells can be tested with a single modulator Thus, a single conventional microtiter plate can test approximately 100 (for example, 96) modulators. If 1536 well plates are used, then a single plate can easily test from about 1000 to about 1500 different compounds. It is also possible to test multiple compounds in each well of the plate. It is possible to test several different plates per day; Selection tests of up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention. More recently, microfluidic strategies for reagent handling have been developed.

The molecule of interest can be attached to the solid state component directly or indirectly, through covalent or non-covalent bonds, for example through a label. The marker can be any of a variety of components. In general, a molecule that binds to the marker (a marker binder) is fixed to a solid support, and the labeled molecule of interest (for example, the taste transduction molecule of interest) is fixed to the solid support by interaction of the marker and the marker binder.

Based on known molecular interactions, well described in the literature, a series of markers and marker binders can be used. For example, when a label has a natural binder, for example, biotin, protein A, or protein G, it can be used together with the appropriate label binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies against molecules with natural binders, such as biotin, and appropriate marker binders are also widely available (see, SIGMA Immunochemicals, 1998 catalog of SIGMA, St. Louis, MO).

Similarly, any haptic or antigenic compound can be used in combination with an appropriate antibody to form a marker / marker binder pair. Thousands of specific antibodies are commercially available, and many other antibodies are described in the literature. For example, in a usual configuration, the label is a first antibody and the label binder is a second antibody that recognizes the first antibody. In addition to antibody-antigen interactions, receptor ligand interactions are also appropriate as pairs of markers and marker binder, for example, agonists and antagonists of cell membrane receptors (e.g., cell-ligand receptor interactions, such as transferrin , c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, the cadherein family, the integrin family, the selectin family, and the like; see, for example, Pigott and Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and poisons, viral epitopes, hormones (for example, opiates, steroids, etc.), intracellular receptors (for example, involved in the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D ; peptides), drugs, lectins, sugars, nucleic acids (in linear and cyclic polymer configuration), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, and polyacetates, can also form an appropriate label or a marker binder. Many other marker / marker binder pairs are also useful in the assay systems described herein, as will be apparent to those skilled in the art after analysis of this description.

The usual connectors, such as peptides, polyethers and the like, can also act as markers, and include polypeptide sequences, such as poly-gly sequences of between about 5 and 200 amino acids. These flexible connectors are known to those skilled in the art. For example, polyethylene glycol connectors are available from Shearwater Polymers, Inc., Huntsville, Alabama. These connectors optionally have amide bonds, sulfhydryl bonds, or heterofunctional bonds.

Marker binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are usually derivatized or functionalized by exposing all

or a part of the substrate to a chemical reagent that fixes a chemical group on the surface, which is reactive with a part of the marker binder. For example, groups that are suitable for binding to a longer chain part will include amine, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The essential of such solid phase biopolymer matrices is well described in the literature; see, for example, Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963) (which describes solid phase synthesis, for example, of peptides); Geysen et al., J. Immun. Meth., 102: 259-274 (1987) (which describes the synthesis of solid phase components on rods); Frank and Doring, Tetrahedron, 44: 6031-6040 (1988) (which describes the synthesis of various peptide sequences on cellulose discs); Fodor et al., Science, 251: 767-777 (1991); Sheldon et al., Clinicla Chemistry, 39 (4): 718-719 (1993); and Kozal et al., Nature Medicine, 2 (7): 753-759 (1996) (all describe matrices of biopolymers attached to solid substrates). Non-chemical strategies for fixing marker binders to substrates include other common methods, such as heat, UV radiation crosslinking, and the like.

3. Cell based assays

In a preferred treatment embodiment, a combination of T1R proteins or polypeptides is co-expressed transiently or stably in a eukaryotic cell in an unmodified form or as chimeric receptors, variants or truncated with, or preferably without, a heterologous chaperone sequence. which facilitates its maturation and acts for transport directed through the secretory route. These T1R polypeptides can be expressed in any eukaryotic cell, such as HEK-293 cells. Preferably, the cells comprise a functional G protein, for example, GaI5 or the previously identified chimeric G protein, or other G protein that is capable of coupling the chimeric receptor to an intracellular signaling pathway or to a signaling protein, such as phospholipase C In addition, a cell that stably coexpress T1R1 / T1R3 or T1R2 / T1R3 will preferably be produced, since it has been found that said cells (as shown in the experimental examples) show enhanced responses to flavor stimuli (relative to cells that express transiently the same combination of T1R). The activation of T1R receptors in said cells can be detected using any conventional method, such as detecting changes in intracellular calcium by detecting Fluo-4 dependent fluorescence in the cell. This essay is the basis of the experimental findings presented in this application.

Activated GPCR receptors are often substrates for kinases that phosphorylate the C-terminal tail of the receptor (and probably also other sites). Thus, the activators will stimulate the transfer of 32P from radiolabelled ATP to the receiver, which can be tested with a scintillation counter. Phosphorylation of the C-terminal tail will stimulate the binding of arrestin-like proteins and interfere with the binding of G proteins. For a general analysis of signal transduction in GPCR and of methods for testing signal transduction see, for example, Methods in Enzymology, vols. 237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature, 10: 349: 117127 (1991), Bourne et al., Nature, 348: 125-132 (1990); Pitcher et al., Annu. Rev. Biochem., 67: 653-692 (1998).

T1R modulation can be tested by comparing the response of T1R polypeptides treated with a putative T1R modulator with the response of an untreated control sample or a sample containing a known "positive" control. These putative T1R modulators may include molecules that inhibit or activate the activity of the T1R polypeptide. In one embodiment, control samples (not treated with activators or inhibitors) are assigned a relative T1R activity value of 100. Inhibition of a T1R polypeptide is achieved when the T1R activity value in relation to the control is approximately 90%, optionally 50%, optionally 25-0%. Activation of a T1R polypeptide is achieved when the T1R activity value in relation to the control is 110%, optionally 150%, 200-500%, or 1000-2000%.

Changes in ion flow can be assessed by determining changes in ionic polarization (i.e., electrical potential) of the cell or membrane that expresses a T1R polypeptide. One means of determining changes in cell polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage setting and patch fixation techniques (see, for example, the "cell-bound" mode ", The" outside "mode, and the" whole cell "mode, for example, Ackerman et al., New Engl. J. Med., 336: 1575-1595 (1997)). Whole cell currents are conveniently determined using the standard. Other known assays include: radiolabelled ion flow assays and fluorescence assays that employ voltage sensitive dyes (see, for example, Vestergarrd-Bogind et al., J. Membrane Biol., 88: 67-75 (1988); Gonzales and Tsien, Chem. Biol., 4: 269-277 (1997); Daniel et al., J. Pharmacol. Meth., 25: 185-193 (1991); Holevinsky et al., J. Membrane Biology, 137: 59-70 (1994)).

