Analysis method for g protein-coupled receptors

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 technology]

[0002] As members of a living organism, cells recognize changes in the physiological conditions around them and change their behavior. Individual organisms adapt to these changes by using their senses to detect changes in the external environment. The basis of the biological mechanism is for individual life units to respond appropriately to endogenous information chemicals such as hormones and neurotransmitters, or exogenous information chemicals such as taste substances. These chemical receptors are carried out by receptor proteins in individual tissue cells. In recent years, many organisms lacking specific receptor genes have been created, demonstrating the necessity of receptors for physiological functions. In addition, various drugs that target receptors are being used to regulate physiological functions. However, there are still many receptors whose functions are unknown.

[0003] A typical receptor in living organisms is the G protein-coupled receptor (GPCR). It is characterized by a seven-transmembrane structure, which transitions to an activated structure upon binding an agonist, and basically transmits signals through interaction with G proteins in the cell. There are approximately 800 types of GPCRs in humans, of which approximately half were known to be not directly involved in physiological functions related to the senses. Information on human GPCRs, excluding olfactory receptors and vomeronasal receptors, can be obtained from the 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 considered to be target molecules for 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 recognizing pheromones (Non-Patent Document 2). Only one functional analysis of VN1R1 in cultured cells has been reported in humans, and this report shows that it recognizes low-molecular-weight volatile compounds (Non-Patent Document 2). In addition, a large-scale survey in Sweden investigated what kind of differences in properties are caused by genetic polymorphisms of VN1R1, and as a result, it was shown that differences were observed in the sexual characteristics of women, suggesting the potential function of VN1R1 (Non-Patent Document 3). There have been no reports of functional analysis, such as identification of the substances that are recognized, for the other VN1R2 to 5.

[0005] Taste receptors, TAS1R (taste 1 receptor), are a gene family found as GPCRs expressed on the tongue. There are three types of genes in the human genome: TAS1R1, TAS1R2, and TAS1R3, all of which are classified as Class C GPCRs. In the taste buds of the tongue, TAS1R1 and TAS1R2 are expressed in different taste cells, and both co-express TAS1R3. It is known that Class C GPCRs, including metabotropic glutamate receptors, 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 perceive umami and sweet substances, respectively. It has been reported that by expressing the TAS1R2 / TAS1R3 complex in cultured cells and screening, a substance that functions as an allosteric modulator of the complex and enhances sweetness has been identified (Non-Patent Document 4). Similarly, by expressing the TAS1R1 / TAS1R3 complex, it is possible to obtain evaluation results showing that inosinic acid, which enhances umami taste, enhances the activity of the complex (Non-Patent Document 5). If these TAS1R proteins can be obtained as more stable proteins or expressed in larger amounts on the membrane of cultured cells, it will be possible to efficiently identify materials that enhance umami taste or 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 types of genes in the human genome. It is classified as a Class A type GPCR and has been shown to recognize various bitter substances, but functional analysis of human TAS2R41, TAS2R42, TAS2R45, TAS2R48, and TAS2R60 has not been successful, and it is unknown what substances they recognize. Patent Document 1 discloses that TAS2R is fused with G protein as a device for efficient functional analysis in order to enable bitterness evaluation and identification of bitterness-modulating substances using TAS2R. Expression of TAS2R is not limited to the tongue, but is also found in the respiratory system, circulatory system, and nervous system, and it has been shown to play various physiological roles in addition to receiving bitterness 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 receptors (TAARs) are GPCRs of the Class A family, and in humans they consist of six different genes. TAAR1, which was first discovered, has been shown to be responsible for the recognition of neurotransmitters in the brain. For this reason, the relationship between TAAR1 and schizophrenia, depression, addiction, and Parkinson's disease has been investigated, and there are efforts to use its agonists to treat neuropathic pain (Patent Document 2). On the other hand, other TAARs, except for TAAR1, are highly expressed in the olfactory epithelium, and have been shown to play a role in selectively and sensitively recognizing volatile amines and generating the sense of smell (Non-Patent Document 7).

[0008] Mas-related G protein-coupled receptor (Mrgpr) is a GPCR of the Class A family, and there are 10 types of genes in humans. Its expression is observed in the sensory nervous system and related tissues such as the peripheral skin, and it detects itching and pain by recognizing agonists (Non-Patent Document 8). In addition, in mice, sensory nerve cells expressing MrgprB4 are involved in pleasant tactile sensations, and efforts to search for pleasant emotion enhancers targeting MrgprB4 have been disclosed (Patent Document 3). In recent years, it has been suggested that MrgprE and MrgprF of the Mrgprs are expressed in various tissues other than sensory nerves, such as the ileum, and play a variety of physiological functions, but there have been few reports of agonists (Non-Patent Document 9).

[0009] Generally, the most widely used method for functional analysis of GPCRs is the method of expressing them in cultured cells, which is simple. A wide variety of analytical methods have been devised for this purpose. Nevertheless, there are still many GPCRs for which functional analysis has not been successful and it is unclear what molecules they recognize as ligands. GPCRs for which functional analysis has not been successful are generally written as GPR (G-protein-coupled receptor) X (X is an arbitrary number). One of the reasons for the delay in identifying ligands for these GPR groups is that even if one tries to express the target GPCR in cultured cells, cultured cells, which are different from the original tissue cells, lack the factors that allow the GPCR to be stably expressed on the cell membrane. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Patent No. 6924590 [Patent Document 2] Special Publication No. 2019-517524 [Patent Document 3] JP 2019-118340 A [Non-patent literature]

[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

[0012] In order to analyze G protein-coupled receptors (GPCRs), a method for efficiently expressing GPCRs is required. [Means for solving the problem]

[0013] The present inventors have intensively investigated methods for efficiently expressing GPCRs. As a result, the present inventors have found that by converting GPCRs into consensus fragments, membrane expression of GPCRs in cultured cells can be improved compared to the original GPCRs. There have been no known examples of converting GPCRs into consensus fragments, except for olfactory receptors.

[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 having 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 a target GPCR (excluding olfactory receptors) has been altered to an amino acid residue of the consensus amino acid sequence at a position corresponding to the amino acid residue; 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 a GPCR encoded by an ortholog of the GPCR of interest in a vertebrate; method. 2) A method for expressing a GPCR polypeptide, comprising: expressing in a cell a GPCR polypeptide having an amino acid sequence in which at least one amino acid residue in the amino acid sequence of a target GPCR that differs from a consensus amino acid sequence has been altered to an amino acid residue in the consensus amino acid sequence at a position corresponding to the amino acid residue; The GPCR polypeptide comprises an amino acid sequence in which at least one amino acid residue in the amino acid sequence of human TAAR6 shown in SEQ ID NO: 36 that differs from the consensus amino acid sequence shown in SEQ ID NO: 143 has been modified to an amino acid residue in the consensus amino acid sequence at a position corresponding thereto. method. 3) A method for measuring a response of a GPCR of interest, comprising: Measuring the response of a GPCR polypeptide expressed by the method of 1) or 2); The method includes: 4) A method for searching for a ligand for a target GPCR, comprising: Measuring the response of the GPCR polypeptide expressed by the method described in 1) or 2) in the presence of a test substance; and selecting a test substance to which the GPCR polypeptide responded; The method includes: 5) A method for evaluating and / or selecting a regulator 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 of 1) or 2); and measuring the response of said GPCR polypeptide to said ligand; The method includes: 6) A method for evaluating taste, comprising: adding a test substance to the GPCR polypeptide expressed by the method of 1) or 2); and measuring the response of said GPCR polypeptide to said test substance; wherein the GPCR polypeptide is a taste receptor polypeptide. method. 7) A method for evaluating and / or selecting an odor suppressant for 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 of 1) or 2); and measuring the response of said GPCR polypeptide to said ligand; wherein the GPCR polypeptide is a trace amine associated receptor polypeptide. method. 8) A method for evaluating and / or selecting an odor suppressant for a ligand of a GPCR of interest, comprising: adding a test substance to the GPCR polypeptide expressed by the method of 1) or 2); and measuring the response of said GPCR polypeptide to said test substance, wherein said GPCR polypeptide is a trace amine associated receptor polypeptide. method. 9) A modified GPCR polypeptide, comprising: an amino acid sequence in which at least one amino acid residue that is different from a consensus amino acid sequence in the amino acid sequence of a target GPCR (excluding olfactory receptors) has been modified to an amino acid residue of the consensus amino acid sequence at a corresponding position; 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 a GPCR encoded by an ortholog of the GPCR of interest in a vertebrate; Modified GPCR Polypeptides. 10) A modified GPCR polypeptide, comprising: The amino acid sequence of human TAAR6 shown in SEQ ID NO: 36 is modified by replacing at least one amino acid residue different from the consensus amino acid sequence shown in SEQ ID NO: 143 with an amino acid residue of the consensus amino acid sequence at a corresponding position. Modified GPCR Polypeptides. 11) A polynucleotide encoding the modified GPCR polypeptide according to 9) or 10). 12) A vector or a DNA fragment comprising the polynucleotide according to 11). 13) A transformed cell containing the vector or DNA fragment described in 12). Effect of the Invention

[0015] The present invention provides a method for efficiently expressing a GPCR, which can be used to elucidate the function of the GPCR and identify agents that regulate its function. [Brief description of the drawings]

[0016] [Figure 1] Membrane expression level of GPCR (taste receptor). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0018] In this specification, "G-protein-coupled receptor (GPCR)" is a general term for receptors that have a seven-transmembrane structure, transition to an activated structure upon binding to a ligand, and basically transmit signals via interaction with intracellular G proteins. Examples of GPCRs include vomeronasal receptors, taste receptors, trace amine-associated receptors, Mas-related G protein-coupled receptors, and GPRX (X is an arbitrary number) with unknown ligands, but are not limited to these.

[0019] As used herein, the term "vomeronasal receptor" refers to VN1R (vomeronasal 1 receptor). For example, there are five types of VN1R genes in the human genome.

[0020] In this specification, the term "taste receptor" refers to a receptor that receives taste molecules in a living organism, 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. Of these, TASR1 and TAS1R3 form a complex to function as an umami substance receptor, and TAS1R2 and TAS1R3 form a complex to 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 Mrpgr genes in the human genome (MAS1, MASL1, MrgprD, MrgprE, MrgprF, MrgprG, MrgprX1-4).

