JP2005500318A - T1R hetero-oligomeric taste receptors and cell lines expressing said receptors and their use for the identification of taste compounds - Google Patents

Abstract

The present invention relates to the discovery that T1R receptors aggregate to form functional taste receptors. In particular, it has been discovered that co-expression of T1R1 and T1R3 provides a taste receptor that responds to umami stimulants including glutamate monosodium salt. It has also been discovered that co-expression of T1R2 and T1R3 receptors results in taste receptors that respond to sweetness stimulants including natural and artificial sweeteners. The invention also relates to the use of hetero-oligomeric taste receptors comprising T1R1 / T1R3 and T1R2 / T1R3 in assays to identify compounds that respond to umami and sweet taste stimuli, respectively. Furthermore, the present invention relates to the construction of cell lines that stably or temporarily co-express T1R1 and T1R3 or a combination of T1R2 and T1R3 under constitutive or inducible conditions. Also provided is the use of these cell lines in assays using cells to identify umami and sweetness modulating compounds, particularly high throughput assays that detect receptors using fluorometric imaging. Finally, since the present invention inhibits sweetness and umami taste according to certain compounds, such as lactisol according to the human T1R2 / T1R3 and T1R1 / T1R3 receptors, these receptors are the only sweet and umami tastes. It relates to the discovery that it is suggested to be a receptor.

Description

【Technical field】
[0001]
This application is a US Provisional Application No. 60 / 300,434, filed Jun. 26, 2001, and U.S. Utility Application, filed Jul. 3, 2001, all of which are incorporated herein by reference in their entirety. 09 / 897,427, US provisional application 60 / 304,749, filed July 13, 2001, US provisional application 60 / 310,493, filed August 8, 2001, November 21, 2001. US Provisional Application No. 60 / 331,771, filed December 14, 2001, US Provisional Application No. 60 / 339,472, filed December 14, 2001, and US Application No. 10 / 035,045, filed January 3, 2002. Claims priority to US Provisional Application No. 60 / 372,090 filed Apr. 15, 2002 and US Provisional Application No. 60 / 374,143 filed Apr. 22, 2002.
[0002]
The present invention relates in part to the discovery that T1R receptors aggregate to form functional taste receptors. In particular, it has been discovered that co-expression of T1R1 and T1R3 provides a taste receptor that responds to umami stimulants including glutamate monosodium salt. It has also been discovered that co-expression of T1R2 and T1R3 receptors results in taste receptors that respond to sweetness stimulants including natural and artificial sweeteners. The invention also relates to the use of hetero-oligomeric taste receptors comprising T1R1 / T1R3 and T1R2 / T1R3 in assays to identify compounds that respond to umami and sweet taste stimuli, respectively.
[0003]
Furthermore, the present invention relates to the construction of cell lines that stably or temporarily co-express T1R1 and T1R3 or a combination of T1R2 and T1R3 under constitutive or inducible conditions.
[0004]
Also provided is the use of these cell lines in assays using cells to identify umami and sweetness modulating compounds, particularly high throughput screening assays that detect receptor activity using fluorometric imaging.
[Background]
[0005]
The taste system provides sensory information about the chemical composition of the outside world. Mammals are thought to have at least five basic tastes: sweet, bitter, sour, salty and umami. For example, Kawamura et al. , Introduction to Umami: A Basic Teste (1987), Kinnamon et al. Ann. Rev. Physiol. 54, 715-31 (1992), Lindemann, Physiol. Rev. 76, 718-66 (1996), Stewart et al. Am. J. et al. Physiol. 272, 1-26 (1997). Each taste is thought to be mediated by a separate protein receptor or multiple receptors expressed in taste receptor cells found on the surface of the tongue (Lindmann, Physiol. Rev., Vol. 76, 718-716). Page (1996)). Taste receptors that recognize bitterness, sweetness and umami taste belong to the G protein coupled receptor (GPCR) superfamily (Hoon et al., Cell, 96, 451 (1999), Adler et al. Cell, 100, 693 (2000)). (Other types of taste are thought to be mediated by ion channels.)
[0006]
G protein coupled receptors mediate many other physiological functions such as endocrine function, exocrine function, heart rate, lipolysis and carbohydrate metabolism. Biochemical analysis and molecular cloning of many such receptors have revealed many basic principles regarding the function of these receptors. For example, US Pat. No. 5,691,188 states that upon ligand binding to a GPCR, this receptor replaces the bound GDP by GTP on the surface of the Gα subunit, followed by the Gα subunit from the Gβ and Gγ subunits. It describes how it undergoes a conformational change that leads to activation of the heterotrimeric G protein by promoting dissociation. Free Gα subunits and Gβγ complexes activate downstream elements of various signaling pathways.
[0007]
The present invention relates to a three-member T1R class of taste-specific GPCRs. Previously, the T1R receptor was postulated to function as a taste receptor (Hoon et al., Cell, 96, 541-51 (1999), Kitagawa et al., Biochem Biophys Res. Commun, 283, 236-42 (2001), Max et al., Nat. Genet., 28, 58-63 (2001), Montmayeur et al., Nat.Neurosci., 4, 41-8 (2001). Sainz et al., J. Neurochem. 77, 896-903 (2001)), Nelson et al. (2001) recently showed that rat T1R2 and T1R3 act together to recognize sweetness stimuli. The present invention relates to two inventions. First, as with rat T1R2 / T1R3, human T1R2 and T1R3 act together to recognize sweetness stimuli. Second, human T1R1 and T1R3 act together to recognize umami stimuli. Therefore, T1R2 / T1R3 is considered to function as a sweet receptor, and T1R1 / T1R3 is considered to function as an umami receptor in mammals. A possible explanation for the interdependence of the functions of T1R1 and T1R3 and of the functions of T1R2 and T1R3 is the structurally relevant GABA._BReceptors (Jones et al., Nature, 396, 5316-22 (1998), Kaupmann et al., Nature, 396, 683-7 (1998), White et al., Nature, 396, 679-82 (1998), Kuner et al., Science, 283, 74-77 (1999)), T1Rs function as a heterodimeric complex. . However, this interdependence of function is necessary but may reflect a transient interaction that ultimately results in a functionally independent monomer or homomultiple bond receptor.
[0008]
Identification and characterization of taste receptors that function as sweet and umami receptors are important because they facilitate the use of these receptors in assays to identify compounds that modulate (enhance or inhibit) sweet and umami tastes It is. These compounds appear to be useful for improving the taste and taste of foods, beverages, drugs for human or animal consumption. In particular, assays using functional sweet taste receptors may lead to the discovery of new sweeteners.
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0009]
To that end, it is an object of the present invention to provide a family of mammalian G protein-coupled receptors, referred to herein as T1Rs, that mediate taste.
[0010]
It is another object of the present invention to provide such T1Rs fragments or variants that retain activity, eg, activated and / or bound by a sweet or umami stimulant.
[0011]
It is another object of the present invention to provide nucleic acid sequences or molecules encoding such T1Rs, fragments or variants thereof.
[0012]
Such T1Rs or fragments or mutations thereof operatively linked to at least one regulatory sequence such as a promoter, enhancer or other sequence involved in positive or negative gene transcription and / or translation and / or protein export It is another object of the present invention to provide an expression vector comprising a nucleic acid sequence encoding the body.
[0013]
Human or non-human cells that functionally express at least one such T1Rs or a fragment or variant thereof, and preferably a combination of T1Rs or a fragment or variant thereof, eg, mammalian, yeast, insect or insect cells Is another object of the present invention.
[0014]
It is another object of the present invention to provide a T1R fusion protein or polypeptide comprising at least a fragment of at least one such T1Rs.
[0015]
At least 50%, preferably 75%, 85%, 90%, 95%, 96%, 97%, 98 and nucleic acid sequences having the hT1R nucleic acid sequence identified below and one of its conservatively modified variants It is another object of the invention to provide isolated nucleic acid molecules that encode a T1R polypeptide comprising a nucleic acid sequence that is% or 99% identical.
[0016]
At least 35-50%, preferably 60%, 75%, 85%, 90%, 95%, and an amino acid sequence selected from one group of T1R amino acid sequences specified below and conservatively modified variants thereof , 96%, 97%, 98% or 99% of the same amino acid sequence, and a fragment length of at least 20, preferably 40, 60, 80, 100, 150, 200 or 250 amino acids It is a further object of the invention to provide isolated nucleic acid molecules that encode. In some cases, the fragment may be an antigenic fragment that binds to an anti-T1R antibody.
[0017]
It is a further object of the invention to provide an isolated polypeptide comprising a variant of said fragment in which there are at most 10, preferably 5, 4, 3, 2 or 1 amino acid residue changes.
[0018]
It is another object of the present invention to provide T1R1 / T1R3 combinations wherein T1R1 and / or T1R3 are variants or fragments, and T1R2 / T1R3 combinations wherein T1R2 and / or T1R3 are variants or fragments.
[0019]
It is another object of the present invention to provide agonists or antagonists of such T1Rs or fragments or variants thereof.
[0020]
It is another object of the present invention to provide a PDZ domain interacting peptide (referred to herein as PDZIP) that can promote surface expression of endogenous plasma membrane proteins, particularly GPCRs such as T1Rs. . It is also an object of the present invention to provide vectors containing PDZIP, host cells that express such vectors, and methods using PDZIP to promote surface expression.
[0021]
It is a preferred object of the present invention to provide assays for identifying taste modulating compounds, particularly sweet and umami modulating compounds, particularly high throughput assays. In such assays, it is preferred to use a combination of T1Rs or a fragment or variant thereof, or a gene encoding such a T1Rs or fragment or variant disclosed herein. Most preferably, such a combination comprises hT1R1 / hT1R3 and hT1R2 / hT1R3.
[0022]
It is a particularly preferred embodiment of the present invention to identify compounds that modulate T1R1 / T1R3 or T1R2 / T1R3 taste receptors, eg, enhance the ability of these receptors to respond to taste stimuli. For example, as described below, it has been discovered that 5'-IMP or 5'-GMP enhances umami (T1R1 / T1R3) responsiveness to L-glutamate. These modulatory compounds enhance the activity of various sweet taste or umami stimulants and increase the taste and / or lower the specific sweet taste or umami inducer compounds whose activity is enhanced by the taste regulator specified by the subject assay At the same concentration, it induces the same taste.
[0023]
Preferred assays for evaluating said one or more compounds for taste comprising contacting one or more compounds with at least one disclosed T1Rs, fragment or variant thereof, preferably a combination of human T1Rs. It is a further object of the present invention to provide.
[0024]
Contacting one or more compounds with a combination comprising hT1R2 and hT1R3 or a complex comprising a fragment, chimera or variant of hT1R2 and / or hT1R3 in a mammal, preferably a human It is a more specific object of the present invention to provide a method of screening for their ability to enhance, simulate, inhibit and / or modulate sweetness perception.
[0025]
Tasting one or more compounds in a mammal, preferably a human, comprising contacting one or more compounds with a combination comprising hT1R1 and hT1R3 or a complex comprising a fragment, chimera or variant of hT1R1 and hT1R3, It is another specific object of the present invention to provide a method of screening for their ability to specifically enhance, simulate, inhibit and / or modulate umami perception.
[0026]
Producing cells co-expressing hT1R2 and hT1R3 or fragments, variants or chimeras for use in identifying compounds that enhance, simulate, inhibit and / or modulate taste, particularly sweetness perception Is a specific purpose.
[0027]
Producing cells that co-express hT1R1 and hT1R3 or fragments, variants or chimeras thereof for use in assays to identify compounds that enhance, simulate, inhibit and / or modulate taste, particularly umami perception Other specific purposes.
[0028]
It is another object of the present invention to produce non-human animals that are genetically modified to express or not express one or more T1Rs.
[0029]
It is another object of the present invention to use compounds identified as flavoring ingredients in food and beverage compositions using assays using T1Rs or combinations thereof. In particular, compounds that interact with hT1R2 and / or hT1R3 as sweetness inhibitors, potentiators, regulators or mimics in food and beverage compositions and hT1R1 and / or as umami inhibitors, potentiators, regulators or mimics It is an object of the present invention to use compounds that interact with hT1R3.
[0030]
For example, it is another object of the present invention to use T1Rs, particularly T1Rs other than humans, to identify compounds that modulate the taste of animal feed formulations for fish farming.
[0031]
a eukaryotic organism that stably co-expresses hT1R1 / hT1R3 or hT1R2 / hT1R3, preferably a mammalian or insect cell line, a G protein that, when expressed in association with T1R2 / T1R3 or T1R1 / T1R3, produces a functional taste receptor; For example, it is a preferred object of the present invention to provide a preferably HEK-292 cell line that also expresses Gα15 or other G proteins.
[0032]
It is another preferred object of the present invention to provide eukaryotic cell lines, preferably mammalian or insect cells, that stably express T1R1 / T1R3 or T1R2 / T1R3, preferably hT1R1 / hT1R3 or hT1R2 / hT1R3. In a preferred embodiment, such cells include HEK-292 cells that stably express Gα15 or other G proteins that bind to T1R1 / T1R3 or T1R2 / T1R3 to produce a functional umami or sweet receptor.
[0033]
Assays using HEK-292 or other cell lines that stably or transiently express T1R1 / T1R3 or T1R2 / T1R3 under constitutive or inducing conditions to identify compounds that modulate umami or sweetness, preferably high throughput It is also an object of the present invention to provide a performance assay.
[0034]
Based on the ability of a compound to affect the binding of lactisol (a sweet taste inhibitor) or a structurally related substance to the T1R1 / T1R3 (umami) taste receptor, the T1R1 / T1R3 umami receptor is enhanced and simulated It is another specific object of the present invention to identify compounds that inhibit and / or modulate.
[Means for Solving the Problems]
[0035]
The present invention relates to the discovery that when co-expressed, various combinations of T1Rs result in functional taste receptors that respond to taste stimuli. In particular, the present invention relates to the discovery that co-expression of T1R2 and T1R3 receptors results in hetero-oligomeric taste receptors that respond to sweet taste stimuli. The present invention also relates to the discovery that T1R1 and T1R3 are co-expressed to become taste receptors that respond to umami stimulants such as monosodium glutamate.
[0036]
The invention also relates to cell lines that co-express T1R1 and T1R3, preferably in humans, or T1R2 and T1R3, preferably in humans. In preferred embodiments, these cell lines express large amounts of receptors constitutively or inducibly. These cell lines include cells that transiently or stably express T1R1 and T1R3 or T1R2 and T1R3.
[0037]
The present invention also relates to assays using T1R2 / T1R3 taste receptors or T1R1 / T1R3 receptors to identify compounds that modulate sweetness or umami, particularly high throughput screening assays, preferably high throughput cells. The assay used is provided. The present invention also provides an assay comprising a taste test to confirm that these compounds modulate sweetness or umami.
BEST MODE FOR CARRYING OUT THE INVENTION
[0038]
The present invention thus provides a functional taste receptor, preferably a human taste receptor resulting from a combination of different T1Rs, preferably T1R1 / T1R3 or T1R2 / T1R3, as well as a functional taste receptor by coexpression, ie Corresponding isolated nucleic acid sequences or fragments, chimeras or variants thereof that produce a sweet taste receptor (T1R2 / T1R3) or an umami receptor (T1R1 / T1R3) are provided.
[0039]
As reported in the literature, members of the T1R family of taste cell-specific GPCRs are known and all described herein by Hoon et al. Cell, 96, 541-551 (1999), International Publication No. 00/06592, International Publication No. 00/06593, and US Patent Application No. 09 / 799,629.
[0040]
More particularly, the present invention relates to co-expression of offspring taste cell specific GPCRs. These nucleic acids and their encoded receptors are referred to as members of the “T1R” family of taste cell specific GPCRs. In certain embodiments of the invention, co-expressed T1R family members include rT1R1, rT1R2, rT1R3, mT1R1, mT1R2, mT1R3, hT1R1, hT1R2, and hT1R3. Without wishing to be bound by theory, these taste cell specific GPCRs are components of the taste transduction pathway, and the taste of sweet and umami stimulants and / or other taste stimulants that exhibit other types of tastes. It is thought to be involved in detection.
[0041]
It has been established herein that T1R family members act together with other T1R family members to function as sweet and umami receptors. As disclosed in more detail in the experimental examples below, it has been demonstrated that heterologous cells that co-express hT1R2 and hT1R3 are selectively activated by sweetness stimulators in a manner that reflects human sweetness. For example, HEK-293-Gα15 cells co-expressing hT1R2 and hT1R3 respond specifically to cyclamate, sucrose, aspartame, and saccharin, and the dose response of these compounds correlates with the threshold of psychophysical taste detection To do. Thus, cells that co-express hT1R2 and hT1R3 can be used for screening, preferably high throughput screening, to identify compounds that mimic, modulate, inhibit and / or enhance sweet taste.
[0042]
Also, as supported by the data in the experimental examples, cells co-expressing hT1R1 and hT1R3 are selectively expressed by glutamate (glutamate monosodium salt) and 5′-ribonucleotides in a manner that reflects human umami. It was shown to be activated. For example, HEK-293-Gα15 cells that co-express hT1R1 and hT1R3 respond specifically to glutamate, and the dose response of compounds exhibiting this umami taste correlates with its psychophysical taste detection threshold. In addition, 5'-ribonucleotides such as IMP enhance the glutamate response of the T1R1 / T1R3 receptor. This is a synergistic property of umami. Thus, cells that co-express hT1R1 and hT1R3 can be used for screening, preferably high throughput screening, to identify compounds that mimic, modulate, inhibit and / or enhance umami sensation.
[0043]
Furthermore, as demonstrated by experimental data in the Examples, cells that stably and inducibly co-express T1R1 and T1R3 respond selectively to the umami stimulants L-glutamate and L-aspartate. Compounds that modulate (enhance or inhibit) umami stimulation at a concentration of T1R1 / T1R3 receptors at concentrations according to experimental data in the much higher examples, which respond only weakly to other L-amino acids. It was shown that further evidence was obtained that it could be used for the identified assay.
[0044]
Also, as evidenced by experimental data in the Examples, cell lines co-expressing T1R1 / T1R3 or T1R2 / T1R3 respond to umami or sweet stimulants, respectively, and a quantitative dose response is observed for T1R1 / Further support the conclusion that T1R3 or T1R2 / T1R3 receptors can be used to identify receptor agonists and antagonists such as MSG substitutes, umami inhibitors, novel artificial and natural sweeteners and sweetness inhibitors It was shown that.
[0045]
It was also shown that the sweet taste inhibitor lactisol inhibits both the T1R2 / T1R3 sweet receptor and the T1R1 / T1R3 umami receptor, as supported by the data in the experimental examples. Thus, assays that screen for compounds that affect the binding of lactisol to T1R2 / T1R3 or T1R1 / T1R3 can be used to identify compounds that enhance, simulate, modulate or inhibit sweetness or umami. It is suggested. The fact that lactisole inhibits both T1R1 / T1R3 and T1R2 / T1R3 receptors, these receptors share a common subunit to which lactisole and potentially other taste regulators bind. It is suggested. This therefore suggests that some compounds that enhance, simulate, regulate or inhibit sweetness have a similar effect on umami and vice versa.
[0046]
Furthermore, as supported by the data in the experimental examples, T1Rs, ie, cell lines stably co-expressing T1R1 / T1R3 or T1R2 / T1R3, have a variety of sweet tastes when assayed by automated fluorescence imaging. It has been shown to respond very effectively to umami stimulants, ie, to a greater extent than transiently transfected cells. Therefore, these cell lines are particularly suitable for high throughput screening assays to identify compounds that modulate, inhibit, mimic or enhance sweetness and umami. However, the present invention also includes assays using cells that transiently express T1R or a combination thereof.
[0047]
Furthermore, the application is that some T1Rs work together, specifically T1R1 / T1R3 and T1R2 / T1R3, and such receptor combinations are used in assays, preferably high throughput assays. However, it should be noted that the subject invention also includes assays for use with T1R1, T1R2 and T1R3 alone or in combination with other proteins such as other GPCRs.
[0048]
In that regard, it is speculated that the sweet taste receptor consists only of T1R2 and / or the umami receptor consists only of T1R1, and that the T1R3 receptor probably functions to promote surface expression of T1R2 or T1R1. .
[0049]
Alternatively, it is assumed that the sweet and umami receptors are composed solely of T1R3 that is differentially processed under the control of T1R1 and / or T1R2. This type of receptor expression appears to be similar to RAMP-dependent processing of calcitonin-related receptors.
[0050]
Compounds identified by the T1R assay can be used to modulate the taste of food and beverages. Suitable assays, described in further detail below, include, as an example, whole cells, including direct binding assays using one of a combination of different T1R receptors, chimeras or fragments thereof, particularly fragments comprising an N-terminal ligand binding domain. Assays and biochemical assays. Examples of assays suitable for use in the present invention are described in more detail below, but are known in the field of GPCRs.
[0051]
Quantify binding of various compounds or mixtures of compounds to T1R taste receptors or combinations of T1R taste receptors or other heterologous (non-T1R) proteins, eg, T1R receptors expressed with other GPCRs, or T1R An assay can be designed to quantify the activation of cells expressing taste receptors. This can be achieved by taste receptors that are stably or transiently expressed in heterologous cells such as HEK-293, CHO and COS cells.
[0052]
The assay will preferably use cells that also (preferably stably) express G protein such as Gα15 or Gα16 or other promiscuous G protein or G protein variants or endogenous G protein. In addition, Gβ and Gγ proteins may also be expressed in the assay.
[0053]
The effect of a compound on sweetness or umami taste using a cell or composition expressing or containing a receptor or a combination of receptors identified above may include calcium sensitive dyes, use of voltage sensitive dyes, cAMP assays, fluorescently labeled ligands or^ThreeIt can be examined by various means including direct binding assays using radioligands such as H-glutamate or transcription assays (using appropriate reporters such as luciferase or β-lactamase).