The effects of the test compounds on the function of the polypeptides can be measured by studying any of the parameters described above. Any suitable physiological change that affects GPCR activity can be used to evaluate the influence of a test compound on the polypeptides of this invention. When functional consequences are determined using intact or animal cells, a variety of effects can also be measured, such as transmitter release, hormone release, transcriptional changes against known and uncharacterized genetic markers (e.g. Northern blots). ), changes in cell metabolism, such as cell growth or changes in pH, and changes in second intracellular messengers, such as Ca2 +, IP3, cGMP, or cAMP.

Preferred assays for GPCR include cells that are charged with ion or voltage sensitive dyes to indicate receptor activity. Assays to determine the activity of such receptors can also use agonists and antagonists of other G-protein coupled receptors as controls to evaluate the activity of the compounds tested. In tests to identify modulating compounds (eg, agonists, antagonists), changes in the level of ions in the cytoplasm or membrane voltage will be controlled using an ion-sensitive fluorescent indicator or membrane voltage, respectively. Among the ion-sensitive indicators and voltage probes that can be used are those described in the 1997 Molecular Probes catalog. For G-protein coupled receptors, promiscuous proteins, such as Ga15 and Ga16 can be used in the chosen assay (Wilkie et al., Proc. Natl. Acad. Sci. USA, 88: 10049-10053 (1991)).

Activation of the receptor initiates subsequent intracellular events, for example, an increase in the second messengers. Activation of some G-protein coupled receptors stimulates the formation of inositol triphosphate (IP3) through phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge and Irvine, Nature, 312: 315-321 (1984)). IP3, in turn, stimulates the release of intracellular calcium ion stores. Thus, a change in cytoplasmic calcium ion levels, or a change in the levels of second messengers, such as IP3, can be used to evaluate the function of the G-protein coupled receptor. The cells expressing said G-protein coupled receptors. they may show higher levels of cytoplasmic calcium as a result of the contribution of calcium release from intracellular reserves and the entry of extracellular calcium through ionic channels in the plasma membrane.

In a preferred embodiment, the activity of the T1R polypeptide is measured by stable or transient coexpression of T1R genes, preferably stable, in a heterologous cell with a promiscuous G protein that binds the receptor to a phospholipase C signal transduction pathway ( see Offermanns and Simon, J. Biol. Chem., 270: 15175-15180 (1995)). In a preferred embodiment, the cell line is HEK-293 (which normally does not express T1R genes) and the promiscuous G protein is Ga15 (Offermanns and Simon, supra). Taste transduction modulation is tested by measuring changes in intracellular Ca2 levels, which change in response to modulation of the T1R signal transduction pathway through the administration of a molecule that is associated with the polypeptides of T1R Changes in Ca2 + levels are optionally measured using fluorescent Ca2 + indicator dyes and fluorometric imaging.

In another embodiment, the hydrolysis of phosphatidylinositol (PI) can be analyzed according to US Patent 5,436,128.

Briefly, the assay involves labeling cells with 3H-myoinositol for 48 hours or more. The labeled cells are treated with a test compound for one hour. The treated cells are lysed and extracted in chloroform-methanol-water after which the inositol phosphates are separated by ion exchange chromatography and quantified by scintillation counting. Folding stimulation is determined by calculating the proportion of cpm in the presence of an agonist, at cpm in the presence of a control buffer. Similarly, the inhibition of folding is determined by calculating the proportion of cpm in the presence of an antagonist, at cpm in the presence of a control buffer (which may or may not contain an agonist).

Other receptor assays may involve the determination of the level of intracellular cyclic nucleotides, for example, cAMP or cGMP. In cases where receptor activation results in a decrease in cyclic nucleotide levels, it may be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, for example, forskolin, before adding a receptor activating compound. to the cells in the assay. In one embodiment, changes in intracellular cAMP or cGMP can be measured using immunoassays. The method described in Offermanns and Simon, J. Bio. Chem., 270: 15175-15180 (1995) can be used to determine the level of cAMP. In addition, the method described in Felley-Bosco et al., Am. J. Resp. Cell and Mol. Biol., 11: 159-164 (1994) can be used to determine the level of cGMP. In addition, an assay kit for measuring cAMP and / or cGMP is described in US Patent 4,115,538.

In another embodiment, transcription levels can be measured to assess the effects of a test compound on signal transduction. A host cell containing T1R polypeptides of interest is contacted with a test compound for a sufficient time for any interaction to take place, and then the level of gene expression is measured. The amount of time for such interactions to occur can be determined empirically, such as establishing a development over time and measuring the level of transcription as a function of time. The amount of transcription can be measured using any method known to those skilled in the art for being suitable. For example, mRNA expression of the protein of interest can be detected using Northern blots, or its polypeptide products can be identified using immunoassays. Alternatively, transcription-based assays using indicator genes can be employed as described in US Patent 5,436,128. The indicator genes can be, for example, chloramphenicol acetyltransferase, luciferase, beta-galactosidase, beta-lactamase, and alkaline phosphatase. In addition, the protein of interest can be used as an indirect indicator by binding to a second indicator, such as the green fluorescent protein (see, for example, Mistili and Spector, Nature Biotechnology, 15: 961-964 (1997)).

The amount of transcription is then compared with the amount of transcription in the same cell in the absence of the test compound, or it can be compared with the amount of transcription in a substantially identical cell lacking the T1R polypeptide or polypeptides of interest. A substantially identical cell can be derived from the same cells from which the recombinant cell was prepared but has not been modified by the introduction of heterologous DNA. Any difference in the amount of transcription indicates that the test compound has somehow altered the activity of the T1R polypeptides of interest.

4. Transgenic non-human animals expressing chemosensory receptors

Non-human animals that express a combination of T1R taste receptor sequences can also be used for receptor assays. This expression can be used to determine whether a test compound specifically binds to a mammalian taste transmembrane receptor complex in vivo by contacting a non-human animal stably or transiently transfected with nucleic acids encoding chemosensory receptors, or regions of ligand binding of these, with a test compound, and determining whether the animal reacts with the test compound by specific binding to the receptor polypeptide complex.

Animals transfected or infected with the vectors of the invention are particularly useful for assays to identify and characterize taste stimuli that can bind to a specific receptor or receptor sets. These animals infected with a vector expressing human taste receptor sequences can be used for in vivo selection of taste stimuli and their effect on, for example, cell physiology (for example, on taste neurons), on the CNS, or behavior. Alternatively, stable cell lines that express a T1R or a combination of T1R can be used as donors for nucleic acid transfer to cloned transgenic animals produced that stably express a specific T1R or combination of T1R. The methods for using nucleic acid transfer to produce cloned animals that express a desired heterologous DNA are the subject of several US patents granted to the University of Massachusetts (registered by Advanced Cell Technology, Inc.) and Roslin Institute (registered by Geron Corp.).

