Method for analyzing G protein-coupled receptor

Abstract

To provide a method allowing g protein-coupled receptors to express and function.SOLUTION: Provided is a method for expressing a GPCR polypeptide. The method comprises expressing in a cell a GPCR polypeptide consisting of an amino acid sequence in which at least one amino acid residue that differs from a consensus amino acid sequence in the amino acid sequence of the target GPCR (excluding olfactory receptors) is modified to an amino acid residue in the consensus amino acid sequence at the corresponding position. Here, the consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the GPCR of interest and the amino acid sequence of GPCR encoded by an orthologue of the GPCR of interest in a vertebrate.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for analyzing G protein-coupled receptors.

Background Art

[0002] As constituent members of a living body, cells recognize changes in the surrounding physiological conditions and change their behavior. An individual organism takes adaptive actions using the senses to detect changes in the external environment. The proper response of individual living units to endogenous information chemicals such as hormones and neurotransmitters, or exogenous information chemicals such as taste substances, forms the basis of the biological mechanism. These chemical receptors are receptor proteins possessed by individual tissue cells. In recent years, many individual organisms lacking specific receptor genes have been produced, and the necessity of receptors in physiological functions has been demonstrated. In addition, various drugs targeting receptors are being put to practical use as regulators of physiological functions. On the other hand, there are still many receptors with unknown functions.

[0003] A typical receptor possessed by organisms is the G-protein-coupled receptor (GPCR). This is characterized by a seven-transmembrane structure. By binding an agonist, it transitions to an active structure and basically transmits signals through interaction with intracellular G proteins. In humans, there are approximately 800 types of GPCRs, and about half of them were previously known to be not directly involved in the physiological functions in the body related to sensation. Information on human GPCRs except for olfactory receptors and vomeronasal receptors can be obtained from a database (G protein-coupled receptors (IUPHAR / BPS Guide to PHARMACOLOGY, http: / / www.guidetopharmacology.org / GRAC / FamilyDisplayForward?familyId=694)). As of November 2017, 134 GPCRs are the target molecules of approved drugs in the United States (Non-Patent Document 1). In addition, receptors that were thought to be involved only in sensation have actually been shown to be expressed in tissues other than sensory tissues and to be involved in specific physiological functions in the body.

[0004] For example, the vomeronasal 1 receptor (VN1R) is a GPCR of the Class A family, and there are five types of genes in humans. Analysis of homologous genes in mice predicts that it is expressed in the nasal cavity and is responsible for pheromone recognition (Non-Patent Document 2). For VN1R1 in humans, only one example of functional analysis in cultured cells has been reported, and this report shows that it can recognize low-molecular-weight volatile compounds (Non-Patent Document 2). Also, in a large-scale survey in Sweden, as a result of investigating what differences in properties occur due to genetic polymorphisms of VN1R1, it was shown that there are differences in the sexual characteristics of women, suggesting the potential function of VN1R1 (Non-Patent Document 3). For the other VN1R2 - 5, there are no reported examples of functional analysis such as identification of the substances they recognize.

[0005] The taste receptor TAS1R (taste 1 receptor) is a gene family discovered as a GPCR expressed in the tongue. There are three genes, TAS1R1, TAS1R2, and TAS1R3, in the human genome, all of which are classified as Class C type GPCRs. In the taste of the tongue, TAS1R1 and TAS1R2 are expressed in different taste cells, and both co-express TAS1R3. Class C type GPCRs, including the metabotropic glutamate receptor, are known to function by forming dimers, and TAS1R is no exception. When TAS1R3 is co-expressed with either TAS1R1 or TAS1R2, HEK293 cells respond to taste substances. The TAS1R1 / TAS1R3 complex and the TAS1R2 / TAS1R3 complex each receive umami and sweet substances. By expressing the TAS1R2 / TAS1R3 complex in cultured cells and performing screening, substances that function as allosteric modulators for the complex and enhance sweetness have been reported (Non-Patent Document 4). Similarly, by expressing the TAS1R1 / TAS1R3 complex, it is also possible to obtain an evaluation result that inosinic acid, which enhances umami, enhances the activity of the complex (Non-Patent Document 5). If these TAS1R proteins can be obtained in a more stable form or expressed in a larger amount on the membrane of cultured cells, it will be possible to efficiently identify materials that enhance umami and sweetness.

[0006] TAS2R (taste 2 receptor), which is also a taste receptor, is a gene family discovered as a bitter taste receptor expressed on the tongue, and there are 25 kinds of genes in the human genome. It is classified as a Class A type of GPCR and has been shown to recognize various bitter substances. However, functional analysis of human TAS2R41, TAS2R42, TAS2R45, TAS2R48, and TAS2R60 has not been successful, and it is unclear what substances they recognize. Patent Document 1 discloses that in order to enable bitter taste evaluation and identification of bitter taste regulators using TAS2R, a fusion protein with a G protein is used as a device for efficient functional analysis of TAS2R. The expression of TAS2R is not limited to the tongue, but is also observed in the respiratory, circulatory, and nervous systems, and has been shown to play various physiological roles in addition to receiving bitter taste on the tongue (Non-Patent Document 6). In addition, expression is also observed in cancerous tissue cells such as ovarian cancer and prostate cancer.

[0007] Trace amine associated receptor (TAAR) is a GPCR of the Class A family and consists of 6 kinds of genes in humans. Regarding TAAR1, which was first discovered, it has been shown to be responsible for the recognition of neurotransmitters in the brain. Therefore, the relationship between TAAR1 and schizophrenia, depression, poisoning, and Parkinson's disease has been studied, and there are efforts to use its agonist for the treatment of neuropathic pain (Patent Document 2). On the other hand, other TAARs except TAAR1 are highly expressed in the olfactory epithelium, have been shown to selectively and highly sensitively recognize volatile amines, and play a role in generating the sense of smell (Non-Patent Document 7).

[0008] The Mas-related G-protein-coupled receptor (Mrgpr) is a GPCR of the Class A family, and there are 10 genes in humans. Its expression is observed in the peripheral sensory nervous system such as the skin and its related tissues, and it senses itching and pain by recognizing agonists (Non-Patent Document 8). Also, in mice, based on the fact that sensory neurons expressing MrgprB4 are involved in pleasant touch, efforts to search for mood-improving agents targeting MrgprB4 have been disclosed (Patent Document 3). More recently, among Mrgprs, MrgprE and MrgprF are suggested to be expressed in various tissues other than sensory neurons, such as the ileum, and to play diverse physiological functions, but there are few reported examples of agonists (Non-Patent Document 9).

[0009] Generally, for the functional analysis of GPCRs, the method of expressing them in cultured cells is simple and most widely used. A very wide variety of devices have been made for the analysis method. Still, there are many that have not yet succeeded in functional analysis. GPCRs that have not succeeded in functional analysis and whose ligands are unknown are generally denoted as GPR (G-protein-coupled receptor) X (X is an arbitrary number). One of the reasons for the slow progress in ligand identification for these GPR groups is that even when trying to express the target GPCR in cultured cells, cultured cells different from the original tissue cells lack factors for stably expressing the GPCR on the cell membrane.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0012] In analyzing G protein-coupled receptors (GPCRs), there is a need for a method to efficiently express GPCRs.

Means for Solving the Problems

[0013] The present inventor has intensively studied a method capable of efficiently expressing GPCRs. As a result, the present inventor has found that by consensusization of GPCRs, the membrane expression of GPCRs in cultured cells can be improved as compared with the original GPCRs. Examples of consensusization of GPCRs excluding olfactory receptors have not been known so far.

[0014] Therefore, the present invention provides the following 1) to 13). 1) A method for expressing a GPCR polypeptide, comprising: expressing in a cell a GPCR polypeptide comprising an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence in the amino acid sequence of a target GPCR (excluding olfactory receptors) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, wherein the consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequences of GPCRs encoded by orthologs of the target GPCR in vertebrates, method. 2) A method for expressing a GPCR polypeptide, comprising: expressing in a cell a GPCR polypeptide comprising an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence in the amino acid sequence of a target GPCR is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, The GPCR polypeptide comprises an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: 143 is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position in the amino acid sequence represented by SEQ ID NO: 36 of human TAAR6. Method. 3) A method for measuring the response of a GPCR of interest, comprising: measuring the response of the GPCR polypeptide expressed by the method according to 1) or 2). A method comprising the above. 4) A method for screening for a ligand of a GPCR of interest, comprising: measuring the response of the GPCR polypeptide expressed by the method according to 1) or 2) in the presence of a test substance; and selecting the test substance to which the GPCR polypeptide responds. A method comprising the above. 5) A method for evaluating and / or selecting a modulator of ligand recognition of a GPCR of interest, comprising: adding a test substance and a ligand of the GPCR of interest to the GPCR polypeptide expressed by the method according to 1) or 2); and measuring the response of the GPCR polypeptide to the ligand. A method comprising the above. 6) A method for evaluating taste, comprising: adding a test substance to the GPCR polypeptide expressed by the method according to 1) or 2); and measuring the response of the GPCR polypeptide to the test substance, wherein the GPCR polypeptide is a taste receptor polypeptide. Method. 7) A method for evaluating and / or selecting an inhibitor of the odor of a ligand of a GPCR of interest, comprising: adding a test substance and a ligand of the GPCR of interest to the GPCR polypeptide expressed by the method according to 1) or 2); and measuring the response of the GPCR polypeptide to the ligand. comprising a GPCR polypeptide which is a trace amine-associated receptor polypeptide, Method. 8) A method for evaluating and / or selecting an inhibitor of the odor of a ligand of a target GPCR, 1) adding a test substance to a GPCR polypeptide expressed by the method according to 1) or 2), and measuring the response of the GPCR polypeptide to the test substance, comprising a GPCR polypeptide which is a trace amine-associated receptor polypeptide, Method. 9) A modified GPCR polypeptide, which consists of an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence in the amino acid sequence of the target GPCR (except for olfactory receptors) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, wherein the consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequences of GPCRs encoded by orthologs of the target GPCR in vertebrates, Modified GPCR polypeptide. 10) A modified GPCR polypeptide, which consists of an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence shown by SEQ ID NO: 143 in the amino acid sequence shown by SEQ ID NO: 36 of human TAAR6 is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, Modified GPCR polypeptide. 11) A polynucleotide encoding the modified GPCR polypeptide according to 9) or 10). 12) A vector or DNA fragment containing the polynucleotide according to 11). 13) A transformed cell containing the vector or DNA fragment according to 12).

Advantages of the Invention

[0015] The present invention provides a method capable of efficiently expressing GPCR. By using such GPCR, it is possible to contribute to the elucidation of the function of GPCR and the identification of function regulators.

Brief Description of the Drawings

[0016]

Figure 1

Modes for Carrying Out the Invention

[0017] All patent documents, non-patent documents, and other publications cited in this specification are hereby incorporated by reference in their entirety.

[0018] In this specification, the "G-protein-coupled receptor (GPCR)" refers to a receptor having a seven-transmembrane structure that transitions to an activated structure by binding a ligand and basically transmits a signal through interaction with an intracellular G protein. Examples of GPCR include vomeronasal receptors, taste receptors, trace amine-related receptors, Mas-related G protein-coupled receptors, etc., and also GPRX (X is any number) with an unknown ligand, but are not limited thereto.

[0019] In this specification, the "vomeronasal receptor" refers to VN1R (vomeronasal 1 receptor). For example, there are five types of VN1R genes in the human genome.

[0020] As used herein, the term "taste receptor" refers to a receptor that receives taste molecules in a living body, and includes umami or sweet receptors belonging to the TAS1R (taste 1 receptor) family that receive umami or sweet molecules, and bitter receptors belonging to the TAS2R (taste 2 receptor) family that receive bitter molecules. For example, there are three types of TAS1R genes in the human genome. Among them, TASR1 and TAS1R3 form a complex and function as an umami substance receptor, and TAS1R2 and TAS1R3 form a complex and function as a sweet substance receptor. In addition, there are 25 types of TAS2R genes in the human genome.

[0021] In the present invention, the term "trace amine-associated receptor" refers to TAAR (trace amine associated receptor). For example, there are six types of TAAR genes in the human genome.

[0022] As used herein, the term "Mas-related G protein-coupled receptor" refers to Mrpgr (Mas-related G-protein-coupled receptor). MAS is a G protein-coupled receptor that binds to angiotensin. For example, there are 10 types of Mrpgr genes in the human genome (MAS1, MASL1, MrgprD, MrgprE, MrgprF, MrgprG, MrgprX1-4).

[0023] As used herein, the term "olfactory receptor" refers to Olfactory receptor or Odorant receptor. Based on criteria such as overall sequence homology, prediction of seven transmembrane regions, and whether it has a conserved partial amino acid sequence, about 400 olfactory receptor genes are predicted in the human genome.

[0024] As used herein, the term "GPCR polypeptide" refers to a GPCR or a polypeptide having an equivalent function thereto. A polypeptide having an equivalent function to a GPCR refers to a polypeptide that can be expressed on the cell membrane, similar to a GPCR, is activated by ligand binding, and upon activation, has a function of promoting GDP / GTP exchange of the G protein α subunit to which it is coupled, such as a function of transmitting a signal into the cell.

[0025] As used herein, when a GPCR polypeptide "functionally expresses" in a cell, it means that the expressed GPCR polypeptide functions as a receptor for the corresponding ligand in the cell.

[0026] As used herein, the term "agonist" refers to a substance that binds to and activates a receptor. On the other hand, as used herein, the term "antagonist" refers to a substance that binds to a receptor but does not activate the receptor or inhibits the response of the receptor to an agonist.

[0027] As used herein, "receptor agonism" refers to binding to a receptor and activating the receptor.

[0028] As used herein, "odor cross adaptation (or olfactory cross adaptation)" with respect to a target odor refers to a phenomenon in which an individual is pre-exposed to an odor of a substance different from the causative agent of the target odor, becomes accustomed to that odor, and as a result, the olfactory sensitivity to the causative agent of the target odor decreases or changes. The inventors have previously clarified that "odor cross adaptation" is a phenomenon based on receptor agonism (International Publication No. WO 2016 / 194788). That is, in "odor cross adaptation", the receptor for the causative agent of the target odor responds to a causative agent of a different odor prior to responding to the causative agent of the target odor, and then becomes desensitized, so that when subsequently exposed to the causative agent of the target odor, only a low response can be elicited, resulting in a decrease or alteration in the intensity of the target odor recognized by the individual. The mechanism of odor cross adaptation caused by such receptor behavior is also referred to herein as "odor cross adaptation by receptor agonism".

[0029] As used herein, "inhibition by receptor antagonism" of a target odor refers to suppressing the response of the receptor to a substance having the target odor by an antagonist, and as a result, suppressing the target odor recognized by the individual.