[0054]
Assays that can be used with one or more T1Rs according to the present invention include, by way of example, assays using genetic selection of live cells, assays using whole cell or membrane fragments or purified T1R protein, such as cAMP and IP3. Assays that use complex second messengers, assays that detect the transfer of arrestin to the cell surface, assays that detect loss of receptor expression on the cell surface by the ligand under test (intracellular uptake), direct ligand binding assays, Competitive binding assays using inhibitors, assays using in vitro translated proteins, assays that detect conformational changes upon ligand binding (eg, evidenced by proteolysis, fluorescence or NMR), flies, T1R or T1R combinations, such as insects or mice Assay using behavioral assays using transgenic animals other than current human, the cells infected with recombinant viruses containing T1R gene, and the like.
[0055]
Determine the X-ray crystal structure of a T1R or T1R fragment (or a combination of T1Rs or a combination of T1R and other proteins) and bind to and / or enhance, mimic, inhibit or modulate a particular T1R receptor or receptor combination Structure-based assays that are used to predict compounds by molecular modeling methods are also within the scope of the present invention. More specifically, the present invention determines the crystal structure of T1R1 / T1R3 (preferably hT1R1 / hT1R3) and / or T1R2 / T1R3 (preferably hT1R2 / hT1R3) and identifies molecules that modulate T1R receptor activity. Including the use of such crystal structures in structure-based design methods.
[0056]
The invention is particularly carried out using one or more full-length T1R receptors or T1R1, T1R2 or T1R3 cells, preferably cells expressing the N-terminal domain, such as mammals, yeast, insects or other heterologous cells. Biochemical assays. The effect of a compound in such an assay may be competitive binding assays such as radioactive glutamate or IMP, fluorescence (eg, fluorescence polarization, FRET) or GTPy³⁵It can be measured using an S binding assay. As mentioned, in preferred embodiments, such assays use cell lines that stably co-express appropriate G proteins such as T1R1 / T1R3 or T1R2 / T1R3 and Gα15. Other suitable G proteins include the chimeric and mutant G proteins disclosed in US Application Nos. 09 / 984,292 and 60 / 243,770, which are incorporated herein by reference in their entirety. .
[0057]
In addition, modified receptors can be constructed with improved properties, such as enhanced surface expression or G protein binding. These T1R variants can be incorporated into cell-based and biochemical assays.
[0058]
It is envisioned that this discovery regarding human T1Rs can be extended to other animal species, such as rodents, pigs, monkeys, dogs and cats and possibly non-mammals such as fish. In this regard, several fish T1R fragments are identified in Example 1 below. Thus, the present invention has application in the screening of compounds for animal feed formulations.
[0059]
The invention further uses various allelic variants of various T1Rs and combinations thereof, thereby inducing a specific taste in an individual expressing the allelic variant or a specific taste in all individuals Screening to allow identification of compounds to be performed. Such compounds can generally be used to improve the taste of food.
[0060]
Since T1R-encoding nucleic acid is specifically expressed in taste cells, it is also a useful probe for identification of taste cells. For example, T1R polypeptide and protein probes can be used to identify taste cells that are present in the leaf-like, circumscribed, and epigastric nipples, as well as those that are present in the geshmax trifen, oral cavity, gastrointestinal epithelium and epiglottis. In particular, methods of detecting T1Rs can be used to identify taste cells that are sensitive to sweet and / or umami stimuli or other taste stimuli that exhibit other types of taste. For example, cells that stably or transiently express T1R2 and / or T1R3 would be expected to respond to sweetness stimuli from the tests herein. Similarly, cells expressing T1R1 and / or T1R3 would be expected to respond to umami stimuli. Nucleic acids encoding the T1R proteins and polypeptides of the invention are genetically engineered, amplified and synthesized by the methods disclosed in WO 00/035374, which is incorporated herein by reference in its entirety. And / or can be isolated from various sources expressed recombinantly. A list of T1Rs that can be expressed by the present invention is given in the Examples. However, it should be emphasized that the present invention includes the expression and use of such T1R sequences and particularly other specific T1Rs or fragments, variants or chimeras constructed based on T1Rs of other animal species. .
[0061]
As disclosed, important aspects of the invention include multiple screening for modulators of these taste cell specific GPCRs, such as activators, inhibitors, stimulators, potentiators, agonists and antagonists. It is a method. Such modulators of taste transmission are useful for the modulation of taste signal transduction pathways. These screening methods can be used to identify agonists and antagonists with high affinity for taste cell activity. These modulating compounds can then be used in the food industry to individually adjust the taste, for example to adjust the sweetness and / or umami of the food.
[0062]
The present invention corrects the lack of previous understanding of sweetness and umami as it can identify specific T1Rs and T1R receptor combinations that mediate the taste of sweetness and umami. Thus, in general, this application addresses the discovery of the inventors regarding the T1R class of taste-specific G protein coupled receptors and their specific functions in taste and a better understanding of these findings and the molecular basis of taste. Related to the relationship.
[0063]
The molecular basis of sweetness and umami (taste of monosodium glutamate) is mysterious. Recently, a three-member class of taste-specific G protein-coupled receptors called T1Rs has been identified. GABA structurally related to overlapping expression patterns of T1R_BProof that the receptor is a heterodimer suggests that T1Rs function as a heterodimeric receptor. In the following examples, the present invention demonstrates the functional co-expression of human T1R1, T1R2 and T1R3 in heterologous cells, ie cells that co-express T1R1 and T1R3 are activated by umami stimulation and co-express T1R2 and T1R3. It is described that cells are activated by a sweet stimulus. The activity of T1R1 / T1R3 and T1R2 / T1R3 showed a correlation with the psychophysical detection threshold. Furthermore, 5'-ribonucleotide IMP was found to enhance the T1R2 / T1R3 response to glutamate (umami synergistic properties). These findings indicate that specific and distinct combinations of specific T1Rs and T1Rs function as sweet and umami receptors.
[0064]
Human bitterness, sweetness and umami perception are thought to be mediated by G protein-coupled receptors (Lindmann B., physiol. Res. 76, 718-66 (1996)). Recently, evaluation of the human genome revealed a bitter taste receptor of the T2R class (Adler et al., Cell, 100, 613-702 (2000), Chandrasgekar et al., Cell, 100, 703). 11 (2000), Matsunami et al., Nature, 404, 601-604 (2000)), no sweet and umami receptors were identified. Recently, other classes of candidate taste receptors, T1Rs, have been identified. T1Rs were first identified by large-scale sequencing of a subtracted cDNA library from rat taste tissue (which identified T1R1), followed by T1R1-based degenerate PCR (which led to the identification of T1R2) (Hoon et al., Cell, 96, 541-551 (1999)). Recently, the inventors and others have identified T1R3, the third and possibly final member of the T1R family in the human genome data bank (Kitagawa et al., Biochem Biophys. Res Commun. Vol. 283, No. 1). 236-42 (2001), Max et al., Nat. Genet., 28, No. 1, 58-63 (2001), Sainz et al., J. Neurochem. No. 3, 896-903 (2001), Montmayeur et al., Nat. Neurosci., 4, 492-8 (2001)). Effectively, mouse T1R3 is mapped to a genomic segment containing Sac, a locus that affects sweetness in mice (Fuller et al., J. Hered. 65, 33-6 (1974), Li et al., Mamm. Genome, Vol. 12, pp. 13-16 (2001)). Therefore, T1R3 was predicted to function as a sweet taste receptor. Recent high-resolution genetic map studies have reinforced the relationship between mouse T1R3 and Sac (Fuller TC, J. Hered. 65, 1, 33-36 (1974), Li et. al., Mammal.Genome, Vol. 12, No. 1, pages 13-16 (2001).
[0065]
Interestingly, all C family receptors (metabolic glutamate receptors, GABA, which have been functionally expressed so far)_BReceptors, calcium-sensing receptors (Conigave AD, Quinn SJ, Brown EM, Proc Natl Acad Sci USA, 97, 4814-9 (2000)) and fish olfactory receptors (Speca DJ et al., Neuron, 23, 487-98 (1999)) was shown to be activated by amino acids. These common features raise the possibility that T1Rs recognize amino acids and that T1Rs is involved in the detection of glutamate in addition to sweet amino acids. Alternatively, since the selective expression in rat taste tissue and the receptor activation threshold are similar to the glutamate psychophysical detection threshold, the transcriptional variant of mGluR4 metabotropic glutamate receptor is the umami receptor. It was proposed (Caudhari et al., Nat. Neurosci. Vol. 3, pp. 113-119 (2000)). This hypothesis is that the expression level of the mGluR4 mutant in the taste tissue is extremely low, and the glutamate taste of mGluR4 knockout mice is virtually unchanged (Chaudhari and Roper, Ann. NY Acad. Sci. 855, 398). ~ 406 (1998)). Furthermore, it is unlikely that a taste receptor variant lacking almost half of the globular N-terminal glutamate binding domain, as well as most of the residues that form the glutamate binding pocket of the wild type receptor (Kunishima et al. , Nature, 407, 971-7 (2000)).
[0066]
Comparative analysis of T1R expression patterns in rodents showed that T1R2 and possibly T1R1 are co-expressed with T1R3, respectively (Hoon et al., Cell 96, 541-51 (1999), Kitagawa) et al., Biochem Biophy.Res.Commun., 283, 236-242 (2001), Max et al., Nat. Genet., 28, 58-63 (2001), Montmayeur et al. Nat. Neurosci., 4, 492-8 (2001), Sainz et al., J. Neurochem, 77, 896-903 (2001)). Furthermore, dimerization has occurred as a common theme for C family receptors. That is, metabotropic glutamate and calcium-sensing receptors are homodimers (Romomano et al., J. Biol. Chem. 271, 28612-6 (1996), Okamoto et al., J. Biol. Chem., 273, 13089-96 (1998), Han et al., J. Biol.Chem., 274, 100008-13 (1999), Bai et al., J. Biol. 273, 23605-10 (1998)), structurally related GABA._BThe receptor is a heterodimer (Jones et al., Nature, 396, 674-9 (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 that human T1R2 functions as a sweet receptor together with human T1R3 and human T1R1 functions as an umami receptor together with human T1R3 by functional co-expression of T1Rs in heterologous cells.
[0067]
The findings described herein are particularly important because the development of improved artificial sweeteners has previously been hampered by the absence of sweetness assays. In fact, five commonly used commercial sweeteners that all activate hT1R2 / hT1R3 have been unexpectedly discovered. Similarly, there is no assay to identify compounds that modulate umami other than sensory testing, which is a laborious method. As demonstrated by the experimental results described below, human sweet and umami receptors have been identified and assays for these receptors have been identified, specifically stabilizing functional T1R taste receptors, ie sweet or umami receptors. These problems have now been more or less solved, as assays have been developed that use cells that are expressed in E. coli.
[0068]
Based on that, the present invention provides an assay for detecting and characterizing taste modulating compounds that act as reporter molecules of the sweet taste and umami taste effects of the taste modulating compounds such that the T1R family acts in taste buds. Specifically provided are assays that identify compounds that individually modulate, simulate, enhance, and / or inhibit sweetness and umami tastes, and these are within the scope of the present invention. Methods for assaying compounds that affect the activity of GPCRs and in particular GPCR activity are well known and applicable to the T1R family members of the invention and combinations of their functions. Appropriate assays were identified above.
[0069]
In particular, subject GPCRs can be used in assays to measure changes in ligand binding, ion concentration, membrane potential, current, ion flow rate, transcription, receptor-ligand interaction, second messenger concentration, for example, in vitro and in vivo. Can be used. In other embodiments, T1R family members can be expressed recombinantly in cells and modulation of taste conversion via GPCR activity is²⁺Concentration and cAMP, cGMP or IP_ThreeCan be assayed by measuring changes in intracellular messengers such as
[0070]
In certain assays, a T1R polypeptide domain, eg, an extracellular, transmembrane or intracellular domain, is fused to a heterologous polypeptide, thereby forming a chimeric polypeptide, eg, a chimeric protein having GPCR activity. ing. The use of fragments of T1R1, T1R2 or T1R3 that contain an N-terminal ligand binding domain is particularly envisaged. Such proteins are useful, for example, in assays to identify ligands, agonists, antagonists or other modulators of T1R receptors. For example, T1R polypeptides can be expressed in eukaryotic cells as chimeric receptors having heterologous chaperone sequences that promote maturation and targeting by plasma membrane trafficking or secretory pathways. The optional heterologous sequence may be a PDZ domain interacting peptide such as a C-terminal PDZIP fragment (SEQ ID NO: 1). PDZIP is an ER export signal and according to the present invention has been shown to promote surface expression of heterologous proteins such as the T1R receptors described herein. More specifically, in one aspect of the invention, PDZIP facilitates proper targeting of problematic membrane proteins such as olfactory receptors, T2R taste receptors, and T1R taste receptors described herein. Can be used for
[0071]
Such chimeric T1R receptors can be expressed in any eukaryotic cell such as HEK-293 cells. The cell is a G protein, preferably G_{α 15}Or G_{α 16}It is preferred to include promiscuous G proteins such as or other types of promiscuous G proteins that can link a wide range of GPCRs to intracellular signaling pathways or signaling proteins such as phospholipase C. Such chimeric receptors in such cells can be detected using standard methods such as by detecting changes in intracellular calcium by detecting FURA-2-dependent fluorescence in the cells. it can. If preferred host cells do not express the appropriate G protein, US application Ser. No. 60 / 243,770, filed Oct. 29, 2001, which is incorporated herein by reference in its entirety. And a gene encoding a promiscuous G protein as described in US application Ser. No. 09 / 989,497 filed Nov. 21, 2001 can be transfected into their host cells.
[0072]
Another method for assaying modulators of taste conversion includes a T1R polypeptide, a portion thereof, ie, an extracellular domain, a transmembrane region or a combination thereof, or a chimeric protein comprising one or more domains of a T1R family member Oocytes or tissue culture cells expressing T1R polypeptides, fragments or fusion proteins; phosphorylation and dephosphorylation of T1R family members; G proteins that bind to GPCRs; ligand binding assays; voltage, membrane potential and conductance changes; Ion flux assays; including changes in intracellular second messengers such as cGMP, cAMP and inositol triphosphate (IP3) as well as in vitro ligand binding assays using changes in intracellular calcium concentration.
[0073]
Furthermore, the present invention provides methods for detecting T1R nucleic acid and protein expression that allow for the study of modulation of taste conversion and the specific identification of taste receptor cells. T1R family members also provide useful nucleic acid probes for paternity and forensic research. The T1R gene is also useful as a nucleic acid probe to identify taste receptor cells such as leaf-like, rod-like, tangled, geshmax-trifen and epiglottis taste receptor cells. T1R receptors can also be used to generate monoclonal and polyclonal antibodies useful for identifying taste receptor cells.
[0074]
Functionally, T1R polypeptides are thought to be involved in taste transmission and contain seven related transmembrane G protein-coupled receptors that can interact with G proteins to mediate taste signaling. (See, eg, Fong, Cell Signal, Vol. 8, 217 (1996), Baldwin, Curr. Opin. Cell Biol., 6, 180 (1994)). Structurally, the nucleotide sequence of a T1R family member encodes an associated related polypeptide comprising an extracellular domain, seven transmembrane domains and a cytoplasmic domain. Related T1R family genes of other animal species are at least about 50 nucleotides in length, optionally 100, 200, 500 or optionally a T1R nucleic acid sequence disclosed in the Examples herein or a conservatively modified variant thereof. T1R polypeptides that share at least about 50% and optionally 60%, 70%, 80%, or 90% nucleotide sequence identity over a region of longer nucleotide length, or disclosed in the Examples below Or a conservatively modified variant thereof and an amino acid region of at least 25 amino acids in length, optionally 50-100 amino acids in length, at least about 35-50% and optionally 60%, 70%, 80% or 90 Encodes polypeptides that share% amino acid sequence identity.
[0075]
Several common amino acid sequences or domains characteristic of T1R family members have also been identified. For example, T1R family members are typically at least about 50%, optionally 55%, 60%, 65%, 70%, 75%, 80% with T1R consensus sequences 1 and 2 (SEQ ID NOs: 2 and 3, respectively). 85%, 90%, 95-99% or higher identity sequences. Thus, these conserved domains can be used to identify members of the T1R family by identity, specific hybridization or amplification, or specific binding by antibodies raised against the domain. T1R consensus sequences include, for example, the following sequences:
T1R family consensus sequence 1: (SEQ ID NO: 2)
(TR) C (FL) (RQP) R (RT) (SPV) (VERKT) FL (AE) (WL) (RHG) E
T1R family consensus sequence 2: (SEQ ID NO: 3)
(LQ) P (EGT) (NRC) YN (RE) A (RK) (CGF) (VLI) T (FL) (AS) (ML)
[0076]
These consensus sequences include those found in the T1R polypeptides described herein, but other organisms' T1R family members may be about 75% or more of the consensus sequences specifically described herein. It is expected to contain a consensus sequence with identity.
[0077]
Specific regions of T1R nucleotide and amino acid sequences can be used to identify polymorphic variants, interspecies homologues and alleles of T1R family members. This identification may be performed in vitro, eg under stringent hybridization conditions or by PCR (eg, using primers encoding the T1R consensus sequence identified above) or for comparison with other nucleotide sequences. This can be done using sequence information in a computer system. Different alleles of the T1R gene within a single species population may also be useful in determining whether allelic sequence differences control taste differences between population members. Classical PCR-type amplification and cloning techniques are useful for isolating new T1Rs, for example, when synonymous primers are sufficient to detect related genes across species.
[0078]
In general, polymorphic variants and alleles of T1R family members can be identified by comparing amino acid sequences of about 25 amino acids or more, eg, 50-100 amino acids. An amino acid identity of approximately at least 35-50%, and optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99% or more is generally that the protein is a T1R family member. Indicates polymorphic variant, interspecies homologue or allele. Sequence comparison can be performed using any of the following sequence comparison algorithms. Antibodies that specifically bind to a T1R polypeptide or a conserved region thereof can also be used to identify alleles, interspecies homologs, and polymorphic variants.
[0079]
Polymorphic variants, interspecies homologues and alleles of the T1R gene can be confirmed by examining taste cell-specific expression of putative T1R genes or proteins. In general, a T1R polypeptide having an amino acid sequence disclosed herein is a positive control in comparison to a putative T1R polypeptide to verify the identity of a polymorphic variant or allele of a T1R family member Can be used as Polymorphic variants, alleles and interspecies homologues are expected to retain the seven transmembrane structures of the G protein coupled receptor. For further details, reference is made to WO 00/06592, which discloses the related T1R family member GPCR-B3s and is incorporated herein by reference in a manner consistent with this disclosure. The GPCR-B3 receptors are referred to herein as rT1R1 and mT1R1. See also WO 00/06593, which also discloses a related T1R family member, GPCR-B4s, the contents of which are incorporated herein by reference in a manner consistent with this disclosure. GPCR-B4 receptors are referred to herein as rT1R2 and mT1R2. As mentioned above, the present invention relates to T1R or combinations of T1R, such as hT1R2 / hT1R3 or hT1R1 / hT1R3, to identify molecules that modulate T1R receptor activity and thereby modulate sweetness and / or umami. Also included are structure-based assays using the X-ray crystal structure of
[0080]
The present invention also provides assays, preferably high throughput assays, for identifying molecules that enhance, mimic, inhibit and / or modulate T1R receptors. In some assays, specific domains of T1R family members are used in combination with specific domains of other T1R family members, eg, extracellular, transmembrane or intracellular domains or regions. In other embodiments, the extracellular domain, transmembrane region or combination thereof is bound to a solid substrate, eg, bound to a ligand, agonist, antagonist or T1R polypeptide and / or modulates its activity. Can be used to separate other molecules that can
[0081]
Various conservative mutations and substitutions are assumed to be within the scope of the present invention. For example, performing amino acid substitutions using known protocols of recombinant gene technology such as PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells and in vitro transcription is well known in the art. Within the level of technology. Mutants can then be screened for activity.
[0082]
Definition
As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
[0083]
“Taste cells” include sensory epithelial cells, such as lobe, scab and umbilical cells, organized into groups to form the taste buds of the tongue (eg, Roper et al., Ann. Rev. Neurosci). 12 (pp. 329-353 (1989)). Taste cells are also found in the palate and other tissues such as the esophagus and stomach.
[0084]
“T1R” refers to one or more members of a family of G protein-coupled receptors that are expressed in taste cells, such as leafy, rod-shaped, and circumscribing cells and palatal and esophageal cells (eg, all by reference). Is incorporated herein by reference, see Hoon et al., Cell, 96, 541-551 (1999)). Members of this family are also referred to as GPCR-B3 and TR1 in WO 00/06592 and GPCR-B4 and TR2 in WO 00/06593. GPCR-B3 is referred to herein as rT1R1, and GPCR-B4 is referred to as rT1R2. Taste receptor cells can be identified based on morphology (eg, Roper, supra, see) or by expression of proteins that are specifically expressed in taste cells. T1R family members have the ability to act as receptors for sweetness conversion or to differentiate between various other types of tastes. Representative T1R sequences including hT1R1, hT1R2 and hT1R3 are shown in the examples below.
[0085]
“T1R” nucleic acids encode a family of GPCRs having seven transmembrane regions with “G protein coupled receptor activity”. For example, they bind to G proteins in response to extracellular stimuli and upon stimulation of enzymes such as phospholipase C and adenylate cyclase, IP3, cAMP, cGMP and Ca²⁺(For example, see Fong, supra, and Baldwin, supra for a description of the structure and function of GPCRs). A single taste cell contains many distinct T1R polypeptides.