Means for infecting / expressing nucleic acids and vectors, individually or in the form of libraries, are well known in the art. A variety of parameters of individual cells, organs or whole animals can be measured by a variety of means. The T1R sequences of the invention can be coexpressed, for example, in animal taste tissues by transport with an infectious agent, for example, an adenovirus expression vector.

Endogenous taste receptor genes may remain functional and their wild-type (native) activity may still be present. In other situations, when it is desirable that all taste receptor activity is produced by the introduced exogenous hybrid receptor, the use of an inactivated line is preferred. Methods for the constitution of non-human transgenic animals, in particular transgenic mice, and the selection and preparation of recombinant constructs to generate transformed cells, are well known in the art.

The constitution of an "inactivated" cell and animal ("knockout") is based on the premise that the level of expression of a particular gene in a mammalian cell can be reduced or abrogated completely by introducing a new sequence into the genome of DNA that acts to interrupt some portion of the DNA sequence of the gene that you want to repress. In addition, a "gene trap insertion" can be used to alter a host gene, and mouse embryonic precursor cells (ES) can be used to produce inactivated transgenic animals (see, for example, Holzschu, Transgenic Res., 6:97 -106 (1997)). The insertion of the exogenous sequence is generally performed by homologous recombination between complementary nucleic acid sequences. The exogenous sequence is some portion of the target gene to be modified, such as an exonic, intronic or transcriptional regulatory sequence, or any genomic sequence that is capable of affecting the level of expression of the target gene, or combinations thereof. The localization of genes by homologous recombination in pluripotential embryonic precursor cells allows to precisely modify the genomic sequence of interest. Any technique can be used to create, select, propagate an inactivated animal, for example, see Bijvoet, Hum. Mol. Genet., 7: 53-62 (1998); Moreadith, J. Mol. Med., 75: 208-216 (1997); Tojo, Cytotechnology, 19: 161-165 (1995); Mudgett, Methods Mol. Biol., 48: 167-184 (1995); Longo, Transgenic Res.,

6: 321-328 (1997); U.S. Patent Nos. 5,616,491; 5,464,764; 4,631,153; 5,487,992; 5,627,059; 5,272,071; WO 91/09955; WO 93/09222; WO 96/29411; WO 95/31560; WO 91/12650.

The nucleic acids of the invention can also be used as reagents to produce "inactivated" human cells and their progeny. Similarly, the nucleic acids of the invention can also be used as reagents to produce "inactivations" in mice. The sequences of the human or rat T1R gene can replace the orthologous T1R in the mouse genome. In this way, a mouse that expresses a human or rat T1R is produced. This mouse can then be used to analyze the function of human or rat T1R, and to identify ligands for said T1R.

to. Modulators

The compounds tested as modulators of a member of the T1R family can be any small chemical compound, or a biological entity, such as a protein, a nucleic acid or a lipid. Examples thereof include 5'-IMP and 5'-GMP. Essentially, any chemical compound can be used as a potential ligand or modulator in the assays of the invention, although compounds that are soluble in aqueous solutions are tested more frequently. Assays can be designed to select large chemical libraries by automating the test steps and providing compounds from any convenient source; These tests are generally carried out in parallel (for example, in microtiter formats or on microtiter plates in robotic tests). It will be appreciated that chemical libraries can be synthesized by one of many chemical reactions (eg, Senomyx patented chemistry). In addition, there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and Similar.

In a preferred embodiment, high performance selection methods involve providing a combinatorial peptide or chemical library containing a large number of compounds that potentially affect taste (potential modulating compounds or ligands). Said "combinatorial chemical libraries" or "ligand libraries" are then selected in one or more assays, as described herein, to identify those members of the library (in particular subclasses or chemical species) that show the desired characteristic activity . The compounds identified in this way can serve as conventional "starting compounds", or they can be used themselves as potential or actual taste modulators.

Preferably, these libraries will be selected against cells or cell lines that stably express T1R2 / T1R3 and preferably a suitable G protein, for example, Ga15. As shown in the following examples, these stable cell lines show very pronounced responses to taste stimuli, for example, sweet or sweet taste stimuli. However, cells and cell lines that express transiently T1R2 / T1R3 can also be used in these assays.

A combinatorial chemical library is a collection of various chemical compounds generated by chemical synthesis or biological synthesis, combining a series of chemical "basic components," such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide library, is formed by combining a set of chemical basic components (amino acids) in any possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound) . Thousands to millions of chemical compounds can be synthesized by said combinatorial mixture of chemical basic components.

The preparation and selection of combinatorial chemical libraries is well known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, for example, US Patent No. 5,010,175; Furka, Int. J. Pept. Prot. Res., 37: 487-493 (1991); and Houghton et al., Nature, 354: 8488 (1991)). Other chemistries can also be used to generate libraries of chemical diversity. These chemicals include, but are not limited to: peptoids (for example, PCT publication No. WO 91/19735), encoded peptides (for example, PCT publication WO 93/20242), random bio-oligomers (for example, PCT publication WO 92/00091 ), benzodiazepines (for example, US Patent No. 5,288,514), diversomers, such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Natl. Acad. Sci. USA, 90: 6909-6913 (1993)) , vinilist polypeptides (Hagihara et al., J. Amer. Chem. Soc., 114: 6568 (1992)), non-peptide peptidomimetics with glucose framework (Hirschmann et al., J. Amer. Chem. Soc., 114: 9217-9218 (1992)), similar organic synthesis of small compound libraries (Chen et al., J. Amer. Chem. Soc., 116: 2661 (1994)), oligocarbamates (Cho et al., Science, 261: 1303 (1993)), peptidyl phosphonates (Campbell et al., J. Org. Chem., 59: 658 (1994)), nucleic acid libraries (Ausubel, Berger and Sambrook, all of them cited above), bibliotec peptide nucleic acid ace (US Patent No. 5,539,083), antibody libraries (Vaughn et al., Nature Biotechnology, 14 (3): 309-314 (1996); and PCT / US96 / 10287), carbohydrate libraries (Liang et al., Science, 274: 15201522 (1996)); and US Patent 5,593,853), libraries of small organic molecules (benzodiazepines, Baum, C&EN, January 18, p. 33 (1993)); thiazolidinones and metathiazanones (US Patent 5,549,974); pyrrolidines (U.S. Patent 5,525,735 and 5,519,134); morpholino compounds (US Patent 5,506,337); benzodiazepines (document 5,288,514) and the like.