[0030] As used herein, the identity of nucleotide sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-41). Specifically, it is calculated by performing an analysis with a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) with a Unit size to compare (ktup) of 2.

[0031] As used herein, the term "amino acid residue" refers to the 20 amino acid residues that make up proteins, alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0032] As used herein, an amino acid modification may be represented by [original amino acid, position, modified amino acid] according to the recognized one-letter amino acid abbreviations of IUPAC. For example, the modification of histidine at position 43 to arginine is denoted as "H43R".

[0033] In this specification, the "corresponding position" on an amino acid sequence can be determined by aligning the target sequence and the reference sequence (in the present invention, the amino acid sequence of the original GPCR) so as to give the maximum homology. The alignment of amino acid sequences can be performed using a known algorithm, and the procedure is known to those skilled in the art. For example, the alignment can be carried out by using the Clustal W multiple alignment program (Thompson, J.D. et al, 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which is a revised version of Clustal W, can also be used. Clustal W, Clustal W2, and Clustal omega can be used, for example, on the Clustal website [www.clustal.org] operated by University College Dublin, the website of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), or the website of the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]) operated by the National Institute of Genetics. The position of the target sequence aligned with any position of the reference sequence by the above alignment is regarded as the "corresponding position" to the arbitrary position.

[0034] A person skilled in the art can further finely adjust the alignment of the amino acid sequences obtained above so as to optimize it. Such an optimal alignment is preferably determined in consideration of the similarity of the amino acid sequences, the frequency of inserted gaps, and the like. Here, the similarity of amino acid sequences refers to the ratio (%) of the number of positions where identical or similar amino acid residues are present in both sequences when two amino acid sequences are aligned to the total number of amino acid residues in the full length. Similar amino acid residues mean amino acid residues that have similar properties to each other in terms of polarity and charge among the 20 amino acids constituting proteins and cause so-called conservative substitutions. Groups consisting of such similar amino acid residues are well known to those skilled in the art. For example, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; leucine and isoleucine, etc. can be mentioned respectively, but are not limited thereto. In addition, the alignment of the amino acid sequences obtained above can be further finely adjusted so as to optimize it, for example, based on amino acids or amino acid motifs that are highly conserved among GPCRs.

[0035] In this specification, "operably linked" between a control region such as a promoter and a gene means that the gene and the control region are linked so that the gene can be expressed under the control of the control region. The procedure for "operably linking" a gene and a control region is well known to those skilled in the art.

[0036] In this specification, "upstream" and "downstream" with respect to a gene refer to upstream and downstream in the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is present on the 3' side of the promoter in the DNA sense strand, and upstream of the gene means the region on the 5' side of the gene in the DNA sense strand.

[0037] As used herein, "homolog" refers to homologous genes derived from a common ancestor. "Ortholog", also called "orthologue", refers to homologs that diverged during speciation, exist in different species, and have the same or similar functions. In one example, the ortholog used in the present invention can be a GPCR gene of a species different from the species from which the target GPCR, which contains the same name as the target GPCR gene, is derived among the homologous genes of the target GPCR gene. When the nomenclature of a GPCR of a certain species is different from the nomenclature in the species from which the target GPCR is derived, the ortholog can be a GPCR gene having high homology with the target GPCR gene in that certain species, preferably the GPCR gene having the highest homology. Alternatively, it can be a GPCR gene known to be an ortholog of the target GPCR gene in that certain species. In another example, the ortholog used in the present invention can be a GPCR gene among the above orthologs that is suggested by phylogenetic analysis to have diverged during speciation.

[0038] As shown in the examples described below, the present inventors modified the amino acid sequence of a human GPCR based on a consensus amino acid sequence derived from the amino acid sequence of the human GPCR and the amino acid sequence of the GPCR encoded by a specific ortholog of the human GPCR, and when the obtained GPCR polypeptide was expressed in cells, it was found that the membrane expression of the GPCR polypeptide in the cells increased compared to the human GPCR before modification (Table 7, Figure 1). That is, the stability of the GPCR polypeptide during expression is improved compared to the human GPCR before modification. As used herein, the GPCR before modification is referred to as the "original GPCR", modifying the amino acid sequence of the GPCR based on the consensus amino acid sequence is referred to as "consensusization", and the consensusized GPCR may be referred to as the "consensus GPCR" or the "modified GPCR polypeptide".

[0039] Therefore, the consensusization of GPCRs is useful for expressing GPCRs, particularly those GPCRs for which functional analysis has been inefficient due to insufficient membrane expression in cultured cells, on the cell membrane of cells. Thus, in one aspect, the present invention provides a method for expressing a GPCR polypeptide. Since the expression method of the present invention enables improvement of GPCR expression (for example, increased expression, stabilized expression), the method is preferably a method for improving the expression of a GPCR polypeptide. The method includes expressing in a cell a GPCR polypeptide consisting of an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence in the amino acid sequence of the target GPCR is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, wherein the consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates.

[0040] In the expression method of the present invention, the target GPCR is not particularly limited and may be a GPCR of any species, preferably a mammalian GPCR, more preferably a human GPCR. The target GPCR may be a GPCR for which functional analysis using cultured cells by conventional methods is possible or an inefficient GPCR, but the method of the present invention is more preferably applied to GPCRs for which functional analysis using cultured cells by conventional methods is inefficient.

[0041] In the expression method of the present invention, the GPCR encoded by the ortholog of the target GPCR in vertebrates is preferably a GPCR selected from GPCRs encoded by orthologs of the target GPCR in mammals, birds, reptiles, amphibians, and fish, more preferably a GPCR selected from GPCRs encoded by orthologs of the target GPCR in mammals, birds, reptiles, and amphibians, and even more preferably a GPCR encoded by an ortholog of the target GPCR in mammals. The ortholog is not particularly limited, but an ortholog with a higher homology to the target GPCR gene is preferably used. Here, mammals refer to the biological species belonging to the class Mammalia of the phylum Chordata, and it is known that about 5,500 species exist. Examples of mammals include humans, chimpanzees, bonobos, gorillas, Sumatran orangutans, northern white-cheeked gibbons, drills, geladas, rhesus monkeys, Anubis baboons, sooty mangabeys, green monkeys, high-crowned colobus monkeys, Angolan colobus monkeys, Garnett's galagos, golden-bellied mouse lemurs, Coquerel's sifakas, Philippine tarsiers, mice, rats, rabbits, cats, dogs, foxes, raccoons, weasels, tigers, cheetahs, bears, seals, sea lions, otters, horses, zebras, camels, pigs, wild boars, cows, goats, sheep, deer, giraffes, hippos, elephants, pangolins, moles, bats, etc., but are not limited thereto. Birds (Aves) refer to the biological species belonging to the class Aves of the phylum Chordata. Examples of birds include chickens, ducks, geese, ostriches, emus, turkeys, pheasants, pigeons, parrots, canaries, fir trees, sparrows, mynas, quails, cranes, etc., but are not limited thereto. Reptiles (Reptilia) refer to the biological species belonging to the class Reptilia of the phylum Chordata. Examples of reptiles include turtles, lizards, crocodiles, iguanas, chameleons, geckos, snakes, etc., but are not limited thereto. Amphibians (Amphibia) refer to the biological species belonging to the class Amphibia of the phylum Chordata. Examples of amphibians include frogs, newts, Japanese giant salamanders, etc., but are not limited thereto. Fish is a general term referring to the biological species belonging to the classes Myxini, Petromyzontida, Chondrichthyes, and Osteichthyes of the phylum Chordata.Examples of fish include, but are not limited to, Conger myriaster, Gymnothorax kidako, sharks, rays, tuna, skipjack, salmon, trout, cod, sea bream, yellowtail, horse mackerel, and mackerel.

[0042] In a preferred example of this embodiment, the ortholog of the target GPCR in vertebrates is a gene that includes the same name as the target GPCR gene among the GPCR genes possessed by a biological species belonging to vertebrates. Such an ortholog can be selected, for example, by the following procedure. Using the amino acid sequence of the target GPCR as a query sequence, perform a database search against a publicly known database such as NCBI BLAST. From the resulting group of homologous genes (for example, the top 500 genes, preferably the top 250 genes, more preferably the top 100 genes, and even more preferably the top 50 genes), select a gene that includes the same name as the target GPCR gene. Even more preferably, select a gene that encodes a GPCR having a certain amino acid sequence identity, for example, 65% or more amino acid sequence identity, with the target GPCR. In addition, when multiple genes derived from the same biological species are selected as orthologs, only one gene with the highest homology to the target GPCR gene may be selected. For example, when the target GPCR is a human GPCR and multiple genes of a biological species other than human are selected as orthologs, the gene of that biological species with the highest homology to the target human GPCR gene may be selected. Also, when the nomenclature of the GPCR of a certain biological species is different from the nomenclature in the biological species from which the target GPCR is derived, as an ortholog, a gene having high homology, preferably the highest homology, with the target GPCR gene in that biological species may be selected. Alternatively, a gene known to be an ortholog of the target GPCR gene in that biological species may be selected.

[0043] The number of types of GPCRs encoded by orthologs of the target GPCR in vertebrates is at least 2 types, preferably at least 5 types, more preferably at least 11 types, still more preferably at least 15 types, still more preferably at least 30 types, and still more preferably 100 types, as the number of receptors. On the other hand, the upper limit of the number of types is the total number of all types of orthologs of the target GPCR in vertebrates, and this number of types is preferably 500 types or less, more preferably 400 types or less, and still more preferably 300 types or less, as the number of receptors. The number of types of GPCRs encoded by orthologs of the target GPCR in vertebrates can be, as the number of receptors, for example, 2 types to the total number of all types of orthologs of the target GPCR in vertebrates, 5 types to the total number of all types of orthologs of the target GPCR in vertebrates, 11 types to the total number of all types of orthologs of the target GPCR in vertebrates, 5 to 500 types, 5 to 400 types, 5 to 300 types, 11 to 500 types, 11 to 400 types, 11 to 300 types, 15 to 300 types, 30 to 300 types, 100 to 300 types.

[0044] In the expression method of the present invention, the "consensus amino acid sequence" is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequences of GPCRs encoded by orthologs of the target GPCR in vertebrates. Specifically, the "consensus amino acid sequence" is an amino acid sequence consisting of consensus residues identified according to the following criteria (i) to (iii) from an alignment of the amino acid sequence of the target GPCR and the amino acid sequences of GPCRs encoded by orthologs of the target GPCR in vertebrates. (i) At each amino acid position of the alignment, (i-i) When there is 1 type of amino acid residue that is different from the amino acid residue of the target GPCR and has an occurrence frequency of 50% or more, that amino acid residue is identified as the consensus residue. (i-ii) When there are 2 types of amino acid residues with an occurrence frequency of 50%, the amino acid residue of the target GPCR is identified as the consensus residue. (i-iii) If there is an amino acid residue in the GPCR of interest and there is no amino acid residue with an occurrence frequency of 40% or more, it is identified as having no consensus residue. (i-iv) If there is no amino acid residue in the GPCR of interest and there is an amino acid residue with an occurrence frequency of 60% or more, the amino acid residue with the highest occurrence frequency is identified as the consensus residue. If there are two or more amino acid residues with the highest occurrence frequency, the amino acid residue with the smallest molecular weight among these amino acid residues is identified as the consensus residue. (i-v) If it does not fall under any of (i-i) to (i-iv) above, the amino acid residue of the GPCR of interest is identified as the consensus residue. (ii) When the consensus residue is identified according to the criteria in (i) above, if the most N-terminal consensus residue is a consensus residue at the N-terminal of the GPCR of interest or at a position corresponding to a position C-terminal thereto and is not a methionine residue, the consensus residue N-terminal to the consensus residue consisting of the methionine residue closest to the N-terminal is changed to having no consensus residue. (iii) When the consensus residue is identified according to the criteria in (i) above, if the most N-terminal consensus residue is a consensus residue at a position corresponding to a position N-terminal to the N-terminal of the GPCR of interest and is not a methionine residue, trace back one amino acid position at a time from the position of the consensus residue in the alignment to the N-terminal until a methionine residue appears. Identify the amino acid residue with the highest occurrence frequency as the consensus residue. If there are two or more amino acid residues with the highest occurrence frequency, identify the amino acid residue with the smallest molecular weight among these amino acid residues as the consensus residue.

[0045] Here, the "occurrence frequency" is shown as the percentage of the number of occurrences of a specific amino acid residue at each amino acid position in the alignment of amino acid sequences with respect to the number of amino acid sequences subjected to the alignment. The alignment of amino acid sequences can be performed by a known algorithm.

[0046] For (i-i) of criterion (i), it is the criterion when there is an amino acid residue at the amino acid position in the alignment of the amino acid sequence of the target GPCR and the amino acid sequences of the GPCRs encoded by the orthologs of the target GPCR in vertebrates. At this time, if there is one amino acid residue that is different from the amino acid residue of the target GPCR and has an occurrence frequency of 50% or more, the amino acid residue with an occurrence frequency of 50% or more is identified as the consensus residue at that position. On the other hand, if the occurrence frequencies of amino acid residues other than the amino acid residue of the target GPCR are all less than 50%, in accordance with criterion (i-v), the amino acid residue of the target GPCR is identified as the consensus residue at that position.

[0047] For (i-ii) of criterion (i), it is the criterion when there is an amino acid residue at the amino acid position in the alignment of the amino acid sequence of the target GPCR and the amino acid sequences of the GPCRs encoded by the orthologs of the target GPCR in vertebrates. At this time, if there are two amino acid residues with an occurrence frequency of 50%, one of the two amino acid residues must be the amino acid residue of the target GPCR, and the amino acid residue of the target GPCR is identified as the consensus residue at that position.

[0048] For (i-iii) of criterion (i), it is the criterion when there is an amino acid residue at the amino acid position in the alignment of the amino acid sequence of the target GPCR and the amino acid sequences of the GPCRs encoded by the orthologs of the target GPCR in vertebrates. At this time, if there are no amino acid residues with an occurrence frequency of 40% or more, it is identified that there is no consensus residue at that position. On the other hand, if the occurrence frequency of no amino acid residue is less than 40%, when it meets the above criterion (i-i), the consensus residue at that position is identified according to that criterion, and when it does not meet the criterion, the amino acid residue of the target GPCR is identified as the consensus residue at that position in accordance with criterion (i-v).