[0023] As used herein, the term "olfactory receptor" refers to an olfactory receptor or an odorant receptor. Based on criteria such as overall sequence homology, predicted seven transmembrane domains, and whether or not the gene has a conserved partial amino acid sequence, approximately 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 a function equivalent thereto. A polypeptide having a function equivalent to a GPCR refers to a polypeptide that can be expressed on a cell membrane, like a GPCR, that is activated by ligand binding, and that, upon activation, has the function of transmitting a signal into a cell, such as the function of promoting GDP / GTP exchange of the coupled G protein α-subunit.

[0025] As used herein, the phrase "functionally expressing" a GPCR polypeptide in a cell 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, whereas the term "antagonist" refers to a substance that binds to a receptor but does not activate the receptor or suppresses 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] In this specification, "odor cross-adaptation (or olfactory cross-adaptation)" with respect to a target odor refers to a phenomenon in which olfactory sensitivity to a target odor causative substance is reduced or changed by receiving the odor of a substance other than the target odor causative substance in advance and becoming accustomed to the odor. The present inventors have previously revealed that "odor cross-adaptation" is a phenomenon based on receptor agonism (International Publication No. 2016 / 194788). That is, in "odor cross-adaptation", a receptor for a target odor causative substance responds to a different odor causative substance prior to responding to the target odor causative substance, and then desensitizes, so that even if it is later exposed to the target odor causative substance, it can only respond poorly, resulting in a reduction or alteration of 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 as "odor cross-adaptation due to receptor agonism" in this specification.

[0029] As used herein, "suppression by receptor antagonism" of a target odor refers to suppressing the receptor response to a substance having the target odor with an antagonist, thereby suppressing the target odor recognized by an individual.

[0030] In the present specification, the identity of nucleotide sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-41). Specifically, the identity is calculated by performing analysis with a unit size to compare (ktup) of 2 using the search homology program of the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development).

[0031] As used herein, the term "amino acid residue" refers to the 20 types of amino acid residues constituting 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, amino acid modifications may be designated by the accepted IUPAC one-letter amino acid abbreviations: [original amino acid, position, modified amino acid]. For example, a modification of histidine at position 43 to arginine is designated "H43R."

[0033] In the present specification, the "corresponding position" on the amino acid sequence can be determined by aligning the target sequence with the reference sequence (the amino acid sequence of the original GPCR in the present invention) 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 performed 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 are revised versions of Clustal W, can be used. Clustal W, Clustal W2 and Clustal omega are available, for example, on the Clustal website operated by University College Dublin [www.clustal.org], the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), and the website of the DNA Data Bank of Japan operated by the National Institute of Genetics (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]). The position of the target sequence aligned to any position of the reference sequence by the above-mentioned alignment is considered to be a "position corresponding to" the any position.

[0034] Those skilled in the art can further fine-tune the alignment of the amino acid sequences obtained above to optimize it. Such an optimal alignment is preferably determined taking into consideration the similarity of the amino acid sequences, the frequency of gaps to be inserted, and the like. Here, the similarity of the amino acid sequences refers to the ratio (%) of the number of positions at which identical or similar amino acid residues exist in both sequences when the two amino acid sequences are aligned to the total number of amino acid residues. The similar amino acid residues refer to amino acid residues that have similar properties in terms of polarity and charge among the 20 types of amino acids that constitute proteins, and that cause so-called conservative substitution. Such groups of similar amino acid residues are well known to those skilled in the art, and examples thereof include, but are not limited to, 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. In addition, the amino acid sequence alignment obtained above can be further refined to optimize, for example, based on amino acids or amino acid motifs that are highly conserved among GPCRs.

[0035] As used herein, the term "operably linked" between a control region such as a promoter and a gene means that the gene and the control region are linked in such a way that the gene can be expressed under the control of the control region. Procedures for "operably linked" between a gene and a control region are well known to those skilled in the art.

[0036] In this specification, "upstream" and "downstream" of a gene refer to the upstream and downstream of 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 a gene means the 5' region of the gene in the DNA sense strand.

[0037] In this specification, "homolog" refers to a homologous gene derived from a common ancestor. "Ortholog", also called "ortholog", refers to a homolog diverged during speciation, exists in different biological species, and has the same or similar function. In one example, the ortholog used in the present invention may be a GPCR gene of a biological species different from the biological species from which the GPCR of interest is derived, which contains the same name as the GPCR gene of interest, among the homologous genes of the GPCR gene of interest. When the nomenclature of GPCRs of a certain biological species is different from the nomenclature of the biological species from which the GPCR of interest is derived, the ortholog may be a GPCR gene having high homology with the GPCR gene of interest in the certain biological species, preferably the GPCR gene having the highest homology. Alternatively, it may be a GPCR gene known to be an ortholog of the GPCR gene of interest in the certain biological species. In another example, the ortholog used in the present invention may be a GPCR gene of the above orthologs that is suggested to have diverged during speciation by phylogenetic tree analysis.

[0038] As shown in the Examples 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 a GPCR encoded by a specific ortholog of the human GPCR, and expressed the resulting GPCR polypeptide in a cell, and found that the membrane expression of the GPCR polypeptide in the cell was increased compared to the human GPCR before modification (Table 7, Figure 1). In other words, the stability of the GPCR polypeptide during expression is improved compared to the human GPCR before modification. In this specification, the GPCR before modification is referred to as the "original GPCR", modifying the amino acid sequence of a GPCR based on a consensus amino acid sequence is referred to as "making it into a consensus", and the consensus-made GPCR may be referred to as the "consensus GPCR" or "modified GPCR polypeptide".

[0039] Thus, the above-mentioned consensus of GPCR is useful for expressing GPCR, particularly GPCR whose functional analysis has been inefficient so far due to insufficient membrane expression in cultured cells, in the cell membrane of a cell. Thus, in one aspect, the present invention provides a method for expressing a GPCR polypeptide. The expression method of the present invention enables improvement of expression of GPCR (e.g., increased expression, stabilization of expression), and thus the method is preferably a method for improving expression of 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 different from a consensus amino acid sequence in the amino acid sequence of a target GPCR is modified to an amino acid residue of the consensus amino acid sequence at a position corresponding thereto, and 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 a GPCR encoded by an orthologue of the target GPCR in a vertebrate.

[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, and more preferably a human GPCR. The target GPCR may be a GPCR whose function can be analyzed using cultured cells by a conventional method, or an inefficient GPCR, but the method of the present invention is more preferably applied to a GPCR whose function can be analyzed using cultured cells by a conventional method inefficiently.

[0041] In the expression method of the present invention, the GPCR encoded by the orthologue of the GPCR of interest in a vertebrate is preferably a GPCR selected from GPCRs encoded by the orthologue of the GPCR of interest in a mammal, a bird, a reptile, an amphibian, or a fish, more preferably a GPCR encoded by the orthologue of the GPCR of interest in a mammal, a bird, a reptile, or an amphibian, and even more preferably a GPCR encoded by the orthologue of the GPCR of interest in a mammal. The orthologue is not particularly limited, but an orthologue having a high homology with the GPCR of interest is preferable. Here, the term "mammals" refers to a species of organisms belonging to the class Mammalia in the phylum Vertebrates, and it is known that there are approximately 5,500 species currently in existence. Mammals include, but are not limited to, humans, chimpanzees, bonobos, gorillas, Sumatran orangutans, northern white-streaked gibbons, drills, gelada baboons, rhesus macaques, Anubis baboons, sooty mangabeys, green monkeys, red colobus monkeys, Angolan colobus monkeys, garnet galagos, gray mouse lemurs, coquerel's sifakas, Philippine tarsier, mice, rats, rabbits, cats, dogs, foxes, raccoon dogs, weasels, tigers, cheetahs, bears, sea lions, seals, fur seals, horses, rhinos, camels, pigs, wild boars, cows, goats, sheep, deer, giraffes, hippos, elephants, pangolins, moles, and bats. Birds (Aves) refer to species of animals belonging to the phylum Vertebrates and class Aves. Birds include, but are not limited to, chickens, ducks, geese, turkeys, ostriches, pheasants, pigeons, parrots, canaries, Bengalese finches, hummingbirds, manakins, quails, and flycatchers. Reptiles (Reptilia) refer to species of organisms belonging to the phylum Vertebrata and class Reptilia. Reptiles include, but are not limited to, turtles, lizards, crocodiles, iguanas, chameleons, geckos, and snakes. Amphibians (Amphibia) refer to species of organisms belonging to the phylum Vertebrata and class Amphibia. Amphibians include, but are not limited to, frogs, newts, and salamanders. Fish (Fish) is a general term referring to species of organisms belonging to the phylum Vertebrata and classes Hagfish, Lamprey, Chondrichthyes, and Osteichthyes.Examples of fish include, but are not limited to, hagfish, lamprey, sharks, rays, tuna, bonito, salmon, trout, cod, sea bream, flounder, yellowtail, horse mackerel, and mackerel.

[0042] In a preferred example of this embodiment, an ortholog of a GPCR of interest in a vertebrate is a gene having the same name as the GPCR of interest among 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 GPCR of interest as a query sequence, a database search is performed against a known database such as BLAST of NCBI. From the resulting group of homologous genes (for example, top 500 genes, preferably top 250 genes, more preferably top 100 genes, and even more preferably top 50 genes), a gene having the same name as the GPCR of interest is selected. More preferably, a gene encoding a GPCR having a certain amino acid sequence identity, for example 65% or more amino acid sequence identity, with the GPCR of interest is selected. 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 from a certain biological species other than human are selected as orthologs, the gene from the certain biological species with the highest homology to the target human GPCR gene may be selected. In addition, when the nomenclature of GPCR in a certain biological species is different from the nomenclature in the biological species from which the target GPCR is derived, a gene with high homology to the target GPCR gene in the certain biological species, preferably the gene with the highest homology, may be selected as the ortholog. Alternatively, a gene known to be an ortholog of the target GPCR gene in the certain biological species may be selected.