Combination library preparation devices are commercially available (see, for example, 357 MPS, 390 MPS (Advanced Chem Tech, Louisville, KY), Symphony (Rainin, Woburn, MA), 433A (Applied Biosystems, Foster City , CA), 9050 Plus (Millipore, Bedford, MA)). In addition, numerous combinatorial libraries are themselves available in the market (see, for example, ComGenex, Princeton, NJ; Tripos, Inc., St. Louis, MO; 3D Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD; etc.).

In one aspect of the description, T1R modulators can be used in any food product, pastry product, pharmaceutical composition or ingredient thereof in order to modulate the flavor of the product, composition or ingredient in a desired manner. For example, T1R modulators that enhance the sweet taste sensation can be added to sweeten a product or composition; T1R modulators can be added that enhance the sensation of umami flavor to foods to increase the tasty taste. Alternatively, T1R antagonists can be used to block the sweet and / or umami taste.

b. Kits

T1R genes and their counterparts are useful tools for identifying chemosensory receptor cells, for paternity and forensic determinations, and for studying taste transduction. Specific reagents of members of the T1R family that specifically hybridize with T1R nucleic acids, such as T1R probes and primers, and specific T1R reagents that specifically bind to a T1R polypeptide, for example, T1R antibodies, are used to study the expression of taste cells and the regulation of taste transduction.

Nucleic acid assays to detect the presence of DNA and RNA of a member of the T1R family in a sample include numerous techniques known to those skilled in the art, such as Southern blot analysis, Northern blot analysis, blot transfers. points, RNAse protection, S1 analysis, amplification techniques, such as PCR, and in situ hybridization. In in situ hybridization, for example, the target nucleic acid is released from its cellular environment so that it is available for hybridization within the cell, while cell morphology is retained for subsequent interpretation and analysis. The following articles provide an overview of the technique of in situ hybridization: Singer et al., Biotechniques, 4: 230-250 (1986); Haase et al., Methods in Virology, vol. VII, pp. 189-226 (1984); and Nucleic Acid Hybridization: A Practical Approach (Names et al., eds., 1987). In addition, a T1R polypeptide can be detected with the various immunoassay techniques described above. The test sample is generally compared to a positive control (for example, a sample that expresses a recombinant T1R polypeptide) and a negative control.

The present description also provides kits for selecting modulators of members of the T1R family. These kits can be prepared from readily available materials and reagents. For example, these kits may comprise one or more of the following materials: T1R nucleic acids or proteins, reaction tubes, and instructions for testing T1R activity. Optionally, the kit contains a biologically active T1R receptor, or a cell line that stably or transiently expresses a taste receptor that contains biologically active T1R. A wide variety of kits and components can be prepared according to the present description depending on the intended user of the kit and the specific needs of the user.

Examples

These examples are only intended to be illustrative.

In the protein sequences presented herein, the one letter code X or Xaa refers to any of the twenty usual amino acid residues. In the DNA sequences presented herein, the one letter code N or n refers to any of the four usual nucleotide bases, A, T, C or

G.

Example 1: Production of hT1R expression constructs without introns

Intrusion constructs of hT1R without introns were cloned by a combination of cDNA-based and genomic DNA-based methods. To generate the full-length hT1R1 expression construct, two 5 'coding exons identified in a cloned hT1R1 interval (registration no. AL159177) were combined by PCR overlap, and then joined to a 5' testis cDNA clone. -truncated. The hT1R2 expression construct was generated from a partially sequenced genomic range of hT1R2. Two missing 5 ’exons of hT1R2 were identified by random library selection of the cloned genomic range using probes derived from the corresponding rat coding sequence. The coding exons were then combined by PCR overlap to produce the full length expression construct. The expression construct of hT1R3 was generated by PCR overlapping from a genomic range of sequenced hT1R3 (registration no. AL139287). Rat T1R3 was isolated from a cDNA library derived from rat gustatory tissue using an exon fragment of rT1R3 generated by a degenerated PCR based on hT1R3. The partial hT1R1 cDNA, the rT1R2 cDNA, and the partial hT1R2 genomic sequences were obtained from Dr. Charles Zuker (University of California, San Diego).

The nucleic acid and amino acid sequences for the cloned T1R sequences identified above, as well as other full-length and partial T1R sequences are indicated below:

SEQ ID NO: 4 Amino acid sequence of rT1R3

SEQ ID NO: 5 Amino acid sequence of hT1R1 SEQ ID NO: 6 Amino acid sequence of hT1R2

SEQ ID NO: 7 Amino acid sequence of hT1R3 SEQ ID NO: 8 Nucleic acid sequence of hT1R1 SEQ ID NO: 9 Nucleic sequence of hT1R3

SEQ ID NO: 10 Nucleic acid sequence of hT1R2 SEQ ID NO: 11 Nucleic acid sequence of rT1R3

In addition, the following conceptual translations, corresponding to the C-terminal of two orthologous pairs of the fish T1Rs, are derived from fragments of unpublished genomic sequences are provided. The Fugu T1RA derived from the “frame 164” record; Fugu T1RB derived from the LCP61711 registry; Tetradon T1RA derived from registration AL226735; Tetradon T1RB was derived from the AL222381 registry. Ambiguities in conceptual translations ("X") result from ambiguities in the database sequences.

SEQ ID NO: 12

T1RA Fugu SEQ ID NO: 13 10 T1RA Tetradon SEQ ID NO: 14 T1RB Fugu SEQ ID NO: 15 T1RB Tetradon

In addition, the registration number and bibliographic citations in relation to mouse and rat T1Rs and their allelic variants in the public domain are indicated below:

rT1R1 (registration number AAD18069) (Hoon et al., Cell, 96 (4): 541-551 (1999)); rT1R2 (registration number AAD18070) (Hoon et al., Cell, 96 (4): 541-559 (1999)); mT1R1 (registration number AAK39437); mT1R2 (registration number AAK39438); mT1R3 (registration number AAK55537) (Max et al., Nat. Genet., 28 (I): 58-63 (2001)); rT1R1 (registration number AAK7092) (Li et al., Mamm. Genome, 12 (I): 13-16 (2001)); mT1R1 (registration number NP114073); mT1R1 (registration number AAK07091) (Li et

10 al., Mamm. Genome, 12 (I): 13-16 (2001)); rT1R2 (registration number AAD18070) (Hoon et al., Cell, 96 (4): 541-551 (1999)); mT1R2 (registration number NP114079); mT1R3 (registration number AAK39436); mT1R3 (registration number BAB47181) (Kitagawa et al., Biochem. Biophys. Res. Commun., 283 (I): 236-242 (2001)); mT1R3 (registration number NP114078); mT1R3 (registration number AAK55536) (Max et al., Nat. Genet., 28 (I): 58-63 (2001)); and mT1R3 (registration number AAK01937).