[0086]
Thus, the term “T1R” family includes (1) a T1R polypeptide, preferably at least about 35-50% over the window of about 25 amino acids, optionally 50-100 amino acids, preferably as shown in Example 1. Having amino acid sequence identity, optionally about 60, 75, 80, 85, 90, 95, 96, 97, 98 or 99% amino acid sequence identity; (2) the T1R polypeptide sequence disclosed in Example 1 and Specifically binds to an antibody raised against an immunogen comprising an amino acid preferably selected from the group consisting of its conservatively modified variants, and (3) carried out under stringent hybridization conditions A hive specifically with a sequence selected from the group consisting of the T1R nucleic acid sequence and conservatively modified variants thereof contained in Example 1 Encoded by a soy nucleic acid molecule (having a size of at least 100, optionally at least about 500-1000 nucleotides), or (4) an amino acid sequence selected from the group consisting of the T1R amino acid sequences set forth in Example 1 and at least about 35 Refers to polymorphic variants, alleles, mutants and interspecies homologs that contain sequences that are -50% identical.
[0087]
Topologically, certain chemosensory GPCRs consist of an “N-terminal domain”, an “extracellular domain”, a “transmembrane domain” that includes seven transmembrane regions and corresponding cytoplasmic and extracellular loops, “ It has a “cytoplasmic domain” and a “C-terminal domain” (eg, Hoon et al., Cell, 96, 541-551 (1999), Buck & Axel, Cell, 65, 175-187 (1991). Year)). 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 (eg, Stryer, Biochemistry (3rd edition, (1988), see also any of a number of sequence analysis programs using the Internet such as found at dot.imgen.bcm.tmc.edu). Such domains are useful for the production of chimeric proteins and for the in vitro assays of the invention, eg, ligand binding assays.
[0088]
Thus, “extracellular domain” refers to a domain of T1R polypeptide that protrudes from the cell membrane and is exposed to the extracellular surface of the cell. Such domains generally comprise an “N-terminal domain” that is exposed on the extracellular surface of the cell, and optionally a portion of the extracellular loop of the transmembrane domain that is exposed on the extracellular surface of the cell, ie, It may include loops between the transmembrane regions 2 and 3, between the transmembrane regions 4 and 5, and between the transmembrane regions 6 and 7.
[0089]
The “N-terminal domain” starts at the N-terminus and extends to a region near the beginning of the first transmembrane. More specifically, in one embodiment of the invention, this domain begins at the N-terminus and ends at approximately the conserved glutamic acid position at amino acid position 563 plus or minus about 20 amino acids. These extracellular domains are useful for soluble and solid phase in vitro ligand binding assays. In addition, the transmembrane regions described below can also bind to ligands along with the extracellular domain and are therefore useful for in vitro ligand binding assays.
[0090]
A “transmembrane domain” comprising seven “transmembrane regions” refers to a domain of T1R polypeptide within the plasma membrane, which may also include the corresponding cytoplasm (intracellular) and extracellular loops. In one embodiment, this region is a domain of a T1R family member that begins with a conserved glutamic acid residue at amino acid position 563 plus or minus about 20 amino acids and ends at an approximately conserved tyrosine amino acid residue at 812 plus or minus about 10 amino acid positions. Corresponding to Seven transmembrane regions and extracellular and cytoplasmic loops are described in Kyte & Doolittle, J. et al. Mol. Biol. 157, 105-32 (1982) or Stryer, supra, and can be identified using standard methods.
[0091]
A “cytoplasmic domain” is a domain of a T1R polypeptide that faces the inside of a cell, eg, a “C-terminal domain” and an intracellular loop of a transmembrane domain, eg, an intracellular loop between the transmembrane regions 1 and 2, the membrane It refers to the intracellular loop between the transmembrane regions 3 and 4 and the intracellular loop between the transmembrane regions 5 and 6. “C-terminal domain” refers to the region that is usually in the cytoplasm, spanning the last transmembrane domain and the C-terminus of the protein. In one embodiment, this region begins with approximately conserved tyrosine amino acid residues at 812 plus or minus about 10 amino acid positions and continues to the C-terminus of the polypeptide.
[0092]
“Ligand binding region” or “ligand binding domain” refers to a sequence 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 comprise an N-terminal domain, and optionally a portion of a transmembrane domain, such as the extracellular loop of the transmembrane domain. The ligand binding region can bind to a ligand and, more specifically, to a compound that enhances, mimics, inhibits and / or modulates taste, eg, sweet taste or umami taste.
[0093]
The phrase “heteromultimer” or “heteromultimer complex”, related to the T1R receptor or polypeptide of the present invention, refers to at least one T1R receptor and other receptors, generally other T1R receptors. Refers to functional binding to a body polypeptide (or other non-T1R receptor polypeptide as an alternative). For clarity, the functional interdependencies of T1Rs are described herein as reflecting their possible function as heterodimeric taste receptor complexes. However, as discussed above, functional interdependencies or may reflect indirect interactions. For example, T1R3 may function simply to promote surface expression of T1R1 and T1R2, which can act independently as taste receptors. Alternatively, functional taste receptors may consist of only T1R3 that is differentially processed under the control of T1R1 or T1R2, similar to RAMP-dependent processing of calcium-related receptors.
[0094]
“Functional effects”, a phrase related to assays for testing compounds that modulate T1R family member-mediated taste conversion, refer to indirect or direct receptor effects such as functional, physical and chemical. Includes measurement of parameters under influence. It includes in vitro, in vivo and ex vivo ligand binding, changes in ion flux, membrane potential, current, transcription, G protein binding, GPCR phosphorylation and dephosphorylation, conformational changes based assay, signaling , Receptor-ligand binding, second messenger concentration (eg cAMP, cGMP, IP3 or intracellular Ca²⁺And other physiological effects such as increased or decreased neurotransmitter or hormone release.
[0095]
“Measuring a functional effect” in connection with an assay is an assay for compounds that increase or decrease the parameters of T1R family members indirectly or directly, eg, parameters under functional, physical and chemical effects. means. Such functional effects can be determined by means known to those skilled in the art, such as spectroscopic properties (eg fluorescence, absorbance, refractive index), hydrodynamic (eg shape), chromatographic or dissolution properties, patches Clamping, voltage sensitive dye, whole cell current, radioisotope efflux, induction marker, change in oocyte T1R gene expression; tissue culture cell T1R expression; transcriptional activation of T1R gene; ligand binding assay; voltage, membrane potential and Conductance change; ion flux assay; change in intracellular second messengers such as cAMP, cGMP and inositol triphosphate (IP3); change in intracellular calcium concentration; neurotransmitter release, conformation assay, etc. be able to.
[0096]
“Inhibitors,” “activators,” and “modulators” of T1R genes or proteins are inhibitors, activation or modulator molecules identified using in vitro and in vivo assays of taste conversion, such as ligands, agonists Used to refer to substances, antagonists and their homologues and mimetics.
[0097]
Inhibitors, for example, compounds that bind, partially or completely inhibit stimulation, reduce, interfere with, delay activation, inactivate, desensitize or down-regulate taste transmission, such as antagonists It is. Activators are, for example, compounds that bind, stimulate, increase, open, activate, promote, enhance activation, sensitize or up-regulate taste transmission, such as agonists. Modulators are, for example, compounds that alter the interaction of receptors with extracellular proteins that bind to activators or inhibitors (eg, ebnerin and other members of the hydrophobic carrier family); G proteins; kinases ( For example, rhodopsin kinases and homologs of beta-adrenergic receptor kinases involved in receptor inactivation and desensitization); and arrestins that also inactivate and desensitize receptors. Modulators can include genetically modified variants of T1R family members, such as variants with altered activity, and natural and synthetic ligands, antagonists, agonists, small chemical molecules, and the like. Such assays for inhibitors and activators, for example, express T1R family members in cells or cell membranes and administer putative regulatory compounds in the presence or absence of seasonings, eg, sweeteners. And then measuring the functional impact on taste conversion as described above. A sample or assay containing a T1R family member treated with a possible activator, inhibitor or modulator is compared to a control sample containing no inhibitor, activator or modulator to determine the degree of modulation. Assume that a positive control sample (eg, a sweetener without added modulator) has a relative T1R activity value of 100%.
[0098]
A negative control sample (eg, a buffer without the addition of a taste stimulant) has a relative T1R activity value of 0%. Inhibition of T1R is achieved when mixing the positive control sample with the modulator results in a T1R activity value of about 80%, optionally 50% or 25-0% compared to the positive control value. The activation of T1R by the modulator alone is such that the T1R activity value is 10%, 25%, 50%, 75%, in some cases 100%, in some cases 150%, in some cases 200-500% compared to the positive control value. Or achieved when higher than 1000-3000%.
[0099]
The terms “purified”, “substantially purified” and “isolated”, as used herein, refer to others in which the compounds of the invention are usually bound in their natural state. Of different compounds, so that “purified”, “substantially purified” and “isolated” objects are at least 0. 0% by weight of the mass of a given sample. 5%, 1%, 5%, 10% or 20% and more preferably at least 50% or 75%. In a preferred embodiment, these terms refer to a compound of the invention comprising at least 95% by weight percent of the mass of a given sample. As used herein, the terms “purified”, “substantially purified” and “isolated” when referring to nucleic acids or proteins are naturally found in the body of a mammal, particularly a human. It refers to a state of purification or concentration that is different from that present. (1) Purified from other bound structures or compounds, or (2) Naturally present in mammals, particularly humans, including bonds with structures or compounds that are not normally bound in mammals, particularly humans A greater degree of purification or concentration than is within the meaning of “isolated”. The nucleic acids or proteins or classes of nucleic acids or proteins described herein can be separated by various methods or steps known to those skilled in the art, or else they are usually bound in nature. Can be combined with other structures or compounds.
[0100]
The term “nucleic acid” or “nucleic acid sequence” refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in single or double stranded form. The term includes nucleic acids, ie, oligonucleotides that contain known analogs of natural nucleotides. This term also includes nucleic acid-like structures with a synthetic backbone (see, eg, Oligonucleotides and Analogues, a Practical Approach, Ed. F. Eckstein, Oxford Universe Press, (1991), Antisense Strategies N. Aness. of Sciences, Volume 600, edited by Baserga et al. (NYAS 1992), Milligan, J. Med. Chem. Volume 36, pp. 1923-1937 (1993), Antisense Research and Applications (1993, CRC Pres). WO 97/03211, WO 96/39154, Mata , Toxicol.Appl.Pharmacol.Vol.144, 189-197 (1997), Strauss-Soukup, Biochemistry, Vol.36, 8692-2869 (1997), Samstag, Antisense Nucleic Acid 6 153-156 (1996)).
[0101]
Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, synonymous codon substitutions) and complementary sequences as well as explicitly indicated sequences. Specifically, synonymous codon substitution is accomplished, for example, by generating a sequence in which the third position of one or more selected codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991), Ohtsuka et al., J. Biol. Chem. 260, 2605-2608 (1985), Rossolini et. al., Mol. Cell. Probes, Vol. 8, pp. 91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
[0102]
The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers.
[0103]
A “plasma membrane translocation domain” or simply “translocation domain”, when incorporated into a polypeptide coding sequence, “evacates” or “transfers” a hybrid (“fusion”) protein with greater efficiency than without a domain. Means a polypeptide domain capable of For example, a “translocation domain” can be obtained from the bovine rhodopsin receptor polypeptide, ie, the amino terminus of the 7-transmembrane receptor. However, all mammalian rhodopsins may be used as other translocation facilitating sequences are possible. Thus, the translocation domain is particularly efficient at translocating the 7-transmembrane fusion protein to the plasma membrane, and proteins containing amino terminal translocation domains (eg, taste receptor polypeptides) are more efficient than without the domain. It is often transported to the plasma membrane. However, the use of other translocation domains may be preferred if the N-terminus of the polypeptide is active upon binding, as in the case of the T1R receptor of the present invention. For example, PDZ domain interacting peptides as described herein can be used.
[0104]
“Transfer domains”, “ligand binding domains” and chimeric receptor compositions described herein also have “analogs” or “conservative variants” having structures and activities substantially corresponding to exemplary sequences. And “mimetics” (peptide mimetics). Thus, a “conservative variant” or “analog” or “mimetic” is defined as a change in which substantially changes the structure and / or activity of a polypeptide (of a conservative variant). A polypeptide having a modified amino acid sequence that does not change. These are conservatively modified variants of the amino acid sequence, i.e., amino acid substitutions, additions or deletions of residues that are not critical to protein activity, or even substitutions of important amino acids substantially alter the structure and / or activity. Substitution of amino acids with residues having similar properties such as acidic, basic, positively or negatively charged, polar or nonpolar, etc.
[0105]
In particular, “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, or essentially the same sequence if the nucleic acid does not encode an amino acid sequence. Point to. Because of the synonym of the genetic code, a large number of functionally identical nucleic acids encode a given protein.
[0106]
For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
[0107]
Such nucleic acid variants are “silent variants,” which are one species of conservatively modified variants. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), can be modified to yield a functionally identical molecule. You will recognize. Thus, each silent variant of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
[0108]
Conservative substitution tables describing functionally similar amino acids are well known in the art. For example, one illustrative guideline for selecting conservative substitutions is as follows (an illustrative substitution following the original residue). 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 glu or glu; met / leu or tyr or ile; phe / met or leu or tyr; ser / thr; thr / ser; trp / tyr; try / trp or phe; ile or leu. Another exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions: 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) (eg, Creighton, Proteins, W See also H. Freeman and Company (1984), Schultz and Schimmer, Principles of Protein Structure, Springer-Vrlag (1979)). One skilled in the art will recognize that the above substitution is not the only possible substitution. For example, all charged amino acids can be considered as conservative substitutions for each other, whether 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 the encoded sequence can also be considered “conservatively modified variants”. .
[0109]
The terms “mimetic” and “peptidomimetic” refer to synthetic compounds having substantially the same structural and / or functional properties of a polypeptide, eg, a transfer domain, ligand binding domain or chimeric receptor of the present invention. The mimetic may be composed entirely of non-natural analogs by amino acid synthesis, or may be a chimeric molecule partially composed of natural peptide amino acids and partially non-natural analogs of amino acids. The mimetics can also incorporate any amount of natural amino acid conservative substitutions as long as the substitution does not substantially alter the mimetic's structure and / or activity.
[0110]
As in the case of a polypeptide of the invention that is a conservative variant, routine experimentation has shown that the mimetic is within the scope of the invention, ie, its structure and / or function has changed substantially. You will be able to determine if it is not. The polypeptide mimetic composition may comprise non-naturally occurring structural components that generally belong to the following three structural groups: A) a residue linkage group other than a natural amide bond (“peptide bond”) linkage, b) a non-natural residue in place of a naturally occurring amino acid residue, or c) a secondary structure mimic, ie Residues that induce or stabilize secondary structures such as β-turns, γ-turns, β-sheets, α-helical conformations, etc. A polypeptide can be characterized as a mimetic when all or part of its residues are linked by chemical means other than natural peptide bonds. Individual peptidomimetic residues are peptide bonds such as glutaraldehyde, N-hydroxysuccinimide ester, difunctional maleide, N, N′-dicyclohexylcarbodiimide (DCC) or N, N′-diisopropylcarbodiimide (DIC). They can be linked by other chemical bonds or coupling means. A linking group that may be substituted for a traditional amide bond (“peptide bond”) linkage is, for example, ketomethylene (eg, —C (═O) —CH instead of —C (═O) —NH—.₂-), Aminomethylene (CH₂-NH), ethylene, olefin (CH = CH), ether (CH₂-O), thioether (CH₂-S), tetrazole (CN_Four), Thiazoles, retroamides, thioamides or esters (e.g., Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, "Peptide Backbone Mofk. Year)). Peptides can also be characterized as mimetics by including all or some non-natural residues in place of naturally occurring amino acid residues. Non-natural residues are well described in the scientific and patent literature.
[0111]
A “label” or “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful labels include³²P, fluorescent dye, electron-dense reagent, enzyme (eg, as commonly used in ELISA), biotin, digoxigenin, or, for example, can be made detectable by incorporating a radioactive label into the peptide, Alternatively, there are haptens and proteins used to detect antibodies that specifically react with peptides.
[0112]
A “labeled nucleic acid probe or oligonucleotide” is a covalent bond to a label, either by a linker or chemical bond, or an ion so that the presence of the probe can be detected by detecting the presence of the label bound to the probe. , Van der Waals, non-covalently bonded by electrostatic or hydrogen bonding.
[0113]
As used herein, a “nucleic acid probe or oligonucleotide” is a target nucleic acid of complementary sequence by one or more types of chemical bonds, usually by complementary base pairing, usually by formation of hydrogen bonds. Defined as a nucleic acid capable of binding to As used herein, a probe may contain natural (ie, A, G, C or T) or modified bases (7-deazaguanosine, inosine, etc.). Furthermore, the base in the probe may be linked by a bond other than a phosphodiesterase bond as long as it does not interfere with hybridization. Thus, for example, a probe may be a peptide nucleic acid in which the constituent bases are linked to peptide bonds rather than phosphodiesterase bonds. It will be appreciated by those skilled in the art that a probe can bind to a target sequence that lacks complete complementarity with the probe sequence due to the stringency of the hybrid conditions. In some cases, the probe may be directly labeled with isotopes, chromophores, luminophores, chromogens, etc., or indirectly labeled with biotin, and the streptavidin complex may be bound later. By testing for the presence or absence of the probe, the presence or absence of the selected sequence or subsequence can be detected.
[0114]
When used with a portion of a nucleic acid, the term “heterologous” indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids with two or more sequences from unrelated genes arranged to make a nucleic acid of novel function, eg, a nucleic acid having a promoter from one source and a coating region from another source generally Recombinantly produced. Similarly, a heterologous protein refers to a protein (eg, a fusion protein) that contains two or more subsequences that are not found in the same relationship to each other in nature.
[0115]
A “promoter” is defined as an array of nucleic acid sequences that directs transcription of a nucleic acid. As used herein, a promoter contains the necessary nucleic acid sequences near the transcription start site, such as the TATA element in the case of the polymerase type II promoter. A promoter also optionally includes distal enhancer or repressor elements that can be located thousands of base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
[0116]
An “inducible” promoter is a promoter that is active under environmental or developmental control. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as an array of promoters or transcription factor binding sites) and a second nucleic acid sequence, thereby controlling nucleic acid expression control. A sequence induces transcription of the nucleic acid corresponding to the second said sequence.
[0117]
As used herein, “recombinant” refers to polynucleotides synthesized in vitro or otherwise manipulated (eg, “recombinant polynucleotides”) in cells or biological systems. A method of using a recombinant polynucleotide to produce a gene product, or a polypeptide (“recombinant protein”) encoded by the recombinant polynucleotide. “Recombinant means” refers to a variety of expression cassettes or vectors for the expression, eg, inducible or constitutive expression of a fusion protein comprising a nucleic acid that is amplified using the transfer domain of the invention and the primers of the invention. Also included are ligation reactions of various coding regions or domains or promoter sequences from the source.
[0118]
As used herein, “stable cell line” refers to a cell line that stably, ie, over time, expresses a heterologous nucleic acid sequence, ie, a T1R or G protein. In a preferred embodiment, such a stable cell line is a linearized vector comprising a T1R expression structure, ie T1R1, T1R2 and / or T1R3, suitable cells, generally mammalian cells such as HEK-293 cells. Will be transfected and produced. Most preferably, such a stable cell line is co-transfected with two linearized plasmids expressing hT1R1 and hT1R3 or hT1R2 and hT1R3, and these genes stably integrated by appropriate selection methods. Will be produced by generating a cell line with. More preferably, the cell line is G_{α 15}G proteins such as are also expressed stably.
[0119]
The phrase “selectively (or specifically) hybridizes” refers to stringent hybridization when a particular nucleotide sequence is present in a complex mixture (eg, whole cell or library DNA or RNA). It refers to a molecule under conditions that binds, duplexes or hybridizes only to the sequence.
[0120]
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence in a complex mixture of nucleic acids but not to other sequences. The exact conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. The broad guidelines on nucleic acid hybridization are Tijssen, Technologies in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, “Overview of Principles of the World.” In general, stringent conditions are selected as temperatures about 5-10 ° C. below the melting point (Tm) of the specific sequence at a defined ionic strength pH. Tm is the temperature (under defined ionic strength, pH and concentration) at which 50% of the probe complementary to the target hybridizes with the target sequence at equilibrium (when the target sequence is present in excess, the equilibrium at Tm) 50% of the probe is occupied). Strict conditions include a pH of 7.0 to 8.3 with a salt concentration of about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salt), and a probe with a short temperature (eg, , 10-50 nucleotides) and at least about 60 ° C. for long probes (eg, longer than 50 nucleotides). Stringent 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 background, optionally 10 times background hybridization. The strict conditions of a specific example are as follows. 50% formamide, 5 × SSC and 1% SDS, incubated at 42 ° C., or 5 × SSC, 1% SDS, 65 ° C., washed with 0.2 × SSC, 0.1% SDS at 65 ° C. Such hybridization and washing steps can be performed, for example, over 1, 2, 5, 10, 15, 30, 60 minutes or longer minutes.
[0121]
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially related if the polypeptides they encode are substantially related. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon synonym allowed by the genetic code. In such cases, the nucleic acids generally hybridize under moderately stringent hybridization conditions. Specific examples of “moderately stringent hybridization conditions” include hybridization at 37 ° C. in a buffer of 40% formamide, 1M NaCl, 1% SDS, and washing at 45 ° C. with 1 × SSC. . Such hybridization and washing steps can be performed, for example, over 1, 2, 5, 10, 15, 30, 60 minutes or longer minutes. Positive hybridization is at least twice background. One skilled in the art will recognize that similar stringency conditions can be obtained using alternative hybridization and wash conditions.
[0122]
“Antibody” refers to a polypeptide comprising a framework region of an immunoglobulin gene or fragments thereof that specifically binds to and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes and a large number of variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which further define immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.
[0123]
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that primarily plays a role in antigen recognition. The terms “variable light chain” (VL) and “variable heavy chain” (VH) refer to these light and heavy chains respectively.
[0124]
A “chimeric antibody” is a class, effector function and / or species in which (a) the constant region or part thereof is altered, substituted or exchanged, resulting in different or altered antigen binding sites (variable regions). Or is linked to a completely different molecule that imparts novel properties to the chimeric antibody, such as enzymes, toxins, hormones, growth factors, drugs, etc., or (b) the variable region or one of them An antibody molecule that has been altered or replaced or replaced with a variable region having a different or altered antigen specificity.