[0043] The number of types of GPCRs encoded by orthologs of a GPCR of interest in a vertebrate, in terms of the number of receptors, is at least 2, preferably at least 5, more preferably at least 11, even more preferably at least 15, even more preferably at least 30, and even more preferably 100. On the other hand, the upper limit of the number of types is the total number of orthologs of a GPCR of interest in a vertebrate, and said number, in terms of the number of receptors, is preferably 500 or less, more preferably 400 or less, and even more preferably 300 or less. The number of types of GPCRs encoded by orthologs of a GPCR of interest in a vertebrate, in terms of the number of receptors, can be, for example, from 2 types to the total types of orthologs of a GPCR of interest in a vertebrate, from 5 types to the total types of orthologs of a GPCR of interest in a vertebrate, from 11 types to the total types of orthologs of a GPCR of interest in a vertebrate, from 5 to 500 types, from 5 to 400 types, from 5 to 300 types, from 11 to 500 types, from 11 to 400 types, from 11 to 300 types, from 15 to 300 types, from 30 to 300 types, or from 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 a GPCR of interest and the amino acid sequence of a GPCR encoded by an orthologue of the GPCR of interest in a vertebrate. Specifically, the "consensus amino acid sequence" is an amino acid sequence consisting of consensus residues identified from an alignment of the amino acid sequence of a GPCR of interest and the amino acid sequence of a GPCR encoded by an orthologue of the GPCR of interest in a vertebrate according to the following criteria (i) to (iii): (i) at each amino acid position of the alignment, (ii) if there is one amino acid residue that is different from the amino acid residue of the target GPCR and occurs at a frequency of 50% or more, the amino acid residue is identified as a consensus residue; (i-ii) if there are two amino acid residues with an occurrence frequency of 50%, the amino acid residue of the GPCR of interest is identified as a consensus residue; (i-iii) if the amino acid residue is present in the GPCR of interest and the amino acid residue is not present with an occurrence frequency of 40% or more, the amino acid residue is identified as being absent; (i-iv) if the amino acid residue is absent in the GPCR of interest and an amino acid residue is present with an occurrence frequency of 60% or more, the most frequently occurring amino acid residue is identified as the consensus residue, and if two or more types of the most frequently occurring amino acid residues are present, the amino acid residue with the smallest molecular weight among the amino acid residues is identified as the consensus residue; (iv) if none of the above (ii) to (i-iv) apply, identifying the amino acid residue of the target GPCR as a consensus residue; (ii) when the consensus residue is identified according to the above criterion (i), if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminus or further C-terminus of the GPCR of interest and is not a methionine residue, changing the consensus residue N-terminal to no consensus residue, the consensus residue consisting of the methionine residue at the position closest to the N-terminus; (iii) When a consensus residue is identified according to the criterion (i) above, if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminal side of the N-terminus of the GPCR of interest and is not a methionine residue, the amino acid residue that occurs most frequently is identified as the consensus residue by going back one amino acid position at a time N-terminally from the position of the consensus residue in the alignment until a methionine residue appears, and if there are two or more types of amino acid residues that occur most frequently, the amino acid residue with the smallest molecular weight among those amino acid residues is identified as the consensus residue.

[0045] Here, the "occurrence frequency" refers to the number of occurrences of a specific amino acid residue at each amino acid position in the alignment of amino acid sequences, expressed as a percentage relative to the number of amino acid sequences subjected to the alignment. The alignment of amino acid sequences can be performed using a known algorithm.

[0046] Criterion (i)(ii) is a criterion for the case where an amino acid residue is present in the GPCR of interest at an amino acid position in the alignment of the amino acid sequence of the GPCR of interest and the amino acid sequence of a GPCR encoded by an orthologue of the GPCR of interest in a vertebrate. In this case, if there is one amino acid residue that is different from the amino acid residue in the GPCR of interest 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 frequency of all amino acid residues other than the amino acid residue in the GPCR of interest is less than 50%, the amino acid residue in the GPCR of interest is identified as the consensus residue at that position according to criterion (iv).

[0047] Criterion (i) (i-ii) is a criterion for the case where an amino acid residue is present in a GPCR of interest at a certain amino acid position in the alignment of the amino acid sequence of the GPCR of interest and the amino acid sequence of a GPCR encoded by an orthologue of the GPCR of interest in a vertebrate. In this case, if there are two amino acid residues with an occurrence frequency of 50%, one of the two amino acid residues is necessarily an amino acid residue in the GPCR of interest, and the amino acid residue in the GPCR of interest is identified as the consensus residue at that position.

[0048] Criterion (i) (i-iii) is a criterion for the case where an amino acid residue is present in the GPCR of interest at an amino acid position in the alignment of the amino acid sequence of the GPCR of interest and the amino acid sequence of a GPCR encoded by an orthologue of the GPCR of interest in a vertebrate. In this case, if no amino acid residue is present with an occurrence frequency of 40% or more, the position is identified as having no consensus residue. On the other hand, if the occurrence frequency of no amino acid residue is less than 40%, if the above criterion (ii) is met, a consensus residue at that position is identified according to the criterion, and if not, an amino acid residue in the GPCR of interest is identified as a consensus residue at that position according to criterion (iv).

[0049] Criterion (i) (i-iv) is a criterion for the case where an amino acid residue is not present in the GPCR of interest at an amino acid position in the alignment of the amino acid sequence of the GPCR of interest and the amino acid sequence of the GPCR encoded by the orthologue of the GPCR of interest in a vertebrate. In this case, if an amino acid residue is present with an occurrence frequency of 60% or more, the most frequently occurring amino acid residue is identified as the consensus residue at that position, and if there are two or more types of amino acids with the most frequent occurrence, the amino acid residue with the smallest molecular weight among the most frequently occurring amino acids is identified as the consensus residue. For example, if there is one type of amino acid residue with the most frequent occurrence, the one type of amino acid residue is identified as the consensus residue at that position, and if there are two or more types of amino acid residues with the most frequent occurrence, the amino acid residue with the smallest molecular weight among them is identified as the consensus residue at that position. In addition, when the change results in the total length of the consensus amino acid sequence being 10% or more longer toward the N-terminus than the total length of the amino acid sequence of the GPCR of interest, from the viewpoint of maintaining the structure of the GPCR, the consensus residue at the position corresponding to the N-terminus of the GPCR of interest in the consensus amino acid sequence may be changed to a methionine residue, and the consensus residue on the N-terminus side of the methionine residue may be changed to none. In other words, the N-terminus structure of the GPCR of interest may be maintained as it is. On the other hand, if the frequency of occurrence of an amino acid residue is less than 60%, the amino acid residue of the GPCR of interest is identified as the consensus residue at that position according to criterion (iv). In other words, the position is identified as having no amino acid residue.

[0050] When an amino acid position in the alignment of the amino acid sequence of a GPCR of interest and the amino acid sequence of a GPCR encoded by an orthologue of the GPCR of interest in a vertebrate does not fall under any of (ii) to (i-iv) in (i) above, the amino acid residue in the GPCR of interest is identified as the consensus residue at that position. In this case, if no amino acid residue is present in the GPCR of interest, it may be identified as no amino acid residue being present at that position in the consensus amino acid sequence.

[0051] Criterion (ii) is a criterion for the case where, when consensus residues are identified at each amino acid position in the alignment of the amino acid sequence of a GPCR of interest and the amino acid sequence of a GPCR encoded by an orthologue of the GPCR of interest in a vertebrate according to criterion (i) above, the consensus residue located closest to the N-terminus is a consensus residue located at a position corresponding to the N-terminus or C-terminus side of the GPCR of interest and is not a methionine residue. In this case, the consensus residue on the N-terminus side of the consensus residue consisting of the methionine residue located closest to the N-terminus is changed to no consensus residue so that the N-terminus consensus residue becomes a methionine residue, in other words, so that the translation initiation amino acid of the GPCR polypeptide becomes a methionine residue. For example, the consensus residues are confirmed one by one from the N-terminus, and if they are not methionine residues, they are identified as no consensus residue at that position, and this may be repeated until a methionine residue first appears. In addition, when the change results in the total length of the consensus amino acid sequence being 10% or more shorter than the total length of the amino acid sequence of the GPCR of interest, the most N-terminal consensus residue among the consensus residues before the change may be changed to a consensus residue consisting of a methionine residue, from the viewpoint of maintaining the helical structure of the GPCR. In this case, if the most N-terminal consensus residue 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 most N-terminal consensus residue among the consensus residues before the change may not be changed, and an amino acid residue of the GPCR of interest that is N-terminal to the position corresponding to the consensus residue may be used as the consensus residue. In other words, the N-terminal structure of the GPCR of interest may be maintained as it is.

[0052] Criterion (iii) is a criterion for the case where, when a consensus residue is identified at each amino acid position in the alignment of the amino acid sequence of a GPCR of interest and the amino acid sequence of a GPCR encoded by an orthologue of the GPCR of interest in a vertebrate according to criterion (i) above, the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminal side of the N-terminus of the GPCR of interest and is not a methionine residue. In this case, the alignment is confirmed by going back one amino acid position at a time from the position of the most N-terminal consensus residue toward the N-terminus so that the N-terminal consensus residue is a methionine residue, in other words, so that the translation initiation amino acid of the GPCR polypeptide is a methionine residue, and the most frequently occurring amino acid residue is identified as the consensus residue until a methionine residue appears, and when there are two or more types of the most frequently occurring amino acid residues, 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 of the amino acid residues in the amino acid sequence of the target GPCR that is different from the consensus amino acid sequence is modified to an amino acid residue in the consensus amino acid sequence at a position corresponding to the amino acid residue. Here, the term "modification" includes 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, the amino acid sequence of the target GPCR is compared with the consensus amino acid sequence, and if an 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 in the target GPCR may be replaced with an amino acid residue in the consensus amino acid sequence. Alternatively, the amino acid sequence of the target GPCR is compared with the consensus amino acid sequence, and if there is no amino acid residue in the consensus amino acid sequence at a position corresponding to a certain amino acid position in the amino acid sequence of the target GPCR, the amino acid residue at that amino acid position in the target GPCR may be deleted. Alternatively, the amino acid sequence of the target GPCR is compared with the consensus amino acid sequence, and if there is an amino acid residue in the consensus amino acid sequence at a position corresponding to a 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 in the target GPCR. Furthermore, if the amino acid sequence of the target GPCR is compared with the consensus amino acid sequence, and the N-terminus of the consensus amino acid sequence is longer than that of the amino acid sequence of the target GPCR, the N-terminus that is present only in the consensus amino acid sequence may be added to the N-terminus of the amino acid sequence of the target GPCR. Alternatively, the amino acid sequence of a target GPCR can be compared with the consensus amino acid sequence, and if the consensus amino acid sequence has a longer C-terminus than the amino acid sequence of the target GPCR, the C-terminal portion that is only present in the consensus amino acid sequence can be added to the C-terminus of the amino acid sequence of the target GPCR.