Example 2: Alignment of human and rat T1R sequences

15 Cloned T1R sequences selected from those identified above were aligned against the corresponding rat T1R. As shown in Figure 1, human T1R1, human T1R2 and human T1R3 and rat T1R3 aligned with the previously described T1R (rT1R1 having registration number AAD18069, and rT1R2 having registration number AAD18070), Rat mGluR1 metabotropic glutamate receptor (registration No. P23385), and the human calcium detector receptor (registration No. P41180). To make the comparison easier,

20 C-terminals of the mGluR1 and calcium receptors were truncated. The seven potential transmembrane segments are framed in blue. The residues that come into contact with the carbutylate of the glutamate side chain in the crystalline structure of mGluR1 are framed in red, and the residues that come into contact with the a-amino acid residue of glutamate are framed in green. The cysteine residues of the mGluR1 receptor and calcium detector receptor involved in the formation of disulfide bonds between subunits are surrounded by a circle

25 purple These cysteines are not conserved in T1R1 and T1R2, but are located in a degraded region of the alignment that contains a potentially similar T1R3 cysteine residue, also surrounded by a circle.

Example 3: Demonstration by RT-PCR that hT1R2 and hT1R3 are expressed in gustatory tissue

As shown in Figure 2, hT1R2 and hT1R3 are expressed in gustatory tissue: the expression of both genes can be detected by RT-PCR of resected human circumvalated papillae.

30 Example 4: Methods for heterologous expression of T1R in heterologous cells

A derivative of HEK-293 (Chandrashekar et al., Cell, 100 (6): 703-711 (2000)), which stably expresses Ga15, was cultured and maintained at 37 ° C in the middle of Eagle modified by Dulbecco ( DMEM, Gibco BRL) supplemented with 10% FBS, MEM of non-essential amino acids (Gibco BRL) and blasticidine 3 µg / ml. For calcium imaging experiments, cells were first seeded on 24-well tissue culture plates 35 (approximately 0.1 million cells per well), and transfected by lipofection with Mirus Transit-293 (PanVera) . To minimize glutamate-induced and glucose-induced desensitization, the supplemented DMEM was replaced by DMEM with low glucose / GlutaMAX (Gibco BRL) content approximately 24 hours after transfection. Twenty-four hours later, the cells were loaded with the calcium dye Fluo-4 (Molecular Probes), 3 µM in Dulbecco PBS buffer (DPBS, Gibco BRL), for 1.5 hours at room temperature. After replacement with 250 µl of DPBS, stimulation was performed at room temperature by adding 200 µl of DPBS supplemented with flavor stimuli. Calcium mobilization was monitored with an Axiovert S100 TV (Zeiss) microscope using the Imaging Workbench 4.0 (Axon) software. The responses of T1R1 / T1R3 and T1R2 / T1R3 were surprisingly transient (the increase in calcium rarely persisted more than 15 seconds) and asynchronous. Therefore, the number of cells that respond is

45 relatively constant over time; therefore, cellular responses were quantified by a manual count of the number of cells that respond at a fixed time, usually 30 seconds after the stimulus was added.

Example 5: Functions of human T1R2 / T1R3 as a sweet taste receptor

HEK cells stably expressing Ga15 were transiently transfected with human T1R2, T1R3 and T1R2 / T1R3, and tested for the increase in intracellular calcium in response to increasing sucrose concentrations (Figure 3 (a)). In addition, the dose responses of T1R2 / T1R3 were determined for various sweet taste stimuli (Figure 3 (b)). The maximum percentage of cells that respond is different for different sweeteners, and varies from 10-30%. To increase clarity, dose responses were normalized to the maximum percentage of cells that respond. The values in Figure 3 represent the mean � e.e. of four independent responses. Circles on the abscissa axis mark the psychophysical detection thresholds determined by taste testing. Gurmarine (50-fold dilution of an aqueous extract of Gymnema sylvestre 10 g / l filtrate) inhibits the response of T1R2 / T1R3 to 250 mM sucrose, but not the response of the endogenous 12-adrenergic receptor to 20 µM isoproterenol (Figure 3 (b)). Figure 3 (c) contains the normalized response of the cell lines that coexpress T1R2 / T1R3 to different sweeteners (sucrose, aspartame, Dtriptophan and saccharin).

Example 6: Rat T1R2 / T1R3 also acts as a sweet taste receptor

HEK cells stably expressing Ga15 were transiently transfected with hT1R2 / hT1R3, rT1R2 / rT1R3, hT1R2 / rT1R3, and rT1R2 / hT1R3. These transfected cells were then tested for the increase in intracellular calcium in response to 350 mM sucrose, 25 mM tryptophan, 15 mM aspartame, and 0.05% monelin. The results with sucrose and aspartame appear in Figure 4, and indicate that rT1R2 / rT1R3 also acts as a sweet taste receptor. In addition, these results suggest that T1R2 can control the ligand specificity of T1R2 / T1R3.

Example 7: T1R2 / T1R3 responses using an automatic fluorescence based assay

HEK cells stably expressing Ga15 were transiently transfected with hT1R2 and hT1R3. These cells were loaded with the Fluo-4 calcium dye, and their responses to a sweetener were measured using a fluorescence plate reader. Figure 5 contains the cyclamate responses (12.5 mM) for cells expressing hT1R2 / hT1R3 and for cells expressing only hT1R3 (J19-22). The fluorescence results obtained indicate that the responses to these flavor stimuli only occur in cells expressing hT1R2 / hT1R3. Figure 6 contains the standardized dose response curves, the results of which show that hT1R2 and hT1R3 act together as a human taste receptor based on their specific dose interaction with various sweet stimuli. In particular, Figure 6 contains the dose responses for sucrose, tryptophan and various other commercially available sweeteners. These results indicate that T1R2 / T1R3 is a gustatory receptor of human candy, since the order of rank and threshold values obtained in the assay closely resemble the values for human sweet taste.

Example 8: The ligand binding moieties of mGluR1 are conserved in T1R1

As shown in Figure 6, the key ligand binding moieties of mGluR1 are conserved in T1R1. The interaction of glutamate with mGluR1 is shown, highlighting several key residues according to the same color scheme as in Figure 1.