[0125]
An “anti-T1R” antibody is an antibody or antibody fragment that specifically binds to a polypeptide encoded by a T1R gene, cDNA or subsequence thereof.
[0126]
An “immunoassay” is an assay that uses an antibody that specifically binds to an antigen. An immunoassay is characterized by the use of specific binding characteristics of a particular antibody to separate, target and / or quantify the antigen.
[0127]
The phrase “specifically (or selectively) binds” to an antibody, or when referring to a protein or peptide, the phrase “specifically (or selectively) exhibits immunoreactivity” refers to proteins and other organisms. Refers to a binding reaction that determines the presence of a protein in a heterogeneous population of biological entities. Thus, under designated immunoassay conditions, a designated antibody binds to a particular protein at least twice background and does not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, a polyclonal antibody acidified against a T1R family member from a particular animal species such as rat, mouse or human is specifically immunoreactive with a T1R polypeptide or an immunogenic portion thereof and Selection can be made to obtain only polyclonal antibodies that are not immunoreactive with other proteins, except orthologs or polymorphic variants and alleles. This selection can be achieved by excluding antibodies that cross-react with T1R molecules of other animal species or other T1R molecules. Antibodies that recognize only T1R GPCR family members but other GPCRs from other families can also be selected.
[0128]
Various types of immunoassays can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, a solid phase ELISA immunoassay is used in a conventional manner to select antibodies that are specifically immunoreactive with a protein (for a description of the types and conditions of immunoassays that can be used to study specific immunoreactivity, (See, for example, Harlow & Lane, Antibodies, A Laboratory Manual (1988)). In general, a specific or selective reaction is at least twice background signal or noise, more typically more than 10 to 100 times background.
[0129]
The phrase “selectively binds” refers to the ability of a nucleic acid to “selectively hybridize” to others, as defined above, or “selectively (or specific) to a protein, as defined above. (Optionally) refers to the ability of the antibody to bind.
[0130]
The term “expression vector” is intended to constitutively or inducibly express a nucleic acid sequence of the invention in vitro or in vivo in any cell, including prokaryotic, yeast, fungi, plant, insect or mammalian cells. Refers to a recombinant expression system for The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. The expression system may or may not be capable of self-replication, i.e. only induce transient expression in the cell. The term includes recombinant expression cassettes that contain only the minimum elements necessary for transcription of a recombinant nucleic acid.
[0131]
“Host cell” means a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells include prokaryotic cells such as E. coli and eukaryotic cells such as CHO, Hela, HEK-293, etc., e.g. yeast, insect, amphibian, insect or mammalian cells such as cultured cells, explants and in vivo cells. It can be a cell.
[0132]
Isolation and expression of T1R polypeptides
Isolation and expression of the T1Rs of the present invention or a fragment or variant thereof can be carried out as follows. PCR primers can be used to amplify nucleic acids encoding taste receptor ligand binding regions, and a library of these nucleic acids can optionally be generated. The host cells can then be infected or transfected with individual expression vectors or libraries of expression vectors for functional expression of these nucleic acids or libraries. These genes and vectors can be made and expressed in vitro or in vivo. Those skilled in the art can obtain a desired phenotype for altering and controlling the expression of nucleic acids by regulating the expression or activity of genes and nucleic acids (eg, promoters, enhancers, etc.) within the vectors of the present invention. Will recognize. Any of the known methods described for increasing or decreasing expression or activity can be used. The present invention can be practiced in combination with any method or protocol known in the art that is well described in the scientific and patent literature.
[0133]
The nucleic acid sequences of the invention and other nucleic acids used to practice the invention, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids, have been genetically engineered, amplified, and / or Alternatively, it can be isolated from a variety of sources expressed recombinantly. In addition to mammalian cells, recombinant expression systems including, for example, bacterial, yeast, insect or plant systems can be used.
[0134]
Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis methods, for example, as described below. 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), Brommers, Biochemistry, 33, 7886-7896 (1994), Narang, Meth. Enzymol. 68, 90 (1979), Brown, Meth. Enzymol. 68, 109 (1979), Beaucage, Tetra. Lett. Vol. 22, 1859 (1981), U.S. Pat. No. 4,458,066. Double stranded DNA fragments can be obtained by synthesizing complementary strands and annealing the strands together under appropriate conditions, or by adding the complementary strands using a DNA polymerase containing appropriate primer sequences.
[0135]
For example, techniques for the manipulation of nucleic acids such as subcloning, labeled probes, sequencing, hybridization to obtain mutants in the sequence are well described in the scientific and patent literature. For example, Sambrook, ed., Molecular Cloning: a Laboratory Manual (2nd edition), Vols. 1-3, Cold Spring Harbor Laboratory (1989), Current Protocols in Molecular & Surgery, Ins. , New York (1997), Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I, Theory and Theory. Y. (1993).
[0136]
Nucleic acids, vectors, capsids, polypeptides, etc. can be analyzed and quantified by any of a number of common 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 superdiffusion chromatography, Immunological methods such as liquid or gel precipitation reactions, immunodiffusion, immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISA), immunofluorescence assays, Southern analysis, Northern analysis, dot blot analysis Gel electrophoresis (eg, SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling methods, scintillation counting methods and affinity chromatography.
[0137]
Oligonucleotide primers can be used to amplify nucleic acid fragments encoding taste receptor ligand binding regions. The nucleic acids described herein can also be clonal or quantitatively measured using amplification techniques. Amplification methods are also well known in the art, such as polymerase chain reaction, PCR (PCR Protocols, a Guide to Methods and Applications, edited by Innis, Academic Press, NY (1990), PCR Strategies, Innis. Ed., Academic Press, NY (1995)), ligase chain reaction (LCR) (eg, Wu, Genomics, Vol. 4, 560 (1989), Landegren, Science, Vol. 241, 1077, (1988), Barringer, Gene, 89, 117 (1990)), transcription amplification (eg, Kwoh, Proc. Natl. Acad. Sci. USA, 86, 11). 3 (1989)), self-sustained sequence replication (see, eg, Guatelli, Proc. Natl. Acad. Sci. USA, 87, 1874 (1990)), Q beta replicase amplification (eg, Smith, J. Clin. Microbiol. 35, 1477-1491 (1997)), automated Q-beta replicase amplification assays (eg, Burg, Mol. Cell. Probes, 10, 257-271). (See 1996)) and other methods using RNA polymerase (eg, NASBA, Cangene, Mississauga, Ontario), and Berger, Methods Enzymol. 152, 307-316 (1987), Sambrook, Ausubel, US Pat. Nos. 4,683,195 and 4,683,202, Sooknanan, Biotechnology, 13, 563-564 (1995) See also)). The primers can be designed to retain the original sequence of the “donor” seven-transmembrane receptor. Alternatively, the primer is a receptor that results in conservative substitutions (eg, hydrophobic for hydrophobic residues, see discussion above) or functionally mild substitutions (eg, cleavage by peptidases that do not prevent plasma membrane insertion) Amino acid residues can be encoded. Once amplified, the nucleic acids are cloned into any of a variety of vectors, individually or as a library, by methods known in the art, and if desired, using conventional molecular biology methods. be able to. Methods for cloning nucleic acids amplified in vitro are described, for example, in US Pat. No. 5,426,039.
[0138]
Primer pairs can be designed to selectively amplify the ligand binding region of a T1R family member. These regions can be different for different ligands or gustatory substances. Thus, what is the minimal binding area for one gustatory substance may be too limited for another gustatory substance. Thus, binding regions of various sizes including various extracellular domain structures may be amplified.
[0139]
The design paradigm for synonymous primer pairs is well known in the art. For example, the Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) strategic computer program is available at http: // blocks. fhrcc. org / codehop. Accessible as html and linked directly from the BlockMaker multi-sequence alignment site as a known taste receptor ligand binding region for hybrid primer prediction starting from a series of related protein sequences (see, eg, Rose, Nucleic Acids Res. 26, 1628-1635 (1998), Singh, Biotechniques 24, 318-319 (1998)).
[0140]
Means for synthesizing oligonucleotide primer pairs are well known in the art. “Natural” base pairs or synthetic base pairs can be used. For example, the use of artificial nucleobases allows a versatile approach to manipulate primer sequences and obtain more complex mixtures of amplification products. Various families of artificial nucleobases can provide means for synonymous molecular recognition by estimating multiple hydrogen bond orientations due to internal bond rotation. By incorporating these analogs into a single position of the PCR primer, a complex library of amplification products can be obtained. See, for example, Hoops, Nucleic Acids Res. 25, 4866-4871 (1997). Nonpolar molecules can also be used to simulate the shape of natural DNA bases. The non-hydrogen bond shape simulation of adenine can be efficiently and selectively reproduced in contrast to the non-polar shape simulation of thymine (eg Morales, Nat. Struct. Biol. Vol. 5, pages 950-954). 1998)). For example, two synonymous bases include pyrimidine base 6H, 8H-3,4-dihydropyrimido [4,5-c] [1,2] oxazin-7-one or purine base N6-methoxy-2,6- It may be a diaminopurine (see, for example, Hill, Proc. Natl. Acad. Sci. USA, 95, 4258-4263 (1998)). Illustrative synonymous primers of the present invention are the nucleobase analogs 5′-dimethoxytrityl-N-benzoyl-2′-deoxy-cytidine, 3 ′-[(2-cyanoethyl)-(N, N-diisopropyl)] Incorporates phosphoramidites (see above for “P” in sequence). This pyrimidine analog hydrogen binds to purines containing A and G residues.
[0141]
Polymorphic variants, alleles and interspecies homologues that are substantially the same as the taste receptors disclosed herein can be isolated using the nucleic acid probes described above. Alternatively, an expression library can be used by immunologically detecting an expressed homologue using an antiserum or purified antibody raised against a T1R polypeptide that also recognizes and selectively binds the T1R homologue. T1R polypeptides and polymorphic variants, alleles and interspecies homologs thereof can be cloned.
[0142]
A nucleic acid encoding a ligand binding region of a taste receptor can be obtained by amplification (eg, PCR) of an appropriate nucleic acid sequence using a synonymous primer pair. The nucleic acid to be amplified may be genomic DNA of any cell or tissue, or mRNA or cDNA derived from a taste receptor-expressing cell.
[0143]
In one embodiment, a hybrid protein coding sequence can be constructed that includes nucleic acids encoding T1Rs fused to a translocation sequence. Also provided are hybrid T1Rs that include the transfer motifs and gustatory substance binding domains of other families of chemosensory receptors, particularly taste receptors. These nucleic acid sequences are operably linked to transcriptional or translational control elements such as transcription and translation initiation sequences, promoters and enhancers, transcription and translation terminators, polyadenylation sequences, and other sequences useful for transcription of DNA into RNA. Can be made. In the construction of recombinant expression cassettes, vectors and transformants, promoter fragments can be used to induce expression of the desired nucleic acid in all desired cells or tissues.
[0144]
In other embodiments, the fusion protein may include a C-terminal or N-terminal translocation sequence. In addition, the fusion protein may include additional elements, eg, for protein detection, purification or other applications. Detection and purification facilitating domains allow for the purification of metal chelating peptides such as polyhistidine tract, histidine-tryptophan module or other domains, maltose binding proteins, immobilized immunoglobulins, which allow the purification of immobilized metals, for example Or the domain used in the FLAGG extension / affinity purification system (Immunex Corp., Seattle, WA).
[0145]
Factor Xa (see, for example, Ottavi, Biochimie, 80, 289-293 (1998)), subtilisin protease recognition motif (eg, Polyak, Protein Eng. 10, 615-619 (1997) )), Enterokinase (Invitrogen, San Diego, Calif.), Etc. between the translocation domain (for efficient plasma membrane expression) and the remaining newly translated polypeptide can facilitate purification. Useful. For example, one construct contains 6 histidine residues followed by an enterokinase cleavage site, thioredoxin (see, eg, Williams, Biochemistry 34, 1787-1797 (1995)) and the C-terminal transfer domain. It can include a nucleic acid sequence encoding a polypeptide that is linked. The histidine residue facilitates detection and purification, while the enterokinase cleavage site provides a means for purification of the desired protein in the remainder of the fusion protein. Techniques relating to vectors encoding fusion proteins and application examples of fusion proteins are well described in the scientific and patent literature. See, for example, Kroll, DNA Cell Biol. See Volume 12, 441-53 (1993).
[0146]
Expression vectors as individual expression vectors or libraries of expression vectors, including vectors encoding ligand binding domains, can be introduced into the genome or cytoplasm or cell nucleus and are well described in the scientific and patent literature. It can be expressed by conventional techniques. For example, Roberts, Nature, 328, 731 (1987), Berger, supra, Schneider, Protein Expr. Purif. 6435, 10 (1995), Sambrook, Tijssen, Ausbel. Product information from manufacturers of biological reagents and laboratory equipment also provides information on 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.
[0147]
Nucleic acids can be expressed using expression cassettes, vectors or viruses that are stably or transiently expressed in cells (eg, episomal expression systems). Selective markers can be incorporated into expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences. For example, selectable markers can be encoded for episomal maintenance and replication so that integration into the host genome is not necessary. For example, to allow selection of cells transformed with the desired DNA sequence, the marker can be antibiotic resistance (eg, chloramphenicol, kanamycin, G418, blasticidin, hygromycin) or herbicide resistance (eg, chloro Sulflon or Basta) can be encoded (e.g., Blendelet-Rouault, Gene, 190, 315-317 (1997), Aubrecht, J. Pharmacol. Exp. Ther. 281, 992-997. Page (1997)). Since selectable marker genes that confer resistance to neomycin or hygromycin-like substrates can only be used in tissue culture, chemical resistance genes can also be used as selectable markers in vitro and in vivo.
[0148]
The chimeric nucleic acid sequence can encode a T1R ligand binding domain within a seven-transmembrane polypeptide. Since 7-transmembrane receptor polypeptides have similar primary sequences and secondary and tertiary structures, structural domains (eg, extracellular domain, TM domain, cytoplasmic domain, etc.) should be easily identified by sequence analysis Can do. For example, homology modeling, Fourier analysis and helical periodicity detection can identify and characterize the seven domains of the seven transmembrane receptor sequence. A fast Fourier transform (FFT) algorithm was used to evaluate the dominant period that characterizes the hydrophobicity and variation profiles of the analyzed sequences. Periodic detection enhancement and α-helical periodicity index evaluation are described in, for example, Donnelly, Protein Sci. Volume 2, pages 55-70 (1993). Other alignment and modeling algorithms are well known in the art. For example, Peitsch, Receptors Channels Vol. 4, 161-164 (1996), Kyte & Doolittle, J. MoI. Med. Bio. 157, 105-132 (1982), Cronet, Protein Eng. See Volume 6, pages 59-64 (1993).
[0149]
The invention also includes DNA and proteins having the specified nucleic acid and amino acid sequences, as well as DNA fragments, particularly fragments of, for example, 40, 60, 80, 100, 150, 200 or 250 nucleotides or more, and for example , 20, 30, 50, 70, 100 or 150 amino acids or more. Optionally, the nucleic acid fragment can encode an antigenic polypeptide that can bind to an antibody raised against a T1R family member. Furthermore, the protein fragments of the present invention can optionally be antigenic fragments that can bind to antibodies raised against T1R family members.
[0150]
Other GPCRs, preferably 7 transmembrane superfamily, 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 Also contemplated are chimeric proteins bound to another amino acid that represents all or part of the members. These chimeras can be prepared from the receptor of the present invention and other CPCRs, or can be prepared by combining two or more of the T1R receptors of the present invention. In one embodiment, a portion of the chimera corresponds to or is derived from the extracellular domain of a T1R polypeptide of the invention. In other embodiments, a portion of the chimera corresponds to or is derived from one or more of the extracellular and transmembrane domains of a T1R polypeptide described herein, and the remaining portion or portions Are from other GPCRs. Chimeric receptors are well known in the art, and the techniques for making them and the selection and boundaries of G protein-coupled receptor domains or fragments for inclusion therein are also well known. Thus, such knowledge of those skilled in the art can be used to easily make such chimeric receptors. By using such a chimeric receptor, for example, one of the taste-selective properties of the receptor specifically disclosed herein and the well-known receptor used in prior art assay systems. It can be coupled with the signaling properties of other receptors.
[0151]
As noted above, such chimeras that are similar to the natural T1R receptor or a combination or binding of natural T1R receptors bind to and / or are activated by molecules that normally affect sweet taste or umami. I will. A functional chimeric T1R receptor or combination of receptors binds when expressed alone or together with other T1Rs or other GPCRs (which may themselves be chimeric), especially taste stimuli, particularly sweet (T1R2 / 3) Alternatively, it is a molecule activated by umami stimulation (T1R1 / 3). Sweetness-inducing molecules include natural and artificial sweeteners such as sucrose, asteltem, xylitol, and cyclamate. Molecules that cause umami include glutamate and glutamate analogs and other compounds that bind to natural T1R1 and / or T1R3, such as 5'-nucleotides.
[0152]
For example, a ligand binding domain, extracellular domain, transmembrane domain, cytoplasmic domain, N-terminal domain, C-terminal domain, or any combination thereof can be covalently bound to a heterologous protein. For example, the T1R extracellular domain can be covalently bound to the heterologous GPCR transmembrane domain, or the heterologous GPCR extracellular domain can be covalently bound to the T1R transmembrane domain. Other suitable heterologous proteins can be used, such as green fluorescent protein.
[0153]
Host cells for expressing the T1Rs, fragments, chimeras or variants of the invention are also within the scope of the invention. In order to obtain a high level expression of a cloned gene or nucleic acid, such as a cDNA encoding the T1Rs, fragments or variants of the present invention, one of ordinary skill in the art will generally identify the nucleic acid sequence in question as a strong promoter that induces transcription In a transcription / translation terminator and, in the case of a nucleic acid encoding a protein, in a ribosome binding site for translation initiation. Suitable bacterial promoters are well known in the art and are described, for example, in Sambrook et al. However, bacterial or eukaryotic expression systems can be used.
[0154]
Well known methods for introducing a heterologous nucleotide sequence into a host cell can be used. These include calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors, and for introducing cloned genomic DNA, cDNA, synthetic DNA or other heterologous genetic material into host cells Other well-known methods (see, for example, Sambrook et al.). It suffices if the specific procedure used in genetic engineering can successfully introduce at least one nucleic acid molecule into a host cell capable of expressing the T1R, fragment or variant in question.
[0155]
After introducing the expression vector into the cells, the transfected cells are cultured under conditions favorable for expression of the receptor, fragment or variant of interest, and then recovered from the culture using standard methods. Examples of such methods are well known in the art. See, eg, WO 00/06593, which is incorporated herein by reference in a manner consistent with this disclosure.
[0156]
Detection of T1R polypeptide
In addition to detecting T1R genes and gene expression using nucleic acid hybridization methods, immunoassays can also be used to detect T1Rs, eg, to identify taste receptor cells as well as variants of T1R family members. Immunoassays can be used to qualitatively or quantitatively analyze T1Rs. A general overview of applicable techniques can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).
[0157]
1. Antibodies against T1R family members
Methods for producing polyclonal and monoclonal antibodies that specifically react with T1R family members are known to those skilled in the art (eg, Coligan, Current Protocols in Immunology (1991), Harlow & Lane, supra, Goding, Monoclonal Antibodies: See Principles and Practice (2nd edition, 1986) and Kohler & Milstein, Nature, 256, 495-497 (1975)). Such techniques include antibody preparation by selection of antibodies from a library of recombinant antibodies in phage or similar vectors, and polyclonal and monoclonal antibodies from immunized rabbits or mice (see, for example, Huse et al. Science, 246, 1275-1281 (1989), Ward et al., Nature 341, 544-546 (1989)).
[0158]
In order to produce antibodies that specifically react with T1R family members, immunogens containing many T1Rs can be used. For example, a recombinant T1R polypeptide or antigenic fragment thereof can be isolated as described herein. Suitable antigenic regions are, for example, common regions used to identify members of the T1R family. Recombinant proteins can be expressed in eukaryotic or prokaryotic cells as described above and can generally be purified as described above. Recombinant protein is a preferred immunogen for the production of monoclonal or polyclonal antibodies. Alternatively, a synthetic peptide obtained from the sequences disclosed herein and conjugated to a carrier protein can be used as an immunogen. Naturally occurring proteins can also be used in pure or impure form. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure proteins.
[0159]
Methods for producing polyclonal antibodies are known to those skilled in the art. For example, syngeneic breeding mice (eg, BALB / C mice) or rabbits are immunized with the protein using standard adjuvants such as Freund's adjuvant and standard immunization protocols. The animal's immune response to the immunogen preparation is monitored by taking a test bleed to determine the titer of reactivity to T1R. If a suitably high titer of antibody to the immunogen is obtained, blood is collected from the animal and antiserum is prepared. If desired, further fractionation of the antiserum can be performed to concentrate antibody reactive to the protein (see Harlow & Lane, supra).
[0160]
Monoclonal antibodies can be obtained by various techniques known to those skilled in the art. Briefly, spleen cells of animals immunized with the desired antigen can generally be immobilized by fusion with myeloma cells (Kohler & Milstein, Eur. J. Immunol. Vol. 6, 511-519). (See 1976)). Another method of immobilization is transformation with Epstein-Barr virus, oncogenes or retroviruses or other methods well known in the art. Colonies arising from single immobilized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and the yield of monoclonal antibodies produced by such cells is injected into the peritoneal cavity of a vertebrate host It can be increased by various methods including: Alternatively, Huse et al. , Sciences, Vol. 246, 1275-1281 (1989), following the general protocol outlined to isolate DNA sequences encoding monoclonal antibodies or binding fragments thereof by screening a DNA library of human B cells can do.