[0054] The number of amino acid residues to be modified in the amino acid sequence of a GPCR of interest is at least 1, preferably at least 5, and more preferably at least 10, and it is even more preferable that all amino acid residues in the amino acid sequence of a GPCR of interest that differ from the consensus amino acid sequence are modified to amino acid residues in the consensus amino acid sequence at the corresponding positions (i.e., the amino acid sequence of a GPCR of interest is modified to the consensus amino acid sequence). Alternatively, the number of amino acid residues to be modified in the amino acid sequence of a GPCR of interest may be preferably at least 10%, more preferably at least 30%, even more preferably at least 50%, even more preferably at least 70%, even more preferably at least 90%, and even more preferably 100% (i.e., the amino acid sequence of a GPCR of interest is modified to the consensus amino acid sequence) of the number of amino acid residues that differ from the consensus amino acid sequence. If the N-terminus of the consensus amino acid sequence is longer than that of the amino acid sequence of the target GPCR, it is preferable to modify the amino acid sequence by adding the N-terminus that exists only in the consensus amino acid sequence as a whole to the N-terminus of the amino acid sequence of the target GPCR, regardless of the number of amino acid residues. If the N-terminus of the consensus amino acid sequence is shorter than that of the amino acid sequence of the target GPCR, it is preferable to modify the amino acid sequence by deleting the N-terminus that exists only in the amino acid sequence of the target GPCR as a whole from the N-terminus of the amino acid sequence of the target GPCR, regardless of the number of amino acid residues. If the C-terminus of the consensus amino acid sequence is longer than that of the amino acid sequence of the target GPCR, it is preferable to modify the amino acid sequence by adding the C-terminus that exists only in the consensus amino acid sequence as a whole to the C-terminus of the amino acid sequence of the target GPCR, regardless of the number of amino acid residues. If the C-terminus of the consensus amino acid sequence is shorter than that of the amino acid sequence of the target GPCR, it is preferable to modify the amino acid sequence by deleting the C-terminus that exists only in the amino acid sequence of the target GPCR as a whole from the C-terminus of the amino acid sequence of the target GPCR, regardless of the number of amino acid residues. The GPCR polypeptide used in the present invention also includes polypeptides that contain substitutions, deletions, additions, or insertions of one to several (e.g., 1 to 10, 1 to 5, 1 to 3) amino acid residues at amino acid positions other than the target amino acid positions for modification based on the above-mentioned consensus amino acid sequence, so long as the function of the GPCR polypeptide is not impaired.

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

[0056] [Table 1-1]

[0057] [Table 1-2]

[0058] 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 SEQ ID NO: (3) in the amino acid sequence in GPCR in Nos. 1 to 4 (1) in the above Table 1-1 is modified to an amino acid residue in the consensus amino acid sequence at a position corresponding to the amino acid residue in the amino acid sequence in the amino acid sequence in the above Table 1-1 in any one of SEQ ID NOs: 108 to 111 in the above Table 1-1 is a consensus vomeronasal receptor consisting of a consensus amino acid sequence derived from the amino acid sequence in vomeronasal receptor in Nos. 1 to 4 (1) in the above Table 1-1 in any one of SEQ ID NOs: 1 to 4 in the above Table 1-1 in the above Table 1-1 in the above Table 1-1.

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

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

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

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

[0063] A GPCR polypeptide consisting of an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence shown in SEQ ID NO: (3) in the amino acid sequence shown in SEQ ID NO: (2) of GPCR No. 109 to 145 (1) in the above Table 1-2 is modified to an amino acid residue of the consensus amino acid sequence at a position corresponding to the amino acid residue 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 the above Table 1-2, a GPCR polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NO: 276 to 312 in (3) is a consensus GPCR consisting of an amino acid sequence shown in any one of SEQ ID NO: 238 to 274 in (2) of GPCR No. 109 to 145 (1) and a consensus amino acid sequence derived from the amino acid sequence of a GPCR encoded by an orthologue of the GPCR in a vertebrate.

[0064] The term "expression" of the GPCR polypeptide of the present invention means that a translation product is produced from a polynucleotide encoding the polypeptide, and that the translation product is localized in a functional state in the cell membrane, which is the site of action. The GPCR polypeptide of the present invention may be expressed in a cell by a method known in the art. For example, the GPCR polypeptide may be expressed in the cell membrane of a host 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 in the cell membrane of a host cell by transforming the host cell with a vector containing a polynucleotide encoding the polypeptide. The host cell may be any cell capable of functionally expressing an exogenous 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] A polynucleotide encoding a GPCR polypeptide can be obtained using various mutagenesis techniques known in the art. For example, a polynucleotide encoding a GPCR polypeptide can be obtained by modifying a nucleotide sequence encoding an amino acid residue to be modified in a polynucleotide encoding the amino acid sequence of a target GPCR to a nucleotide sequence encoding the modified amino acid residue.

[0066] Introduction of a desired mutation into a polynucleotide encoding the amino acid sequence of a desired GPCR can be carried out using various site-directed mutagenesis methods well known to those skilled in the art. Site-directed mutagenesis can be carried out by any method, such as inverse PCR or annealing. Commercially available site-directed mutagenesis kits (e.g., QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit, both manufactured by Stratagene) can also be used.

[0067] Alternatively, a polynucleotide encoding a GPCR polypeptide can be obtained by genome editing using artificial DNA cleaving enzymes (artificial DNA nucleases or programmable nucleases), DNA synthesis based on the nucleotide sequence, or the like.

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

[0069] In a preferred example, a polynucleotide encoding a GPCR polypeptide consists of a nucleotide sequence shown in any one of SEQ ID NOs: 215 to 234 and 313 to 317. The polynucleotide encodes a GPCR polypeptide consisting of an amino acid sequence shown in 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 DNA fragment. Preferably, the vector is an expression vector. Also preferably, the vector is an expression vector capable of introducing a GPCR polynucleotide into a host cell and expressing the polynucleotide in the host cell. Preferably, the vector may be an extrachromosomal autonomously replicable vector such as a plasmid, or may be an intrachromosomal vector. A specific example of the vector includes, but is 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 introduced polynucleotide encoding a GPCR polypeptide in a host cell into which the vector has been introduced, and examples of the control region include expression control regions such as promoters and terminators, and transcription initiation sites. The type of the control region can be appropriately selected depending on the type of 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 DNA fragments containing a polynucleotide encoding a GPCR polypeptide include PCR-amplified DNA fragments and restriction enzyme-cleaved DNA fragments. Preferably, the DNA fragment may be an expression cassette containing the polynucleotide and a control region operably linked thereto. Examples of control regions that can be used are the same as those in the case of vectors.

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

[0074] To introduce a vector or DNA fragment into a host cell, a general transformation method for mammalian cells, such as electroporation, lipofection, or particle gun method, can be used. Transformed cells into which a vector or DNA fragment of interest has been introduced can be selected using a selection marker. Alternatively, the introduction of a vector or DNA fragment of interest can be confirmed by examining the DNA sequence of the cell.

[0075] The GPCR polypeptide of the present invention is produced from the polynucleotide encoding the GPCR polypeptide contained in the vector or DNA fragment introduced into the cell by the above-mentioned procedure, and is integrated into the cell membrane. Thus, the GPCR polypeptide expressed by the expression method of the present invention is expressed in the cell membrane of a transformed cell 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 fusing a tag such as a FLAG tag to the GPCR polypeptide in advance and using an antibody that specifically recognizes the tag, by known techniques such as flow cytometry.

[0077] The GPCR polypeptide expressed by the expression method of the present invention can be expressed more efficiently on the cultured cell membrane than the original GPCR. Furthermore, the response of the GPCR polypeptide is considered to reflect the response of the original GPCR. Thus, in another aspect, the present invention provides a method for measuring the response of a target GPCR. The method comprises measuring the response of a GPCR polypeptide expressed by the expression method of a GPCR polypeptide of the present invention. The response measurement method of the present invention can improve the efficiency of measuring the response of a target GPCR, and enables the measurement of the response of a target GPCR that could not be functionally analyzed due to insufficient expression in the cell membrane of cultured cells in the past.

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

[0079] In the response measurement method of the present invention, the response of the GPCR polypeptide may be measured by any method known in the art as a method for measuring the response of the GPCR. For example, the measurement may be performed by a method for directly or indirectly measuring the amount of intracellular cAMP. For example, it is known that when a G protein-coupled receptor is activated by a ligand, it couples with a G protein α subunit classified as the Gs family or a G protein α subunit classified as the Gi family in the cell to activate or inhibit adenylate cyclase, thereby fluctuating the amount of intracellular cAMP. On the other hand, when a G protein-coupled receptor is activated by a ligand, it also couples with proteins belonging to the Gq family, such as Gαq, Gα16, and Gα15, in the cell, and can increase the amount of calcium ions in the cell. 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 ELISA and reporter gene assays. Examples of methods for measuring calcium ion concentration include calcium imaging and TGFα shedding assays. The TGFα shedding assay is also effective for measuring the signal when GPCR is coupled to Gα12 and Gα13 in cells. As an example of a method using the behavior of downstream molecules activated by cAMP levels as an indicator, the two-electrode voltage clamp method is also effective for measuring the potential change inside and outside the cell membrane mediated by the cystic fibrosis transmembrane conductance regulator CFTR, which is activated by cAMP signals, in Xenopus oocytes. Any of the above response measurement methods can be used by using a chimeric protein consisting of a G protein α subunit that easily couples with the target GPCR and any other G protein α subunit. For example, taste receptors can efficiently couple with G protein α subunits belonging to the Gi family among various G protein α subunits.In light of this, by using Gα15 (Gα15 / i3, Gα15 / gust) which has 5 amino acids on the C-terminus side of Gαi3 or Gαgust, a signal derived from an activated taste receptor can be detected as an increase in intracellular calcium ion concentration, rather than a decrease in the original amount of intracellular cAMP. In addition, the GPCR polypeptide may be purified from cells in which the GPCR polypeptide is expressed according to the present invention, and then its binding to a ligand may be evaluated. The binding of the purified GPCR polypeptide to a ligand may be evaluated by any method known in the art. For example, a method for measuring the amount of heat generated by the GPCR polypeptide under ligand-added conditions and a method for evaluating a change in the thermal stability of the GPCR polypeptide may be used.