Example 9: Human T1R1 / T1R3 acts as a taste receptor for umami

HEK cells stably expressing Ga15 were transiently transfected with human T1R1, T1R3 and T1R1 / T1R3, and tested for the increase in intracellular calcium in response to increasing concentrations of glutamate (Figure 8 (a)), since 0.5 mM glutamate, at 0.2 mM IMP, and 0.5 mM glutamate plus 0.2 mM IMP (Figure 8 (b)). The dose responses of human T1R1 / T1R3 for glutamate were determined in the presence and absence of 0.2 mM IMP (Figure 8 (c)). The maximum percentages of responding cells were approximately 5% for glutamate and approximately 10% for glutamate plus IMP. To increase clarity, dose responses were normalized to the maximum percentage of cells that respond. The values represent the mean � e.e. of four independent responses. Circles on the abscissa axis mark the detection thresholds determined by taste testing.

Example 10: PDZIP as an export sequence

The sequence of six PDZIP residues (SVSTW (SEQ ID NO: 1)) was condensed with the C-terminal of hT1R2, and the chimeric receptor (i.e., hT1R2-PDZIP) was transfected into a HEK-293 host cell. The surface expression of hT1R2 was then monitored using immunofluorescence and FACS scan data. As shown in Figures 9A and 9B, the inclusion of the PDZIP sequence increases the surface expression of hT1R2-PDZIP relative to hT1R2. More specifically, Figure 9A shows an immunofluorescence staining of myc-labeled hT1R2 demonstrating that PDZIP significantly increases the amount of hT1R2 protein on the plasma membrane. Figure 6B shows the FACS analysis data showing the same result: cells expressing hc1R2 labeled with myc are indicated with the dashed line, and cells expressing hT1R2-PDZIP labeled with myc are indicated with the continuous line. In particular, Figure 10A shows stable Ga15 host cells not transfected in HBS buffer, Figure 10B shows stable Ga15 host cells transfected with hT1R2-PDZIP in the group of sweeteners # 5 (saccharin, sodium cyclamate, acesulfame K, and aspartame, 20 mM each in HBS buffer), Figure 10C shows stable Ga15 host cells transfected with T1R3-PDZIP in the # 5 sweetener pool, and Figure 10D shows stable Ga15 host cells co-transfected with hT1R2-PDZIP / hT1R3-PDZIP in sweetener group no.

5. In addition, Figures 10E-10H show the dose-dependent response of stable Ga15 host cells co-transfected with hT1R2 / hT1R3 against sucrose (E: 0 mM in HBS buffer; F: 30 mM; G: 60 mM; and H : 250 mM). Figures 10I-10L show the responses of stable Ga15 host cells co-transfected with hT1R2 / hT1R3 against individual sweeteners (I: aspartame (1.5 mM); J: acesulfame K (1 mM); K: neotame (20 mM); L: sodium cyclamate (20 mM)). As demonstrated by the calcium images in Figure 10, both hT1R2 and hT1R3 are required for activities activated by the sweet stimulus.

Example 11: Generation of cell lines that stably coexpress T1R1 / T1R3 or T1R2 / T1R3

Human cell lines that stably coexpress human T1R2 / T1R3 or human T1R1 / T1R3 were generated by transfecting linearized PEAK10 derived vectors (Edge Biosystems) and vectors derived from pCDNA 3.1 / ZEO (Invitrogen), which respectively contain a construct of hT1R1 expression

or hT1R2 (plasmid SAV2485 for T1R1, SAV2486 for T1R2) and hT1R3 (plasmid SXV550 for T1R3) in a cell line expressing Ga15. Specifically, stable T1R2 / T1R3 cell lines were co-transfected SAV2486 and SXV550 linearized in a HEK-293 cell line from Aurora Bioscience stably expressing Ga15. Stable T1R1 / T1R3 cell lines were produced by transfecting SAV2485 and SXV550 linearized on the same HEK-293 cell line that stably expresses Ga15. After transfections with SAV2485 / SXV550 and SAV2486 / SXV550, the puromycin-resistant and zeocin-resistant colonies were selected, expanded and tested by calcium imaging for responses to sweet or umami stimuli. Cells were selected in puromycin 0.0005 mg / ml (CALBIOCHEM) and zeocin 0.1 mg / ml (Invitrogen) at 37 ° C in DMEM with low glucose content supplemented with GlutaMAX, 10% dialyzed FBS, and 0.003 mg blasticidine / ml Resistant colonies expanded, and their responses to sweet taste stimuli were evaluated by fluorescence microscopy. For automatic fluorimetric imaging with a VIPR-II instrument (Aurora Biosciences), stable T1R2 / T1R3 cells were first seeded on 96-well plates (approximately 100,000 cells per well). Twenty-four hours later the cells were loaded with the calcium dye Fluo-3-AM (Molecular Probes), 0.005 mM in PBS for 1 hour at room temperature. After substitution with 70 µl of PBS, stimulation was carried out at room temperature by adding 70 µl of PBS supplemented with flavor stimuli. Fluorescence responses (480 nm excitation and 535 nm emission) from 20 to 30 seconds after compound addition were averaged, corrected for background fluorescence measured before compound addition, and normalized to response to 0.001 mM ionomycin (CALBIOCHEM), a calcium ionophore.

Then it was observed that when these cell lines are exposed to sweet stimuli or umami, in active clones generally 80-100% of cells respond to taste stimuli. Unexpectedly, the magnitude of individual cell responses was markedly greater than that of transiently transfected cells.

Based on this observation, the inventors tested the activity of T1R stable cell lines by automatic fluorescence imaging using the Aurora Biosciences VIPR instrument as described above. The responses of two T1R1 / T1R3 cell lines and a T1R2 / T1R3 cell line are shown in Figure 11 and Figure 12, respectively.

Notably, the combination of an increasing number of responding cells and increasing response magnitudes resulted in a 10-fold increase in activity relative to transiently transfected cells (as a comparison, the percentage of response to ionomycin of transiently transfected cells with T1R2 / T1R3 is approximately 5% under optimal conditions). In addition, responses to the doses obtained for T1R2 / T1R3 and T1R1 / T1R3 stably expressed correlate with human taste detection thresholds. The robust T1R activity of these stable cell lines suggests that they are very suitable for use in the high-performance selection of chemical libraries to identify compounds, for example, small molecules, that modulate the sweet or umami taste receptor and that, by therefore, modulate, potentiate, block or imitate the sweet or umami flavor.

Example 12: Generation of cell lines that coexpress T1R1 / T1R3 in an inducible way and selectively respond to umami taste stimuli

HEK-293 T1R1 / T1R3 cell lines stably expressing the umami taste receptor shows robust enhanced activity relative to transiently transfected cells. However, a disadvantage is that they can lose activity rapidly during cell propagation.

