Cultured cell expressing modified vasopressin receptor

Abstract

To provide a method that enables measurement of AVP with high sensitivity and without influence from DDAVP, as well as to provide an AVP receptor.SOLUTION: The present invention employs a cultured cell in which a wild-type or mutant form of the platypus V2 receptor, a high-sensitivity receptor, is expressed. Moreover, receptor activation by DDAVP is reduced by introducing a point mutation into the amino acid sequence of the platypus V2 receptor.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a method and kit for measuring arginine vasopressin (AVP), which enables the diagnosis of pituitary diseases caused by AVP. [Background technology]

[0002] AVP is a peptide hormone synthesized in the hypothalamus, transported to the posterior pituitary gland, and secreted. AVP acts on vasopressin receptors (V2 receptors) present in renal collecting duct cells, promoting water reabsorption and reducing urine volume, and is therefore also known as antidiuretic hormone (ADH).

[0003] Central diabetes insipidus is a disease caused by a complete or partial deficiency in AVP secretion, resulting in symptoms such as polydipsia and polyuria due to impaired AVP secretion and impaired reabsorption of urine in the kidney. Syndrome of inappropriate antidiuretic hormone secretion (SIADH) is known as a disease caused by abnormal vasopressin secretion, which is caused by increased vasopressin secretion due to central nervous system disease or ectopic vasopressin production from tumors. Nephrogenic diabetes insipidus, which exhibits symptoms similar to central diabetes insipidus, is also known, due to decreased sensitivity to vasopressin caused by mutations in the V2 receptor.

[0004] It is widely known that measuring plasma AVP concentration is useful for diagnosing central diabetes insipidus. For example, radioimmunoassay (RIA) is known as a method for measuring plasma AVP concentration. Specifically, cold ethanol is added to a patient's plasma to extract AVP, and the resulting sample is centrifuged and dried to obtain a measurement sample. Isotope-labeled AVP is added to the sample, and the sample is reacted with an anti-AVP antibody to measure the plasma AVP concentration through a competitive inhibition reaction between the isotope-labeled AVP contained in the sample and plasma-derived AVP (Non-Patent Document 1). However, the RIA described in Non-Patent Document 1 requires a total of about three days from the AVP extraction step to the completion of measurement, and therefore simplification and speedup of the measurement have been desired.

[0005] Recently, there have been known examples of applying the measurement of intracellular cyclic adenosine monophosphate concentration associated with activation of V2 receptors by AVP to the measurement of AVP concentration. Specifically, a bioassay system is known in which Chinese hamster ovary (CHO) cells are incubated in the presence of a test sample, and the amount of cAMP produced by stimulation of human V2R by AVP contained in the sample is detected by calcium ion-mediated luminescence (Patent Document 1).

[0006] More recently, amino acid residue substitutions in V2R have been investigated, and Non-Patent Document 2 reports on F229V and R137C / L mutants. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Re-table No. 2012-086756 [Non-patent literature]

[0008] [Non-Patent Document 1] AVP Kit "Yamasa" package insert, revised June 2021 (4th edition), https: / / www.info.pmda.go.jp / downfiles / ivd / PDF / 800075_22700AMX00611000_A_04_03.pdf [Non-patent document 2] Scientific Reports (2020) 10:9111 Summary of the Invention [Problem to be solved by the invention]

[0009] While investigating methods for measuring AVP concentration, the present inventors investigated an assay system using cultured cells expressing V2 receptors and found that the sensitivity of the assay system is highly dependent on the receptor used. Because the AVP concentration in blood samples is very low, ranging from 0.1 to several pg / mL, a highly sensitive assay system is desirable for accurate measurement. Furthermore, DDAVP (1-deamino-8-D-arginine-vasopressin), which is used as a therapeutic agent for central diabetes insipidus, activates the V2 receptor, thereby interfering with the measurement of AVP concentration in samples. Therefore, it is desirable to be able to eliminate the influence of DDAVP when measuring AVP concentration in patient blood samples. [Means for solving the problem]