[0080] As described above, the GPCR polypeptide expressed by the expression method of the present invention can be expressed more efficiently on the cultured cell membrane than the original GPCR, and the response of the GPCR polypeptide is considered to reflect the response of the original GPCR, so that the GPCR polypeptide can be used to search for a ligand of the original GPCR. Therefore, 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 responded. The ligand searching method of the present invention can improve the efficiency of searching for a ligand of a target GPCR, and enables the search for a ligand of a target GPCR that could not be functionally analyzed due to insufficient expression on the cell membrane of cultured cells in the past.

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

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

[0083] In the ligand screening method of the present invention, a test substance is applied to a GPCR polypeptide expressed by the expression method of the present invention. Means for applying a test substance to a GPCR polypeptide include, but are not limited to, a method of adding the test substance to a medium for culturing cells expressing the GPCR polypeptide.

[0084] In the ligand screening method of the present invention, a test substance is added to a GPCR polypeptide, and then the response of the GPCR polypeptide to the test substance is measured. The method for measuring the response can be the same as that of the response measurement method of the present invention.

[0085] Then, the test substance is evaluated based on the measured response of the GPCR polypeptide. The test substance that causes the response of the GPCR polypeptide can be determined to be the ligand of the GPCR polypeptide, i.e., the ligand of the original GPCR. Therefore, in the ligand screening method of the present invention, the test substance to which the GPCR polypeptide responded is selected as the ligand of the original GPCR.

[0086] Suitably, the response of the GPCR polypeptide to the test substance can be evaluated by comparing the response of the GPCR polypeptide to which the test substance has been added (test group) with the response of the GPCR polypeptide in a control group. The control group can 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, the GPCR polypeptide before the addition of the test substance, etc. If 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 the ligand of the original GPCR.

[0087] Therefore, in one embodiment of the ligand discovery method of the present invention, the response of the GPCR polypeptide in the presence and absence of a 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 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 of 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 of the original GPCR.

[0088] As described above, the GPCR polypeptide expressed by the expression method of the present invention can be expressed more efficiently on a cultured cell membrane than the original GPCR, and the response of the GPCR polypeptide is considered to reflect the response of the original GPCR. Therefore, the GPCR polypeptide can be used to evaluate and / or select a substance that controls the recognition of a ligand in the original GPCR. Therefore, in another aspect, the present invention provides a method for evaluating and / or selecting a regulator of the recognition of a ligand of a target GPCR. The method includes adding a test substance and a target ligand to a 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, a preferred ligand is a pheromone substance. In another example, when a taste receptor polypeptide is used as the GPCR polypeptide, a preferred ligand is a taste substance (bitter substance, umami substance, or sweet substance). The ligand of the target GPCR is preferably a ligand selected by the ligand screening method of the present invention. According to the method for evaluating and / or selecting a regulator of the present invention, it is possible to efficiently evaluate or select a substance capable of selectively suppressing or enhancing the recognition of a target ligand.

[0089] The evaluation and / or selection of a regulatory agent of the present invention may be an in vitro or ex vivo method.

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

[0091] The GPCR polypeptide and its form used in the regulator evaluation and / or selection method of the present invention are the same as the GPCR polypeptide and its form used in the response measurement method of the present invention.

[0092] In the method for evaluating and / or selecting a regulator of the present invention, a test substance and a target ligand are applied to a GPCR polypeptide expressed by the expression method of the present invention. Means for applying the test substance and the target ligand to a GPCR polypeptide include, but are not limited to, a method in which the test substance and the target ligand are added to a medium for culturing cells expressing the GPCR polypeptide.

[0093] In the method of evaluating and / or selecting a regulator of the present invention, a test substance and a target ligand are added to a GPCR polypeptide, and then the response of the GPCR polypeptide to the ligand is measured. The method of measuring the response can be the same as that of the response measurement method of the present invention.

[0094] A test substance that inhibits the response of the GPCR polypeptide to the ligand is then detected based on the measured response. The detected test substance is selected as an inhibitor of recognition of the ligand. Alternatively, a test substance that enhances the response of the GPCR polypeptide to the ligand is detected based on the measured response. The detected test substance is selected as an enhancer of 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 to which the test substance has been added (test group) to the ligand with the response of the control group to the ligand. 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 a test substance has been added, the GPCR polypeptide before the addition of the test substance, and cells in which the GPCR polypeptide is not expressed. Preferably, the method for evaluating and / or selecting an inhibitor of the present invention includes measuring the activity of the GPCR polypeptide to the ligand in the presence and absence of a test substance.

[0096] For example, if the response in the test group is more suppressed 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, 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 preferably suppressed to 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, 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 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, i.e., 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 preferably by 120% or more, more preferably by 150% or more, and even more preferably by 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 regulator 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 GPCR of interest is a pheromone substance, and a pheromone suppressor or a pheromone enhancer is evaluated and / or selected as a regulator of ligand recognition. In another embodiment of the method for evaluating and / or selecting a regulator of the present invention, the GPCR polypeptide is an umami receptor polypeptide (a combination of consensus TAS1R1 and consensus TAS1R3) expressed by the expression method of the present invention, the ligand of the GPCR of interest is an umami substance, and a umami suppressor or an umami enhancer is evaluated and / or selected as a regulator of ligand recognition. In another embodiment of the method for evaluating and / or selecting a regulator of the present invention, the GPCR polypeptide is a sweet receptor polypeptide (a combination of consensus TAS1R2 and consensus TAS1R3) expressed by the expression method of the present invention, the ligand of the GPCR of interest is a sweet substance, and a sweet suppressor or a sweet enhancer is evaluated and / or selected as a regulator of ligand recognition. In another embodiment of the method for evaluating and / or selecting a control 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 GPCR of interest is a bitter substance, and a bitterness suppressor or bitterness enhancer is evaluated and / or selected as a control agent for ligand recognition.

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

[0100] Therefore, in one embodiment, the substance selected by the evaluation and / or selection method of the present invention for a regulator of ligand recognition may be an active ingredient of the regulator of ligand recognition. Alternatively, the substance selected by the evaluation and / or selection method of the present invention for a regulator of ligand recognition may be contained in a compound or composition for controlling the recognition of the ligand as an active ingredient for controlling the recognition of the ligand. Alternatively, the substance selected by the evaluation and / or selection method of the present invention for a regulator of ligand recognition may be used for the manufacture of the regulator of ligand recognition, or for the manufacture of a compound or composition for controlling the recognition of the ligand. The substance can suppress or enhance the recognition of the target ligand.

[0101] As described above, the GPCR polypeptide expressed by the expression method of the present invention can be expressed more efficiently on a cultured cell membrane than the original GPCR, and the response of the GPCR polypeptide is considered to reflect the response of the original GPCR, so that the taste of a test substance can be evaluated using a taste receptor polypeptide as the GPCR polypeptide. Therefore, 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, and 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 absence or the degree of a taste (e.g., umami, sweetness, or bitterness) is desired. The test substance may be a naturally occurring substance, or a substance artificially synthesized by a chemical or biological method, or may be a compound, composition, or mixture.

[0103] The GPCR polypeptide and its form used in the taste evaluation method of the present invention are the same as the GPCR polypeptide and its form used in the response measurement method of the present invention, so long as the GPCR polypeptide is a taste receptor polypeptide.

[0104] In the taste evaluation method of the present invention, a test substance is applied to a GPCR polypeptide expressed by the expression method of the present invention. Means for applying a test substance to a GPCR polypeptide include, but are not limited to, a method of adding the test substance to a medium for culturing cells expressing the GPCR polypeptide.

[0105] In the taste evaluation method of the present invention, a test substance is added to a GPCR polypeptide, and then the response of the GPCR polypeptide to the test substance is measured. The method for measuring the response can be the same as that of the response measurement method of the present invention.

[0106] The test substance is then evaluated based on the measured response of the GPCR polypeptide.The test substance that causes the response of the GPCR polypeptide can be evaluated as a substance that exhibits taste (e.g., umami, sweet, or bitter).The greater the intensity of the response caused by the test substance, the greater the degree of taste of the test substance is evaluated (e.g., more umami, more sweet, or more bitter).

[0107] Preferably, the response of the GPCR polypeptide to the test substance can be evaluated by comparing the response of the GPCR polypeptide to which the test substance is added (test group) with the response of the GPCR polypeptide in a control group. The control group can 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, etc. If the response in the test group is higher than that in the control group, the test substance is evaluated as a substance that exhibits 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 a 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 that exhibits 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 that exhibits 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 that exhibits a taste.

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

[0110] As described above, the GPCR polypeptide expressed by the expression method of the present invention can be expressed more efficiently on the cultured cell membrane than the original GPCR, and the response of the GPCR polypeptide is considered to reflect the response of the original GPCR, so that a substance that suppresses the odor recognition of a ligand (an odorant, specifically an amine) in the original GPCR can be evaluated and / or selected using a 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, i.e., causes a change in the ligand response of the original GPCR, and 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, i.e., causes a change in the ligand response of the original GPCR, and as a result, the odor of the ligand can be selectively suppressed based on odor cross-adaptation due to receptor agonism.

[0111] Therefore, in another aspect, the present invention provides a method for evaluating and / or selecting an odor suppressant for a ligand of a target GPCR. The method includes measuring the response of the GPCR polypeptide after addition of a test substance, the GPCR polypeptide being 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 odor suppressant for the target ligand. That is, a test substance that suppresses the response of the GPCR polypeptide is selected as an odor suppressant for the ligand based on receptor antagonism, and a test substance that enhances the response of the GPCR polypeptide is selected as an odor suppressant for the ligand based on odor cross-adaptation due to receptor agonism. The ligand of the target GPCR is preferably a ligand selected by the ligand screening method of the present invention. According to the method for evaluating and / or selecting odor suppressors of the present invention, substances that can selectively suppress the odor of a target ligand can be efficiently evaluated or selected, and even when the target ligand is a ligand of a GPCR that could not be functionally analyzed in the past due to insufficient expression in the cell membrane of cultured cells, substances that can selectively suppress the odor of the ligand can be evaluated or selected.