In addition, these findings support the inventors' hypothesis that (i) T1R1 / T1R3 is the umami taste receptor, and (ii) the robust T1R1 / T1R3 cell lines, preferably the stable T1R1 / T1R3 cell lines and / or inducible, can be used in assays, preferably for high-performance selection of chemical libraries to identify new umami flavor modulators. Modulators that enhance the umami flavor can be used.

To solve the instability of stable T1R1 / T1R3 cell lines, HEK-Ga15 cells have been modified to induce T1R1 / T1R3 inducible expression using the GeneSwitch system (Invitrogen). The zeocin-resistant expression vectors derived from pGene for T1R1 and T1R3 (plasmid SXV603 for T1R1 and SXV611 for T1R3) and a puromycin-resistant pSwitch vector carrying the GeneSwitch protein (plasmid SXV628) were linearized and co-transfected on the line. HEK-Ga15 cell. Zeocin and puromycin resistant colonies were selected, expanded, induced with varying amounts of mifepristone, and tested by calcium imaging for responses to umami flavor stimuli.

The inducible expression of T1R1 / T1R3 results in robust activity. For example, about 80% of induced cells, but only about 10% of transiently transfected cells, respond to Lglutamate. More specifically, the zeocin-resistant expression vectors derived from pGene expressing human T1R1 and human T1R3, and a puromycin-resistant pSwitch vector carrying the GeneSwitch protein were linearized and co-transfected into HEK-Ga15 cells. Cells were selected in 0.5 µg / ml puromycin (CALBIOCHEM) and 100 µg / ml zeocin (Invitrogen) at 37 ° C in Dulbecco's modified Eagle's medium supplemented with GlutaMAX (10% dialyzed FBS, and 3 ug basticidine / ml) Resistant colonies expanded, and their responses to umami taste stimuli were determined after induction with 10-10 M mifepristone by fluorescence microscopy following the methods of Li et al., PNAS, 99 (7): 4692-4696 (2002 ).

For automatic fluorometric imaging on a FLIPR (Molecular Device) instrument, the cells of a clone (called clone I-17) were seeded in 96-well plates (approximately 80,000 cells per well) in the presence of 10-10 M mifepristone and incubated for 48 hours. The cells were then loaded with the Fluo-4-AM (Molecular Probes) calcium dye, 3 µM in PBS for 1.5 hours at room temperature.

After substitution with 50 µl of PBS, stimulation was carried out at room temperature by adding 50 µl of PBS supplemented with different stimuli. In contrast to the previous transient expression systems of the T1R1 / T1R3 umami receptor, which require quantification of the activity of the T1R1 / T1R3 receptor by individual counting of the responding cells (Li et al., PNAS, 99 (7) : 4692-4696 (2002)) (due to the low activity of the receptor in them), this inducible expression system produces a clone I-17 that has a substantially greater activity that allows quantifying the activity of the receptor by determining the maximum increase in fluorescence (480 nm excitation and 535 nm emission) added from all cell imaging fields. The maximum fluorescence of four independent determinations was averaged, corrected for the background fluorescence measured before the compound was added and normalized to the response against 0.002 mM ionomycin (CALBIOCHEM).

These results are contained in Figure 13. In particular, Figure 13 contains a dose-response curve determined for L-glutamate in the presence and absence of 0.2 mM IMP. In the figure, each value represents the average of the maximum added fluorescence (corrected for background fluorescence) for four independent determinations. These dose response curves correspond to those determined for cells transiently transfected with T1R1 / T1R3.

Umami T1R1 / T1R3 taste receptor selectivity was also evaluated by a selection with different L-amino acids. The results obtained indicate that T1R1 / T1R3 is selectively activated by Umami-flavored L-amino acids (L-glutamate and L-aspartate).

The results of experiments in which the responses of clone I-17 were obtained when tested in the presence of different L-amino acids appear in Figure 14 and Figure 15. Figure 14 shows the results of an experiment in which the line I-17 cell was contacted with different L-amino acids at a concentration of 10 mM in the presence and in the absence of 1 mM IMP.

Figure 15 contains a dose-response curve for active amino acids determined in the presence of 0.2 mM IMP. Each value represents the average of four independent determinations.

The results obtained in these experiments support the specificity and selectivity of the umami taste receptor by umami flavor stimuli. While the L-glutamate and L-aspartate flavor stimuli significantly activate the T1R1 / T1R3 receptor at different concentrations (see Figures 14 and 15), the other Lamino acids that activate the human T1R1 / T1R3 receptor only activate the receptor weakly and at concentrations much older.

Therefore, these results support the selectivity of the T1R1 / T1R3 receptor by umami flavor stimuli and the suitability of this inducible stable expression system for use in high performance assays using automatic fluorometric imaging tools to identify compounds that activate the umami taste receptor, for example L-glutamate and L-aspartate, or that potentiate the activity of L-glutamate to activate the umami taste receptor, for example 5'-IMP or 5'-GMP, or block activation of the umami taste receptor by umami flavor stimuli, such as L-glutamate and L-aspartate.

Compounds identified using these assays have a potential application as flavorings in food and beverage compositions to mimic or block umami flavor stimuli.

Example 13: Lactisol inhibits the activities of the human T1R2 / T1R3 and T1R1 / T1R3 receptor, and the sweet and umami taste

It was believed that lactisol, an aralkylcarboxylic acid, was a selective sweet taste inhibitor (see, for example, Lindley (1986), US Patent 4,567,053; and Schiffman et al., Chem. Senses, 24: 439-447 (1999)). The responses of transiently transfected HEK-Ga15 cells with T1R2 / T1R3 to 150 mM sucrose were measured in the presence of varying concentrations of lactisol. Lactisol inhibits the activity of human T1R2 / T1R3 with an IC50 of 24 µM.

The gustatory receptor of the umami T1R1 / T1R3 and the sweet T1R2 / T1R3 can share a common subunit. Therefore, the theory has been reached that lactisol, which inhibits the sweet taste receptor T1R2 / T1R3, can have a similar effect on the taste taste receptor of the umami T1R1 / T1R3. The inventors of the present invention tested the effect of lactisol on the response of human T1R1 / T1R3 to 10 mM L-glutamate. As with the sweet taste receptor T1R2 / T1R3, lactisol also inhibits T1R1 / T1R3 with an IC50 of 165 µM. It is likely that inhibition by lactisol reflects an antagonism in T1R receptors rather than, for example, a non-specific inhibition of Ga15-mediated signaling, because the response of muscarinic acetylcholine receptors was not inhibited by lactisol.