[0010] The present inventors have conducted studies using V2 receptors derived from various mammals and found that the sensitivity of an assay system using cultured cells expressing V2 receptors depends on the V2 receptor used. By comparing V2 receptors derived from various animals, they have discovered a highly sensitive receptor. Furthermore, by introducing a point mutation into the amino acid sequence of the platypus V2 receptor, which was the most sensitive of the newly discovered highly sensitive receptors, they were able to reduce receptor activation by DDAVP, thereby completing the present invention. The present invention provides the following:

[0011] [1] A cultured cell expressing any one of the following proteins: (1) a protein consisting of the amino acid sequence of SEQ ID NO: 1 (platypus V2 receptor); (2) A protein having an amino acid sequence of SEQ ID NO: 1, which has at least one mutation selected from the group consisting of phenylalanine (F) corresponding to position 126, asparagine (N) corresponding to position 126, tyrosine (Y) corresponding to position 126, alanine (A) corresponding to position 129, N corresponding to position 205, an amino acid other than A corresponding to position 214, and an amino acid other than glutamic acid (E) corresponding to position 218; (3) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of the protein according to (1) or (2) and having AVP receptor activity. (4) A protein having an amino acid sequence having at least 75% identity with the amino acid sequence of the protein according to (1) or (2), with the proviso that the portion corresponding to positions 219 to 332 or 333 has at least 95% identity, and having AVP receptor activity; (5) A protein, wherein the portion corresponding to positions 334 to 339 in the amino acid sequence of the protein according to (1) to (4) is the amino acid sequence of SEQ ID NO: 19. [2] Any one of the following proteins: (2) A protein having an amino acid sequence of SEQ ID NO: 1, which has at least one mutation selected from the group consisting of phenylalanine (F) corresponding to position 126, asparagine (N) corresponding to position 126, tyrosine (Y) corresponding to position 126, alanine (A) corresponding to position 129, N corresponding to position 205, an amino acid other than A corresponding to position 214, and an amino acid other than glutamic acid (E) corresponding to position 218; (3') A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of the protein set forth in SEQ ID NO: 1 or (2) (excluding the amino acid sequence of SEQ ID NO: 1), and having AVP receptor activity. (4') a protein having an amino acid sequence having at least 75% identity with the amino acid sequence of the protein set forth in SEQ ID NO: 1 or (2) (excluding the amino acid sequence of SEQ ID NO: 1), with the proviso that the portion corresponding to positions 219 to 332 or 333 has at least 95% identity, and having AVP receptor activity; (5) A protein in which the portion corresponding to positions 334 to 339 in the amino acid sequence of the protein described in (2), (3'), and (4') is the amino acid sequence of SEQ ID NO: 19. [3] The cultured cell according to [1], which further expresses a cAMP biosensor. [4] A polynucleotide encoding the protein described in [2]. [5] A kit for measuring AVP, comprising the cultured cells according to [1] or [3]. [6] The kit according to [5], which is an in vitro diagnostic pharmaceutical. [7] A method for analyzing AVP levels in a sample, comprising the steps of: (a) adding a sample to cultured cells expressing AVP receptors and incubating; In this case, the AVP receptor is selectively activated by AVP, while its activation by 1-deamino-8-D-arginine vasopressin (DDAVP) is inhibited; (b) analyzing the activation level of AVP receptors after incubation; (c) A method for analyzing the AVP level attenuated by the influence of DDAVP based on the obtained analytical results, wherein the AVP receptor is a protein defined in [1] or a protein described in [2]. [Effects of the Invention]