[0049] For (i)-(iv) of reference (i), when there is no amino acid residue at the amino acid position in the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates, it is a reference. At this time, if an amino acid residue is present with a frequency of 60% or more, the amino acid residue with the highest frequency of occurrence is identified as the consensus residue at that position. When there are two or more amino acids with the highest frequency of occurrence, the amino acid residue with the smallest molecular weight among the amino acids with the highest frequency of occurrence is identified as the consensus residue. For example, if there is one amino acid residue with the highest frequency of occurrence, the one amino acid residue is identified as the consensus residue at that position. If there are two or more amino acid residues with the highest frequency of occurrence, the amino acid residue with the smallest molecular weight among them may be identified as the consensus residue at that position. In addition, when the total length of the consensus amino acid sequence becomes 10% or more longer on the N-terminal side than the total length of the amino acid sequence of the target GPCR due to the above change, from the perspective of maintaining the structure of the GPCR, the consensus residue at the position corresponding to the N-terminal of the target GPCR in the consensus amino acid sequence is set as a methionine residue, and the consensus residues on the N-terminal side of the methionine residue may be changed to none. That is, the N-terminal structure of the target GPCR may be maintained as it is. On the other hand, if the frequency of occurrence of the amino acid residue is less than 60%, the amino acid residue of the target GPCR is identified as the consensus residue at that position according to reference (i)-(v). That is, it is identified that there is no amino acid residue at that position.

[0050] When none of (i)-(iv) of (i) above apply at the amino acid position in the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates, the amino acid residue of the target GPCR is identified as the consensus residue at that position. At this time, if there is no amino acid residue in the target GPCR, it may be identified that there is no amino acid residue at that position in the consensus amino acid sequence.

[0051] Criterion (ii) is a criterion when, according to the above criterion (i), a consensus residue is identified at each amino acid position in the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates, and the consensus residue located most N-terminally among the consensus residues is a consensus residue at a position corresponding to the N-terminus of the target GPCR or a position C-terminal thereto and is not a methionine residue. At this time, so that the N-terminal consensus residue becomes a methionine residue, in other words, so that the start amino acid of translation of the GPCR polypeptide becomes a methionine residue, the consensus residue N-terminal to the consensus residue consisting of the methionine residue closest to the N-terminus among the consensus residues is changed to no consensus residue. For example, the consensus residues are confirmed one by one from the N-terminus. If it is not a methionine residue, it is identified as having no consensus residue at that position, and this may be repeated until a methionine residue appears for the first time. In addition, when the full length of the consensus amino acid sequence becomes 10% or more shorter than the full length of the amino acid sequence of the target GPCR due to the above change, from the viewpoint of maintaining the helix structure of the GPCR, the consensus residue most N-terminal among the consensus residues before the change may be changed to a consensus residue consisting of a methionine residue. At this time, if the consensus residue most N-terminal among the consensus residues before the change is asparagine, serine or threonine involved in glycosylation and / or membrane translocation, from the viewpoint of maintaining the structure of the GPCR, the consensus residue most N-terminal among the consensus residues before the change is not changed, and the amino acid residue of the target GPCR N-terminal to the position corresponding to the consensus residue may be used as the consensus residue. That is, the N-terminal structure of the target GPCR may be maintained as it is.

[0052] Criterion (iii) is a criterion where, when identifying consensus residues at each amino acid position in the alignment of the amino acid sequence of the target GPCR and the amino acid sequence of the GPCR encoded by the ortholog of the target GPCR in vertebrates according to the above criterion (i), the most N-terminal consensus residue is a consensus residue at a position corresponding to a position N-terminal to the N-terminus of the target GPCR and is not a methionine residue. At this time, so that the N-terminal consensus residue becomes a methionine residue, in other words, so that the start amino acid of the translation of the GPCR polypeptide becomes a methionine residue, the alignment is confirmed by going back one amino acid position at a time from the position of the most N-terminal consensus residue to the N-terminal side until a methionine residue appears. The amino acid residue with the highest frequency of occurrence is identified as the consensus residue. When there are two or more amino acid residues with the highest frequency of occurrence, the amino acid residue with the smallest molecular weight among the amino acid residues is identified as the consensus residue.

[0053] The amino acid sequence consisting of the consensus residues thus identified is the consensus amino acid sequence. The GPCR polypeptide used in the present invention is a modified GPCR polypeptide consisting of an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence in the amino acid sequence of the target GPCR is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position. Here, "modification" is a concept including any of substitution, deletion, addition, and insertion. The difference between the amino acid sequence of the target GPCR and the consensus amino acid sequence can be found, for example, by aligning the amino acid sequences using a known algorithm and comparing the two amino acid sequences. For example, when comparing the amino acid sequence of the target GPCR with the consensus amino acid sequence, if the amino acid residue at a certain amino acid position in the amino acid sequence of the target GPCR is different from the amino acid residue at the corresponding position in the consensus amino acid sequence, the amino acid residue of the target GPCR may be substituted with the amino acid residue of the consensus amino acid sequence. Alternatively, when comparing the amino acid sequence of the target GPCR with the consensus amino acid sequence, if there is no amino acid residue at the position corresponding to a certain amino acid position in the amino acid sequence of the target GPCR in the consensus amino acid sequence, the amino acid residue at that amino acid position of the target GPCR may be deleted. Alternatively, when comparing the amino acid sequence of the target GPCR with the consensus amino acid sequence, if there is an amino acid residue in the consensus amino acid sequence at the position corresponding to the position where there is no amino acid residue in the amino acid sequence of the target GPCR, the amino acid residue of the consensus amino acid sequence may be inserted at that amino acid position of the target GPCR. Incidentally, when comparing the amino acid sequence of the target GPCR with the consensus amino acid sequence, if the consensus amino acid sequence is longer at the N-terminus than the amino acid sequence of the target GPCR, the N-terminal portion that exists only in the consensus amino acid sequence may be added to the N-terminus of the amino acid sequence of the target GPCR. Alternatively, when comparing the amino acid sequence of the target GPCR with the consensus amino acid sequence, if the consensus amino acid sequence is longer at the C-terminus than the amino acid sequence of the target GPCR, the C-terminal portion that exists only in the consensus amino acid sequence may be added to the C-terminus of the amino acid sequence of the target GPCR.

[0054] The number of amino acid residues modified in the amino acid sequence of the target GPCR is at least 1, preferably at least 5, more preferably at least 10, and more preferably all of the amino acid residues different from the consensus amino acid sequence in the amino acid sequence of the target GPCR are modified to the amino acid residues of the consensus amino acid sequence at the corresponding positions (i.e., the amino acid sequence of the target GPCR is modified to the consensus amino acid sequence). Alternatively, the number of amino acid residues modified in the amino acid sequence of the target GPCR may be preferably at least 10%, more preferably at least 30%, still more preferably at least 50%, still more preferably at least 70%, still more preferably at least 90%, still more preferably 100% (i.e., the amino acid sequence of the target GPCR is modified to the consensus amino acid sequence) of the number of amino acid residues different from the consensus amino acid sequence. In addition, if the N-terminus of the consensus amino acid sequence is longer than that of the amino acid sequence of the target GPCR, regardless of the number of amino acid residues, it is preferable to modify by adding the N-terminal portion that exists only in the consensus amino acid sequence to the N-terminus of the amino acid sequence of the target GPCR as a whole. If the N-terminus of the consensus amino acid sequence is shorter than that of the amino acid sequence of the target GPCR, regardless of the number of amino acid residues, it is preferable to modify by deleting the N-terminal portion that exists only in the amino acid sequence of the target GPCR from the N-terminus of the amino acid sequence of the target GPCR as a whole. If the C-terminus of the consensus amino acid sequence is longer than that of the amino acid sequence of the target GPCR, regardless of the number of amino acid residues, it is preferable to modify by adding the C-terminal portion that exists only in the consensus amino acid sequence to the C-terminus of the amino acid sequence of the target GPCR as a whole. If the C-terminus of the consensus amino acid sequence is shorter than that of the amino acid sequence of the target GPCR, regardless of the number of amino acid residues, it is preferable to modify by deleting the C-terminal portion that exists only in the amino acid sequence of the target GPCR from the C-terminus of the amino acid sequence of the target GPCR as a whole. As the GPCR polypeptide used in the present invention, as long as its function is not impaired, one to several (for example, 1 to 10, 1 to 5, 1 to 3) amino acid residues are substituted, deleted, added or inserted at amino acid positions other than the amino acid positions targeted for modification based on the above consensus amino acid sequence. A polypeptide containing such modifications is also included in the present invention.

[0055] In a preferred example of this embodiment, the GPCR polypeptide is composed of an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence represented by the sequence number (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position in the amino acid sequence represented by the sequence number (2) of the GPCR in (1) of Tables 1-1 and 1-2 below. More preferably, the GPCR polypeptide is a GPCR polypeptide composed of the amino acid sequence represented by any of sequence numbers 108 to 214 and 275 to 312 in (3) of Tables 1-1 and 1-2 below, or a combination of a GPCR polypeptide composed of the amino acid sequence represented by sequence number 112 or 113 in (3) of Table 1-1 below and a GPCR polypeptide composed of the amino acid sequence represented by sequence number 114. Incidentally, the GPCR polypeptide composed of the amino acid sequence represented by any of sequence numbers 108 to 112, 114 to 120, 138 to 145, 167, 183, 187, 192 and 209 has a highly improved membrane expression compared to the original GPCR. In Tables 1-1 and 1-2, the GPCRs of (1) from No. 1 to 145 are human GPCRs, and Accession No. indicates the Accession No. in GenBank.

[0056]

Table 1-1

[0057]

Table 1-2

[0058] In the amino acid sequence represented by the sequence number (2) of the GPCR of (1) from No. 1 to 4 in Table 1-1 above, at least one amino acid residue different from the consensus amino acid sequence represented by the sequence number (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and the GPCR polypeptide consisting of the modified amino acid sequence is a vomeronasal receptor polypeptide. In Table 1-1 above, the vomeronasal receptor polypeptide consisting of the amino acid sequence represented by any one of the sequence numbers 108 to 111 of (3) is a consensus vomeronasal receptor consisting of a consensus amino acid sequence derived from the amino acid sequence represented by any one of the sequence numbers 1 to 4 of (2) of the vomeronasal receptors of (1) from No. 1 to 4 and the amino acid sequence of the vomeronasal receptor encoded by the ortholog of the vomeronasal receptor in vertebrates.

[0059] In the amino acid sequence represented by SEQ ID NO: (2) of the GPCR of (1) from No. 5 to 30 in Table 1-1 above, at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and the GPCR polypeptide consisting of the resulting amino acid sequence is a taste receptor polypeptide. Among them, in the amino acid sequence represented by SEQ ID NO: (2) of the GPCR of (1) of No. 5, at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and the GPCR polypeptide consisting of the resulting amino acid sequence, and in the amino acid sequence represented by SEQ ID NO: (2) of the GPCR of (1) of No. 7, at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and the GPCR polypeptide consisting of the resulting amino acid sequence form a umami taste receptor complex. Also, in the amino acid sequence represented by SEQ ID NO: (2) of the GPCR of (1) of No. 6, at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and the GPCR polypeptide consisting of the resulting amino acid sequence, and in the amino acid sequence represented by SEQ ID NO: (2) of the GPCR of (1) of No. 7, at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and the GPCR polypeptide consisting of the resulting amino acid sequence form a sweet taste receptor complex. On the other hand, in the amino acid sequence represented by SEQ ID NO: (2) of the GPCR of (1) from No. 8 to 30, at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and the GPCR polypeptide consisting of the resulting amino acid sequence is a bitter taste receptor polypeptide.In Table 1-1 above, the taste receptor polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 112 to 137 in (3) is a consensus taste receptor consisting of the amino acid sequence represented by any of SEQ ID NOs: 5 to 30 in (2) of the taste receptor in (1) of Nos. 5 to 30 and the consensus amino acid sequence derived from the amino acid sequence of the taste receptor encoded by the ortholog of the taste receptor in vertebrates.

[0060] In the amino acid sequence represented by the SEQ ID NO in (2) of the GPCR in (1) of Nos. 31 to 36 in Table 1-1 above, the GPCR polypeptide consisting of the amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence represented by the SEQ ID NO in (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position is a trace amine-related receptor polypeptide. In Table 1-1 above, the trace amine-related receptor polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 138 to 143 in (3) is a consensus trace amine-related receptor polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 31 to 36 in (2) of the trace amine-related receptor polypeptide in (1) of Nos. 31 to 36 and the consensus amino acid sequence derived from the amino acid sequence of the trace amine-related receptor polypeptide encoded by the ortholog of the trace amine-related receptor polypeptide in vertebrates. Among them, the trace amine-related receptor polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 143 is a consensus trace amine-related receptor consisting of the consensus amino acid sequence obtained by maintaining the N-terminal structure of the target trace amine-related receptor polypeptide as it is in the above criterion (ii).

[0061] In the amino acid sequence represented by SEQ ID NO: (2) of the GPCRs of No. 37 to 46 in Table 1-1 above, a GPCR polypeptide consisting of an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position thereof is a Mas-related G protein-coupled receptor polypeptide. In Table 1-1 above, the Mas-related G protein-coupled receptor polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 144 to 153 of (3) is a consensus Mas-related G protein-coupled receptor consisting of the amino acid sequence represented by any one of SEQ ID NOs: 37 to 46 of (2) of the GPCRs of No. 37 to 46 and the consensus amino acid sequence derived from the amino acid sequence of the Mas-related G protein-coupled receptor encoded by the ortholog of the Mas-related G protein-coupled receptor in vertebrates.

[0062] In the amino acid sequence represented by SEQ ID NO: (2) of the GPCRs of No. 47 to 108 in Tables 1-1 and 1-2 above, a GPCR polypeptide consisting of an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position thereof is a GPR polypeptide. In Tables 1-1 and 1-2 above, the GPR polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 154 to 214 and 275 of (3) is a consensus GPR consisting of the amino acid sequence represented by any one of SEQ ID NOs: 47 to 107 and 237 of (2) of the GPRs of No. 47 to 108 and the consensus amino acid sequence derived from the amino acid sequence of the GPR encoded by the ortholog of the GPR in vertebrates.

[0063] In the amino acid sequence represented by the sequence number (2) of the GPCR of No. 109 to 145 in Table 1-2 above, at least one amino acid residue different from the consensus amino acid sequence represented by the sequence number (3) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and the GPCR polypeptide consisting of the resulting amino acid sequence is a GPCR polypeptide other than a vomeronasal receptor polypeptide, a taste receptor polypeptide, a trace amine-related receptor polypeptide, a Mas-related G protein-coupled receptor polypeptide, a GPR polypeptide, and an olfactory receptor polypeptide. In Table 1-2 above, the GPCR polypeptide consisting of the amino acid sequence represented by any of the sequence numbers 276 to 312 of (3) is a consensus GPCR consisting of a consensus amino acid sequence derived from the amino acid sequence represented by any of the sequence numbers 238 to 274 of (2) of the GPCR of No. 109 to 145 and the amino acid sequence of the GPCR encoded by the ortholog of the GPCR in vertebrates.