[0112] The evaluation and / or selection of odor suppressants of the present invention may be an in vitro or ex vivo method.

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

[0114] The GPCR polypeptide and its form used in the odor suppressor evaluation and / or selection method of the present invention are similar to the GPCR polypeptide and its form used in the response measurement method of the present invention, so long as the GPCR polypeptide is a trace amine-related receptor polypeptide.

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

[0116] In the odor suppressant evaluation and / or selection method of the present invention, a test substance is added to a GPCR polypeptide, and then the response of the GPCR polypeptide to the test substance is measured. The method for measuring the response can be the same as the response measurement method of the present invention.

[0117] In a first embodiment, the method for evaluating and / or selecting an odor suppressant of the present invention includes adding a test substance and a target ligand to a GPCR polypeptide expressed by the expression method of the present invention, and measuring the response of the GPCR polypeptide to the ligand. The method for applying the ligand to the GPCR polypeptide can be the same as the method for applying the test substance. Then, 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 odor suppressant for 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 to which the test substance has been added (test group) to the ligand with the response of the control group to the ligand. 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 test substance has been added, the GPCR polypeptide before the addition of the test substance, and cells in which the GPCR polypeptide is not expressed. Preferably, the odor inhibitor evaluation and / or selection method of the present invention in the first embodiment includes measuring the activity of the olfactory GPCR polypeptide to the ligand in the presence and absence of a test substance.

[0119] For example, if the response in the test group is more suppressed 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, 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 preferably suppressed to 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, 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 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, i.e., a substance that suppresses the response of the original GPCR to the ligand.

[0120] In a second embodiment, the method for evaluating and / or selecting an odor suppressant of the present invention comprises 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 odor suppressant of the ligand.

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

[0122] The effect 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 a test substance has been added with the response in a control group. Examples of the control group include those described above. Preferably, the method for evaluating and / or selecting an odor suppressor of the present invention in the second embodiment includes measuring the activity of the GPCR polypeptide in the presence and absence of a test substance. Also preferably, the method for evaluating and / or selecting an odor suppressor of the present invention in the second embodiment includes measuring the response of cells expressing and non-expressing the GPCR polypeptide to the ligand in the presence of a 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 preferably by 120% or more, more preferably by 150% or more, and even more preferably by 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 target ligand by an individual by suppressing the response of GPCR to the target ligand. Therefore, the test substance identified by the above procedure can be selected as an odor suppressant of the ligand. The substance selected as an odor suppressant of the target ligand by the odor suppressant evaluation and / or selection method of the present invention can suppress the odor of the ligand by suppressing the response of GPCR to the ligand.

[0125] Therefore, in one embodiment, the substance selected by the evaluation and / or selection method of the ligand odor suppressor of the present invention can be the active ingredient of the ligand odor suppressor. Alternatively, the substance selected by the evaluation and / or selection method of the ligand odor suppressor of the present invention can be contained in a compound or composition for suppressing the ligand odor as an active ingredient for suppressing the ligand odor. Alternatively, the substance selected by the evaluation and / or selection method of the ligand odor suppressor of the present invention can be used for producing the ligand odor suppressor or for producing a compound or composition for suppressing the ligand odor. According to this substance, the odor of the target ligand can be deodorized without causing the discomfort caused by the strong odor of the fragrance, which occurred in the conventional deodorant or deodorization method using a fragrance, or the problem of suppressing other odors.

[0126] The following compositions, manufacturing methods, uses or methods are further disclosed herein as exemplary embodiments of the present invention, but the present invention is not limited to these embodiments.

[0127] [1] A method for expressing a GPCR polypeptide, comprising the steps of: expressing in a cell a GPCR polypeptide having 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 a target GPCR (excluding olfactory receptors) has been altered to an amino acid residue of the consensus amino acid sequence at a position corresponding to the amino acid residue; 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 a GPCR encoded by an ortholog of the GPCR of interest in a vertebrate; method. [2] The method described in [1], which is a method for improving expression of a GPCR polypeptide. [3] A method for functionalizing a target GPCR, comprising the steps of: expressing in a cell a GPCR polypeptide having 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 a target GPCR (excluding olfactory receptors) has been altered to an amino acid residue of the consensus amino acid sequence at a position corresponding to the amino acid residue; 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 a GPCR encoded by an ortholog of the GPCR of interest in a vertebrate; method. [4] The method according to any one of [1] to [3], wherein the ortholog is selected from orthologs in mammals, birds, reptiles, amphibians and fish, preferably selected from orthologs in mammals, birds, reptiles and amphibians, more preferably an ortholog in a mammal. [5] The method according to any one of [1] to [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) to (iii): (i) at each amino acid position of the alignment, (ii) if there is one amino acid residue that is different from the amino acid residue of the target GPCR and occurs at a frequency of 50% or more, the amino acid residue is identified as a consensus residue; (i-ii) if there are two amino acid residues with an occurrence frequency of 50%, the amino acid residue of the GPCR of interest is identified as a consensus residue; (i-iii) if the amino acid residue is present in the GPCR of interest and the amino acid residue is not present with an occurrence frequency of 40% or more, the amino acid residue is identified as being absent; (i-iv) if the amino acid residue is absent in the GPCR of interest and an amino acid residue is present with an occurrence frequency of 60% or more, the most frequently occurring amino acid residue is identified as the consensus residue, and if two or more types of the most frequently occurring amino acid residues are present, the amino acid residue with the smallest molecular weight among the amino acid residues is identified as the consensus residue; (iv) if none of the above (ii) to (i-iv) apply, identifying the amino acid residue of the target GPCR as a consensus residue; (ii) when the consensus residue is identified according to the above criterion (i), if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminus or further C-terminus of the GPCR of interest and is not a methionine residue, changing the consensus residue N-terminal to no consensus residue, the consensus residue consisting of the methionine residue at the position closest to the N-terminus; (iii) When a consensus residue is identified according to the criterion (i) above, if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminal side of the N-terminus of the GPCR of interest and is not a methionine residue, the amino acid residue that occurs most frequently is identified as the consensus residue by going back one amino acid position at a time N-terminally from the position of the consensus residue in the alignment until a methionine residue appears, and if there are two or more types of amino acid residues that occur most frequently, the amino acid residue with the smallest molecular weight among those amino acid residues is identified 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 at least 2, preferably at least 5, more preferably at least 11, even more preferably at least 15, even more preferably at least 30, and even more preferably at least 100 types of GPCRs encoded by orthologs of the target GPCR in the vertebrate. [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] to [8], wherein the GPCR polypeptide comprises an amino acid sequence in which at least one amino acid residue different from the consensus amino acid sequence shown in SEQ ID NO: (3) in the amino acid sequence shown in SEQ ID NO: (2) of the GPCR shown in (1) in Tables 2-1 and 2-2 below has been modified to an amino acid residue of the consensus amino acid sequence at a corresponding position.

[0128] [Table 2-1]

[0129] [Table 2-2]

[0130]

[10] The method according to [9], wherein the GPCR polypeptide preferably consists of an amino acid sequence shown in any one of SEQ ID NOs: 108 to 142, 144 to 214, and 275 to 312.

[11] A method for expressing a GPCR polypeptide, comprising the steps of: expressing in a cell a GPCR polypeptide having an amino acid sequence in which at least one amino acid residue in the amino acid sequence of a target GPCR that differs from a consensus amino acid sequence has been altered to an amino acid residue in the consensus amino acid sequence at a position corresponding to the amino acid residue; The GPCR polypeptide comprises an amino acid sequence in which at least one amino acid residue in the amino acid sequence of human TAAR6 shown in SEQ ID NO: 36 that differs from the consensus amino acid sequence shown in SEQ ID NO: 143 has been altered to an amino acid residue in the consensus amino acid sequence at a position corresponding to the amino acid residue, and preferably comprises the amino acid sequence shown in SEQ ID NO: 143. method.

[12] The method described in

[11] , which is a method for improving expression of a GPCR polypeptide.

[13] A method for functionalizing a target GPCR, comprising the steps of: expressing in a cell a GPCR polypeptide having an amino acid sequence in which at least one amino acid residue in the amino acid sequence of a target GPCR that differs from a consensus amino acid sequence has been altered to an amino acid residue in the consensus amino acid sequence at a position corresponding to the amino acid residue; The GPCR polypeptide comprises an amino acid sequence in which at least one amino acid residue in the amino acid sequence of human TAAR6 shown in SEQ ID NO: 36 that differs from the consensus amino acid sequence shown in SEQ ID NO: 143 has been altered to an amino acid residue in the consensus amino acid sequence at a position corresponding to the amino acid residue, and preferably comprises the amino acid sequence shown in SEQ ID NO: 143. method.

[14] The method according to any one of [1] to

[13] , wherein the GPCR polypeptide is expressed in the cell membrane of the cell.

[15] The method according to any one of [1] to

[14] , preferably further comprising expressing RTP1S in the cell.

[16] The method according to any one of [1] to

[15] , wherein the cell is a HEK293 cell.

[0131]

[17] A method for measuring a response of a target GPCR, comprising: Measuring the response of the GPCR polypeptide expressed by the method according to any one of [1] to

[16] ; The method includes:

[18] A method for searching for a ligand of a target GPCR, comprising the steps of: Measuring the response of the GPCR polypeptide expressed by the method according to any one of [1] to

[16] in the presence of a test substance; and selecting a test substance to which the GPCR polypeptide responded; The method includes:

[19] The method of

[18] , further comprising measuring the response of the GPCR polypeptide in the absence of a test substance.

[20] The method described in

[19] , wherein a test substance is selected that preferably 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] The method described in

[19] , wherein a test substance is selected that preferably increases the response of the GPCR polypeptide in the presence of the test substance by a statistically significant amount compared to the response in the absence of the test substance.

[22] A method for evaluating and / or selecting a regulator of ligand recognition of a target GPCR, comprising: adding a test substance and a ligand of the GPCR of interest to the GPCR polypeptide expressed by the method according to any one of [1] to

[16] ; and measuring the response of said GPCR polypeptide to said ligand; The method includes:

[23] The method according to

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

[18] to

[21] .

[24] The method of any one of

[22] or

[23] , further comprising identifying a test substance that controls, preferably inhibits or enhances, the response of the GPCR polypeptide 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 to which the test substance has not been added.