[0161]
Monoclonal antibodies and polyclonal sera are collected and titered against the immunogenic protein in an immunoassay, eg, a solid phase immunoassay using an immunogen immobilized on a solid support. In general, polyclonal antisera with a titer of 104 or higher are selected and competitive binding immunoassays for their cross-reactivity to non-T1R polypeptides or other T1R family members or other related proteins of other organisms. Use and test. Specific polyclonal antisera and monoclonal antibodies typically have a value of at least about 0.1 mM, more usually about 1 pM, optionally at least about 0.1 pM or better, and optionally at least 0.01 pM or better. It binds with Kd.
[0162]
Once T1R family member specific antibodies are obtained, individual T1R proteins and protein fragments can be detected by various immunoassays. For a review of immunological and immunoassay methods, see Basic and Clinical Immunology (Edited by States & Terr, 7th Edition, 1991). Furthermore, the immunoassays of the present invention can be performed in any of several configurations that are extensively reviewed in Enzyme Immunoassay (Maggio, 1980) and Harlow & Lane, supra.
[0163]
2. Immunological binding assay
T1R proteins, fragments and variants can be detected and / or quantified using any of a number of well-recognized immunological binding assays (eg, US Pat. No. 4,366,241). 4,376,110, 4,517,288 and 4,837,168). For reviews of general immunoassays, see Methods in Cell Biology: Antibodies in Cell Biology, Volume 37 (Asai, 1993), Basic and Clinical Immunology (see Stites & Terr, 7th Edition, 1991). Immunological binding assays (or immunoassays) generally use antibodies that specifically bind to optimal proteins or antigens (in this case, T1R family members or antigenic subsequences thereof). Antibodies (eg, anti-T1R) can be produced by any of a number of means well known to those of skill in the art and as described above.
[0164]
Also in an immunoassay, a labeling substance for specifically binding and labeling a complex formed by an antibody and an antigen is often used. The labeling substance may itself be one of the parts comprising the antibody / antigen complex. Therefore, the labeling substance may be a labeled T1R polypeptide or a labeled anti-T1R antibody. Alternatively, the labeling substance may be a third moiety such as a second antibody that specifically binds to the antibody / T1R complex (the second antibody is against the antibody of the animal species from which the first antibody is obtained). Generally specific). Other proteins that can specifically bind to the immunoglobulin constant region, such as protein A or protein G, can also be used as the labeling substance. These proteins show strong non-immunogenic reactivity with immunoglobulin constant regions of various animal species (see, for example, Kronal et al., J. Immunol. 111, 1401-1406 (1973), Akerstrom). et al., J. Immunol., 135, 2589-2542 (1985)). The labeling substance can be modified with a detectable moiety such as biotin to which other molecules such as streptavidin can specifically bind. Various detectable moieties are well known to those skilled in the art.
[0165]
Throughout the assay, incubation and / or washing steps are required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the incubation time depends on the assay format, antigen, amount of solution, concentration, etc. Typically, the assay can be performed at a temperature range such as 10 ° C. to 40 ° C., but is performed at ambient temperature.
[0166]
A.Non-competitive assay format
Immunoassays for detecting T1R polypeptides in a sample can be competitive or noncompetitive. A non-competitive immunoassay is an assay that directly measures the amount of antigen. In one preferred “sandwich” assay, for example, anti-T1R antibodies can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies capture the T1R polypeptide present in the test sample. The T1R polypeptide is so immobilized and then bound by a labeling substance such as a second T1R antibody bearing a label. Alternatively, the second antibody may lack a label, but is then bound by a labeled third antibody specific for the antibody of the animal species from which the second antibody is obtained. The second or third antibody is generally modified with a detectable moiety such as biotin to which other molecules such as streptavidin can specifically bind to obtain a detectable moiety. ing.
[0167]
B.Competitive assay format
In a competitive assay, the amount of T1R polypeptide present in a sample is displaced from the anti-T1R antibody (competitively desorbed) by an unknown T1R polypeptide present in the sample (exogenous) T1R polypeptide. It is measured indirectly by measuring the amount of peptide. In one competitive assay, a known amount of T1R polypeptide is added to the sample and the sample is then contacted with an antibody that specifically binds to T1R. The amount of exogenous T1R polypeptide that binds to the antibody is inversely proportional to the concentration of 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 is determined by measuring the amount of T1R polypeptide present in the T1R / antibody complex or by measuring the amount of remaining uncomplexed protein. The amount of T1R polypeptide can be detected by supplying a labeled T1R molecule.
[0168]
A hapten inhibition assay is another preferred competitive assay. In this assay, a known T1R polypeptide is immobilized on a solid substrate. A known amount of anti-T1R antibody is added to the sample, and then the sample is contacted with immobilized T1R. The amount of anti-T1R antibody bound to the known immobilized T1R 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 antibody or the fraction of antibody remaining in solution. Detection can be performed directly when the antibody is labeled, or indirectly by subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
[0169]
C.Cross-reactivity measurement
Competitive binding immunoassays can also be used to measure cross-reactivity. For example, proteins that are at least partially encoded by the nucleic acid sequences disclosed herein can be immobilized on a solid support. Proteins that compete for binding of antisera to immobilized antigen (eg, T1R polypeptides and homologs) are added to the assay. The ability of the added protein to compete for binding of the antiserum to the immobilized protein is compared to the ability of the T1R polypeptide encoded by the nucleic acid sequences disclosed herein to compete with itself. The percentage reactivity of the above proteins is calculated by standard calculation method. Antisera showing less than 10% cross-reactivity with each of the above added proteins are selected and pooled. Cross-reactive antibodies are optionally removed from the pooled antisera by immunoabsorption with the considered additive protein, eg, a less relevant homolog. In addition, peptides containing amino acid sequences representing conserved motifs used to identify members of the T1R family can be used to determine cross-reactivity.
[0170]
Immunoabsorbed and pooled antisera are used in the competitive binding immunoassay described above to convert a second protein, possibly an allelic or polymorphic variant of a T1R family member, to an immunogenic protein (ie, the present To the T1R polypeptide encoded by the nucleic acid sequence disclosed herein). To make this comparison, each of the two proteins is analyzed over 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. The amount of the second protein required to inhibit 50% of binding is less than 10 times the amount of protein encoded by the nucleic acid sequences disclosed herein required to inhibit 50% of binding. In some cases, the second protein is said to specifically bind to a polyclonal antibody raised against T1R.
[0171]
Antibodies raised against T1R conserved motifs can also be used to prepare antibodies that specifically bind only to T1R family GPCRs, but can be used for other family GPCRs. Can not.
[0172]
Polyclonal antibodies that specifically bind to a particular member of the T1R family can be prepared by removing cross-reactive antibodies with other T1R family members. A species-specific polyclonal antibody can be prepared in the same manner. For example, antibodies specific for human T1R1 can be prepared by removing orthologous sequences, eg, antibodies that are cross-reactive with rat T1R1 or mouse T1R1.
[0173]
D.Other assay formats
Western blot (immunoblot) analysis is used to detect and quantify the presence of T1R polypeptide in the sample. This technique generally separates the sample protein by gel electrophoresis based on molecular weight, transfers the separated protein to an appropriate solid support (nitrocellulose filter, nylon filter or derivatized nylon filter), and converts the sample to T1R polypeptide. Incubating with an antibody that specifically binds. An anti-T1R polypeptide antibody specifically binds to a T1R polypeptide on a solid support. These antibodies can be directly labeled with a labeled antibody that specifically binds to the anti-T1R antibody (eg, sheep anti-mouse antibody) or can be detected later.
[0174]
Other assay formats include liposome immunoassay (LIA) using liposomes designed to bind to specific molecules (eg, antibodies) and release encapsulation reagents or markers. The released chemical is detected by standard techniques (see Monroe et al., Amer. Clin. Prod. Rev. 5, 34-41 (1986)).
[0175]
E.Reduce non-specific binding
One skilled in the art will recognize that it is often desirable to minimize specific binding in an immunoassay. In particular, if the assay uses an antigen or antibody immobilized on a solid substrate, it is desirable to minimize the amount of non-specific binding to the substrate. Means for reducing such non-specific binding are well known to those of skill in the art. In general, this approach involves coating a substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), skim milk powder and gelatin are widely used, with milk powder being most preferred.
[0176]
F.Sign
The particular label or detectable group used in the assay is not an important aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group may be a substance having a detectable physical or chemical property. Such detectable labels are well developed in the field of immunoassays and, in general, most labels useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the present invention include magnetic beads (eg, DYNABEADS ™), fluorescent dyes (eg, fluorescein isothiocyanate, Texas red, rhodamine, etc.), radioactive labels (eg,^ThreeH,¹²⁵I,¹⁴C,³⁵S), colorimetry such as enzymes (eg horseradish, alkaline phosphate and others commonly used in ELISA) colloidal gold or colored glass or plastic beads (eg polystyrene, polypropylene, latex, etc.) There are signs.
[0177]
The label can be coupled directly or indirectly to the desired component of the assay by methods known in the art. As noted above, a wide range of labels can be used, but the label is selected depending on the sensitivity required, ease of conjugation with the compound, stability requirements, available equipment and disposal regulations.
[0178]
Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (eg, biotin) is covalently bound to the molecule. The ligand then binds to other molecules (eg, streptavidin) that are inherently detectable, such as detectable enzymes, fluorescent compounds or chemiluminescent compounds, or that are covalently linked to the signal system. The ligands and their labels can be used in appropriate combination with an antibody that recognizes the T1R polypeptide or a second antibody that recognizes the anti-T1R.
[0179]
The molecule can also be coupled directly to the signal generating compound, eg, complexed with an enzyme or fluorophore. The enzymes in question as labels are mainly hydrolases, in particular phosphatases, esterases and glycosidases or oxidases, in particular peroxidases. Examples of the fluorescent compound include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds include luciferin and 2,3-dihydrophthalazinedione, such as luminol. See US Pat. No. 4,391,904 for a review of various labeling or signal generating systems that can be used.
[0180]
Means of detecting the label are well known to those skilled in the art. Thus, for example, when the label is a radioactive label, the detection means is a photographic film as in a scintillation counter or autoradiography. When the label is a fluorescent label, the label can be detected by exciting the fluorescent dye with light of an appropriate wavelength and detecting the generated fluorescence. Fluorescence can be detected visually by photographic film by use of electronic detectors such as charge coupled devices (CCDs) or photomultiplier tubes. Similarly, enzyme labels can be detected by detecting the resulting reaction product using an appropriate substrate for the enzyme. Finally, simple colorimetric labels can be detected by simply observing the color associated with the label. Thus, in various dipstick assays, the bound gold often appears pink, while the various composite beads appear bead color.
[0181]
Some assay formats do not require the use of labeled compounds. For example, an agglutination assay can be used to detect the presence of the target antibody. In this case, the antigen-coated particles are aggregated with a sample containing the target antibody. In this format, no compound needs to be labeled and the presence of the target antibody is detected by simple visual inspection.
[0182]
Modulator detection
Described below are compositions and methods for investigating whether a test compound specifically binds to the T1R receptor of the present invention in vitro and in vivo. Many aspects of cell physiology can be monitored to assess the effects of ligand binding to the T1R polypeptides of the invention. These assays can be performed on intact cells that express chemosensory receptors, on permeabilized cells, or on membrane fragments produced by standard methods or newly synthesized proteins in vitro.
[0183]
In vivo, taste receptors bind to taste substances and initiate the conversion of chemical stimuli into electrical signals. Activated or inhibited G proteins then alter the properties of target enzymes, channels and other effector proteins. Some examples include cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by stimulating G proteins, activation of phospholipase C by Gq and other cognate G proteins and various channels by Gi and other G proteins Adjustment. Downstream results such as the production of diacylglycerol and IP3 by phospholipase C and the consequent calcium mobilization by IP3 can also be considered.
[0184]
The T1R protein or polypeptide of the assay is preferably selected from polypeptides having a T1R polypeptide sequence selected from those disclosed in Example 1 or fragments or conservatively modified variants thereof. Optionally, the fragments and variants may be antigenic fragments and variants that bind to the anti-T1R antibody. Optionally, fragments and variants may be bound to or activated by sweeteners or umami taste substances.
[0185]
Alternatively, the T1R protein or polypeptide of the assay may be derived from a eukaryotic host cell and may be of the T1R polypeptide sequence disclosed in Example 1 or a fragment thereof or a conservatively modified variant and amino acid sequence. It may contain amino acid subsequences with identity. Generally, amino acid sequence identity is at least 35-50%, or optionally 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. Optionally, the T1R protein or polypeptide of the assay may comprise a T1R protein domain, such as an extracellular domain, a transmembrane region, a transmembrane domain, a cytoplasmic domain, a ligand binding domain. Furthermore, as described above, a T1R protein or domain thereof can be covalently linked to a heterologous protein to create a chimeric protein for use in the assays described herein.
[0186]
Modulators of T1R receptor activity are tested using recombinant or naturally occurring T1R proteins or polypeptides as described above. A T1R protein or polypeptide can be isolated recombinantly or naturally occurring, co-expressed in a cell, co-expressed in a membrane obtained from the cell, and co-expressed in a tissue or animal. For example, tongue sections, cells separated from the tongue, transformed cells, or membranes can be used. Modulation can be tested using one of the in vitro or in vivo assays described herein.
[0187]
For example, as disclosed in the experimental examples below, certain 5¹Nucleotide, eg, 5¹IMP or 5¹GMP enhances the activity of L-glutamate and activates the umami receptor or activates the umami receptor by an umami stimulant such as LL-glutamate or L-aspartate. Inhibit.
[0188]
1. In vitro binding assay
Taste conversion can also be examined in vitro by a soluble or solid state reaction using the T1R polypeptides of the present invention. In certain embodiments, a T1R ligand binding domain can be used for in vitro soluble or solid state reactions to assay for ligand binding.
[0189]
For example, it is predicted that the N-terminal domain of T1R is involved in ligand binding. More specifically, T1Rs belong to the GPCR subfamily characterized by a large, approximately 600 amino acid extracellular N-terminal segment. These N-terminal segments are thought to form a ligand binding domain and are therefore useful in biochemical assays to identify T1R agonists and antagonists. The ligand binding domain may be formed by additional portions of the extracellular domain, such as the extracellular loop of the transmembrane domain.
[0190]
In vitro assays have been used for other GPCRs related to T1Rs, such as metabotropic glutamate receptors (eg, Han and Hampson, J. Biol. Chem. 274, 10008-10013 (1999)). See). These assays involve measurement of displacement of radioactive or fluorescently labeled ligands, changes in intrinsic fluorescence or changes in protein susceptibility, and the like.
[0191]
Ligand binding to a heteromultimeric complex of a T1R polypeptide of the invention can be tested in solution, optionally in a bilayer membrane bound to a solid phase, in a lipid monolayer, or in a vesicle. it can. Modulator binding can be tested, for example, using changes in spectroscopic properties (eg, fluorescence, absorbance, refractive index), hydrodynamic (eg, shape), chromatography, or solubility properties.
[0192]
In another embodiment of the invention, GTPγ³⁵The S assay can be used. As described above, activation of GPCR leads to G protein complex G_αThe subunits are stimulated to exchange bound GDP with GTP. Ligand-mediated stimulation of G protein exchange activity is achieved by adding radiolabeled GTPγ to G protein in the presence of a putative ligand.³⁵It can be measured in a biochemical assay that measures S binding. In general, the membrane containing the chemosensory receptor in question is mixed with the G protein complex. Possible inhibitors and / or activators and GTPγ³⁵Add S to the assay and add GTPγ to G protein³⁵S binding is measured. Binding can be measured by other means known in the art including liquid scintillation counting or scintillation proximity assay (SPA). Other assay formats can utilize fluorescently labeled GTPγS.
[0193]
2. Fluorescence polarization assay
In other embodiments, fluorescence binding (“FP”) based assays can be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzyme activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation or electrophoresis. These assays are performed directly in solution in real time and do not require an immobilized phase. Since the measurement of polarization is rapid and does not destroy the sample, the polarization value can be measured repeatedly and after addition of the reagent. In general, this technique can be used to measure polarization values from low picomolar to micromolar. This section describes how fluorescence polarization is used to measure ligand binding to the T1R polypeptides of the present invention in a simple and quantitative manner.
[0194]
When a fluorescently labeled molecule is excited by plane polarized light, the molecule emits light having a degree of polarization that is inversely proportional to its molecular rotation. Large fluorescently labeled molecules remain relatively stationary in the excited state (4 nanoseconds for fluorescein) and the polarization of light remains relatively constant between excitation and emission. Small fluorescently labeled molecules rotate rapidly during the excited state, and the polarization changes significantly between excitation and emission. Thus, small molecules have low polarization values and large molecules have high polarization values. For example, a single-stranded fluorescein labeled oligonucleotide has a relatively low polarization value, but has a higher polarization value when hybridized with a complementary strand. When FP is used to detect and monitor gustatory substance binding that activates or inhibits the chemosensory receptors of the present invention, fluorescently labeled gustatory substances or autofluorescent gustatory substances can be used.
[0195]
The fluorescence polarization (P) is defined as follows.
[0196]
[Expression 1]
[0197]
Here, Π is the intensity of the radiated light parallel to the excitation light surface, Int ⊥ is the intensity of the radiated light perpendicular to the excitation light surface, and P is the ratio of the light intensity and is a dimensionless number. For example, Beacon ™ and Beacon 2000 ™ can be used in connection with these assays. Such systems generally represent polarized light in milli-polarized units (1 polarized unit = 1000 mP unit).
[0198]
The relationship between molecular rotation and size is described by the Perrin equation. The reader has written a detailed explanation of this equation, Jolley M. E. (1991), Journal of Analytical Toxicology, pages 236-240. In summary, the Perrin equation describes that the polarization is directly proportional to the rotational relaxation time, which is the time required for the molecule to rotate an angle of about 68.5 °. The rotational relaxation time is related to viscosity (η), absolute temperature (T), molecular volume (V), and gas constant (R) by the following equation:
[0199]
[Expression 2]
[0200]
The rotational relaxation time is small (about 1 nanosecond) for small molecules (eg, fluorescein) and large (about 100 nanoseconds) for large molecules (eg, immunoglobulins). If the viscosity and temperature are kept constant, the rotational relaxation time, and hence the polarization, is directly related to the molecular volume. The change in molecular volume may be due to changes in interaction, dissociation, polymerization, degradation, hybridization or conformation of the fluorescently labeled molecule with other molecules. For example, fluorescence polarization has been used to measure enzymatic cleavage of large fluorescently labeled polymers by proteases, DNases and RNases. Fluorescence polarization has also been used to measure equilibrium binding in protein / protein interactions, antibody / antigen binding and protein / DNA binding.
[0201]
A.Solid state and soluble high throughput assays
In other embodiments, the invention provides solubility assays using hetero-oligomeric T1R polypeptide complexes or cell or tissue co-expressed T1R polypeptides. Preferably, the cells stably co-express functional T1R1 / T1R3 (umami) taste receptors or T1R2 / T1R3 (sweet) taste receptors. In other embodiments, the invention provides for binding a T1R polypeptide or a cell or tissue that expresses a T1R polypeptide to a solid phase substrate or a taste stimulant and contacting the T1R receptor with an appropriate label or T1R receptor. In contrast, solid-phase based in vitro assays in a high-throughput format that detect binding using antibodies produced against.
[0202]
The high throughput assay of the present invention can screen thousands of modulators or ligands per day. In particular, each well of the microtiter plate can be used to perform an independent assay for the potential modulator selected, or 5-10 wells can be used if a concentration or incubation time effect is observed. One modulator can be tested. Thus, an assay can be performed for about 100 (eg, 96) modulators on a single standard microtiter plate. When using a 1536 well plate, the assay can be easily performed for about 1000 to about 1500 compounds on a single plate. It is possible to assay multiple compounds in each plate well. Assays can be performed using several different plates per day, and using the integrated system of the present invention, up to about 6,000-20,000 compounds can be screened by assay. Recently, a microfluidic approach to reagent manipulation has been developed.
[0203]
The molecule of interest can be bound to the solid state component directly or indirectly via a covalent or non-covalent moiety, eg, via a tag. The tag can be any of a variety of components. In general, a molecule that binds to the label (label binder) is bound to a solid support, and the labeled molecule in question (eg, taste-changing molecule in question) is bound to the solid support by the interaction of the label and the label binder. .
[0204]
Numerous labels and label binders can be used based on known molecular interactions well documented in the literature. For example, if the label comprises a natural binder such as biotin, protein A or protein G, it can be used with an appropriate label binder (avidin, streptavidin, neutravidin, immunoglobulin Fc portion, etc.). . Antibodies against molecules with natural binders such as biotin are also widely available and are suitable label binders (see SIGMA Immunochemicals 1998 catalog, SIGMA (St. Louis, MO)).
[0205]
Similarly, a haptenic or antigenic compound can be combined with a suitable antibody to produce a label / label binder pair. Thousands of specific antibodies are commercially available, and many other antibodies are described in the literature. For example, in one common arrangement, 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 suitable as label and label binder pairs. For example, cell membrane receptor agonists and antagonists (eg, cell receptors such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies) -Ligand interactions, as well as the cadherin family, integrin family, selectin family etc. see eg Pigot & Power, The Adhesion Molecular Facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (eg, opiates, steroids, etc.), intracellular receptors (eg, mediate the action of various small ligands including steroids, thyroid hormones, retinoids and vitamin D; peptides) Drugs, lectins, sugars, nucleic acids (linear and cyclic polymer arrangements), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.
[0206]
Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides and polyacetates can also form suitable labels or label binders. Many other label / label binder pairs are also useful in the assay systems described herein, as will be apparent to those of skill in the art upon reviewing this disclosure.
[0207]
General linkers such as peptides and polyethers can also serve as labels, and examples include polypeptide sequences such as a poly-gly sequence of about 5 to 200 amino acids. Such flexible linkers are known to those skilled in the art. For example, poly (ethylene glycol) linkers are available from Shearwater Polymers, Inc. (Huntsville, Alabama). These linkers may optionally have amide bonds, sulfhydryl bonds, or heterofunctional group bonds.