[0064] "Expression" of the GPCR polypeptide of the present invention means that a translation product is produced from a polynucleotide encoding the polypeptide, and the translation product is localized in the cell membrane, which is its action site, in a functional state. The GPCR polypeptide of the present invention may be expressed in cells by a method known in the art. For example, the GPCR polypeptide can be expressed on the cell membrane of the cell by introducing a vector containing a polynucleotide encoding the polypeptide into the host cell, or by introducing a DNA fragment containing a polynucleotide encoding the polypeptide into the genome of the host cell. Preferably, the GPCR polypeptide is expressed on the cell membrane of the host cell by transforming the host cell with a vector containing a polynucleotide encoding the polypeptide. The host cell may be any cell that can functionally express a foreign GPCR. Specific examples of cells include, but are not limited to, human embryonic kidney cells (HEK293 cells), Chinese hamster cells (CHO cells), monkey cells (COS cells), isolated olfactory nerve cells, Xenopus oocytes, insect cells, yeast, or bacteria.

[0065] The polynucleotide encoding the GPCR polypeptide can be obtained using various mutagenesis techniques known in the art. For example, the polynucleotide encoding the GPCR polypeptide can be obtained by modifying the nucleotide sequence encoding the amino acid residue to be modified in the polynucleotide encoding the amino acid sequence of the target GPCR into the nucleotide sequence encoding the modified amino acid residue.

[0066] The introduction of the target mutation into the polynucleotide encoding the amino acid sequence of the target GPCR can be carried out using various site-directed mutagenesis methods well-known to those skilled in the art. The site-directed mutagenesis method can be carried out by any method such as, for example, the inverse PCR method or the annealing method. It is also possible to use commercially available site-directed mutagenesis kits (for example, Stratagene's QuickChange II Site-Directed Mutagenesis Kit, QuickChange Multi Site-Directed Mutagenesis Kit, etc.).

[0067] Alternatively, the polynucleotide encoding the GPCR polypeptide can also be obtained by genome editing using artificial DNA nucleases or Programmable nuclease, DNA synthesis based on its nucleotide sequence, and the like.

[0068] A polynucleotide encoding a GPCR polypeptide may include single-stranded or double-stranded DNA, cDNA, RNA, or other artificial nucleic acids. The DNA, cDNA, and RNA may be chemically synthesized. The polynucleotide may also include the nucleotide sequence of the untranslated region (UTR) in addition to the open reading frame (ORF). The polynucleotide may also have codons optimized according to the type of cells for expressing the GPCR. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0069] In a preferred example, the polynucleotide encoding the GPCR polypeptide consists of the nucleotide sequence represented by any of SEQ ID NOs: 215 to 234 and 313 to 317. Each of the polynucleotides encodes a GPCR polypeptide consisting of the amino acid sequence represented by SEQ ID NOs: 108 to 112, 114 to 120, 138 to 145, 167, 183, 187, 192, and 209, respectively.

[0070] The obtained polynucleotide encoding the GPCR polypeptide can be incorporated into a vector or a DNA fragment. Preferably, the vector is an expression vector. Also preferably, the vector is an expression vector that can introduce the GPCR polynucleotide into a host cell and express the polynucleotide in the host cell. Preferably, the vector may be an extrachromosomal vector such as a plasmid that can autonomously replicate and grow, or a vector that can be integrated into the chromosome. Specific examples of vectors include, but are not limited to, pME18S.

[0071] The vector preferably contains a polynucleotide encoding a GPCR polypeptide and a control region operably linked thereto. The control region is a sequence for expressing the polynucleotide encoding the introduced GPCR polypeptide in a host cell into which the vector has been introduced, and examples thereof include expression regulatory regions such as promoters and terminators, and transcription start points. The type of the control region can be appropriately selected according to the type of the vector. If necessary, the vector or DNA fragment may further have a drug resistance gene such as ampicillin as a selection marker. The control region and the selection marker gene may be those originally contained in the vector, or may be incorporated into the vector together with or separately from the polynucleotide encoding the GPCR polypeptide.

[0072] Examples of the DNA fragment containing the polynucleotide encoding the GPCR polypeptide include a PCR-amplified DNA fragment and a restriction enzyme-digested DNA fragment. Preferably, the DNA fragment can be an expression cassette containing the polynucleotide and a control region operably linked thereto. Examples of the control region that can be used are the same as those in the case of the vector.

[0073] Preferably, to promote the cell membrane expression of a GPCR polypeptide, a polynucleotide encoding an RTP (receptor-transporting protein) (also referred to herein as the RTP gene) is introduced into a cell together with a polynucleotide encoding the GPCR polypeptide. Alternatively, preferably, to promote the cell membrane expression of a GPCR polypeptide, a polynucleotide encoding an REEP (receptor expression enhancing protein) (also referred to herein as the REEP gene) is introduced into a cell together with a polynucleotide encoding the GPCR polypeptide. For example, a vector containing the RTP gene or the REEP gene and a polynucleotide encoding the GPCR polypeptide may be constructed and introduced into a host cell, or a vector containing the RTP gene or the REEP gene and a vector containing a polynucleotide encoding the GPCR polypeptide may be introduced into a host cell respectively. Examples of RTP include RTP1S, RTP3, and RTP4, and an example of RTP1S is human RTP1S. Human RTP1S is registered in GenBank as AY562235 and is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 236, which is encoded by a gene having the nucleotide sequence of SEQ ID NO: 235. Examples of REEP include REEP1 and REEP2, and an example of REEP1 is human REEP1, and an example of REEP2 is human REEP2.

[0074] For the introduction of a vector or a DNA fragment into a host cell, common transformation methods for mammalian cells, such as electroporation, lipofection, particle gun method, etc. can be used. The transformed cells into which the target vector or DNA fragment has been introduced can be selected using a selection marker. Alternatively, the introduction of the target vector or DNA fragment can also be confirmed by examining the sequence of the cell's DNA.

[0075] The GPCR polypeptide of the present invention is produced from a polynucleotide encoding the GPCR polypeptide of the present invention contained in the vector or DNA fragment introduced into cells by the above procedure and is incorporated into the cell membrane. Therefore, the GPCR polypeptide expressed by the expression method of the present invention is expressed on the cell membrane of transformed cells genetically engineered to express the GPCR polypeptide.

[0076] The cell membrane expression (amount) of the GPCR polypeptide of the present invention can be measured, for example, by a known method such as flow cytometry using an antibody that specifically recognizes a tag such as a FLAG tag previously fused to the GPCR polypeptide.

[0077] The GPCR polypeptide expressed by the expression method of the present invention can be efficiently expressed on the cultured cell membrane as compared with the original GPCR. Also, the response of the GPCR polypeptide is considered to reflect the response of the original GPCR. Therefore, in another aspect, the present invention provides a method for measuring the response of a target GPCR. The method includes measuring the response of a GPCR polypeptide expressed by the expression method of the GPCR polypeptide of the present invention. According to the response measurement method of the present invention, the response measurement efficiency of the target GPCR can be improved, and it becomes possible to measure the response of a target GPCR that could not be functionally analyzed due to insufficient expression on the cell membrane of conventional cultured cells.

[0078] The GPCR polypeptide used in the response measurement method of the present invention may be any GPCR polypeptide expressed by the expression method of the present invention. The GPCR polypeptide is as described above. Specific examples of the GPCR polypeptide preferably include a GPCR polypeptide consisting of an amino acid sequence represented by any of SEQ ID NOs: 108 to 214 and 275 to 312, or a combination of a GPCR polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 112 or 113 and a GPCR polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 114. The GPCR polypeptide can be used in any form as long as its responsiveness to a ligand is not lost. For example, the GPCR polypeptide can be used in the form of a transformed cell expressing the GPCR polypeptide or its culture, the membrane of the transformed cell having the GPCR polypeptide, an artificial lipid bilayer having the GPCR polypeptide, and the like. Preferably, as the GPCR polypeptide, a transformed cell expressing the GPCR polypeptide or its culture is used.

[0079] In the response measurement method of the present invention, the measurement of the response of the GPCR polypeptide may be performed by any method known in the art as a method for measuring the response of GPCR. For example, it may be performed by a method for directly or indirectly measuring the amount of intracellular cAMP. For example, when a G protein-coupled receptor is activated by a ligand, it is known to vary the amount of intracellular cAMP by activating or suppressing adenylate cyclase in conjugation with a G protein α subunit classified into the Gs family or a G protein α subunit classified into the Gi family in the cell. On the other hand, when a G protein-coupled receptor is activated by a ligand, it can also conjugate with a protein belonging to the Gq family such as Gαq, Gα16, Gα15 in the cell and increase the amount of intracellular calcium ions. Therefore, the response of the GPCR polypeptide can be measured by using the amount of intracellular calcium ions or the behavior of downstream molecules activated through them as an index. Examples of methods for measuring the amount of cAMP include the ELISA method and reporter gene assay. Examples of methods for measuring the calcium ion concentration include the calcium imaging method and TGFα shedding assay. The TGFα shedding assay is also effective for measuring the signal when the GPCR is conjugated with Gα12 and Gα13 in the cell. Further, as an example of a method using the behavior of downstream molecules activated through the amount of cAMP as an index, the two-electrode membrane potential clamping method for measuring the potential change across the cell membrane through the cystic fibrosis transmembrane conductance regulator CFTR activated by the cAMP signal in Xenopus oocytes is also effective. By using a chimeric protein of a G protein α subunit that is likely to conjugate with the target GPCR and any other G protein α subunit, any of the above response measurement methods can be used. For example, taste receptors can efficiently conjugate with those belonging to the Gi family among various G protein α subunits.Based on this, by using Gα15 (Gα15 / i3, Gα15 / gust) having the C-terminal 5 amino acids of Gαi3 or Gαgust, signals derived from activated taste receptors can be detected as an increase in intracellular calcium ion concentration rather than a decrease in the original intracellular cAMP level. Further, after purifying the GPCR polypeptide from cells expressing the GPCR polypeptide according to the present invention, the binding property with a ligand may be evaluated. The method for evaluating the binding property of the purified GPCR polypeptide with a ligand may be performed by any method known in the art. For example, a method of measuring the heat generated from the GPCR polypeptide under ligand addition conditions, and a method of evaluating the change in the thermal stability of the GPCR polypeptide can be mentioned.

[0080] As described above, the GPCR polypeptide expressed by the expression method of the present invention can be efficiently expressed on the cultured cell membrane as compared with the original GPCR, and the response of the GPCR polypeptide is considered to reflect the response of the original GPCR. Therefore, a ligand of the original GPCR can be searched for using the GPCR polypeptide. Thus, in another aspect, the present invention provides a method for searching for a ligand of a target GPCR. The method includes measuring the response of the GPCR polypeptide expressed by the expression method of the present invention in the presence of a test substance, and selecting the test substance to which the GPCR polypeptide responds. According to the ligand search method of the present invention, the ligand search efficiency of the target GPCR can be improved, and it enables the search for a ligand of a target GPCR that could not be functionally analyzed because the expression in the cell membrane of conventional cultured cells was not sufficient.

[0081] The test substance used in the ligand search method of the present invention is not particularly limited as long as it is a substance for which it is desired to confirm whether it is a ligand of the target GPCR. The test substance may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, etc., or may be a compound, a composition or a mixture.

[0082] The GPCR polypeptide used in the ligand search method of the present invention and its form are the same as the GPCR polypeptide and its form used in the response measurement method of the present invention.

[0083] In the ligand search method of the present invention, a test substance is applied to the GPCR polypeptide expressed by the expression method of the present invention. As a means of applying the test substance to the GPCR polypeptide, examples include a method of adding the test substance to the medium in which the cells expressing the GPCR polypeptide are cultured, but it is not particularly limited.

[0084] In the ligand search method of the present invention, following the addition of the test substance to the GPCR polypeptide, the response of the GPCR polypeptide to the test substance is measured. As a method for measuring the response, the same method as the response measurement method of the present invention can be used.

[0085] Next, based on the measured response of the GPCR polypeptide, the test substance is evaluated. A test substance that causes a response of the GPCR polypeptide can be determined to be a ligand of the GPCR polypeptide, that is, a ligand of the original GPCR. Therefore, in the ligand search method of the present invention, a test substance to which the GPCR polypeptide has responded is selected as a ligand of the original GPCR.

[0086] Preferably, the response of the GPCR polypeptide to the test substance can be evaluated by comparing the response of the GPCR polypeptide (test group) to which the test substance has been added with the response of the GPCR polypeptide in the control group. Examples of the control group include the GPCR polypeptide to which no test substance has been added, the GPCR polypeptide to which a control substance has been added, the GPCR polypeptide to which a lower concentration of the test substance has been added, and the GPCR polypeptide before the addition of the test substance. When the response in the test group is higher than that in the control group, the GPCR polypeptide is evaluated as having responded to the test substance, and the test substance is selected as a ligand of the original GPCR.

[0087] Thus, in one embodiment of the ligand discovery method of the present invention, the responses of the GPCR polypeptide in the presence and absence of the test substance are measured, and then it is determined whether the response in the presence of the test substance is higher than the response in the absence of the test substance. If the response in the presence of the test substance is higher, the test substance is selected as a ligand. In a preferred embodiment, if the response intensity of the GPCR polypeptide in the presence of the test substance is preferably 120% or more, more preferably 150% or more, and even more preferably 200% or more compared to the absence of the test substance, the test substance is selected as a ligand for the original GPCR. In another preferred embodiment, if the response intensity of the GPCR polypeptide in the presence of the test substance is statistically significantly increased compared to the absence of the test substance, the test substance is selected as a ligand for the original GPCR.

[0088] In addition, as described above, the GPCR polypeptide expressed by the expression method of the present invention can be efficiently expressed on the cultured cell membrane as compared with the original GPCR, and the response of the GPCR polypeptide is considered to reflect the response of the original GPCR. Therefore, a substance that controls the recognition of a ligand in the original GPCR can be evaluated and / or selected using the GPCR polypeptide. Accordingly, in another aspect, the present invention provides a method for evaluating and / or selecting a controlling agent for the recognition of a ligand of a target GPCR. The method includes adding a test substance and a target ligand to the GPCR polypeptide expressed by the expression method of the present invention, and measuring the response of the GPCR polypeptide to the ligand. In one example, since the vomeronasal receptor is considered to function as a pheromone receptor, when a vomeronasal receptor polypeptide is used as the GPCR polypeptide, the ligand is preferably a pheromone substance. In another example, when a taste receptor polypeptide is used as the GPCR polypeptide, the ligand is preferably a taste substance (bitter substance, umami substance, or sweet substance). As the ligand of the target GPCR, a ligand selected by the ligand searching method of the present invention is preferable. According to the method for evaluating and / or selecting a controlling agent of the present invention, a substance that can selectively suppress or enhance the recognition of a target ligand can be efficiently evaluated or selected.

[0089] The evaluation and / or selection of the controlling agent of the present invention can be a method performed in vitro or ex vivo.