Then, the inventors of the present invention evaluated the effect of lactisol on human umami taste. The taste thresholds were determined in the presence of lactisol 1 and 2 mM for the Umami L-glutamate flavor stimulus with or without 0.2 mM IMP, the sweet taste stimuli sucrose and D-tryptophan, and the salt salty flavor stimulus of sodium, following the methods of Schiffman et al. (Chem. Senses, 24: 439-447 (1989)). Milimolar concentrations of lactisol dramatically increased the detection thresholds for sweet and umami taste stimuli, but not for salty taste stimuli. These results appear in Figure 16.

To conclude, (i) these findings also support the inventors' hypothesis that T1R1 / T1R3 is the only gustatory receptor of umami, and (ii) that the T1R1 / T1R3 and T1R2 / T1R3 receptors can share a lactisol binding domain structurally related.

Claims (18)

1.-A taste receptor comprising at least one T1R2 polypeptide and at least one T1R3 polypeptide, wherein said taste receptor specifically binds to and / or is activated by a sweet taste stimulus, wherein said T1R2 polypeptide It is selected from the group consisting of:

(to)

 a polypeptide having an amino acid sequence at least 85% identical to SEQ ID NO: 6, a fragment thereof having a length of at least 40 amino acids, or a fragment of an amino acid sequence that is at least 90% identical to SEQ ID NO: 6 and having a length of at least 20 amino acids;

(b)

 a polypeptide encoded by a polynucleotide that, under stringent hybridization conditions, hybridizes with the reverse complement of SEQ ID NO: 10; Y

(C)

a polypeptide encoded by a polynucleotide that, under stringent hybridization conditions, hybridizes with the reverse complement of a polynucleotide encoding the polypeptide of SEQ ID NO: 6;

and wherein said T1R3 polypeptide is selected from the group consisting of:

(d)

 a polypeptide having an amino acid sequence at least 85% identical to SEQ ID NO: 7, a fragment thereof having a length of at least 40 amino acids, or a fragment of an amino acid sequence that is at least 90% identical to SEQ ID NO: 7 and having a length of at least 20 amino acids;

(and)

 a polypeptide encoded by a polynucleotide that, under stringent hybridization conditions, hybridizes with the reverse complement of SEQ ID NO: 9; Y

(F)

 a polypeptide encoded by a polynucleotide that, under stringent hybridization conditions, hybridizes with the reverse complement of a polynucleotide encoding the polypeptide of SEQ ID NO: 7.

2. The receptor of claim 1, wherein said T1R2 polypeptide and said T1R3 polypeptide are derived from the same or different species. 3. The receptor of claim 1, wherein the T1R2 polypeptide and / or the T1R3 polypeptide are derived from a mammal, a fish, a reptile, an amphibian or a bird, and are preferably selected from the group consisting of hT1R2, hT1R3, rT1R2, rT1R3, mT1R2, mT1R3. 4. The receptor of claim 1, wherein at least one of the T1R polypeptides is coupled to amino acids that represent all or part of another receptor coupled to protein G. 5. The receptor of claim 4, wherein all or part of another G-protein coupled receptor comprises an extracellular, transmembrane or intracellular domain. 6. A composition containing a receptor according to claim 4 or claim 5. 7. An expression vector comprising the nucleic acid sequence encoding the taste receptor according to any one of claims 1 to 5, which is operatively linked to at least one regulatory sequence. 8. A host cell comprising an expression vector according to claim 7, which expresses a receptor according to any of claims 1 to 5. 9. The host cell of claim 8, which is selected from the group consisting of a HEK-293 cell, a COS cell, a CHO cell, and Xenopus oocytes. 10. The host cell of claim 8, which expresses, stably or transiently, said receptor, and also optionally expresses a G protein, preferably a promiscuous G protein. 11. A method for identifying compounds that modulate the perception of taste by identifying compounds that bind, activate, inhibit and / or modulate a receptor according to any one of claims 1 to 5. 12. The method of claim 11, wherein the receptor is expressed in vitro in a cell according to any one of claims 8 to 10. 13. The method of claim 12, wherein said compound is identified by its effect on receptor internalization, receptor phosphorylation, or arrestine translocation. 14. The method of claim 11, wherein said compound is identified by its effect on the activation of G protein by said receptor, the conformation of the receptor, the changes in the intracellular concentration of a second messenger, the potential for cell membrane, transcription assays with reporter genes, or intracellular calcium concentration. 15. The method of claim 12, wherein said compound agonizes or antagonizes the T1R2 / T1R3 receptor. 16. The method of claim 15, wherein the cell inducibly expresses the receptor, and wherein the method is used to select a compound that competes with IMP, GMP or its analogs for binding with the taste receptor of sweet T1R2 / T1R3. 17. The method of claim 15, which is a high performance screening assay that employs automatic fluorometric imaging instrumentation. 18. The method of claim 11, wherein said compound is identified by its effect on a non-human animal that expresses the transgenic T1R receptors of any one of claims 1 to 5.  Figure 1: Catalog of human and rat T1Rs   Figure 2: hT1R2 and hT1R3 are expressed in the human tongue epithelium. The specific cDNA amplification products can be amplified from cDNA prepared from resected human circumvalated papillae. Figure 3: Human T1R2 / T1R3 acts as a sweet taste receptor Figure 4: T1R2 can control the specificity of the T1R2 / T1R3 ligand Figure 7: The key ligand binding moieties of mGluR1 are conserved in T1R1 Figure 8: Human T1R1 / T1R3 acts as a taste receptor for umami Figure 9: PDZIP facilitates the expression of human T1R2 on the surface.  A. Immunofluorescence staining of Myc-labeled hT1R2 indicates that PDZIP significantly increases the amount of human T1R2 protein on the plasma membrane. B. FACS analysis data demonstrates the same result. Human T1R2 labeled with Myc: green line; C. Human T1R2 labeled with Myc with PDZIP: black line. Figure 10: Calcium imaging data demonstrate the responses of hT1R2 / hT1R3 to a series of sweet stimuli 5 Figure 16: Lactisol inhibits the receptors of the sweet T1R2 / T1R3 and the umami T1R1 / T1R3 and the sweet and umami taste. (Left panel) HEK-G cell responses are shown 15 transiently transfected with T1R1 / T1R3 (circles) to 10 mM L-glutamate, and HEK-Ga15 cells transiently transfected with T1R2 / T1R3 (square) to 150 mM sucrose, in the presence of varying concentrations of lactisol. (Right panel) increases in number of times in taste detection thresholds are shown 10 in the presence of 1 and 2 mM lactisol for sweet, sucrose and D-tryptophan flavor stimuli, umami, L-glutamate (MSG) and L-glutamate plus 0.2 mM IMP, and sodium chloride stimuli. The detection thresholds were determined following the method of Schiffman et al.