[0208]
The label binder is affixed to the solid substrate using a variety of currently available methods. Solid substrates are generally derivatized or functionalized by exposing all or part of the substrate to a chemical reagent that immobilizes to the surface a chemical group that is reactive with a portion of the labeled binder. For example, suitable groups for attachment to longer chain moieties include hydroxyl, thiol and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize various surfaces such as glass surfaces. The structure of such a solid phase biopolymer array is fully described in the literature. For example, Merrifield, J. et al. Am. Chem. Sic. 85, 2149-2154 (1963) (eg, describing solid phase synthesis of peptides), Geysen et al. J. et al. Immun. Meth. 102, 259-274 (1987) (describes the synthesis of solid phase components on pins), Frank & Doring, Tetrahedron, 44, 6031640 (1988) (synthesis of various peptide sequences on cellulose discs. Description), Fodor et al. Science, Vol. 251, 767-777 (1991), Sheldon et al. , Clinical Chemistry, Vol. 39, No. 4, 718-719 (1993) and Kozal et al. , Nature Medicine, Vol. 2, No. 7, 757759 (1996), which describes an array of biopolymers all fixed to a solid substrate. As a non-chemical method for fixing the label binder to the substrate, there are other general methods such as cross-linking by heat and UV light.
[0209]
3. Cell-based assays
In a preferred embodiment of the treatment, the T1R protein or polypeptide combination includes or preferably does not include a heterologous chaperone sequence that facilitates its mutation and targeting in eukaryotic cells, in an unmodified form, or by a secretory pathway. Co-expressed temporarily or stably as a chimera, mutant or truncated receptor. Such T1R polypeptides can be expressed in any eukaryotic cell such as HEK-293 cells. Cells may be functional G proteins, such as Gα15 or previously identified chimeric G proteins, or other G proteins that can bind chimeric receptors to intracellular signaling pathways or signaling proteins such as phospholipase C. It is preferred to include a protein. Also, since such cells were found to exhibit a strong response to taste stimulants (related to cells that transiently express the same T1R combination) (as shown in the experimental examples), T1R1 / T1R3 Alternatively, it is preferable to generate cells that stably express T1R2 / T1R3. Such activation of T1R receptors in cells can be detected using standard methods such as by detecting Fluo-4-dependent fluorescence in the cells and detecting changes in intracellular calcium. it can. Such an assay is the basis for the experimental findings presented in this application.
[0210]
Activated GPCR receptors are kinase substrates that often phosphorylate the C-terminal part of the receptor (and possibly other sites as well). Thus, the activator is from radiolabeled ATP to the receptor.³²It will promote the transfer of P. This can be assayed with a scintillation counter. C-terminal phosphorylation promotes arrestin-like protein binding and prevents G protein binding. For a general review of GPCR signaling and methods of assaying signaling, see, for example, Methods in Enzymology, Vol. 237 and Vol. 238 (1994) and Vol. 96 (1983), Borne et al. Nature, 349, 117-27 (1991), Bourne et al. Nature, 348, 125-32 (1990), Pitcher et al. Annu. Rev. Biochem. 67, 653-92 (1998).
[0211]
T1R modulation can be assayed by comparing the response of a T1R polypeptide treated with a putative T1R modulator to the response of an untreated control sample or a sample containing a known “positive” control. Such putative T1R modulators may be molecules that inhibit or activate T1R polypeptide activity. In one embodiment, a control sample (not treated with an activator or inhibitor) has a relative T1R activity value of 100%. Inhibition of T1R polypeptide is achieved when the T1R activity value relative to the control is about 90%, optionally 50%, optionally 25-0%. Activation of the T1R polypeptide is achieved when the T1R activity value relative to the control is 110%, optionally 150%, 200-500% or 1000-2000%.
[0212]
Changes in ion flux can be assessed by measuring changes in ionic polarization (ie, potential) of cells or membranes expressing T1R polypeptides. One means of measuring the change in cell polarization is to measure the change in current (and thereby measure the change in polarization) by voltage-clamping and patch-clamping methods (eg, “cell adhesion” mode, “ (Refer to "inside-inverted" mode and "whole cell" mode, Ackerman et al., New Engl. J Med. 336, 1575-1595 (1997)). Total cell current is conveniently measured using standard methods. Other known assays include radiolabeled ion flux assays and fluorescent assays using voltage-sensitive dyes (eg, Vestergard-Bogind et al., J. Membrane Biol. 88, 67-75 (1988)). ), Gonzales & Tsien, Chem. Biol., 4, 269-277 (1997), Daniel et al., J. Pharmacol. Meth., 25, 185-193 (1991), Holvinsky et al., J. Membrane Biology, 137, 59-70 (1994)).
[0213]
The effect of the test compound on the function of the polypeptide can be measured by examining any of the above parameters. Appropriate physiological changes that affect GPCR activity can be used to assess the effect of test compounds on the polypeptides of the invention. When functional results are examined using intact cells or animals, transmitter release, hormone release, transcriptional changes to known and unidentified genetic markers (Northern blot) and Ca²⁺Various effects such as changes in intracellular second messengers such as IP3, cGMP or cAMP can also be measured.
[0214]
Preferred assays for GPCRs are directed to cells loaded with ions or voltage sensitive dyes that report receptor activity. Assays for measuring the activity of such receptors can also use known agonists and antagonists of other G protein-coupled receptors as controls for assessing the activity of the tested compounds. In assays to identify modulatory compounds (eg, agonists, antagonists), changes in cytoplasmic ion concentration or membrane potential are monitored with an ion sensitive or membrane potential fluorometer, respectively. The main ones of ion sensitivity indicators and potential probes that can be used are disclosed in the Molecular Probes 1997 catalog (Molecular Probes 1997 Catalog). For G protein-coupled receptors, promiscuous G proteins such as Gα15 and Gα16 can be used in the most appropriate assays (Wilkie et al., Proc. Nat′1 Acad. Sci. Vol. 88, 10049-10053. Page (1991)).
[0215]
Receptor activation initiates subsequent intracellular events, such as an increase in second messengers. Activation of some G protein coupled receptors stimulates the generation of inositol triphosphate (IP3) by phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge & Irvin, Nature, 312, 315-21 (1984). Year)). IP3 then stimulates the release of intracellular stored calcium ions. Thus, changes in cytosolic calcium ion concentration or changes in second messenger concentrations such as IP3 can be used to assess G protein-coupled receptor function. Cells expressing such G protein coupled receptors show an increase in cytoplasmic calcium concentration due to calcium release from intracellular stores and the contribution of extracellular calcium influx through plasma membrane ion channels.
[0216]
In a preferred embodiment, a T1R gene that co-expresses T1R polypeptide activity stably or transiently, preferably stably, in a heterologous cell comprising a promiscuous G protein that links the receptor to the phospholipase C signaling pathway. (See Offermanns & Simon, J. Biol. Chem. 270, 15175-15180 (1995)). In a preferred embodiment, the cell line is HEK-293 (usually does not express the T1R gene) and the promiscuous G protein is Gα15 (Offermanns & Simon, supra). The regulation of taste transduction is intracellular Ca that changes in response to regulation of the T1R signaling pathway by administration of a molecule that binds to a T1R polypeptide.²⁺Test by measuring the change in concentration. Ca²⁺The change in concentration may be due to fluorescence Ca²⁺Measure by indicator dye and fluorescence analysis imaging.
[0217]
In other embodiments, the hydrolysis of phosphatidylinositol (PI) can be analyzed according to US Pat. No. 5,436,128, incorporated herein by reference. Briefly, the assay involves labeling the cells with 3H-myoinositol for over 48 hours. The labeled cells are treated with the test compound for 1 hour. The treated cells are lysed and extracted with chloroform-methanol-water, then inositol phosphate is separated by ion exchange chromatography and quantified by scintillation counting. The stimulation ratio is determined by calculating the ratio of cpm in the presence of agonist and cpm in the presence of buffer. Similarly, the inhibition ratio is determined by calculating the ratio of cpm in the presence of antagonist to cpm in the presence of a buffer (which may or may not contain an agonist).
[0218]
Other receptor assays can include measuring the concentration of intracellular cyclic nucleotides, such as cAMP or cGMP. Where receptor activation results in a decrease in cyclic nucleotides, the cells may be exposed to a substance that increases the intracellular cyclic nucleotide concentration, such as forskolin, prior to adding the receptor activating compound to the cells in the assay. It seems preferable. In one embodiment, changes in intracellular cAMP or cGMP can be measured using an immunoassay. Offermanns & Simon, J.M. Bio. Chem. 270, 15175-15180 (1995) can be used to measure the cAMP concentration. Also, Felley-Bosco et al. Am. J. et al. Resp. Cell and Mol. Biol. The concentration of cGMP can be measured using the method described in Vol. 11, pages 159-164 (1994). In addition, an assay kit for measuring cAMP and / or cGMP is described in US Pat. No. 4,115,538, incorporated herein by reference.
[0219]
In other embodiments, the level of transcription can be measured to assess the effect of a test compound on signal transduction. After contacting the host cell containing the T1R polypeptide of interest with a test compound for a time sufficient to effect interaction, the level of gene expression is measured. The length of time that such interaction occurs can be determined experimentally, such as by passing time and measuring the level of transcription as a function of time. The amount of transcription can be measured using methods known to be appropriate to those skilled in the art. For example, mRNA expression of the protein of interest may be detected using Northern blots, or their peptide products may be identified using immunoassays. Alternatively, a transcription-based assay using a reporter gene can be used as described in US Pat. No. 5,436,128, incorporated herein by reference. The reporter gene may be, for example, chloramphenicol acetyltransferase, luciferase, β-galactosidase, β-lactamase and alkaline phosphatase. In addition, the protein in question can be used as an indirect reporter by binding to a second receptor such as green fluorescent protein (eg Mitili & Spector, Nature Biotechnology, Vol. 15, pages 961-964 (1997)). See).
[0220]
The amount of transcription can then be compared to the amount of transcription in the same cell in the absence of the test compound or to the amount of transcription in substantially the same cell that does not contain the T1R polypeptide of interest. . Substantially the same cells can be obtained from the same cells prepared by recombinant cells but not modified by introduction of heterologous DNA. A difference in the amount of transcription indicates that the test compound altered the activity of the T1R polypeptide in question in some way.
[0221]
4). Non-human transgenic animals expressing chemosensory receptors
Non-human animals expressing combinations of T1R taste receptor sequences of the invention can also be used in receptor assays. Such expression can be achieved by contacting a non-human animal stably or transiently transfected with a nucleic acid encoding a chemosensory receptor or a ligand binding region thereof with a test compound such that the test compound is a mammalian transmembrane receptor. To determine whether the animal specifically binds to the complex in vivo and to determine whether the animal reacts to the test compound by specifically binding to the receptor polypeptide complex. Can do.
[0222]
Animals transfected or infected with the vectors of the present invention are particularly useful in assays for identifying and characterizing taste stimulants that can bind to specific receptors or combinations thereof. Such vector-infected animals that express human taste receptor sequences can be used for in vivo screening of taste stimulators and their effects on, for example, cell physiology (eg, on taste neurons), CNS, or behavior . Alternatively, stable cell lines expressing T1R or combinations thereof can be used as nucleic acid transport donors to produced clonal transgenic animals that stably express a particular receptor or combination thereof. Methods for using nucleic acid transporters to produce cloned animals that express the desired heterologous DNA are described by the University of Massachusetts (licensed to Advanced Cell Technology, Inc.) and Roslin Institute (licensed to Geron Gorp.). Is the subject of several issued US patents.
[0223]
Means for infecting / expressing nucleic acids and vectors individually or as a library are well known in the art. Various individual cells, organs or whole animal parameters can be measured by various means. The T1R sequences of the invention can be co-expressed in animal taste tissue, for example, by delivery using an infectious agent such as an adenovirus expression vector.
[0224]
Endogenous taste receptor genes can still function and wild-type (native) activity may still exist. In other situations, the use of a knockout line is preferred when it is desired that all taste receptor activity be due to an introduced exogenous hybrid receptor. Methods for selecting and preparing recombinant constructs to generate transgenic animals other than humans, particularly transgenic mice, and transformed cells are well known in the art.
[0225]
“Knockout” cell and animal construction is the introduction of new DNA sequences into the genome that serve to block the level of expression of certain genes in mammalian cells from inhibiting certain portions of the DNA sequence of the gene. It is based on the premise that it can be reduced or eliminated completely. In addition, host genes can be disrupted using “gene trap insertion” and knockout transgenic animals can be produced using mouse embryonic stem cells (ES) (eg, Holzschu, Transgenic Res, Vol. 6, 97-106 (1997)). The insertion of exogenous sequences is generally by homologous recombination between complementary nucleic acid sequences. An exogenous sequence is a portion of the target gene to be modified, such as an exon, intron or transcriptional regulatory sequence, or a genomic sequence or combination thereof that can affect the level of expression of the target gene. Gene targeting by homologous recombination in pluripotent embryonic stem cells makes it possible to precisely modify the genomic sequence in question. Any technique can be used to create, screen and propagate knockout animals. For example, Bijvoet, Hum. Mil. Genet. 7, p. 53-62 (1998), Moreadith, J. et al. 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 to 328 (1997), U.S. Pat. Nos. 5,616,491, 5,464,764, 5,631,153, 5,487,992, and 5,627. No. 5,059, No. 5,272,071, International Publication No. 91/09955, International Publication No. 93/09222, International Publication No. 96/29411, International Publication No. 95/31560, International Publication No. 91/12650. See issue.
[0226]
The nucleic acids of the invention can also be used as reagents for producing “knock-out” human cells and their progeny. Similarly, the nucleic acids of the invention can also be used as reagents for producing “knock-in” cells in mice. The human or rat T1R gene sequence can replace the orthologous T1R in the mouse nom. In this way, mice expressing human or rat T1R are produced. The mouse can then be used to analyze the function of human or rat T1Rs to identify such T1Rs ligands.
[0227]
a. Regulatory substance
A compound to be tested as a modulator of a T1R family member may be a small compound or a biological substance such as a protein, nucleic acid or lipid. Examples of these are 5¹IMP and 5¹There is GMP. Although compounds that are soluble in aqueous solutions are very often tested, essentially any compound can be used as a potential modulator or ligand in the assays of the invention. By automating the assay steps and supplying compounds from convenient sources, assays can be designed to screen large chemical libraries. These assays are generally performed in parallel (eg, in a microtiter format on a microtiter plate in a robotic assay). It will be appreciated that chemical libraries can be synthesized by one of many chemical reactions (eg, Senomix exclusive chemistry). In addition, there are many suppliers of compounds such as Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analyika (Buchs, Switzerland).
[0228]
In one preferred embodiment, the high-throughput screening method involves providing a combinatorial chemistry or peptide library containing a number of compounds (possible modulators or ligand compounds) that can affect taste. Such “combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays as described herein to show library members that exhibit the desired specific activity (specific Chemical species or subclass). The compounds thus identified can be normal “lead compounds” or can themselves be used as possible or actual taste regulators.
[0229]
Preferably, such a library comprises a T1R or combination of T1Rs, ie T1R1 / T1R3 or T1R2 / T1R3 and preferably a suitable G protein, eg G_{α 15}Are screened against stably expressing cells or cell lines. As shown in the examples below, such stable compounds exhibit a very pronounced response to taste stimulants, such as umami or sweetness stimulants. However, cells and cell lines that transiently express one or more T1Rs can also be used in such assays.
[0230]
A combinatorial chemical library is a collection of various compounds obtained by chemical or biological synthesis by combining multiple “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide library, combines a series of chemical building blocks in all possible ways for a given compound length (ie, the number of amino acids in a polypeptide compound). It is formed by. By such combinatorial mixing of chemical building blocks, thousands to millions of compounds can be synthesized.
[0231]
The preparation and screening of combinatorial chemical libraries is known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (eg, US Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. Vol. 37, 487). -493 (1991) and Houghton et al., Nature, 354, 84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistry includes, but is not limited to: Peptoids (eg PCT publication number WO 91/19735), encoded peptides (eg PCT publication number WO 93/20242), random bio-oligomers (eg PCT publication number WO 92/00091), benzodiazepines (eg US Pat. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. 90, 6909-6913 (1993)), Bini. Rogue polypeptide (Hagihara et al., J. Amer. Chem. Soc. 114, 6568 (1992)), non-peptidomimetic with glucose scaffold (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)), peptidylphosphonate (Campbell et al., J. Org. Chem. 59, 658 (1994). )), Nucleic acid library (Ausubel, Berger and Sambrook, all supra), peptide nucleic acid library (US Pat. No. 5,539,083), antibody library (Vaughn et al., Nature Biotechnology, Vol. 14) , 3, 309-314 (1996) and PCT / US96 / 10287), carbohydrate library (Liang et al., Science, 274, 1520-1522 (1996)) and US Pat. No. 5,593. 853), small organic molecule library (benzodiazepine, Baum, C & EN, January 18, 33 (1993); thiazolidinone and metathiazanone, US Pat. No. 5,549,974; pinloridine, US Pat. No. 5,525. , 735 and 5,519,134; morpholino compounds, US Pat. No. 5,506,337; benzodiazepines, 5,288,514, etc.).
[0232]
Combinatorial library preparation equipment is commercially available (eg, 357 MPS, 390 MPS (Advanced Chem Tech, Louisville, KY), Symphony (Rainin, Woburn, Mass.), 433A (Applied Biosystems, Foster City, Calif.). 9050 Plus (Millipore, Bedford, Mass.) In addition, many combinatorial libraries are commercially available (eg, ComGenex, Princeton, NJ; Tripos, Inc., St. Louis, MO; 3D Pharmaceuticals, Exton, PA); Biosciences, Mary Land, Columbia, etc.).
[0233]
In one embodiment of the invention, the T1R modulator can be used in foods, confectionery, pharmaceutical compositions or components thereof to thereby adjust the taste of the product, composition or component in the desired manner. For example, a T1R modulator that enhances the sweet taste sensation can be added to sweeten the product or composition, and a T1R modulator that enhances the umami sensation can be added to the food to increase the savory taste. . Alternatively, T1R antagonists can be used to block sweetness and / or umami.
[0234]
b. kit
T1R genes and their homologues are useful tools for the identification of chemosensory receptor cells, forensic medicine and paternity determination, and for the study of taste transduction. T1R family member-specific reagents that specifically hybridize with T1R nucleic acids, such as T1R probes and primers, and T1R-specific reagents that specifically bind to T1R polypeptides, such as T1R antibodies, examine taste cell expression and taste conversion control Used to do.
[0235]
Nucleic acid assays for the presence or absence of T1R family member DNA and RNA in a sample include Southern analysis, Northern analysis, dot blot, RNase protection, S1 analysis, amplification methods such as PCR and in situ hybridization. There are many techniques known to those skilled in the art. In in situ hybridization, for example, the target nucleic acid is released from its cellular environment for use in intracellular hybridization while preserving cell morphology for subsequent interpretation and analysis. The following paper provides an overview of in situ hybridization techniques. Singer et al. Biotechniques, Vol. 4, 230250 (1986), Haase et al. , Methods in Virology, VII, 189-226 (1984) and Nucleic Acid Hybridization: A Practical Approach (Edited by Names et al., 1987). In addition, T1R polypeptides can be detected by the various immunoassay techniques described above. A test sample is generally compared to a positive control (eg, a sample expressing a recombinant T1R polypeptide) and a negative control.
[0236]
The present invention also provides kits for screening for modulators of T1R family members. Such kits can be prepared from readily available materials and reagents. For example, such a kit can include any one or more of the following materials. T1R nucleic acid or protein, reaction tube and T1R activity test instructions. Optionally, the kit may include a cell line that stably or transiently expresses a biologically active T1R comprising a biologically active T1R receptor or a taste receptor. Depending on the intended user of the kit and the specific needs of the user, various kits and components can be prepared according to the present invention.
[0237]
Example
Having described the invention in detail above, the following examples are given to illustrate preferred embodiments. These examples are for purposes of illustration and are not intended to limit the scope of the invention.
[0238]
In the protein sequences shown herein, the one letter code X or Xaa means any of the 20 common amino acid residues. In the DNA sequences shown herein, the one letter code N or n means any of the four common nucleotide bases A, T, C or G.
[Example 1]
[0239]
Production of hT1R expression constructs without introns
The intron-free hT1R expression construct was cloned by a combination of cDNA and genomic DNA methods. To obtain a full-length hT1R1 expression construct, two 5′-encoded exons identified in the clone hT1R1 segment (Accession number AL159177) are combined by PCR overlap and then bound to a 5′-cleaved testis cDNA clone. It was. The hT1R2 expression construct was obtained from a partially sequenced hT1R2 genomic segment. Two missing hT1R2 5 'exons were identified by screening a shotgun library of clonal genomic segments with probes derived from the corresponding rat coding sequences. The encoded exons were then joined by PCR overlap to generate a full length expression construct. The hT1R3 expression construct was obtained by PCR overlap from a sequenced hT1R3 genomic segment (accession number AL139287). Rat T1R3 was isolated from a rat taste tissue-derived cDNA library using the rT1R3 exon fragment obtained by hT1R3 degenerate PCR. Partial hT1R1 cDNA, rT1R2 cDNA, and partial hT1R2 genomic sequence were obtained from Dr. Obtained from Charles Zuker (University of California, San Diego).
[0240]
The nucleic acid and amino acid sequences of the above T1R clone sequences and other full length and partial T1R sequences are shown below.
SEQ ID NO: 4
Amino acid sequence rT1R3
SEQ ID NO: 5
Amino acid sequence hT1R1
SEQ ID NO: 6
Amino acid sequence hT1R2
SEQ ID NO: 7
Amino acid sequence hT1R3
SEQ ID NO: 8
Nucleic acid sequence hT1R1
SEQ ID NO: 9
Nucleic acid sequence hT1R3
SEQ ID NO: 10
Nucleic acid sequence hT1R2
(Sequence Listing 7)
SEQ ID NO: 11
Nucleic acid sequence rT1R3
[0241]
In addition, the following conceptual translations corresponding to the C-termini of two orthologous pairs of fish T1Rs have been obtained and provided from unpublished genomic sequence fragments. The puffer fish T1RA was obtained from the accession number “scaffold 164” and the puffer fish T1RB was obtained from the accession number LPC61711. Tetradon T1RA was obtained from accession number AL226735, and tetradon T1RB was obtained from accession number AL2222381. The ambiguity in the conceptual translation (“X”) is due to the ambiguity in the database sequence.