[0090] The test substance used in the method for evaluating and / or selecting a controlling agent of the present invention is not particularly limited as long as it is a substance desired to be used as a controlling agent for the recognition of a target ligand. The test substance may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, etc., or a compound, a composition or a mixture.

[0091] Regarding the GPCR polypeptide and its forms used in the method for evaluating and / or selecting a control agent of the present invention, they are the same as the GPCR polypeptide and its forms used in the response measurement method of the present invention.

[0092] In the method for evaluating and / or selecting a control agent of the present invention, a test substance and a target ligand are applied to the GPCR polypeptide expressed by the expression method of the present invention. Examples of means for applying a test substance and a target ligand to the GPCR polypeptide include, but are not particularly limited to, a method of adding a test substance and a target ligand to the culture medium for culturing cells expressing the GPCR polypeptide.

[0093] In the method for evaluating and / or selecting a control agent of the present invention, following the addition of a test substance and a target ligand to the GPCR polypeptide, the response of the GPCR polypeptide to the ligand is measured. As a method for measuring the response, the same method as the response measurement method of the present invention can be used.

[0094] Next, based on the measured response, a test substance that suppresses the response of the GPCR polypeptide to the ligand is detected. The detected test substance is selected as an inhibitor of the recognition of the ligand. Alternatively, based on the measured response, a test substance that enhances the response of the GPCR polypeptide to the ligand is detected. The detected test substance is selected as an enhancer of the recognition of the ligand.

[0095] A test substance that suppresses the response of the GPCR polypeptide to the ligand is selected as an inhibitor of the recognition of the ligand. Alternatively, a test substance that enhances the response of the GPCR polypeptide to the ligand is selected as an enhancer of the recognition of the ligand. The effect of the test substance on the response of the GPCR polypeptide to the ligand can be evaluated, for example, by comparing the response of the GPCR polypeptide (test group) to which the test substance is added to the ligand with the response to the ligand in the control group. Examples of the control group include the GPCR polypeptide to which no test substance is added, the GPCR polypeptide to which a control substance is added, the GPCR polypeptide to which a lower concentration of the test substance is added, the GPCR polypeptide before the test substance is added, cells in which the GPCR polypeptide is not expressed, and the like. Preferably, the method for evaluating and / or selecting the inhibitor of the present invention includes measuring the activity of the GPCR polypeptide against the ligand in the presence and absence of the test substance.

[0096] For example, if the response in the test group is suppressed compared to the control group, the test substance can be identified as a substance that suppresses the response of the GPCR polypeptide to the target ligand, that is, a substance that suppresses the response of the original GPCR to the ligand. For example, if the response of the GPCR polypeptide in the test group is suppressed to preferably 60% or less, more preferably 50% or less, and even more preferably 25% or less compared to the control group, the test substance can be identified as a substance that suppresses the response of the GPCR polypeptide to the ligand, that is, a substance that suppresses the response of the original GPCR to the ligand. Alternatively, if the response of the GPCR polypeptide in the test group is statistically significantly suppressed compared to the control group, the test substance can be identified as a substance that suppresses the response of the GPCR polypeptide to the ligand, that is, a substance that suppresses the response of the original GPCR to the ligand.

[0097] Alternatively, for example, if the response in the test group is enhanced compared to the control group, the test substance can be identified as a substance that enhances the response of the GPCR polypeptide to the target ligand, i.e., a substance that enhances the response of the original GPCR to the ligand. For example, if the response of the GPCR polypeptide in the test group is enhanced by preferably 120% or more, more preferably 150% or more, and even more preferably 200% or more compared to the control group, the test substance can be identified as a substance that enhances the response of the GPCR polypeptide to the ligand, i.e., a substance that enhances the response of the original GPCR to the ligand. Alternatively, if the response of the GPCR polypeptide in the test group is statistically significantly enhanced compared to the control group, the test substance can be identified as a substance that enhances the response of the GPCR polypeptide to the ligand, i.e., a substance that enhances the response of the original GPCR to the ligand.

[0098] In one embodiment of the method for evaluating and / or selecting a controlling agent of the present invention, the GPCR polypeptide is a vomeronasal receptor polypeptide expressed by the expression method of the present invention, the ligand of the target GPCR is a pheromone substance, and as a controlling agent for ligand recognition, a pheromone inhibitor or a pheromone enhancer is evaluated and / or selected. In another embodiment of the method for evaluating and / or selecting a controlling agent of the present invention, the GPCR polypeptide is a umami receptor polypeptide (combination of consensus TAS1R1 and consensus TAS1R3) expressed by the expression method of the present invention, the ligand of the target GPCR is a umami substance, and as a controlling agent for ligand recognition, a umami inhibitor or a umami enhancer is evaluated and / or selected. In another embodiment of the method for evaluating and / or selecting a controlling agent of the present invention, the GPCR polypeptide is a sweet receptor polypeptide (combination of consensus TAS1R2 and consensus TAS1R3) expressed by the expression method of the present invention, the ligand of the target GPCR is a sweet substance, and as a controlling agent for ligand recognition, a sweet inhibitor or a sweet enhancer is evaluated and / or selected. In another embodiment of the method for evaluating and / or selecting a controlling agent of the present invention, the GPCR polypeptide is a bitter receptor polypeptide expressed by the expression method of the present invention, the ligand of the target GPCR is a bitter substance, and as a controlling agent for ligand recognition, a bitter inhibitor or a bitter enhancer is evaluated and / or selected.

[0099] The test substance identified by the above procedure is a substance that can control an individual's recognition of the ligand by controlling the response of the GPCR to the target ligand. Therefore, the test substance identified by the above procedure can be selected as a controlling agent for the recognition of the ligand. The substance selected as a controlling agent for the recognition of the target ligand by the method for evaluating and / or selecting a controlling agent of the present invention can control the recognition of the ligand by controlling the response of the GPCR to the ligand.

[0100] Therefore, in one embodiment, a substance selected by the method for evaluating and / or selecting a modulator of ligand recognition of the present invention can be an active ingredient of the modulator of ligand recognition. Alternatively, a substance selected by the method for evaluating and / or selecting a modulator of ligand recognition of the present invention can be contained as an active ingredient for controlling ligand recognition in a compound or composition for controlling ligand recognition. Or alternatively, a substance selected by the method for evaluating and / or selecting a modulator of ligand recognition of the present invention can be used for the production of the modulator of ligand recognition or for the production of a compound or composition for controlling ligand recognition. According to the substance, the recognition of the target ligand can be suppressed or enhanced.

[0101] Also, as described above, the GPCR polypeptide expressed by the expression method of the present invention can be efficiently expressed on the cultured cell membrane as compared with the original GPCR, and the response of the GPCR polypeptide is considered to reflect the response of the original GPCR. Therefore, the taste of a test substance can be evaluated using the taste receptor polypeptide as the GPCR polypeptide. Thus, in another aspect, the present invention provides a method for evaluating taste. The method includes adding a test substance to a GPCR polypeptide expressed by the expression method of the present invention, and measuring the response of the GPCR polypeptide to the test substance, wherein the GPCR polypeptide is a taste receptor polypeptide. According to the taste evaluation method of the present invention, the taste of a test substance can be efficiently evaluated.

[0102] The test substance used in the taste evaluation method of the present invention is not particularly limited as long as it is a substance for which confirmation of the presence or degree of a taste (e.g., umami, sweetness, or bitterness) is desired. The test substance may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, etc., or may be a compound, a composition, or a mixture.

[0103] Regarding the GPCR polypeptide and its forms used in the taste evaluation method of the present invention, as long as the GPCR polypeptide is a taste receptor polypeptide, it is the same as the GPCR polypeptide and its forms used in the response measurement method of the present invention.

[0104] In the taste evaluation method of the present invention, a test substance is applied to the GPCR polypeptide expressed by the expression method of the present invention. As a means of applying the test substance to the GPCR polypeptide, methods such as adding the test substance to the culture medium for culturing the cells expressing the GPCR polypeptide can be mentioned, but it is not particularly limited.

[0105] In the taste evaluation method of the present invention, following the addition of the test substance to the GPCR polypeptide, the response of the GPCR polypeptide to the test substance is measured. As a method for measuring the response, a method similar to the response measurement method of the present invention can be used.

[0106] Next, based on the measured response of the GPCR polypeptide, the test substance is evaluated. A test substance that causes a response of the GPCR polypeptide can be evaluated as a substance presenting a taste (for example, umami, sweetness, or bitterness). The greater the response intensity caused by the test substance, the greater the degree of taste of the test substance (for example, stronger umami, stronger sweetness, or stronger bitterness) is evaluated.

[0107] Preferably, the response of the GPCR polypeptide to the test substance can be evaluated by comparing the response of the GPCR polypeptide (test group) to which the test substance is added with the response of the GPCR polypeptide in the control group. As the control group, the GPCR polypeptide to which no test substance is added, the GPCR polypeptide to which a control substance is added, the GPCR polypeptide to which a lower concentration of the test substance is added, the GPCR polypeptide before adding the test substance, and the like can be mentioned. When the response in the test group is higher than that in the control group, the test substance is evaluated as a substance presenting a taste.

[0108] Therefore, in one embodiment of the taste evaluation method of the present invention, the response of the GPCR polypeptide in the presence and absence of the test substance is measured, and then it is determined whether the response in the presence of the test substance is higher than the response in the absence of the test substance. If the response in the presence of the test substance is higher, the test substance is evaluated as a substance having a taste. In a preferred embodiment, if the response intensity of the GPCR polypeptide in the presence of the test substance is preferably 120% or more, more preferably 150% or more, and even more preferably 200% or more compared to the absence of the test substance, the test substance is evaluated as a substance having a taste. In another preferred embodiment, if the response intensity of the GPCR polypeptide in the presence of the test substance is statistically significantly increased compared to the absence of the test substance, the test substance is evaluated as a substance having a taste.

[0109] In one embodiment of the taste evaluation method of the present invention, the GPCR polypeptide is a umami receptor polypeptide (combination of consensus TAS1R1 and consensus TAS1R3) expressed by the expression method of the present invention, and the taste to be evaluated is umami. In another embodiment of the taste evaluation method of the present invention, the GPCR polypeptide is a sweet receptor polypeptide (combination of consensus TAS1R2 and consensus TAS1R3) expressed by the expression method of the present invention, and the taste to be evaluated is sweetness. In another embodiment of the taste evaluation method of the present invention, the GPCR polypeptide is a bitter receptor polypeptide expressed by the expression method of the present invention, and the taste to be evaluated is bitterness.

[0110] Also, as described above, the GPCR polypeptide expressed by the expression method of the present invention can be efficiently expressed on the cultured cell membrane as compared with the original GPCR, and it is considered that the response of the GPCR polypeptide reflects the response of the original GPCR. Therefore, a substance that suppresses the odor recognition of a ligand (odorant substance, specifically an amine) in the original GPCR can be evaluated and / or selected using the trace amine-related receptor polypeptide as the GPCR polypeptide. A substance that suppresses the response of the GPCR polypeptide causes a change in the ligand response of the GPCR polypeptide, that is, a change in the ligand response of the original GPCR. As a result, the odor of the ligand can be selectively suppressed based on receptor antagonism. On the other hand, a substance that enhances the response of the GPCR polypeptide causes a change in the ligand response of the GPCR polypeptide, that is, a change in the ligand response of the original GPCR. As a result, the odor of the ligand can be selectively suppressed based on cross-adaptation of the odor by receptor agonism.

[0111] Accordingly, in another aspect, the present invention provides a method for evaluating and / or selecting an inhibitor of the odor of a ligand of a target GPCR. The method includes measuring the response of the GPCR polypeptide after addition of a test substance, wherein the GPCR polypeptide is a trace amine-associated receptor polypeptide. Based on the measured response, a test substance that suppresses or enhances the response of the GPCR polypeptide is detected. The detected test substance is selected as an inhibitor of the odor of the target ligand. That is, a test substance that suppresses the response of the GPCR polypeptide is selected as an inhibitor of the odor of the ligand based on receptor antagonism, and a test substance that enhances the response of the GPCR polypeptide is selected as an inhibitor of the odor of the ligand based on cross-adaptation of odor by receptor agonism. As the ligand of the target GPCR, a ligand selected by the ligand search method of the present invention is preferable. According to the method for evaluating and / or selecting an odor inhibitor of the present invention, a substance that can selectively suppress the odor of a target ligand can be efficiently evaluated or selected. Even when the target ligand is a ligand of a GPCR that could not be functionally analyzed because its expression in the cell membrane of conventional cultured cells was insufficient, a substance that can selectively suppress the odor of the ligand can be evaluated or selected.

[0112] The evaluation and / or selection of the odor inhibitor of the present invention can be a method performed in vitro or ex vivo.

[0113] The test substance used in the method for evaluating and / or selecting an odor inhibitor of the present invention is not particularly limited as long as it is a substance desired to be used as an inhibitor of the odor of a target ligand. The test substance may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, etc., or may be a compound, a composition or a mixture.

[0114] Regarding the GPCR polypeptide and its forms used in the method for evaluating and / or selecting an odor inhibitor of the present invention, as long as the GPCR polypeptide is a trace amine-related receptor polypeptide, it is the same as the GPCR polypeptide and its forms used in the response measurement method of the present invention.

[0115] In the method for evaluating and / or selecting an odor inhibitor of the present invention, a test substance is applied to the GPCR polypeptide expressed by the expression method of the present invention. Examples of the means for applying the test substance to the GPCR polypeptide include, but are not particularly limited to, a method of adding the test substance to the culture medium for culturing the cells expressing the GPCR polypeptide.

[0116] In the method for evaluating and / or selecting an odor inhibitor of the present invention, following the addition of the test substance to the GPCR polypeptide, the response of the GPCR polypeptide to the test substance is measured. As the method for measuring the response, a method similar to the response measurement method of the present invention can be used.

[0117] In a first embodiment, the method for evaluating and / or selecting an odor inhibitor of the present invention includes adding a test substance and a target ligand to the GPCR polypeptide expressed by the expression method of the present invention, and measuring the response of the GPCR polypeptide to the ligand. As the method for applying the ligand to the GPCR polypeptide, a method similar to the method for applying the test substance can be used. Then, based on the measured response, a test substance that inhibits the response of the GPCR polypeptide to the ligand is detected. The detected test substance is selected as an odor inhibitor of the ligand.

[0118] In the first embodiment, the test substance that suppresses the response of the GPCR polypeptide to the ligand is selected as an inhibitor of the odor of the ligand based on receptor antagonism. The effect of the test substance on the response of the GPCR polypeptide to the ligand can be evaluated, for example, by comparing the response of the GPCR polypeptide (test group) to which the test substance is added with the response of the ligand in the control group. Examples of the control group include the GPCR polypeptide to which no test substance is added, the GPCR polypeptide to which a control substance is added, the GPCR polypeptide to which a lower concentration of the test substance is added, the GPCR polypeptide before the test substance is added, cells in which the GPCR polypeptide is not expressed, and the like. Preferably, the method for evaluating and / or selecting the odor inhibitor of the present invention in the first embodiment includes measuring the activity of the olfactory GPCR polypeptide against the ligand in the presence and absence of the test substance.