[26] The method described in

[25] , 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 has been added to the test substance is suppressed more than the response of the GPCR polypeptide to the ligand to which the test substance has not been added, or identifying the test substance as a substance that enhances the response of the GPCR polypeptide to the ligand when the response of the GPCR polypeptide to which the test substance has been added to the test substance is enhanced more than the response of the GPCR polypeptide to the ligand to which the test substance has not been added.

[27] A method for evaluating taste, comprising: adding a test substance to the GPCR polypeptide expressed by the method according to any one of [1] to

[16] ; and measuring the response of said GPCR polypeptide to said test substance; wherein the GPCR polypeptide is a taste receptor polypeptide. method.

[28] The method described in

[27] , preferably further comprising measuring the response of the GPCR polypeptide to which the test substance has not been added.

[29] The method described in

[28] , wherein a test substance that increases the response of the GPCR polypeptide to which the test substance has been added to 120% or more of the response of the GPCR polypeptide to which the test substance has not been added is evaluated as a substance that exhibits a taste.

[30] The method described in

[28] , wherein a test substance that statistically significantly increases the response of the GPCR polypeptide to which the test substance has been added compared to the response of the GPCR polypeptide to which the test substance has not been added is evaluated as a substance that exhibits a taste.

[31] The method according to any one of

[27] to

[30] , wherein the taste is preferably umami, sweet, or bitter.

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

[16] ; and measuring the response of said GPCR polypeptide to said ligand; wherein the GPCR polypeptide is a trace amine associated receptor polypeptide. method.

[33] The method according to

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

[18] to

[21] .

[34] The method of

[32] or

[33] , further comprising identifying a test substance that inhibits 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 to which the test substance has not been added.

[36] The method described in

[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 has been added to the ligand is more inhibited than the response of the GPCR polypeptide to the ligand to which the test substance has not been added.

[37] A method for evaluating and / or selecting an odor suppressant of a ligand of a target GPCR, comprising: adding a test substance to the GPCR polypeptide expressed by the method according to any one of [1] to

[16] ; and measuring the response of said GPCR polypeptide to said test substance; wherein the GPCR polypeptide is a trace amine associated receptor polypeptide. method.

[38] The method according to

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

[18] to

[21] .

[39] The method of

[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 described in any one of

[37] to

[39] , further comprising measuring the response of the GPCR polypeptide to which the test substance has not been added.

[41] The method described in

[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 has been added is enhanced compared to the response of the GPCR polypeptide to which the test substance has not been added.

[42] The method according to any one of

[17] to

[41] , wherein the response of the GPCR polypeptide is preferably measured by measuring the amount of intracellular cAMP by ELISA or a reporter gene assay, measuring the amount of calcium ions by calcium imaging or a TGFα shedding assay, or measuring the change in potential inside and outside the cell membrane by a two-electrode voltage clamp method using Xenopus oocytes.

[0132]

[43] A modified GPCR polypeptide, comprising: an amino acid sequence in which at least one amino acid residue that is different from a consensus amino acid sequence in the amino acid sequence of a target GPCR (excluding olfactory receptors) has been modified to an amino acid residue of the consensus amino acid sequence at a corresponding position; 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 a GPCR encoded by an ortholog of the GPCR of interest in a vertebrate; Modified GPCR Polypeptides.

[44] The modified GPCR polypeptide of

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

[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, (ii) if there is one amino acid residue that is different from the amino acid residue of the target GPCR and occurs at a frequency of 50% or more, the amino acid residue is identified as a consensus residue; (i-ii) if there are two amino acid residues with an occurrence frequency of 50%, the amino acid residue of the GPCR of interest is identified as a consensus residue; (i-iii) if the amino acid residue is present in the GPCR of interest and the amino acid residue is not present with an occurrence frequency of 40% or more, the amino acid residue is identified as being absent; (i-iv) if the amino acid residue is absent in the GPCR of interest and an amino acid residue is present with an occurrence frequency of 60% or more, the most frequently occurring amino acid residue is identified as the consensus residue, and if two or more types of the most frequently occurring amino acid residues are present, the amino acid residue with the smallest molecular weight among the amino acid residues is identified as the consensus residue; (iv) if none of the above (ii) to (i-iv) apply, identifying the amino acid residue of the target GPCR as a consensus residue; (ii) when the consensus residue is identified according to the above criterion (i), if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminus or further C-terminus of the GPCR of interest and is not a methionine residue, changing the consensus residue N-terminal to no consensus residue, the consensus residue consisting of the methionine residue at the position closest to the N-terminus; (iii) When a consensus residue is identified according to the criterion (i) above, if the most N-terminal consensus residue is a consensus residue at a position corresponding to the N-terminal side of the N-terminus of the GPCR of interest and is not a methionine residue, the amino acid residue that occurs most frequently is identified as the consensus residue by going back one amino acid position at a time N-terminally from the position of the consensus residue in the alignment until a methionine residue appears, and if there are two or more types of amino acid residues that occur most frequently, the amino acid residue with the smallest molecular weight among those amino acid residues is identified 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 with the amino acid sequences of at least 2, preferably at least 5, more preferably at least 11, even more preferably at least 15, even more preferably at least 30, and even more preferably at least 100 types of GPCRs encoded by orthologs of the target GPCR in the vertebrate.

[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] The modified GPCR polypeptide according to any one of

[43] to

[48] , preferably consisting of an amino acid sequence in which at least one amino acid residue in the amino acid sequence shown in SEQ ID NO: (2) of the GPCR shown in (1) of Tables 2-1 and 2-2 above, which differs from the consensus amino acid sequence shown in SEQ ID NO: (3), has been modified to an amino acid residue of the consensus amino acid sequence at a corresponding position.

[50] The modified GPCR polypeptide according to

[49] , preferably consisting of an amino acid sequence shown in any of SEQ ID NOs: 108 to 142, 144 to 214, and 275 to 312.

[51] A modified GPCR polypeptide, comprising: The amino acid sequence is an amino acid sequence in which at least one amino acid residue in the amino acid sequence of human TAAR6 shown in SEQ ID NO: 36 that differs from the consensus amino acid sequence shown in SEQ ID NO: 143 is modified to an amino acid residue in the consensus amino acid sequence at a position corresponding to the amino acid residue, and preferably the amino acid sequence shown in SEQ ID NO: 143. Modified GPCR Polypeptides.

[52] A polynucleotide encoding the modified GPCR polypeptide according to any one of

[43] to

[51] .

[53] The polynucleotide according to

[52] , which consists of a base sequence shown in any one of SEQ ID NOs: 215 to 234 and 313 to 317.

[54] A vector or DNA fragment comprising the polynucleotide described in

[52] or

[53] .

[55] A transformed cell containing the vector or DNA fragment described in

[54] .

[56] The transformed cell according to

[55] , preferably further comprising a vector or a DNA fragment containing a polynucleotide encoding RTP1S.

[57] The transformed cell described in

[55] or

[56] , wherein the cell is a HEK293 cell. EXAMPLES

[0133] The present invention will be described more specifically below with reference to examples.

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

[0135] [Table 3-1]

[0136] [Table 3-2]

[0137] [Table 3-3]

[0138] [Table 3-4]

[0139] [Table 4]

[0140] For the GPCRs No. 1 to 145 in Table 4, alignment analysis of the identified gene group was performed using ClustalW. Based on the alignment results, a consensus receptor was designed using Jalview. In the alignment, when one type of amino acid residue different from the amino acid residue of the reference amino acid sequence and having an occurrence frequency of 50% or more exists at a position corresponding to each amino acid position of the original receptor amino acid sequence of the reference receptor, the amino acid residue of the reference amino acid sequence was modified to the amino acid residue. Even when one type of amino acid residue different from the amino acid residue of the reference amino acid sequence and having an occurrence frequency of 50% exists at a position corresponding to each amino acid position of the original receptor amino acid sequence of the reference receptor, if the occurrence frequency of the amino acid residue of the reference amino acid sequence is also 50%, the amino acid residue of the reference amino acid sequence was not modified. In the alignment, when a deletion exists at a position corresponding to each amino acid position of the original receptor amino acid sequence of the reference receptor and having an occurrence frequency of 40% or more, the amino acid residue of the reference amino acid sequence was modified to a deletion. For example, in the case of TAAR1, a deletion occurs at a position corresponding to the amino acid position of the C-terminus with a frequency of 40% or more, so the amino acid residues of the reference amino acid sequence were modified to be deleted. Furthermore, in the case of TAAR1, TAAR2, MAS1L, GPR135, and ADGRG4, a deletion occurs at a position corresponding to the start methionine position in the human sequence with a frequency of 40% or more, so the amino acid residues of the reference amino acid sequence were modified to be deleted, and the first methionine in the amino acid sequence after modification by the above procedure was selected as the start methionine, and the amino acid sequence before it was deleted. However, in this method, when the consensus residue on the N-terminal side of the consensus residue consisting of the methionine residue closest to the N-terminus was changed to no consensus residue, the resulting total length of the amino acid sequence of the consensus receptor was 10% or more shorter than the total 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 consensus residue asparagine on the most N-terminal side was an important amino acid residue for glycosylation and membrane translocation, the consensus residue was not changed, and the N-terminal structure of the original receptor on the N-terminal side of the position corresponding to the consensus residue was maintained as it is. On the other hand, in the alignment, when an amino acid with an occurrence frequency of 60% or more is present 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. When there are two or more types of the most conserved amino acid, the amino acid with the smallest molecular weight was inserted. For example, in the case of TAS2R1, an amino acid with an occurrence frequency of 60% or more is present on the N-terminal side of the position corresponding to the amino acid position of the N-terminus, so an amino acid residue was added to the N-terminal side of the N-terminus of the reference amino acid sequence. The topology of the receptors was confirmed using the Transmembrane Hidden Markov Model (TMHMM). The designed receptors must have a seven-transmembrane structure. For GPCRs No. 1-5, 7-13, 31-38, 60, 76, 80, 85 and 102 in Table 4, the DNA sequences encoding the various receptor polypeptides were obtained by DNA synthesis after optimizing the base sequence codons corresponding to the amino acid sequences for expression in human cultured cells. EcoRI and XhoI sites were added to both ends of this base sequence, and it was recombined with EcoRI and XhoI sites created downstream of the Flag-Rho tag sequence on the pME18S vector. A gene encoding human RTP1S, which translocates GPCR proteins made in cultured cells onto the cell membrane, was inserted into the EcoRI and XhoI sites of another pME18S vector to create the pME18S-RTP1S vector.