SEQ ID NO: 12
T1RA puffer fish
SEQ ID NO: 13
T1RA Tetradon
SEQ ID NO: 14
T1RB puffer fish
SEQ ID NO: 15
T1RB Tetradon
[0242]
In addition, accession numbers and reference citations for mouse and rat T1Rs and allelic variants in the public domain are provided below.
rT1R1 (accession number AAD18069) (Hoon et al., Cell, Vol. 96, No. 4, pp. 541-51 (1999))
rT1R2 (accession number AAD18070) (Hoon et al., Cell, Vol. 96, No. 4, pp. 541-59 (1999))
mrT1R1 (Accession number AAK39437); mT1R2 (Accession number AAK39438); mT1R3 (Accession number AAK55537) (Max et al., Nat. Genet. 28, No. 1, 58-63 (2001)); rT1R1 (Acceptance) No. AAK7092) (Li et al., Mamm. Genome, Vol. 12, No. 1, pages 13-16 (2001); mT1R1 (accession number NP114073); mTR1 (accession number AAK07091) (Li et al., Mamm Genome, Vol. 12, No. 1, pages 13-16 (2001); rT1R2 (accession number AAD18070) (Hoo et al., Cell, 9664, 541-551 (1999); ; MT1R3 ( (Accession number AAK39436); mT1R3 (accession number BAB47181); (Kitagawa et al., Biochem. Biophys. Res. Comm. No. 283, No. 1, pages 236-42 (2001)); MT1R3 (accession number AAK55536) (Max et al., Nat. Genet. 28, No. 1, pages 58-63 (2001)); and mT1R3 (accession number AAK01937).
[Example 2]
[0243]
Sequence alignment of human and rat T1Rs
The clone T1R sequence selected from above was aligned to the corresponding rat T1Rs. As shown in FIG. 1, previously described T1Rs of human T1R1, human T1R2 and human T1R3 and rat T1R3 (rT1R1 with accession number AAD18069 and rT1R2 with accession number AAD18070), rat mGluR1 metabotropic glutamate receptor (accession number) Alignment to P23385) and the human calcium detection receptor (accession number P41180) was performed. To clarify the comparison, the C-terminus of mGluR1 and the calcium detection receptor is cut off. Seven possible transmembrane segments are surrounded by a blue frame. Residues that contact glutamate side chain carbyrate in the mGluR1 crystal structure are enclosed in a red frame, and residues that contact the glutamate α-amino acid moiety are enclosed in a green frame. The mGluR1 and calcium detection receptor cysteine residues contained in the intersubunit disulfide-based construct are surrounded in purple. These cysteines are not conserved in T1R1 and T1R2, but are in the degradation region of the alignment containing potentially similar T1R3 cysteine residues, which are also surrounded in purple.
[Example 3]
[0244]
hT1R2 and hT₁R_ThreeThat RT is expressed in taste tissue by RT-PCR
As shown in FIG. 2, hT1R2 and hT1R3 are expressed in taste tissues, and the expression of both genes can be detected by RT-PCR of excised human circumvallate papilla.
[Example 4]
[0245]
Method for heterologous expression of T1Rs in heterologous cells
HEK-293 derivative stably expressing Gα15 (Chandradhekar et al., Cell 100, No. 6, 703-11 (2000)), 10% FBS, MEM non-essential amino acid (Gibco BRL) and 3 μg Grown and maintained at 37 ° C. in Dulbecco's modified Eagle's medium (DMEM, Gibco RRL) supplemented with / ml blasticidin. For calcium imaging experiments, cells are first seeded in 24-well tissue culture plates (approximately 0.1 × 10⁶Cells / well), and transfected by lipofection using Miras Translt-293 (Pan Vera). To minimize glutamate and glucose-induced desensitization, supplemental DMEM was replaced with low glucose DMEM / GlutaMAX (Gibco BRL) approximately 24 hours after transfection. After 24 hours, cells were loaded with 3 μM calcium dye Fluo-4 (Molecular Probes) in Dulbecco's PBS buffer (DPBS, Gibco BRL) for 1.5 hours at room temperature. After replacement with 250 μl DPBS, stimulation was performed by adding 200 μl DPBS supplemented with taste stimulants at room temperature. Calcium mobilization was monitored with an Axiovert S100 TV microscope (Zeiss) using Imaging Workingbench 4.0 software (Axon). The responses of T1R1 / T1R3 and T1R2 / T1R3 were remarkably transient, with the calcium increase rarely lasting more than 15 seconds and asynchronous. Thus, the time course of the number of responding cells is relatively constant, and thus the cellular response is quantified by manually counting the number of responding cells at a certain time, generally 30 seconds after the addition of the stimulating substance. did.
[Example 5]
[0246]
Human T1R2 / T1R3 functions as a sweet receptor
HEK cells stably expressing Gα15 were transiently transfected with human T1R2, T1R3 and T1R2 / T1R3, and measurements were made for increases in intracellular calcium in response to increasing sucrose concentrations (FIG. 3a). ). In addition, T1R2 / T1R3 dose response was measured for several sweetness stimulants (FIG. 3b). The maximum percentage of responding cells varied between 10-30% with different sweeteners. For clarity, the dose response was normalized to the maximum percentage of responding cells. The values in FIG. 3 are the mean ± sd of 4 independent responses. e. It is. The psychophysical taste detection threshold determined by the taste test is indicated by a circle on the X axis. Gulmarin (50-fold diluted solution of filtered 10 g / l Gymnema sylvestre aqueous extract) inhibited the response of T1R2 / T1R3 to 250 mM sucrose but inhibited the response of endogenous β2-adrenergic receptor to 20 μM isoproterenol. (FIG. 3 (b)). FIG. 3 (c) shows the normalized response of T1R2 / T1R3 co-expressing cell lines to various sweeteners (sucrose, aspartame, D-tryptophan and saccharin).
[Example 6]
[0247]
Rat T1R2 / T1R3 also functions as a sweet receptor
HEK cells stably expressing Gα15 were transiently transfected with hT1R2 / hT1R3, rT1R2 / rT1R3, hT1R2 / rT1R3 and rT1R2 / hT1R3. These transfected cells were then measured for an increase in intracellular calcium in response to 350 mM sucrose, 25 mM tryptophan, 15 mM aspartame and 0.05% monelin. The results for sucrose and aspartame are shown in FIG. 4, indicating that rT1R2 / rT1R3 also functions as a sweetness receptor. These results also suggest that T1R2 may regulate T1R2 / T1R3 ligand specificity.
[Example 7]
[0248]
T1R2 / T1R3 response using a fluorescence-based automated assay
HEK cells stably expressing Gα15 were transiently transfected with hT1R2 and hT1R3. These cells were loaded with the calcium dye Fluo-4 and their response to sweeteners was measured using a fluorescent plate reader. FIG. 5 shows the cyclamate (12.5 mM) responses of cells expressing hT1R2 / hT1R3 and cells expressing only hT1R3 (J19-22). The fluorescence results obtained indicate that the response to these taste stimulants occurs only in cells that expressed hT1R2 / hT1R3. FIG. 6 shows the normalized dose response curve. The results indicate that hT1R2 and hT1R3 function together as human taste receptors due to the dose-specific interaction with sweetness stimulants. In particular, FIG. 6 shows the dose response to sucrose, torptophan and various other commercial sweeteners. These results indicate that T1R2 / T1R3 is a human sweet receptor because the rank order and threshold values obtained in the assay closely reflect human sweet taste values.
[Example 8]
[0249]
The ligand binding residue of mGluR1 is conserved in T1R1
As shown in FIG. 6, an important ligand binding residue of mGluR1 is conserved in T1R1. The interaction between glutamate and mGluR1 is shown, with several important residues highlighted according to the same color scheme as in FIG.
[Example 9]
[0250]
Human T1R1 / T1R3 functions as an umami receptor
HEK cells stably expressing Gα15 were transiently transfected with human T1R1, T1R3 and T1R1 / T1R3 to increase the concentration of glutamate (FIG. 8 (a)) and 0.5 mM glutamate, 0.2 mM IMP and Measurements were made of the increase in intracellular calcium in response to 0.5 mM glutamate + 0.2 mM IMP (FIG. 8 (b)). The dose response of human T1R1 / T1R3 was measured for glutamate in the presence and absence of 0.2 mM IMP (FIG. 8 (c)). The maximum percentage of responding cells was about 5% for glutamate and about 10% for glutamate + IMP. For clarity, the dose response was normalized to the maximum percentage of responding cells. Values are the mean ± sd of 4 independent responses. e. It is. The taste detection threshold determined by the taste test is indicated by a circle on the X axis.
[Example 10]
[0251]
PDZIP as an export array
A six residue PDZIP sequence (SVSTW (SEQ ID NO: 1)) was fused to the C-terminus of hT1R2, and then the chimeric receptor (ie hT1R2-PDZIP) was monitored using immunofluorescence and FACS scanning data. As shown in FIGS. 9A and 9B, inclusion of the PDZIP sequence increased the surface expression of hT1R2-PDZIP compared to hT1R2. More specifically, FIG. 9A shows that immunofluorescent staining of myc-labeled hT1R2 demonstrated that PDZIP significantly increased the amount of hT1R2 on the plasma membrane. FIG. 9B shows FACS analysis data showing the same results. Cells expressing myc-labeled hT1R2 are indicated by dotted lines, and cells expressing myc-labeled hT1R2-PDZIP are indicated by solid lines. In particular, FIG. 10A shows untransfected Gα15 stable host cells in HBS buffer, and FIG. HT1R2-PDZIP transfected Gα15 stable host cells in 5 pools (HBS buffer, saccharin, sodium cyclamate acesulfame K and aspartame—20 mM each) are shown in FIG. HT1R3-PDZIP transfected Gα15 stable host cells in 5 pools are shown, sweetener no. Shown are hT1R2-PDZIP / hT1R3-PDZIP co-transfected Gα15 stable host cells in 5 pools. In addition, FIGS. 10E-10H show the dose-dependent response of hT1R2 / hT1R3 co-transfected Gα15 stable host cells to 0 mM; F: 30 mM; G: 60 mM and H: 250 mM in sucrose-E: HBS buffer. FIGS. 10I-10L show the individual sweeteners-I: aspartame (1.5 mM); J: acesulfame K (1 mM); K: neotame (20 mM); L: hT1R2 / hT1R3 protein against sodium cyclamate (20 mM). The response of transfected Gα15 stable host cells is shown. As shown by the calcium image in FIG. 10, both hT1R2 and hT1R3 require stimulation of activity by a sweetness stimulant.
Example 11
[0252]
Generation of cell lines stably co-expressing T1R1 / T1R3 or T1R2 / T1R3
Linearized PEAK10-derived (Edge Biosystems) vectors and pCDNA3.1 /, each containing hT1R1 or hT1R2 expression constructs (plasmid SAV2485 for T1R1, plasmid SAV2486 for T1R2) and hT1R3 (plasmid SXV550 for T1R3), respectively. A cell line stably expressing human T1R1 / T1R3 or human T1R2 / T1R3 was generated by transfecting a ZEO-derived (Invitrogen) vector into a Gα15-expressing cell line. In particular, linearized SAV2486 and SXV550 are designated G_{α 15}A T1R2 / T1R3 stable cell line was generated by co-transfecting the Aurora Bioscience HEK-293 cell line stably expressing. Linearize SAV2485 and SXV550 with G_{α 15}T1R1 / T1R3 stable cell lines were generated by co-transfecting the same HEK-293 cell line stably expressing. Following SAV22485 / SXV550 and SAV2486 / SXV550 transfection, puromycin and zeocin resistant colonies were selected, expanded, and tested for response to sweet or umami stimulants by calcium imaging. Cells were selected with 0.0005 mg / ml puromycin (CALBIOCHEM) and 0.1 mg / ml zeocin (Invitrogen) at 37 ° C. in low glucose DMEM supplemented with GlutaMAX, 10% dialyzed FBS and 0.003 mg / ml blasticidin. . Resistant colonies were expanded and their response to sweetness stimulants was evaluated by fluorescence microscopy. For automated fluorescence analysis imaging using a VIPR-II instrument (Aurora Biosciences), T1R2 / T1R3 stable cells were initially seeded in 96-well plates (approximately 100,000 cells / well). After 24 hours, cells were loaded with 0.005 mM calcium dye fluo-3-AM (Molecular Probes) in PBS for 1 hour at room temperature. After replacing with 70 μl PBS, stimulation was performed by adding 70 μl PBS to which a taste stimulating substance was added at room temperature. Fluorescence (480 nm excitation, 535 nm emission) response 20-30 seconds after compound addition was averaged, corrected for background fluorescence measured before compound addition, and in response to 0.001 mM ionomycin (CALBIOCHEM) (calcium ionophore) Normalized for.
[0253]
When these cell lines were exposed to sweet or umami stimulants, it was observed that for active clones, generally 80-100% of cells responded to taste stimulants. Surprisingly, the magnitude of individual cell responses was significantly greater than that of transiently transfected cells.
[0254]
Based on this finding, the inventors tested the activity of the T1R stable cell line by automated fluorescence analysis imaging using the Aurora Biosciences VIPR-II instrument as described above. The responses of two T1R1 / T1R3 and one T1R2 / T1R3 cell lines are shown in FIGS. 11 and 12, respectively.
[0255]
Of note, the increase in the number of responding cells combined with the increase in the magnitude of the response resulted in a 10-fold increase in activity compared to the transiently transfected cells. (In comparison, the percentage of ionomycin response in cells transiently transfected with T1R2 / T1R3 was about 5% under optimal conditions.) Further, obtained for stably expressed human T1R2 / T1R3 and T1R1 / T1R3. The dose response given correlated with the human taste detection threshold. Since these stable cell lines have robust T1R activity, they modulate sweetness or umami receptors and thus compounds that modulate, enhance, inhibit or mimic sweetness or umami taste, eg small molecules It is suggested that it is well suited for use in high throughput screening of chemical libraries to identify
Example 12
[0256]
Generation of cell lines co-expressing T1R1 / T1R3 that selectively respond to umami stimulants
The T1R1 / T1R3 HEK293 cell line stably expressing the umami receptor shows a robust improvement in activity compared to transiently transfected cells. However, the disadvantage is that it can lose activity rapidly during cell growth.
[0257]
These findings also indicate that (i) T1R1 / TR1R3 is an umami receptor and (ii) a cell line that robustly expresses T1R1 / TR1R3, preferably a stable and / or inducible T1R1 / TR1R3 cell line. Support the inventors' hypothesis that they can be used for high throughput screening of assays, preferably chemical libraries, to identify novel modulators. Modulators that enhance umami may be used.
[0258]
To overcome the instability of the T1R1 / TR1R3 stable cell line, HEK-G_{α 15}Cells were engineered to express T1R1 / TR1R3 by induction using the GeneSwitch system (Invitrogen). A pGene-derived zeocin resistant expression vector of human T1R1 and T1R3 (plasmid SXV603 for T1R1, plasmid SXV611 for T1R3) and a puromycin resistant pSwitch-derived vector (plasmid SXV628) carrying the GeneSwitch protein were linearized and HEK-G_{α 15}Cotoy transfected cell lines. Zeocin and puromycin resistant colonies were selected, expanded, induced with variable amounts of mifepristone, and tested for response to umami stimulants using calcium imaging.
[0259]
Inducible expression of T1R1 / T1R3 resulted in robust activity. For example, about 80% of the induced cells responded to L-glutamic acid, whereas transiently transfected cells responded 10%. More specifically, a pGene-derived zeocin resistant expression vector expressing human T1R1 and human T1R3 and a puromycin resistant pSwitch derived vector carrying GeneSwitch protein are linearized, and G_{α 15}Cotoy transfected cell lines. Cells were selected with 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 μg / ml blasticidin. Expand resistant colonies, 10^-TenTheir response to umami stimulation after induction with M mifepristone was determined by Li et al. , PNAS, Vol. 99, No. 7, pages 4692-4696 (2002).
[0260]
For automated fluorescence analysis imaging using a FLIPR instrument (Molecular Device), cells from one clone (referred to as clone 1-17) were^-Ten96-well plates (approximately 800,000 cells / well) were seeded in the presence of M mifepristone and incubated for 48 hours. The cells were then loaded with 3 μM calcium dye fluo-4-AM (Molecular Probes) in PBS for 1.5 hours at room temperature.
[0261]
After replacement with 50 μl PBS, stimulation was performed by adding 50 μl PBS supplemented with various taste stimulants at room temperature. It was necessary to quantify T1R1 / T1R3 receptor activity by counting responding cells individually (Li et al., PNAS 99, 7, 4692-4696 (2002)) (Receptor activity In contrast to the previous transient T1R1 / T1R3 umami receptor expression system, the subject inducible expression system resulted in a substantially higher activity in clone 1-17, thus increasing the maximum fluorescence over the field of imaged cells It was possible to quantify receptor activity by measuring the total (excitation at 480 nm, emission at 535 nm). Maximum fluorescence in 4 independent measurements was averaged, corrected for background fluorescence measured before compound addition, and normalized to response to 0.002 mM ionomycin (CALBIOCHEM).
[0262]
These results are shown in FIG. In particular, FIG. 13 shows the dose response curve measured for L-glutamate in the presence and absence of 0.2 mM IMP. In the figure, each value is an average value of maximum fluorescence (corrected by background fluorescence) obtained by combining four independent measurements. These dose response curves correspond to those measured for cells transiently transfected with T1R1 / T1R3.
[0263]
The selectivity of umami T1R1 / T1R3 taste receptors was also evaluated by screening with various L-amino acids. The obtained results indicate that T1R1 / T1R3 is selectively activated by umami L-amino acids (L-glutamic acid and L-aspartic acid).
[0264]
The experimental results of the response of 1-17 clones tested in the presence of various L-amino acids are shown in FIGS. FIG. 14 shows the results of experiments in which the 1-17 cell line was contacted with various L-amino acids at a concentration of 10 mM in the presence of 1 mM IMP.
[0265]
FIG. 15 shows a dose response curve of active amino acids measured in the presence of 0.2 mM IMP. Each value is the average of 4 independent measurements.
[0266]
The results obtained in these experiments support the specificity and selectivity of the umami receptor for umami stimulants. The umami stimulants L-glutamate and L-aspartate significantly activated the T1R1 / T1R3 receptor at various concentrations (FIGS. 14 and 15), but other Ls that activated the human T1R1 / T1R3 receptor. -The amino acids only activated the receptor slightly and at higher concentrations.
[0267]
Thus, these results indicate that the selectivity of the T1R1 / T1R3 receptor for umami stimulants and compounds that activate the umami receptor, such as L-glutamate or L-aspartate, or activate the umami receptor Of umami receptors by umami stimulants such as 5'-IMP or 5'-GMP, or L-glutamate and L-aspartate, which enhance the activity of L-glutamate This confirms the suitability of this inducible stable expression system for high throughput assays using automated fluorescent imaging equipment to identify compounds that inhibit
[0268]
Compounds identified using these assays have potential application as seasonings in food and beverage compositions to mimic or inhibit umami stimulants.
Example 13
[0269]
Lactisol inhibits human T1R2 / T1R3 and T1R1 / T1R3 receptor activity and sweet and umami taste
Lactisol, an alkyl carboxylic acid, was believed to be a selective sweetness inhibitor (see, for example, Lindley (1986) US Pat. No. 4,567,053 and Schiffman et al., Chem Senses 24, 439-447 (1999)). HEK-G transfected with T1R2 / T1R3_{α 15}The response of cells to 150 mM sucrose in the presence of various concentrations of lactisol was measured. Lactisol inhibits the activity of human T1R2 / T1R3, and IC₅₀Is 24 μM.
[0270]
T1R1 / T1R3 umami and T1R2 / T1R3 sweet receptors share a common subunit. Therefore, it was hypothesized that lactisol, which inhibits the T1R2 / T1R3 sweet receptor, may have a similar effect on the T1R1 / T1R3 umami receptor. The inventors tested the effect of lactisol on the response of human T1R1 / T1R3 to 10 mM L-glutamic acid. As with the T1R2 / T1R3 sweet receptor, lactisol inhibits the activity of T1R1 / T1R3 and IC₅₀Was 165 μM. Since muscarinic acetylcholine receptors are not inhibited by lactisol, lactisol inhibition is e.g._{α 15}It reflects antagonism at the T1R receptor rather than non-specific inhibition of mediated signaling.
[0271]
The inventors next evaluated the effect of lactisole on human umami. Taste threshold in the presence of 1 and 2 mM lactisol is determined in the presence or absence of 0.2 mM IMP according to the method of Schiffman et al. (Chem. Senses, 24, 439-447 (1898)). The umami taste stimulating substance L-glutamate, the sweetness stimulating substance sucrose and D-tryptophan, and the salty taste stimulating substance sodium chloride were measured. Milliolar lactisol dramatically increased the detection threshold for sweetness and umami stimulants, but not for salty stimulants. These results are shown in FIG.
[0272]
In conclusion, (i) these findings further support the inventors' hypothesis that T1R1 / T1R3 is the only umami receptor, (ii) T1R1 / T1R3 and T1R2 / T1R3 receptors are structurally May share an associated lactisole domain.
[0273]
While the foregoing detailed description describes several embodiments of the present invention, it should be understood that the above description is illustrative only and is not intended to limit the disclosed invention. . The present invention is limited only by the claims.
[Brief description of the drawings]
[0274]
FIG. 1 includes a sequence alignment of rat T1Rs, human calcium sensing receptors and rat metabotropic glutamate receptors.