[0119] For example, when the response in the test group is suppressed more than that in the control group, the test substance can be identified as a substance that suppresses the response of the GPCR polypeptide to the target ligand, that is, a substance that suppresses the response of the original GPCR to the ligand. For example, if the response of the GPCR polypeptide in the test group is suppressed to preferably 60% or less, more preferably 50% or less, and even more preferably 25% or less compared to the control group, the test substance can be identified as a substance that suppresses the response of the GPCR polypeptide to the ligand, that is, a substance that suppresses the response of the original GPCR to the ligand. Alternatively, if the response of the GPCR polypeptide in the test group is statistically significantly suppressed compared to the control group, the test substance can be identified as a substance that suppresses the response of the GPCR polypeptide to the ligand, that is, a substance that suppresses the response of the original GPCR to the ligand.

[0120] In a second embodiment, a method for evaluating and / or selecting an odor inhibitor of the present invention includes adding a test substance to a GPCR polypeptide expressed by the expression method of the present invention, and measuring the response of the GPCR polypeptide to the test substance. Then, based on the measured response, a test substance that enhances the response of the GPCR polypeptide to a target ligand is detected. The detected test substance is selected as an inhibitor of the odor of the ligand.

[0121] A test substance that enhances the response of the GPCR polypeptide can weaken the response of the GPCR when later exposed to the target ligand by first enhancing the response of the GPCR. As a result, based on odor cross-adaptation, the recognition of the odor of the ligand by an individual can be suppressed. Therefore, in the second embodiment, an inhibitor of the odor of the ligand based on odor cross-adaptation by receptor agonism is selected.

[0122] The action of a test substance on the GPCR polypeptide can be evaluated, for example, by comparing the response of the GPCR polypeptide (test group) to which the test substance is added with the response in the control group. Examples of the control group include those described above. Preferably, the method for evaluating and / or selecting an odor inhibitor of the present invention in the second embodiment includes measuring the activity of the GPCR polypeptide in the presence and absence of the test substance. Also preferably, the method for evaluating and / or selecting an odor inhibitor of the present invention in the second embodiment includes measuring the response of cells expressing and not expressing the GPCR polypeptide to the ligand in the presence of the test substance.

[0123] For example, if the response in the test group is enhanced compared to the control group, the test substance can be identified as a substance that suppresses the response of the GPCR polypeptide to the target ligand, i.e., a substance that suppresses the response of the original GPCR to the ligand. For example, if the response of the GPCR polypeptide in the test group is enhanced by preferably 120% or more, more preferably 150% or more, and even more preferably 200% compared to the control group, the test substance can be identified as a substance that suppresses the response of the GPCR polypeptide to the ligand, i.e., a substance that suppresses the response of the original GPCR to the ligand. Alternatively, if the response of the GPCR polypeptide in the test group is statistically significantly enhanced compared to the control group, the test substance can be identified as a substance that suppresses the response of the GPCR polypeptide to the ligand, i.e., a substance that suppresses the response of the original GPCR to the ligand.

[0124] The test substance identified by the above procedure is a substance that can suppress the recognition of the odor of the ligand by an individual by suppressing the response of the GPCR to the target ligand. Therefore, the test substance identified by the above procedure can be selected as an inhibitor of the odor of the ligand. A substance selected as an inhibitor of the odor of a target ligand by the method for evaluating and / or selecting an odor inhibitor of the present invention can suppress the odor of the ligand by suppressing the response of the GPCR to the ligand.

[0125] Therefore, in one embodiment, a substance selected by the method for evaluating and / or selecting an odor inhibitor of the ligand of the present invention can be an active ingredient of an odor inhibitor of the ligand. Alternatively, a substance selected by the method for evaluating and / or selecting an odor inhibitor of the ligand of the present invention can be contained as an active ingredient for suppressing the odor of the ligand in a compound or composition for suppressing the odor of the ligand. Or alternatively, a substance selected by the method for evaluating and / or selecting an odor inhibitor of the ligand of the present invention can be used for the production of an odor inhibitor of the ligand or for the production of a compound or composition for suppressing the odor of the ligand. According to the substance, problems such as discomfort based on the strong odor of the fragrance that occurred in the conventional deodorizing method using a deodorant or fragrance, or the problem of suppressing other odors do not occur, and the odor of the target ligand can be deodorized.

[0126] As exemplary embodiments of the present invention, the following compositions, production methods, uses or methods are further disclosed herein. However, the present invention is not limited to these embodiments.

[0127] 〔1〕A method for expressing a GPCR polypeptide, comprising: expressing in a cell a GPCR polypeptide consisting of an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence in the amino acid sequence of a target GPCR (except for olfactory receptors) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, wherein the consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequences of GPCRs encoded by orthologs of the target GPCR in vertebrates, method. 〔2〕The method according to 〔1〕, which is a method for improving the expression of a GPCR polypeptide. 〔3〕A method for functionalizing a target GPCR, comprising: Expressing in a cell a GPCR polypeptide comprising an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence in the amino acid sequence of a target GPCR (except for olfactory receptors) is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, wherein the consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequences of GPCRs encoded by orthologs of the target GPCR in vertebrates, Method. 〔4〕The method according to any one of 〔1〕~〔3〕, wherein the ortholog is an ortholog selected from orthologs in mammals, birds, reptiles, amphibians and fish, preferably an ortholog selected from orthologs in mammals, birds, reptiles and amphibians, more preferably an ortholog in mammals. 〔5〕The method according to any one of 〔1〕~〔4〕, wherein the consensus amino acid sequence is an amino acid sequence consisting of consensus residues identified from the alignment according to the following criteria (i)~(iii): (i) At each amino acid position of the alignment, (i-i) When there is one amino acid residue that is different from the amino acid residue of the target GPCR and has an occurrence frequency of 50% or more, the amino acid residue is identified as the consensus residue, (i-ii) When there are two amino acid residues with an occurrence frequency of 50%, the amino acid residue of the target GPCR is identified as the consensus residue, (i-iii) When there is an amino acid residue in the target GPCR and there is no amino acid residue with an occurrence frequency of 40% or more, it is identified as having no consensus residue, (i-iv) When there is no amino acid residue in the target GPCR and there is an amino acid residue with an occurrence frequency of 60% or more, the amino acid residue with the highest occurrence frequency is identified as the consensus residue. When there are two or more amino acid residues with the highest occurrence frequency, the amino acid residue with the smallest molecular weight among the amino acid residues is identified as the consensus residue, (i-v) If it does not fall under any of (i-i) to (i-iv) above, identify the amino acid residue of the target GPCR as a consensus residue. (ii) When identifying a consensus residue according to the criteria in (i) above, if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminus of the target GPCR or a position C-terminal thereto and is not a methionine residue, change the consensus residue N-terminal to the consensus residue consisting of the methionine residue closest to the N-terminus to a non-consensus residue. (iii) When identifying a consensus residue according to the criteria in (i) above, if the most N-terminal consensus residue is a consensus residue at a position corresponding to a position N-terminal to the N-terminus of the target GPCR and is not a methionine residue, trace back one amino acid position at a time from the position of the consensus residue of the alignment to the N-terminal side until a methionine residue appears, and identify the amino acid residue with the highest frequency of occurrence as the consensus residue. When there are two or more amino acid residues with the highest frequency of occurrence, identify the amino acid residue with the smallest molecular weight among the amino acid residues as the consensus residue. [6] The method according to any one of [1] to [5], wherein the alignment is an alignment of the amino acid sequence of the target GPCR with the amino acid sequences of GPCRs encoded by orthologs of the target GPCR in the vertebrate, and there are at least two, preferably at least five, more preferably at least eleven, still more preferably at least fifteen, still more preferably at least thirty, still more preferably at least one hundred GPCRs. [7] The method according to any one of [1] to [6], wherein the target GPCR is a human GPCR. [8] The method according to any one of [1] to [7], wherein the target GPCR is a vomeronasal receptor, a taste receptor, a trace amine-associated receptor, a Mas-related G protein-coupled receptor, or a GPR. 〔9〕The method according to any one of 〔1〕~〔8〕, wherein the GPCR polypeptide preferably comprises an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: (3) in the amino acid sequence of the GPCR of (1) in Tables 2-1 and 2-2 below is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position.

[0128]

Table 2-1

[0129]

Table 2-2

[0130] 〔10〕The method according to 〔9〕, wherein the GPCR polypeptide preferably comprises an amino acid sequence represented by any of SEQ ID NOs: 108~142, 144~214, and 275~312. 〔11〕A method for expressing a GPCR polypeptide, comprising expressing in a cell a GPCR polypeptide comprising an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position in the amino acid sequence of the target GPCR, wherein the GPCR polypeptide comprises an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence represented by SEQ ID NO: 143 in the amino acid sequence represented by SEQ ID NO: 36 of human TAAR6 is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and preferably comprises the amino acid sequence represented by SEQ ID NO: 143. Method. 〔12〕The method according to 〔11〕, which is a method for improving the expression of a GPCR polypeptide. 〔13〕A method for functionalizing a target GPCR, Expressing in a cell a GPCR polypeptide comprising an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence in the amino acid sequence of a target GPCR is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, wherein the GPCR polypeptide consists of an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence shown by SEQ ID NO: 143 in the amino acid sequence shown by SEQ ID NO: 36 of human TAAR6 is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and preferably consists of the amino acid sequence shown by SEQ ID NO: 143, Method. 〔14〕The method according to any one of 〔1〕~〔13〕, wherein the GPCR polypeptide is expressed on the cell membrane of the cell. 〔15〕Preferably, the method according to any one of 〔1〕~〔14〕, further comprising expressing RTP1S in the cell. 〔16〕The method according to any one of 〔1〕~〔15〕, wherein the cell is a HEK293 cell.

[0131] 〔17〕A method for measuring the response of a target GPCR, measuring the response of the GPCR polypeptide expressed by the method according to any one of 〔1〕~〔16〕, comprising the method. 〔18〕A method for screening for a ligand of a target GPCR, measuring the response of the GPCR polypeptide expressed by the method according to any one of 〔1〕~〔16〕 in the presence of a test substance, and selecting the test substance to which the GPCR polypeptide responds , comprising the method. 〔19〕Preferably, the method according to 〔18〕, further comprising measuring the response of the GPCR polypeptide in the absence of the test substance. 〔20〕Preferably, the method according to 〔19〕, selecting a test substance that increases the response of the GPCR polypeptide in the presence of the test substance to 120% or more of the response in the absence of the test substance. 〔21〕Preferably, the method according to

[19] , which comprises selecting a test substance that statistically significantly increases the response of the GPCR polypeptide in the presence of the test substance as compared to the response in the absence of the test substance. 〔22〕A method for evaluating and / or selecting a controller of the recognition of a ligand of a target GPCR, comprising: 〔1〕Adding a test substance and a ligand of the target GPCR to a GPCR polypeptide expressed by the method according to any one of [1] to

[16] , and Measuring the response of the GPCR polypeptide to the ligand. A method comprising the above steps. 〔23〕The method according to

[22] , wherein the ligand is a ligand selected by the method according to any one of

[18] to

[21] . 〔24〕The method according to any one of

[22] or

[23] , further comprising identifying a test substance that controls the response of the GPCR polypeptide, preferably a test substance that inhibits or enhances the response, based on the measured response. 〔25〕The method according to any one of

[22] to

[24] , further comprising measuring the response of the GPCR polypeptide to the ligand in the absence of the test substance. 〔26〕When the response of the GPCR polypeptide to the ligand in the presence of the test substance is inhibited as compared to the response of the GPCR polypeptide to the ligand in the absence of the test substance, identifying the test substance as a substance that inhibits the response of the GPCR polypeptide to the ligand, or when the response of the GPCR polypeptide to the ligand in the presence of the test substance is enhanced as compared to the response of the GPCR polypeptide to the ligand in the absence of the test substance, identifying the test substance as a substance that enhances the response of the GPCR polypeptide to the ligand. The method according to

[25] , further comprising the above steps. 〔27〕A method for evaluating taste, comprising: 〔1〕Adding a test substance to a GPCR polypeptide expressed by the method according to any one of [1] to

[16] , and Measuring the response of the GPCR polypeptide to the test substance, comprising a GPCR polypeptide that is a taste receptor polypeptide, method.

[28] Preferably, the method according to

[27] , further comprising measuring the response of the GPCR polypeptide without adding the test substance.

[29] Preferably, a test substance that increases the response of the GPCR polypeptide to which the test substance is added to 120% or more of the response of the GPCR polypeptide to which the test substance is not added is evaluated as a substance having a taste, according to the method of

[28] .

[30] Preferably, a test substance that statistically significantly increases the response of the GPCR polypeptide to which the test substance is added as compared to the response of the GPCR polypeptide to which the test substance is not added is evaluated as a substance having a taste, according to the method of

[28] .

[31] Preferably, the method according to any one of

[27] to

[30] , wherein the taste is umami, sweetness, or bitterness.

[32] A method for evaluating and / or selecting an inhibitor of the odor of a ligand of a target GPCR, [1] Adding a test substance and a ligand of the target GPCR to a GPCR polypeptide expressed by the method according to any one of [1] to

[16] , and measuring the response of the GPCR polypeptide to the ligand, comprising a GPCR polypeptide that is a trace amine-related receptor polypeptide, method.

[33] The method according to

[32] , wherein the ligand is a ligand selected by the method according to any one of

[18] to

[21] .

[34] The method according to

[32] or

[33] , further comprising identifying a test substance that suppresses the response of the GPCR polypeptide based on the measured response.

[35] The method according to any one of

[32] to

[34] , further comprising measuring the response of the GPCR polypeptide to the ligand without adding the test substance. 〔36〕The method according to

[35] , further comprising identifying the test substance as a substance that inhibits the response of the GPCR polypeptide to the ligand when the response of the GPCR polypeptide to which the test substance is added to the ligand is inhibited compared to the response of the GPCR polypeptide to which the test substance is not added to the ligand. 〔37〕A method for evaluating and / or selecting an inhibitor of the odor of a ligand of a target GPCR, comprising: (1) adding a test substance to a GPCR polypeptide expressed by the method according to any one of [1] to

[16] , and (2) measuring the response of the GPCR polypeptide to the test substance, wherein the GPCR polypeptide is a trace amine-associated receptor polypeptide. Method. 〔38〕The method according to

[37] , wherein the ligand is a ligand selected by the method according to any one of

[18] to

[21] . 〔39〕The method according to

[37] or

[38] , further comprising identifying a test substance that enhances the response of the GPCR polypeptide based on the measured response. 〔40〕The method according to any one of

[37] to

[39] , further comprising measuring the response of the GPCR polypeptide without adding the test substance. 〔41〕The method according to

[40] , further comprising identifying the test substance as a substance that inhibits the response of the GPCR polypeptide to the ligand when the response of the GPCR polypeptide to which the test substance is added is enhanced compared to the response of the GPCR polypeptide to which the test substance is not added. 〔42〕Preferably, the response of the GPCR polypeptide is measured by measuring the intracellular cAMP level by ELISA or reporter gene assay, measuring the calcium ion level by calcium imaging or TGFα shedding assay, or measuring the potential change across the cell membrane by the two-electrode voltage clamp method using Xenopus oocytes. The method according to any one of

[17] to

[41] .