[0141] 2) Preparation of cultured cells transformed with the receptor gene 1 For flow cytometry, 3.3 × 10 HEK293 cells suspended in DMEM (Nacalai) were placed in each well of a 6-well dish. 524 hours after seeding with 1000 cells, a reaction solution with the composition shown in Table 5 was prepared, left to stand in a clean bench for 20 minutes, and then added to each well of a 6-well dish. The receptor genes referred to here are the GPCR genes of Nos. 1 to 4, 8 to 13, 31 to 38, 60, 76, 80, 85, and 102 in Table 4. The cells were cultured for 24 hours in an incubator maintained at 37°C and 5% CO2. 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 flow cytometry, 3.3 × 10 HEK293 cells suspended in DMEM (Nacalai) were placed in each well of a 6-well dish. 5 24 hours after seeding with 1000 cells, a reaction solution with the composition shown in Table 6 was prepared, left to stand in a clean bench for 20 minutes, and then added to each well of a 6-well dish. The cells were cultured for 24 hours in an incubator maintained at 37°C and 5% CO2. 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 (flow cytometry) The cells collected using a Cellstripper were treated with anti-FLAG antibody (Cosmo Bio) as the primary antibody for 1 hour on ice. After washing the cells, PE (phycoerythrin)-conjugated anti-mouse IgG (Abcam) was treated as the secondary antibody for 30 minutes on ice. After washing, 0.25 μg of 7-AAD (7-Aminoactinomycin D, Fujifilm Wako Pure Chemical) was added and the average PE signal of the cell population negative for 7-AAD was measured using a flow cytometry system (BD) as an index of the amount of receptors on the cell membrane. In each experimental run, cells expressing FLAG-M2 acetylcholine receptors were used as positive controls (PC) and cells not expressing the receptors were used as negative controls (NC), and the average PE signal was calculated in the same manner as above. The PE signal of NC was standardized to 0% and the PE signal of PC to 100%, and the PE signal (%) of each receptor was calculated as the membrane expression amount (cell surface expression).

[0146] 5) Results The expression level of each receptor on HEK293 cells was analyzed using flow cytometry (above 2) and 4). As shown in Table 7, the average values ​​of three experiments were compared, and as a result, an increase in membrane expression level was observed by the consensus method for all receptors subjected to membrane expression level analysis, 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.

[0147] [Table 7]

[0148] Using flow cytometry (3) and 4) above, we analyzed the expression levels of the original receptor of TAS1R1 and the original receptor of TAS1R3, the consensus receptor of TAS1R1 and the consensus receptor of 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 in HEK293 cells. As shown in Figure 1, it was revealed that the membrane expression level of TAS1R1 increased when a consensus receptor of either of TAS1R1 and TAS1R3 was expressed compared to when the two original receptors of TAS1R1 and TAS1R3 were coexpressed in HEK293 cells. In particular, the highest expression level was observed when both TAS1R1 and TAS1R3 were consensus-modified.

[0149] The Accession Nos. and amino acid sequences of the original GPCRs and the amino acid sequences of the consensus GPCRs in the above Examples are shown in Tables 8-1 and 8-2 below. In addition, DNA sequences encoding consensus GPCRs consisting of the amino acid sequences shown in 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 improving the expression of a target G protein-coupled receptor (GPCR), To express in cells a GPCR polypeptide consisting of an amino acid sequence in which at least 10% of the amino acid residues in the amino acid sequence shown by the sequence number (2) of the target GPCR in Tables 1 and 2 below, which differ from the consensus amino acid sequence shown by the sequence number (3), are modified to the corresponding amino acid residues of the consensus amino acid sequence. Methods including (but excluding methods of surgery or treatment of humans). Table 1 Table 2

2. The method according to claim 1, wherein the GPCR polypeptide comprises an amino acid sequence obtained by modifying at least 50% of the amino acid residues in the amino acid sequence shown by the sequence number (2) of the target GPCR in Tables 1 and 2 above, which differ from the consensus amino acid sequence shown by the sequence number (3), to amino acid residues of the consensus amino acid sequence at the corresponding positions.

3. The method according to claim 1, wherein the GPCR polypeptide comprises an amino acid sequence represented by any of SEQ ID NOs: 113, 121-137, 146-166, 168-182, 184-186, 188-191, 193-208, 210-214, and 275-312.

4. A method for measuring the response of a target GPCR, To express in cells a GPCR polypeptide consisting of an amino acid sequence in which at least 10% of the amino acid residues in the amino acid sequence indicated by the sequence number (2) of the target GPCR in Tables 3 and 4 below that differ from the consensus amino acid sequence indicated by the sequence number (3) have been modified to the corresponding amino acid residues of the consensus amino acid sequence, and To measure the response of the GPCR polypeptide, Methods including (but excluding methods of surgery or treatment of humans). Table 3 Table 4

5. A method for searching for a ligand for a target GPCR, To express in cells a GPCR polypeptide consisting of an amino acid sequence in which at least 10% of the amino acid residues that differ from the consensus amino acid sequence shown by the sequence number Measuring the response of the GPCR polypeptide in the presence of the test substance, and Select the test substance to which the GPCR polypeptide responded. Methods including (but excluding methods of surgery or treatment of humans). Table 5 Table 6

6. A method for evaluating and / or selecting a regulatory agent for the recognition of a target GPCR ligand, To express in cells a GPCR polypeptide consisting of an amino acid sequence in which at least 10% of the amino acid residues in the amino acid sequence shown by the sequence number (2) of the target GPCR in Tables 7 and 8 below, which differ from the consensus amino acid sequence shown by the sequence number (3), are modified to the corresponding amino acid residues of the consensus amino acid sequence. Adding the test substance and the ligand of the target GPCR to the GPCR polypeptide, and Measuring the response of the GPCR polypeptide to the ligand, Methods including (but excluding methods of surgery or treatment of humans). Table 7 Table 8

7. This is a method for evaluating taste, To express in cells a GPCR polypeptide consisting of an amino acid sequence in which at least 10% of the amino acid residues in the amino acid sequence shown by the sequence number (2) of the target GPCR in Tables 9 and 10 below, which differ from the consensus amino acid sequence shown by the sequence number (3), are modified to the corresponding amino acid residues of the consensus amino acid sequence. Adding a test substance to the GPCR polypeptide, and To measure the response of the GPCR polypeptide to the test substance, The GPCR polypeptide is a taste receptor polypeptide, Methods (excluding methods involving surgery or treatment of humans). Table 9 Table 10

8. A method for evaluating and / or selecting an odor inhibitor for a target GPCR ligand, To express in cells a GPCR polypeptide consisting of an amino acid sequence in which at least 10% of the amino acid residues in the amino acid sequence shown by the sequence number (2) of the target GPCR in Tables 11 and 12 below, which differ from the consensus amino acid sequence shown by the sequence number (3), are modified to the corresponding amino acid residues of the consensus amino acid sequence. Adding the test substance and the ligand of the target GPCR to the GPCR polypeptide, and Measuring the response of the GPCR polypeptide to the ligand, The GPCR polypeptide is a trace amine-related receptor polypeptide, Methods (excluding methods involving surgery or treatment of humans). Table 11 Table 12

9. A method for evaluating and / or selecting an odor inhibitor for a target GPCR ligand, To express in cells a GPCR polypeptide consisting of an amino acid sequence in which at least 10% of the amino acid residues in the amino acid sequence shown by the sequence number (2) of the target GPCR in Tables 13 and 14 below, which differ from the consensus amino acid sequence shown by the sequence number (3), are modified to the corresponding amino acid residues of the consensus amino acid sequence. Adding a test substance to the GPCR polypeptide, and To measure the response of the GPCR polypeptide to the test substance, The GPCR polypeptide is a trace amine-related receptor polypeptide, Methods (excluding methods involving surgery or treatment of humans). Table 13 Table 14

10. The method according to any one of claims 4 to 9, wherein the GPCR polypeptide comprises an amino acid sequence obtained by modifying at least 50% of the amino acid residues in the amino acid sequence shown by the sequence number (2) of the target GPCR in Tables 3 to 14 above, which differ from the consensus amino acid sequence shown by the sequence number (3), to amino acid residues of the consensus amino acid sequence at the corresponding positions.

11. The method according to any one of claims 4 to 9, wherein the GPCR polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 113, 121-137, 146-166, 168-182, 184-186, 188-191, 193-208, 210-214, and 275-312.

12. The method according to any one of claims 4 to 9, wherein the response of the GPCR polypeptide is measured by measuring the amount of intracellular cAMP by ELISA or a reporter gene assay, measuring the amount of calcium ions by calcium imaging or a TGFα shedding assay, or measuring the change in potential across the cell membrane by a two-electrode membrane potential fixation method using African clawed frog oocytes.

13. A modified GPCR polypeptide, The amino acid sequence consists of the amino acid sequence in which, in the amino acid sequence shown in the sequence number (2) of the target GPCR in (1) of Tables 15 and 16 below, at least 10% of the amino acid residues that differ from the consensus amino acid sequence shown in the sequence number (3) are modified to the amino acid residues of the consensus amino acid sequence at the corresponding positions. Modified GPCR polypeptide. Table 15 Table 16

14. The modified GPCR polypeptide according to claim 13, wherein the GPCR polypeptide comprises an amino acid sequence in which at least 50% of the amino acid residues that differ from the consensus amino acid sequence shown by the sequence number (3) in the amino acid sequence shown by the sequence number (2) of the target GPCR in Tables 15 and 16 above are modified to amino acid residues of the consensus amino acid sequence at corresponding positions.

15. A modified GPCR polypeptide according to claim 13, comprising an amino acid sequence represented by any one of SEQ ID NOs: 113, 121-137, 146-166, 168-182, 184-186, 188-191, 193-208, 210-214, and 275-312.

16. A polynucleotide encoding a modified GPCR polypeptide according to any one of claims 13 to 15.

17. A vector or DNA fragment comprising the polynucleotide described in claim 16.

18. Transformed cells containing the vector or DNA fragment according to claim 17.

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