FIG. 2 includes results of RT-PCR amplification experiments showing that hT1R2 and hT1R3 are expressed in taste tissues.
FIG. 3. Various sweet stimulants (a) in HEK cells stably expressing Gα15 transiently transfected with human T1R2, T1R3 and T1R2 / T1R3 at various concentrations of sweet stimulants; Dose response of human T1R2 / T1R3 to the substance (b); response of human T1R2 / T1R3 to sucrose in the presence of gurmarin, and response of endogenous β2-adrenergic receptor to isoproterenol in the presence of gurmarin; Functional data (intracellular calcium response) induced by. c includes the normalized response to various sweeteners.
FIG. 4: 350 mM sucrose, 25 mM tryptophan, 15 mM aspertame and 0.05% in HEK cells stably expressing Gα15 transiently transfected with hT1R2 / hT1R3, rT1R2 / rT1R3, hT1R2 / rT1R3 and rT1R2 / hT1R3 Includes intracellular calcium response to monelin.
FIG. 5. Fluorescent plate reaction in which HEK cells stably expressing Gα15 are transiently transfected with hT1R2 and hT1R3 or hT1R3 alone and contacted with a calcium dye Fluo-4 and a sweetness stimulant (12.5 mM cyclamate). Includes the results of the assay using the instrument.
FIG. 6 shows that hT1R2 and hT1R3 together as human sweet receptors based on dose-specific interactions with various sweetness stimulants (trp, cyclamate, sucrose, neotame, asperame, saccharin and Acek) Includes a normalized dose response curve that demonstrates functioning.
FIG. 7 includes structural information regarding mGluR1 and T1R1 indicating that important ligand binding residues are found in these molecules.
FIGS. 8a-c include functional data showing that HEK cells stably expressing Gα15 transiently transfected with T1R1 / T1R3 respond to glutamate in an intracellular calcium-based assay.
a indicates that intracellular calcium increases in response to increasing glutamate concentration.
b shows that intracellular calcium responds to IMP (2 mM), glutamate (0.5 mM) and 0.2 mM IMP.
c shows that T1R1 / T1R3 responds to glutamate in the presence and absence of 0.2 mM IMP.
FIGS. 9a and b are FACS showing the results of an immunofluorescent staining assay using Myc-labeled hT1R2 and that PDZIP peptide (SEQ ID NO: 1) incorporation enhanced expression of T1R (hT1R2) on the plasma membrane, respectively. Includes experimental results.
FIGS. 10a-b include calcium image data showing that hT1R2 / hT1R3 responds to various sweetness stimulants.
FIG. 11 shows the response by automated fluorescence imaging to umami stimulants in cell lines stably expressing hT1R1 / hT1R3.
FIG. 12 shows autofluorescence imaging response to a cell-line sweet stimulant that stably expresses hT1R2 / hT1R3.
FIG. 13: Dose response curves measured using autofluorescence imaging in cell lines inducibly expressing human T1R1 / T1R3 taste receptors for L-glutamate in the presence and absence of 0.2 mM IMP. Indicates.
FIG. 14 shows the response to a panel of L-amino acids of cell lines inducibly expressing human T1R1 / T1R3 taste receptors (1-17 clones). 10 mM of various C-amino acids were tested in the presence and absence of 1 mM IMP.
FIG. 15 shows the response to a panel of L-amino acids of cell lines inducibly expressing human T1R1 / T1R3 taste receptors (1-17 clones). The dose response for the active amino acid was measured in the presence of 0.2 mM IMP.
FIG. 16 shows that lactisol inhibits receptor activity of human T1R2 / T1R3 and human T1R1 / T1R3.

Claims (193)

Consisting of at least one T1R1 polypeptide (or variant, fragment or chimera of said T1R1 polypeptide) and / or at least one T1R3 polypeptide (or variant, fragment or chimera of said T1R3 polypeptide), A receptor that is specifically bound and / or activated by an umami stimulant. 2. The receptor of claim 1 comprising a fragment, variant or chimera of a native hT1R1 polypeptide. 2. The receptor of claim 1 comprising a fragment, variant or chimera of a native hT1R3 polypeptide. The receptor of claim 1, wherein the T1R1 and T1R3 are derived from the same animal species. The receptor according to claim 1, wherein the T1R1 and T1R3 are derived from different animal species. The receptor according to claim 1, wherein the T1R1 and / or T1R3 is derived from mammals, fish, reptiles, amphibians or birds. The receptor according to claim 1, wherein the T1R1 and / or T1R3 is selected from the group consisting of hT1R1, hT1R3, rT1R1, rT1R3, mT1R1, mT1R3 or fragments, mutants or chimeras derived therefrom. A composition comprising the receptor of claim 1. A composition comprising the receptor of claim 2. A composition comprising the receptor of claim 3. A composition comprising the receptor of claim 4. A composition comprising the receptor of claim 5. A composition comprising the receptor of claim 6. A composition comprising the receptor of claim 7. Consisting of at least one T1R1 polypeptide (or variant, fragment or chimera of said T1R1 polypeptide) and / or at least one T1R3 polypeptide (or variant, fragment or chimera of said T1R3 polypeptide), A cell expressing a receptor that is specifically bound and / or activated by an umami stimulant. 16. The cell of claim 15, selected from the group consisting of HEK-293, COS and CHO cells and Xenopus oocytes. The cell according to claim 15, wherein the T1R1 and T1R3 are derived from the same animal species. The cell according to claim 15, wherein the T1R1 and T1R3 are derived from different animal species. The cell according to claim 15, wherein the T1R1 and / or T1R3 is derived from mammals, fish, reptiles, amphibians or birds. The cell according to claim 15, wherein the T1R1 and T1R3 are selected from the group consisting of hT1R1, hT1R3, mT1R1, mT1R3, rT1R1 and rT1R3, or fragments, mutants or chimeras derived therefrom. Consisting of at least one T1R2 polypeptide (or variant, fragment or chimera of said T1R2 polypeptide) and / or at least one T1R3 polypeptide (or variant, fragment or chimera of said T1R3 polypeptide), A receptor that is specifically bound to and / or activated by a sweetness stimulant. 24. The receptor of claim 21, comprising a fragment, variant or chimera of a native hT1R2 polypeptide. 24. The receptor of claim 21, comprising a fragment, variant or chimera of a native hT1R3 polypeptide. The receptor of claim 21, wherein said T1R2 and T1R3 are derived from the same animal species. The receptor according to claim 21, wherein said T1R2 and T1R3 are derived from different animal species. The receptor according to claim 21, wherein said T1R2 and / or T1R3 is derived from mammals, fish, reptiles, amphibians or birds. The receptor according to claim 21, wherein said T1R2 and / or T1R3 is selected from the group consisting of hT1R2, hT1R3, rT1R2, rT1R3, mT1R2, mT1R3 or fragments, variants or chimeras derived therefrom. A composition comprising the receptor of claim 21. 23. A composition comprising the receptor of claim 22. 24. A composition comprising the receptor of claim 23. 25. A composition comprising the receptor of claim 24. 26. A composition comprising the receptor of claim 25. 27. A composition comprising the receptor of claim 26. 28. A composition comprising the receptor of claim 27. Consisting of at least one T1R2 polypeptide (or variant, fragment or chimera of said T1R2 polypeptide) and / or at least one T1R3 polypeptide (or variant, fragment or chimera of said T1R3 polypeptide), A cell that expresses a receptor that is specifically bound to and / or activated by a sweetness stimulating substance. 36. The cell of claim 35, selected from the group consisting of HEK-293, COS and CHO cells and Xenopus oocytes. 36. The cell of claim 35, wherein the T1R2 and T1R3 are derived from the same animal species. 36. The cell of claim 35, wherein the T1R2 and T1R3 are derived from different animal species. 36. The cell of claim 35, wherein said T1R2 and / or T1R3 is derived from a mammal, fish, reptile, amphibian or bird. 36. The cell of claim 35, wherein said T1R2 and T1R3 are selected from the group consisting of hT1R2, hT1R3, mT1R2, mT1R3, rT1R2 and rT1R3, or fragments, variants or chimeras derived therefrom. 28. The receptor according to any one of claims 1 to 7 and 21 to 27, which is bound to a solid phase. 28. A receptor according to any one of claims 1 to 7 and 21 to 27 in solution. 28. A receptor according to any one of claims 1 to 7 and 21 to 27 in a lipid bilayer or vesicle. Identify compounds that modulate taste by identifying compounds that bind, activate, inhibit, enhance and / or modulate one or more receptors according to claims 1-7 and / or 21-27. Method. 45. The method of claim 44, wherein the receptor is bound to a solid phase. 45. The method of claim 44, wherein the receptor is in solution. 45. The method of claim 44, wherein the receptor is in a lipid bilayer or vesicle. 45. The method of claim 44, wherein a receptor binding assay is used to identify the compound. 45. The method of claim 44, wherein an assay based on receptor activity is used to identify the compound. 45. The method of claim 44, wherein the receptor is expressed in a cell. 51. The method of claim 50, wherein the cells are HEK-293, COS or CHO cells. 51. The method of claim 50, wherein the compound is identified by its effect on the cellular uptake of the receptor. 51. The method of claim 50, wherein said compound is identified by its effect on receptor phosphorylation. 51. The method of claim 50, wherein the compound is identified by its effect on arrestin transfer. 45. The method of claim 44, wherein a second messenger assay is used. The method of claim 55, wherein said second messenger is cAMP or IP _3. 51. The method of claim 50, wherein a potential sensitive or calcium sensitive dye is used. 51. The method of claim 50, wherein the cell expresses at least one G protein. 59. The method of claim 58, wherein the G protein is a promiscuous G protein. The method of claim 59, wherein the promiscuous G protein is G _{alpha 15} or G _{alpha 16.} 45. The method of claim 44, wherein the receptor is expressed using a viral vector. 62. The method of claim 61, wherein the viral vector is expressed in mammalian cells. 45. The method of claim 44, wherein the receptor is produced by translation in vitro. 45. The method of claim 44, wherein the receptor is separated as a membrane bound form. 45. The method of claim 44, wherein the compound is identified by its effect on G protein activation by the receptor. 45. The method of claim 44, wherein said compound is identified by its effect on receptor conformation. 68. The method of claim 66, wherein the conformational change is detected by a change in sensitivity to proteolysis. 68. The method of claim 66, wherein the conformational change is detected by NMR spectroscopy. 68. The method of claim 66, wherein the conformational change is detected by fluorescence spectroscopy. 45. The method of claim 44, wherein the compound is identified by its effect on binding of a radiolabeled or fluorescently labeled ligand to the receptor. 72. The method of claim 70, wherein displacement of the labeled compound is measured by fluorescence deflection or FRET assay. 45. The method of claim 44, wherein the method is a high throughput screening assay. 45. The method of claim 44, wherein the receptor activity is associated with a reporter gene. The method according to claim 73, wherein the reporter gene is luciferase, alkaline phosphatase, β-galactosidase, or β-lactamase. 45. The method of claim 44, wherein the receptor is a constitutively active variant. 45. The method of claim 44, wherein expression of the receptor is under the control of a constitutive promoter. 45. The method of claim 44, wherein expression of the receptor is under the control of a regulatable promoter. 45. The method of claim 44, wherein the receptor is fused to a peptide that promotes surface expression. 79. The method of claim 78, wherein the peptide is a PDZ domain interacting peptide. 45. The method of claim 44, wherein the effect of the compound on the receptor is predicted based on the X-ray crystal structure of the receptor. 45. The method of claim 44, wherein said compound is identified by its effect on a non-human animal expressing a natural or transformed T1R receptor. 92. The method of claim 81, wherein the compound is identified by its effect on behavior. 92. The method of claim 81, wherein the compound is identified by its effect on taste receptor cells. 84. The method of claim 81, wherein the non-human animal is a mouse, rat, insect, fish or insect. 45. The method of claim 44, wherein the compound is identified by its effect on yeast cells that express the receptor. 45. The method of claim 44, wherein the compound is identified from a combinatorial library of compounds. 45. The method of claim 44, wherein the compound is identified from a peptide library. 45. The method of claim 44, wherein the compound is identified from a randomized library of small molecules. 45. A method of altering taste in an animal using a compound identified by claim 44. 90. The method of claim 89, wherein the taste is umami. 90. The method of claim 89, wherein the taste is sweet. 90. The method of claim 89, wherein the animal is a human, dog, cat, fish, cow, sheep or pig. 90. The method of claim 89, wherein the compound is formulated in a food, beverage or oral pharmaceutical composition. 28. A method for quantifying the taste of individual compounds or food or beverage compositions using one or more receptors according to claims 1-7 and / or 21-27. At least one T1R1 polypeptide or a variant, fragment or chimera of said T1R1 polypeptide and / or at least one T1R3 polypeptide or a variant, fragment or chimera of said T1R3 polypeptide, specific for umami stimulating substance Stably expressing a receptor to be bound and / or activated. 96. The cell of claim 95, selected from the group consisting of HEK-293, COS and CHO cells and Xenopus oocytes. 96. The cell of claim 95, wherein said T1R1 and T1R3 are derived from the same animal species. 96. The cell of claim 95, wherein said T1R1 and T1R3 are derived from different animal species. 96. The cell of claim 95, wherein said T1R1 and / or T1R3 is derived from a mammal, fish, reptile, amphibian or bird. 96. The cell of claim 95, wherein said T1R1 and T1R3 are selected from the group consisting of hT1R1, hT1R3, mT1R1, mT1R3, rT1R1 and rT1R3, or fragments, variants or chimeras derived therefrom. Claim 95, wherein the cell is a HEK-293 stably expressing the G _{alpha 15.} 96. A composition comprising the cell of claim 95. 99. A composition comprising the cell of claim 96. 98. A composition comprising the cell of claim 97. 99. A composition comprising the cell of claim 98. 100. A composition comprising the cell of claim 99. 101. A composition comprising the cell of claim 100. 102. A composition comprising the cell of claim 101. At least one T1R2 polypeptide or variant, fragment or chimera of said T1R2 polypeptide, and / or at least one T1R3 polypeptide or variant, fragment or chimera of said T1R3 polypeptide, specific for sweet taste stimulants Stably expressing a receptor to be bound and / or activated. 110. The cell of claim 109, selected from the group consisting of HEK-293, COS and CHO cells and Xenopus oocytes. 110. The cell of claim 109, wherein the T1R2 and T1R3 are derived from the same animal species. 110. The cell of claim 109, wherein the T1R2 and T1R3 are derived from different animal species. 110. The cell of claim 109, wherein said T1R2 and / or T1R3 is derived from a mammal, fish, reptile, amphibian or bird. 110. The cell of claim 109, wherein said T1R2 and T1R3 are selected from the group consisting of hT1R2, hT1R3, mT1R2, mT1R3, rT1R2 and rT1R3, or fragments, variants or chimeras derived therefrom. hT1R2, hT1R3 and G _{alpha 15} claim 109, wherein the cells stably expressing. A method of identifying a compound that modulates taste by identifying a compound that binds, activates, inhibits and / or modulates a receptor expressed by a cell stably expressing at least one T1R. 117. The method of claim 116, wherein the cells are bound to a solid phase. 118. The method of claim 117, wherein the cells are in solution. 118. The method of claim 117, wherein said compound is identified using a receptor binding assay. 117. The method of claim 116, wherein the cell is of any one of claims 95-101 and 109-115. 117. The method of claim 116, wherein said compound is identified using an assay based on receptor activity. 117. The method of claim 116, wherein said T1R receptor is expressed in a cell. 117. The method of claim 116, wherein the cell is a HEK-293, COS or CHO cell. 117. The method of claim 116, wherein said compound is identified by its effect on intracellular uptake of a receptor by said cell. 117. The method of claim 116, wherein said compound is identified by its effect on receptor phosphorylation. 117. The method of claim 116, wherein the compound is identified by its effect on metastasis inhibition. 117. The method of claim 116, wherein a second messenger assay is used. The method of claim 127, wherein said second messenger is cAMP or IP _3. 117. The method of claim 116, wherein a voltage sensitive or calcium sensitive dye is used. 117. The method of claim 116, wherein the cell expresses at least one G protein. 131. The method of claim 130, wherein said G protein is a promiscuous G protein. The method of claim 131, wherein the promiscuous G protein is G _{alpha 15} or G _{alpha 16.} 117. The method of claim 116, wherein the receptor is stably expressed using a viral vector. 117. The method of claim 116, wherein the cell is a mammalian cell. 117. The method of claim 116, wherein said compound is identified by its effect on G protein activation by said receptor. 117. The method of claim 116, wherein said compound is identified by its effect on receptor conformation. 137. The method of claim 136, wherein the conformational change is detected by a change in sensitivity to proteolysis. 138. The method of claim 136, wherein the conformational change is detected by NMR spectroscopy. 138. The method of claim 136, wherein the conformational change is detected by fluorescence spectroscopy. 137. The method of claim 136, wherein said compound is identified by its effect on binding of a radiolabeled or fluorescently labeled ligand to said stably expressed receptor. 141. The method of claim 140, wherein displacement of the labeled compound is measured by fluorescence deflection or FRET assay. 117. The method of claim 116, which is a high throughput screening assay. 117. The method of claim 116, wherein the receptor activity is associated with a reporter gene. 144. The method of claim 143, wherein the reporter gene is luciferase, alkaline phosphatase, β-galactosidase, or β-lactamase. 143. The method of claim 140, wherein the receptor is a constitutively active variant. 141. The method of claim 140, wherein expression of the receptor is under the control of a constitutive promoter. 141. The method of claim 140, wherein expression of the receptor is under the control of a regulatable promoter. 145. The method of claim 140, wherein the receptor is fused to a peptide that promotes surface expression. 141. The method of claim 140, wherein the peptide is a PDZ domain interacting peptide. 141. The method of claim 140, wherein the effect of the compound on the receptor is predicted based on the X-ray crystal structure of the receptor. 117. The method of claim 116, wherein said compound is identified by its effect on yeast cells that express said receptor. 117. The method of claim 116, wherein the compound is identified from a combinatorial library of compounds. 117. The method of claim 116, wherein the compound is identified from a peptide library. 117. The method of claim 116, wherein the compound is identified from a randomized library of small molecules. 117. A method of altering taste in an animal using a compound identified in claim 116. The method of claim 155, wherein said taste is umami. The method of claim 155, wherein said taste is sweet. The method of claim 155, wherein said animal is a human, dog, cat, fish, cow, sheep or pig. 165. The method of claim 155, wherein the compound is incorporated into a food, beverage or oral pharmaceutical composition. A cell stably expressing a heterologous nucleic acid sequence encoding at least one T1R according to any one of claims 1 to 7 and 15 to 21 is used to taste individual compounds or food or beverage compositions. How to quantify. A cell line that inducibly expresses human T1R1 / T1R3 umami receptor or T1R2 / T1R3 sweet receptor. 164. The cell line of claim 161, wherein the cell line is a CHO, COS, HEK or BHK cell line. 163. The cell line of claim 162, which is HEK-293. 164. The cell line of claim 161 that expresses G protein. The cell line of claim 164, wherein G protein is G _{alpha 15} or G _{alpha 16.} 163. The cell line of claim 161, which stably expresses the T1R1 / T1R3 receptor. 164. The cell line of claim 161, wherein expression is induced by a GeneSwitch protein. 164. A method using the cell line of claim 161 for identifying a compound that agonizes or antagonizes a T1R1 / T1R3 receptor or a T1R2 / T1R3 receptor. 169. The method of claim 168, which is a binding assay. 169. The method of claim 168, which is a high throughput screening assay. 169. The method of claim 168, which is a fluorometric assay. 169. The method of claim 168, wherein the compound that screens with L-glutamate or L-aspartate for binding to the T1R1 / T1R3 umami receptor is screened. 169. The method of claim 168, wherein the method is a high throughput screening assay using an automated fluorescence analysis imaging instrument. 169. The method of claim 168, wherein the method is used to screen a compound library that enhances or modulates the activity of L-glutamic acid to activate the T1R1 / T1R3 umami receptor. 169. The method of claim 168, wherein the method is used to screen a compound library for compounds that agonize or antagonize a T1R2 / T1R3 sweet receptor. 169. The method of claim 168, wherein the compound that screens with IMP, GMP, or an analog thereof for binding to the T1R1 / T1R3 umami receptor is screened. 169. The method of claim 168, wherein said compound is used to screen a compound library that mimics the activity of IMP, GMP, or an analog thereof and enhances the activity of a T1R1 / T1R3 agonist. 169. The method of claim 168, wherein the method is used to screen a compound library that enhances or modulates sweeteners to activate the T1R2 / T1R3 sweetness receptor. Contacting the T1R1 / T1R3 umami receptor with a sweet taste inhibitor that inhibits both the T1R1 / T1R3 sweet receptor and the T1R2 / T1R3 taste receptor. 179. The method of claim 179, wherein the inhibitor is lactisol. A method of identifying a compound that modulates a T1R1 / T1R3 umami receptor by screening a compound that competes with lactisol for binding to and / or inhibition of the T1R1 / T1R3 umami receptor. 179. The method of claim 179, which is an assay using cells. 184. The method of claim 182, wherein said assay uses a cell line that co-expresses T1R1 and T1R3. 184. The method of claim 183, wherein said cell line is a HEK-G [ _{alpha] 15} cell line. 184. The method of claim 183, wherein said cell line stably expresses said receptor. 184. The method of claim 183, wherein said cell line transiently expresses said receptor. The method of claim 186, wherein said G protein is G _{alpha 15} or G _{alpha 16.} 184. The method of claim 181 which is an assay using cells. 189. The method of claim 188, wherein said assay uses a cell line that co-expresses T1R1 and T1R3. 189. The method of claim 189, wherein said cell line is HEK-G [ _{alpha] 15} said cell line. 189. The method of claim 189, wherein said cell line stably expresses said receptor. 191. The method of claim 191, wherein said cell line transiently expresses said receptor. The method of claim 192, wherein said G protein is G _{alpha 15} or G _{alpha 16.}