[0132]

[43] A modified GPCR polypeptide, comprising at least one amino acid residue different from the consensus amino acid residue at the corresponding position in the amino acid sequence of the target GPCR (excluding olfactory receptors) modified to the amino acid residue of the consensus amino acid sequence at that position, wherein the consensus amino acid sequence is an amino acid sequence derived from an alignment of the amino acid sequence of the target GPCR and the amino acid sequences of GPCRs encoded by orthologs of the target GPCR in vertebrates, Modified GPCR polypeptide.

[44] The modified GPCR polypeptide according to

[43] , wherein the ortholog is an ortholog selected from orthologs in mammals, birds, reptiles, amphibians and fish, preferably an ortholog selected from orthologs in mammals, birds, reptiles and amphibians, more preferably an ortholog in mammals.

[45] The modified GPCR polypeptide according to

[43] or

[44] , wherein the consensus amino acid sequence is an amino acid sequence consisting of consensus residues identified from the alignment according to the following criteria (i) to (iii): (i) At each amino acid position of the alignment, (i-i) When there is one amino acid residue different from the amino acid residue of the target GPCR and having an occurrence frequency of 50% or more, the amino acid residue is identified as the consensus residue, (i-ii) When there are two amino acid residues having an occurrence frequency of 50%, the amino acid residue of the target GPCR is identified as the consensus residue, (i-iii) When there is an amino acid residue in the target GPCR and there is no amino acid residue with an occurrence frequency of 40% or more, it is identified as having no consensus residue, (i-iv) When there is no amino acid residue in the target GPCR and there is an amino acid residue with an occurrence frequency of 60% or more, the amino acid residue with the highest occurrence frequency is identified as the consensus residue. When there are two or more amino acid residues with the highest occurrence frequency, the amino acid residue with the smallest molecular weight among the amino acid residues is identified as the consensus residue. (i-v) If it does not fall under any of (i-i) to (i-iv) above, identify the amino acid residue of the target GPCR as a consensus residue. (ii) When identifying consensus residues according to the criteria in (i) above, if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminus of the target GPCR or a position C-terminal thereto and is not a methionine residue, change the consensus residues N-terminal to the consensus residue consisting of the methionine residue closest to the N-terminus to non-consensus residues. (iii) When identifying consensus residues according to the criteria in (i) above, if the most N-terminal consensus residue is a consensus residue at a position corresponding to a position N-terminal to the N-terminus of the target GPCR and is not a methionine residue, trace back one amino acid position at a time from the position of the consensus residue of the alignment to the N-terminal side until a methionine residue appears, identify the amino acid residue with the highest frequency of occurrence as the consensus residue, and if there are two or more amino acid residues with the highest frequency of occurrence, identify the amino acid residue with the smallest molecular weight among the amino acid residues as the consensus residue.

[46] The modified GPCR polypeptide according to any one of

[43] to

[45] , wherein the alignment is an alignment of the amino acid sequence of the target GPCR and the amino acid sequences of GPCRs encoded by orthologs of the target GPCR in the vertebrate, with at least two, preferably at least five, more preferably at least eleven, still more preferably at least fifteen, still more preferably at least thirty, and still more preferably at least one hundred GPCRs.

[47] The modified GPCR polypeptide according to any one of

[43] to

[46] , wherein the target GPCR is a human GPCR.

[48] The modified GPCR polypeptide according to any one of

[43] to

[47] , wherein the target GPCR is a vomeronasal receptor, a taste receptor, a trace amine-associated receptor, a Mas-related G protein-coupled receptor, or a GPR. [〔49〕Preferably, it is a modified GPCR polypeptide according to any one of 〔43〕to 〔48〕, which comprises an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence represented by the sequence number (3) in the amino acid sequence represented by the sequence number (2) of the GPCR in the above Tables 2-1 and 2-2 is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position.] [〔50〕Preferably, it is the modified GPCR polypeptide according to 〔49〕, which comprises an amino acid sequence represented by any one of sequence numbers 108 to 142, 144 to 214, and 275 to 312.] [〔51〕A modified GPCR polypeptide,] which comprises an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence represented by sequence number 143 in the amino acid sequence represented by sequence number 36 of human TAAR6 is modified to the amino acid residue of the consensus amino acid sequence at the corresponding position, and preferably comprises the amino acid sequence represented by sequence number 143.] [Modified GPCR polypeptide.] [〔52〕A polynucleotide encoding the modified GPCR polypeptide according to any one of 〔43〕to 〔51〕.] [〔53〕The polynucleotide according to 〔52〕, which comprises a base sequence represented by any one of sequence numbers 215 to 234 and 313 to 317.] [〔54〕A vector or DNA fragment containing the polynucleotide according to 〔52〕 or 〔53〕.] [〔55〕A transformed cell containing the vector or DNA fragment according to 〔54〕.] [〔56〕Preferably, the transformed cell according to 〔55〕 further comprises a vector or DNA fragment containing a polynucleotide encoding RTP1S.] [〔57〕The transformed cell according to 〔55〕 or 〔56〕, wherein the cell is a HEK293 cell.] [EXAMPLES]

[0133] Hereinafter, examples are shown to more specifically explain the present invention.]

[0134] Example 1: Preparation and Analysis of Consensus Receptor 1) Preparation of Receptor Gene When designing a consensus receptor, the search for homologous gene candidates of the target receptor gene was performed using NCBI BLAST. Ortholog groups were identified for the obtained gene groups. Specifically, when the human receptor (GPCR) was the target receptor, in the identification of orthologs, from the genes with the highest homology searched by BLAST, the gene with the same name as the target receptor was selected as the ortholog. For example, in the case of the human trace amine-associated receptor TAAR1, using the amino acid sequence of human TAAR1 (NP_612200.1) as the query sequence, 249 genes containing TAAR1 in their names were identified as orthologs from among the top 250 genes with the highest homology searched by BLAST. Alignment analysis and identification of consensus amino acids were performed on the amino acid sequences of these 250 genes (Tables 3-1 to 4) including human TAAR1 as described below. For human TAAR2, TAAR5, TAAR6, TAAR8, and TAAR9, ortholog groups were identified in the same manner. When human TAAR6, TAAR8, or TAAR9 was the target receptor, the identified orthologs were orthologs of primates and mammals of orders other than primates. When TAAR2 or TAAR5 was the target receptor, the identified orthologs were orthologs of primates, mammals of orders other than primates, and birds. When TAAR1 was the target receptor, the identified orthologs were orthologs of primates, mammals of orders other than primates, birds, reptiles, and amphibians. For example, in the case of VN1R1, which is a human vomeronasal receptor, when a BLAST search is performed using the amino acid sequence of human VN1R1 (NP_065684.1) as the query sequence, 22 genes containing vomeronasal type-1 receptor 1 in their names were identified among the top 250 genes with high homology. VN1R is a receptor gene family with high diversity among homologous genes. Among these homologous genes, 7 genes having 65% or more amino acid identity with human VN1R1 were identified as orthologs. Alignment analysis and determination of consensus amino acids were performed on the amino acid sequences of a total of 8 genes including human VN1R1 among these 7 genes as described below. For human VN1R2, VN1R4, and VN1R5, ortholog groups were also identified in the same manner. When VN1R1, VN1R2, VN1R4, or VN1R5 is the target receptor, the identified orthologs were orthologs of mammals (primates). For example, in the case of TAS1R1, which is a human taste receptor (umami taste receptor), 249 genes containing TAS1R1 in their names were identified as orthologs from among the top 250 genes retrieved by BLAST using the amino acid sequence of human TAS1R1 (NP_619642.2) as the query sequence. Alignment analysis and determination of consensus amino acids were performed on the amino acid sequences of a total of 250 genes including human TAS1R1 among these 249 genes as described below. For human TAS1R2 and TAS1R3, ortholog groups were also identified in the same manner. When TAS1R1, TAS1R2, or TAS1R3 is the target receptor, the identified orthologs were orthologs of primates, orthologs of mammals of orders other than primates, orthologs of birds, orthologs of reptiles, orthologs of amphibians, and orthologs of fish. For example, in the case of TAS2R8, which is a human taste receptor (bitter taste receptor), using the amino acid sequence of human TAS2R8 (NP_076407.1) as the query sequence, 90 genes containing TAS2R8 in their names were identified as orthologs from among the top 250 genes with the highest homology retrieved by BLAST. For the amino acid sequences of a total of 91 genes, including human TAS2R8 added to these 90 genes, alignment analysis and identification of consensus amino acids were performed as described below. For human TAS2R16, TAS2R38, TAS2R42, TAS2R45, TAS2R46, TAS2R31, TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7, TAS2R9, TAS2R10, TAS2R13, TAS2R14, TAS2R20, TAS2R30, TAS2R39, TAS2R40, TAS2R43, TAS2R50, and TAS2R60, ortholog groups were similarly identified. When human TAS2R45 was the target receptor, the identified orthologs were primate orthologs. When human TAS2R8, TAS2R16, TAS2R38, TAS2R42, or TAS2R46 was the target receptor, the identified orthologs were primate orthologs and orthologs of mammals of orders other than primates. For example, in the case of MrgprE, which is a human Mas-related G protein-coupled receptor, using the amino acid sequence of human MrgprE (NP_001034254.2) as the query sequence, 72 genes containing MrgprE in their names were identified as orthologs from among the top 250 genes with the highest homology retrieved by BLAST. For the amino acid sequences of a total of 73 genes, including human MrgprE added to these 72 genes, alignment analysis and identification of consensus amino acids were performed as described below. For human MrgprF, MAS1, MAS1L, MRGPRD, MRGPRG, MRGPRX1, MRGPRX2, MRGPRX3, and MRGPRX4, ortholog groups were similarly identified. When human MrgprE or MrgprF was the target receptor, the identified orthologs were primate orthologs and orthologs of mammals of orders other than primates. For example, in the case of human GPR3, the amino acid sequence of human GPR3 (NP_005272.1) was used as the query sequence, and 234 genes containing GPR3 in their names were identified as orthologs from among the top 250 genes with high homology retrieved by BLAST. Alignment analysis and identification of consensus amino acids were performed on the amino acid sequences of a total of 235 genes, including human GPR3 added to these 234 genes, as described below. For human GPRs other than GPR3, GPR17, GPR31, GPR50, GPR65, GPR68, and GPR42 listed in Table 4, ortholog groups were similarly identified. In the case of human GPR17, genes containing "uracil nucleotide / cysteinyl leukotriene receptor", known as an ortholog in its name, were identified as orthologs. Similarly, in the case of human GPR31, genes containing "12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid receptor" in their names, in the case of human GPR50, genes containing "melatonin-related receptor" in their names, in the case of human GPR65, genes containing "psychosine receptor" in their names, and in the case of human GPR68, genes containing "ovarian cancer G-protein coupled receptor 1" in their names were each identified as orthologs. In the case of human GPR42, genes containing "GPR42" in their names and genes containing "free fatty acid receptor 3" in their names (GPCR genes highly homologous to the GPR42 gene) were identified as orthologs. Among human GPCRs other than those described above, in the case of ADGRB1, using the amino acid sequence of human ADGRB1 (NP_001693.2) as the query sequence, 234 genes containing ADGRB1 in their names were identified as orthologs from among the top 250 genes with the highest homology retrieved by BLAST. Alignment analysis and identification of consensus amino acids were performed on the amino acid sequences of a total of 235 genes, including human ADGRB1, among these 234 genes. For human ADGRB2, ADGRB3, and ADGRD1, ortholog groups were similarly identified. In the case of human ADGRA1, genes containing "ADGRA1" in their names and genes containing "GPR123" in their names (GPCR genes with high homology to the ADGRA1 gene) were identified as orthologs. Similarly, in the case of human ADGRA2, genes containing "ADGRA2" in their names and genes containing "GPR124" in their names were identified as orthologs, and in the case of human ADGRA3, genes containing "ADGRA3" in their names and genes containing "GPR125" in their names were identified as orthologs. In the case of human ADGRC1, a gene containing "EGF LAG seven-pass G-type receptor 1" in its name (a GPCR gene with high homology to the ADGRC1 gene) was identified as an ortholog. Similarly, in the case of human ADGRC2, a gene containing "cadherin EGF LAG seven-pass G-type receptor 2" in its name was identified as an ortholog, in the case of human ADGRC3, a gene containing "cadherin EGF LAG seven-pass G-type receptor 3" in its name was identified as an ortholog, in the case of human ADGRD2, a gene containing "adhesion G protein-coupled receptor D2" or "G-protein coupled receptor 144" in its name was identified as an ortholog, in the case of human ADGRE1, a gene containing "adhesion G protein-coupled receptor E1" or "adhesion G protein-coupled receptor E1 isoform 2 precursor" in its name was identified as an ortholog, and in the case of human ADGRE2, a gene containing "adhesion G protein-coupled receptorA gene containing "E2", in the case of human ADGRE3, a gene containing "adhesion G protein-coupled receptor E3" in its name, in the case of human ADGRE5, a gene containing "adhesion G protein-coupled receptor E5" or "CD97 antigen" in its name, in the case of human ADGRF1, a gene containing "adhesion G-protein coupled receptor F1" or "adhesion G-protein coupled receptor F1 isoform 1 precursor" in its name, in the case of human ADGRF3, a gene containing "adhesion G-protein coupled receptor F3" or "G-protein coupled receptor 113" in its name, in the case of human ADGRF4, a gene containing "adhesion G-protein coupled receptor F4" or "G-protein coupled receptor 115" in its name, in the case of human ADGRF5, a gene containing "adhesion G-protein coupled receptor F5" or "G-protein coupled receptor 116" in its name, in the case of human ADGRG1, a gene containing "adhesion G-protein coupled receptor G1" or "adhesion G-protein coupled receptor G1 isoform b precursor" in its name, in the case of human ADGRG2, a gene containing "adhesion G-protein coupled receptor G2" or "G-protein coupled receptor 64" in its name, in the case of human ADGRG4, a gene containing "adhesion G-protein coupled receptor G4" or "G-protein coupled receptor 112" in its name, in the case of human ADGRG5, a gene containing "adhesion G-protein coupled receptor G5" or "G-protein coupled receptorA gene containing "114", for human ADGRG6, a gene containing "adhesion G-protein coupled receptor G6" or "G-protein coupled receptor 126" in the name; for human ADGRG7, a gene containing "adhesion G-protein coupled receptor G7" or "adhesion G-protein coupled receptor G7 isoform 2 precursor" in the name; for human ADGRL1, a gene containing "adhesion G protein-coupled receptor L1" or "adhesion G protein-coupled receptor L1 isoform 1 precursor" in the name; for human ADGRL2, a gene containing "adhesion G-protein coupled receptor L2" or "latrophilin and seven transmembrane domain-containing protein" in the name; for human ADGRL3, a gene containing "adhesion G-protein coupled receptor L3" or "latrophilin-3" in the name; for human ADGRL4, a gene containing "adhesion G-protein coupled receptor L4" or "latrophilin-2 isoform" in the name; for human Chrm-4 / M4R, a gene containing "M4R" in the name; for human F2RL1, a gene containing "proteinase-activated receptor 2" in the name; for human GRM5, a gene containing "metabotropic glutamate receptor 5" in the name; for human APLNR, a gene containing "apelin receptor" in the name; for human CALCRL, a gene containing "calcitonin gene-related peptide type 1" in the name; for human GLP2R, a gene containing "glucagon-like peptide 2 receptor" in the name; for human MC4R, a gene containing "MC4R", "Melanocortin receptorGenes containing "4", "Melanocortin-4 receptor", or "Melanocortin 4 receptor" were identified as orthologs, and in the case of human CCR6, the gene containing "C-C chemokine receptor type 6" in its name was identified as an ortholog. Table 4 shows the total number of genes of each human GPCR and the orthologs identified for that human GPCR as the reference gene number.

[0135]

Table 3-1

[0136]

Table 3-2

[0137]

Table 3-3

[0138]

Table 3-4

[0139]

Table 4

[0140] Regarding the GPCRs numbered 1 to 145 in Table 4, the alignment analysis of the identified gene groups was performed using ClustalW. Based on the alignment results, the consensus receptor was designed using Jalview. In this alignment, when there was one amino acid residue different from the amino acid residue of the reference amino acid sequence at the position corresponding to each amino acid position of the original receptor amino acid sequence of the target receptor used as a reference and the occurrence frequency was 50% or more, the amino acid residue of the reference amino acid sequence was modified to that amino acid residue. In addition, even when there was one amino acid residue different from the amino acid residue of the reference amino acid sequence at the position corresponding to each amino acid position of the original receptor amino acid sequence of the target receptor used as a reference and the occurrence frequency was 50%, and the occurrence frequency of the amino acid residue of the reference amino acid sequence was also 50%, the amino acid residue of the reference amino acid sequence was not modified. In this alignment, when there was a deletion with an occurrence frequency of 40% or more at the position corresponding to each amino acid position of the original receptor amino acid sequence of the target receptor used as a reference, the amino acid residue of the reference amino acid sequence was modified to a deletion. For example, since there was a deletion with an occurrence frequency of 40% or more at the position corresponding to the amino acid position at the C-terminus like TAAR1, the amino acid residue of the reference amino acid sequence was modified to a deletion. Further, for example, since a deletion with an occurrence frequency of 40% or more was observed at the position corresponding to the start methionine position in the human sequence like TAAR1, TAAR2, MAS1L, GPR135, and ADGRG4, the amino acid residue of the reference amino acid sequence was modified to a deletion, and further, in the amino acid sequence after modification by the above procedure, the first methionine was selected as the start methionine, and the amino acid sequence before that was deleted. However, in this method, when TAAR6 changed the consensus residue on the N-terminal side of the consensus residue consisting of the methionine residue closest to the N-terminus to no consensus residue, the full length of the amino acid sequence of the resulting consensus receptor became 10% or more shorter than the full length of the amino acid sequence of the original receptor, so this method was not followed, and instead, the N-terminal structure of the original receptor was maintained as it was.Specifically, since the most N-terminal consensus residue asparagine was an amino acid residue important for glycosylation modification and membrane translocation, the consensus residue was not changed, and the N-terminal structure of the original receptor N-terminal to the position corresponding to the consensus residue was maintained as it was. On the other hand, in the alignment, when an amino acid exists at a frequency of 60% or more at a position corresponding to the deletion position of the original receptor amino acid sequence of the target receptor as a reference, the most conserved amino acid was inserted at the deletion position of the reference amino acid sequence for modification. When there are two or more most conserved amino acids, it was modified to insert the amino acid with the smallest molecular weight. For example, since an amino acid exists at a frequency of 60% or more N-terminal to the position corresponding to the amino acid position at the N-terminus like TAS2R1, an amino acid residue was added N-terminal to the N-terminus of the reference amino acid sequence. For the confirmation of the topology of the receptor in the design, TMHMM (Transmembrane Hidden Markov Model) was used. Each designed receptor needs to have a seven-transmembrane structure. Regarding the GPCRs of No. 1 to 5, 7 to 13, 31 to 38, 60, 76, 80, 85 and 102 in Table 4, the DNA sequences encoding the various designed receptor polypeptides were optimized for the expression in human cultured cells with the base sequence codons corresponding to their amino acid sequences and then obtained by DNA synthesis. EcoRI and XhoI sites were added to both ends of this base sequence, and it was recombined into the EcoRI and XhoI sites created downstream of the Flag-Rho tag sequence on the pME18S vector. The gene encoding human RTP1S, which translocates the GPCR protein produced in cultured cells to the cell membrane, was incorporated into the EcoRI and XhoI sites of another pME18S vector to prepare a pME18S-RTP1S vector.

[0141] 2) Preparation of Cultured Cells Transformed with Receptor Genes 1 For the Flowcytometry method, in each well of a 6 well dish, HEK293 cells suspended in DMEM (Nacalai) were 3.3×10 5Cells were seeded and after 24 hours, a reaction solution with the composition shown in Table 5 was prepared. After standing still in a clean bench for 20 minutes, it was added to each well of a 6-well dish. Here, the receptor genes are the genes of the GPCRs of No. 1 - 4, 8 - 13, 31 - 38, 60, 76, 80, 85, and 102 in Table 4. The cells were cultured in an incubator maintained at 37°C and 5% CO2 for 24 hours. As a control, cells that do not express the receptor (mock) were prepared.

[0142]

Table 5

[0143] 3) Preparation of cultured cells transformed with the receptor gene 2 For the Flowcytometry method, in each well of a 6-well dish, HEK293 cells suspended in DMEM (Nacalai) were seeded at 3.3×10 5 cells. After 24 hours, a reaction solution with the composition shown in Table 6 was prepared. After standing still in a clean bench for 20 minutes, it was added to each well of the 6-well dish. The cells were cultured in an incubator maintained at 37°C and 5% CO2 for 24 hours. As a control, cells that do not express the receptor (mock) were prepared.

[0144]

Table 6

[0145] 4) Measurement of the amount of receptor protein on the cell membrane (Flowcytometry method) The anti-FLAG antibody (Cosmo Bio Co., Ltd.) was allowed to act on the cells recovered using Cellstripper as the primary antibody on ice for 1 hour. After washing the cells, PE (phycoerythrin)-conjugated anti-mouse IgG (Abcam) was allowed to act as the secondary antibody on ice for 30 minutes. After washing, 0.25 μg of 7-AAD (7-Aminoactinomycin D, Fujifilm Wako Pure Chemical Corporation) was added, and the average value of the PE signal of the cell population negative for 7-AAD was measured by a flow cytometry system (BD) as an index of the receptor amount on the cell membrane. In each experimental run, cells expressing the FLAG-M2 acetylcholine receptor were used as the positive control (PC), and cells not expressing the receptor were used as the negative control (NC), and the average value of the PE signal was determined in the same manner as above. Standardization was performed with the PE signal of NC set to 0% and the PE signal of PC set to 100%, and the PE signal (%) of various receptors was calculated as the cell surface expression amount.

[0146] 5) Results The expression levels of each receptor on HEK293 cells were analyzed using the Flowcytometry method (2) and (4) above). As shown in Table 7, as a result of comparing the average values of the three experiments, for all the receptors analyzed for cell surface expression amount, specifically VN1R1, VN1R2, VN1R4, VN1R5, TAS2R8, TAS2R16, TAS2R38, TAS2R42, TAS2R45, TAS2R46, TAAR1, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, MrgprE, MrgprF, GPR31, GPR65, GPR82, GPR88, and GPR171, an increase in the cell surface expression amount was observed by the consensus method.

[0147]

Table 7

[0148] Using flow cytometry (as described in (3) and (4) above), the expression levels of the original receptors of TAS1R1 and TAS1R3, the consensus receptors of TAS1R1 and TAS1R3, the original receptor of TAS1R1 and the consensus receptor of TAS1R3, or the combination of the consensus receptor of TAS1R1 and the original receptor of TAS1R3 on HEK293 cells were analyzed. As shown in Figure 1, compared with the co-expression of the two original receptors of TAS1R1 and TAS1R3 in HEK293 cells, when expressing a receptor in which either one was made consensus, it was revealed that the membrane expression level of TAS1R1 increased. In particular, the highest expression was observed when both TAS1R1 and TAS1R3 were made consensus.

[0149] The Accession No., amino acid sequence of the original GPCR of the above examples, and the amino acid sequence of the consensus GPCR are shown in Tables 8-1 and 8-2 below. Also, the DNA sequences encoding the consensus GPCRs consisting of the amino acid sequences represented by SEQ ID NOs: 108 to 112, 114 to 120, 138 to 145, 167, 183, 187, 192, and 209 are shown in SEQ ID NOs: 215 to 234 and 313 to 317.

[0150]

Table 8-1

[0151]

Table 8-2

Claims

1. A method for enhancing the expression of a target G protein-coupled receptor (GPCR), comprising: expressing in a cell a GPCR polypeptide consisting of an amino acid sequence in which all amino acid residues different from the consensus amino acid sequence represented by SEQ ID NO: (3) in the amino acid sequence represented by SEQ ID NO: (2) of the target GPCR in Table 1 below are modified to the amino acid residues of the consensus amino acid sequence at the corresponding positions; (excluding methods for operating or treating humans). 【Table 1】

2. A method for measuring the response of a target GPCR, comprising: expressing in a cell a GPCR polypeptide consisting of an amino acid sequence in which all amino acid residues different from the consensus amino acid sequence represented by SEQ ID NO: (3) in the amino acid sequence represented by SEQ ID NO: (2) of the target GPCR in Table 2 below are modified to the amino acid residues of the consensus amino acid sequence at the corresponding positions; and measuring the response of the GPCR polypeptide; and (excluding methods for operating or treating humans). 【Table 2】

3. A method for screening for a ligand of a target GPCR, comprising: expressing in a cell a GPCR polypeptide consisting of an amino acid sequence in which all amino acid residues different from the consensus amino acid sequence represented by SEQ ID NO: (3) in the amino acid sequence represented by SEQ ID NO: (2) of the target GPCR in Table 3 below are modified to the amino acid residues of the consensus amino acid sequence at the corresponding positions; measuring the response of the GPCR polypeptide in the presence of a test substance; and selecting a test substance to which the GPCR polypeptide responds; (excluding methods for operating or treating humans). 【Table 3】

4. A method for evaluating and / or selecting a controller for ligand recognition of a target GPCR, comprising: expressing in a cell a GPCR polypeptide consisting of an amino acid sequence in which all amino acid residues different from the consensus amino acid sequence represented by SEQ ID NO: (3) in the amino acid sequence represented by SEQ ID NO: (2) of the target GPCR in Table 4 below are modified to the amino acid residues of the consensus amino acid sequence at the corresponding positions; adding a test substance and a ligand of the target GPCR to the GPCR polypeptide; and measuring the response of the GPCR polypeptide to the ligand; (excluding methods for operating or treating humans). 【Table 4】

5. A method for evaluating taste, comprising expressing in cells a GPCR polypeptide consisting of an amino acid sequence in which all amino acid residues different from the consensus amino acid sequence represented by the sequence number (3) in the amino acid sequence represented by the sequence number (2) of the GPCR for the purpose of (1) in Table 5 below are modified to the amino acid residues of the consensus amino acid sequence at the corresponding positions, adding a test substance to the GPCR polypeptide, and measuring the response of the GPCR polypeptide to the test substance, wherein the GPCR polypeptide is a taste receptor polypeptide (however, excluding methods for operating or treating humans).

6. 【Table 5】 A method for evaluating and / or selecting an inhibitor of odor of a ligand of a GPCR of interest, comprising expressing in cells a GPCR polypeptide consisting of an amino acid sequence in which all amino acid residues different from the consensus amino acid sequence represented by the sequence number (3) in the amino acid sequence represented by the sequence number (2) of the GPCR for the purpose of (1) in Table 6 below are modified to the amino acid residues of the consensus amino acid sequence at the corresponding positions, adding a test substance and a ligand of the GPCR of interest to the GPCR polypeptide, and measuring the response of the GPCR polypeptide to the ligand, wherein the GPCR polypeptide is a trace amine-related receptor polypeptide (however, excluding methods for operating or treating humans).

7. A method for evaluating and / or selecting an inhibitor of odor of a ligand of a GPCR of interest, 【Table 6】 comprising expressing in cells a GPCR polypeptide consisting of an amino acid sequence in which all amino acid residues different from the consensus amino acid sequence represented by the sequence number (3) in the amino acid sequence represented by the sequence number (2) of the GPCR for the purpose of (1) in Table 7 below are modified to the amino acid residues of the consensus amino acid sequence at the corresponding positions, adding a test substance to the GPCR polypeptide, and measuring the response of the GPCR polypeptide to the test substance, wherein the GPCR polypeptide is a trace amine-related receptor polypeptide (however, excluding methods for operating or treating humans).

8. ​ ​ 【Table 7】 ​ The method according to any one of claims 2 to 7, wherein the response of the GPCR polypeptide is measured by measuring the intracellular cAMP amount by ELISA or reporter gene assay, measuring the calcium ion amount by calcium imaging or TGFα shedding assay, or measuring the potential change across the cell membrane by the two-electrode voltage clamp method using Xenopus oocytes.

9. A modified GPCR polypeptide, comprising an amino acid sequence represented by any of SEQ ID NOs: 108 to 112, 114 to 120, 138 to 145, 167, 183, 187, 192, and 209, a modified GPCR polypeptide.

10. A polynucleotide encoding the modified GPCR polypeptide according to claim 9.

11. A vector or DNA fragment comprising the polynucleotide according to claim 10.

12. A transformed cell containing the vector or DNA fragment according to claim 11.

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