[0012] The present invention provides cultured cells or receptor proteins that express a V2 receptor protein that has high AVP receptor activity and reduced activation of the V2 receptor by DDAVP. According to the present invention, AVP contained in a patient's blood sample can be measured with high sensitivity, and by reducing receptor activation by DDAVP, the AVP concentration can be measured accurately. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows the pEC50 (-log10EC50) when HEK293A cells expressing V2 receptors derived from various animals were stimulated with AVP. The vertical axis shows pEC50, and the horizontal axis shows the names of the various animals. [Figure 2] Figure 2 is a schematic diagram showing the mutation sites introduced into V2R in Example 1 of the present specification. In the figure, asterisks indicate the positions of the mutation sites. In a character string consisting of one alphabetic character, two or three digits, and another alphabetic character, the central three-digit number indicates the number of the corresponding amino acid residue in platypus V2R, the alphabet on the left side indicates the amino acid residue in wild-type V2R, and the alphabet on the right side indicates the amino acid residue after mutation. [Figure 3] Figure 3 shows concentration-response curves for wild-type platypus V2R and the platypus V2R mutant (aspartic acid (D) at position 126). The vertical axis shows the cAMP level (RLU) after the addition of each concentration of AVP or DDAVP, normalized to the cAMP level (RLU) after the addition of forskolin. The horizontal axis shows the concentration of AVP or DDAVP. [Figure 4] Figure 4 shows concentration-response curves for wild-type platypus V2R and the platypus V2R mutant (methionine (M) at position 205). The vertical axis shows the cAMP level (RLU) after the addition of each concentration of AVP or DDAVP, normalized to the cAMP level (RLU) after the addition of forskolin. The horizontal axis shows the concentration of AVP or DDAVP. [Figure 5] Figure 5 shows concentration-response curves for wild-type platypus V2R and platypus V2R mutants (D126Y, D126N, M205N, D126Y / M205N, D126N / M205N). The vertical axis shows the cAMP level (RLU) after addition of each concentration of AVP, normalized to the cAMP level (RLU) after addition of forskolin. The horizontal axis shows the AVP concentration. [Figure 6]Figure 6 shows concentration-response curves for wild-type platypus V2R and platypus V2R mutants (D126Y, D126N, M205N, D126Y / M205N, D126N / M205N). The vertical axis shows the cAMP level (RLU) after the addition of each concentration of DDAVP, normalized to the cAMP level (RLU) after the addition of forskolin. The horizontal axis shows the DDAVP concentration. [Figure 7] Figure 7 shows a schematic diagram of a human-platypus chimeric receptor in which part of the amino acid sequence of the platypus V2 receptor has been replaced with that of the human V2 receptor. The dark gray portion of the figure represents the sequence derived from the platypus V2 receptor, and the light gray portion represents the sequence derived from the human V2 receptor. [Figure 8] Figure 8 shows the concentration-response curve for the human-platypus chimeric receptor. The vertical axis shows the cAMP level (RLU) after the addition of each concentration of AVP, normalized to the cAMP level (RLU) after the addition of forskolin. The horizontal axis shows the AVP concentration. hV2R and pV2R represent the full-length V2R derived from human and platypus, respectively. [Figure 9] Figure 9 shows concentration-response curves for chimeric receptors in which the N-terminal domain (NTD) or extracellular loop region (ECL) of platypus V2R was introduced into human V2R. The vertical axis shows the cAMP level (RLU) after the addition of various concentrations of AVP, normalized to the cAMP level (RLU) after the addition of forskolin. The horizontal axis shows the AVP concentration. [Figure 10] Figure 10 shows the concentration-response curve for a chimeric receptor (ChimeraV2R (hECL3)) in which the extracellular loop region 3 of human V2R was introduced into platypus V2R. The vertical axis shows the cAMP level (RLU) after the addition of each concentration of AVP, normalized to the cAMP level (RLU) after the addition of forskolin. The horizontal axis shows the AVP concentration. [Figure 11]11 shows the concentration-response curves for Chimera V2R (hECL3) with D126Y and M205N mutations introduced, and for the Q129X mutant of platypus V2R. The vertical axis shows the cAMP level (RLU) after the addition of each concentration of AVP or DDAVP, normalized to the cAMP level (RLU) after the addition of forskolin. The horizontal axis shows the concentration of AVP or DDAVP. [Figure 12] Figure 12 shows the results of alanine scanning of extracellular loop region 2 of human V2R. The vertical axis represents pEC50, and the horizontal axis represents each mutant. F200, D211, W213, A214, F216, and E218 showed reduced cross-reactivity with DDAVP, suggesting that these residues are involved in ligand interaction. Furthermore, A214 and E218 are considered promising mutations, as their reactivity with AVP was comparable to that of the wild type (WT). [Figure 13] Sequences of SEQ ID NOs: 1 to 6. [Figure 14] Sequences of SEQ ID NOs: 7 to 16. [Figure 15] Sequences of SEQ ID NOs: 17 and 18. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Mutated platypus V2 receptor and cultured cells expressing it] (cultured cells) One aspect of the present invention is a cultured cell expressing a wild-type platypus V2R consisting of the amino acid sequence of SEQ ID NO: 1, or a cultured cell expressing a platypus V2R mutant having mutations at aspartic acid (D) at position 126, alanine (A) at position 129, and methionine (M) at position 205. Another aspect of the present invention is a cultured cell expressing a platypus V2R mutant that maintains the amino acid sequence important for sensitive measurement of AVP concentration, i.e., the portion corresponding to positions 219 to 333, of the sequence of SEQ ID NO: 1. Such a cultured cell is a cultured cell expressing any of the following proteins:

[0015] (1) a protein consisting of the amino acid sequence of SEQ ID NO: 1 (platypus sequence); (2) A protein having an amino acid sequence of SEQ ID NO: 1, which has at least one mutation selected from the group consisting of phenylalanine (F) corresponding to position 126, asparagine (N) corresponding to position 126, tyrosine (Y) corresponding to position 126, alanine (A) corresponding to position 129, N corresponding to position 205, an amino acid other than A corresponding to position 214, and an amino acid other than glutamic acid (E) corresponding to position 218; (3) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence of the protein described in (1) or (2) and that has AVP receptor activity. Preferably, the protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of the protein described in (1) or (2), provided that, in the amino acid sequence of SEQ ID NO: 1, it has at least one mutation selected from the group consisting of phenylalanine (F) at position 126, asparagine (N) at position 126, tyrosine (Y) at position 126, alanine (A) at position 129, N at position 205, an amino acid other than A at position 214, and an amino acid other than glutamic acid (E) at position 218; and has AVP receptor activity; (4) A protein having AVP receptor activity, which has an amino acid sequence that is at least 75% identical to the amino acid sequence of the protein described in (1) or (2), with the proviso that the portion corresponding to positions 219 to 333 has at least 95% identity. Preferably, the protein has an amino acid sequence that is at least 75% identical to the amino acid sequence of the protein described in (1) or (2), with the proviso that the portion corresponding to positions 219 to 333 has at least 95% identity, with the proviso that in the amino acid sequence of SEQ ID NO: 1, the protein has at least one mutation selected from the group consisting of phenylalanine (F) at position 126, asparagine (N) at position 126, tyrosine (Y) at position 126, alanine (A) at position 129, N at position 205, an amino acid other than A at position 214, and an amino acid other than glutamic acid (E) at position 218, and has AVP receptor activity; (5) A protein in which the portion corresponding to positions 334 to 339 in the amino acid sequence of the protein according to (1) to (4) is the amino acid sequence of SEQ ID NO: 19 (PEAPLE, extracellular loop region 3 of human V2R (hECL3)).

[0016] The sequence listing shows the amino acid sequence of the D126Y mutant as SEQ ID NO: 2, the amino acid sequence of the D126N mutant as SEQ ID NO: 3, the amino acid sequence of the M205N mutant as SEQ ID NO: 4, the amino acid sequence of the D126Y / M205N mutant as SEQ ID NO: 5, and the amino acid sequence of the D126N / M205N mutant as SEQ ID NO: 6.

[0017] The term "corresponding to position 126" in (2) above refers to the 126th position in the reference sequence of SEQ ID NO: 1 in (1). However, if one or more amino acids are deleted in the sequence of SEQ ID NO: 1, the position may shift and no longer be 126th, but it still refers to the position corresponding to position 126 in the sequence of SEQ ID NO: 1. The same applies to other items such as (4). Those skilled in the art can appropriately identify corresponding positions by aligning the two sequences in an optimal manner. The same applies to "corresponding to positions 219 to 333."

[0018] When the mutation introduced into V2R involves substitution, deletion, insertion, and / or addition of one or more amino acids, amino acid substitution is preferred because, compared to amino acid deletion or insertion, amino acid substitution can change the constituent amino acids while maintaining the basic three-dimensional structure of the GPCR, making it more suitable for functional modification.

[0019] In one embodiment, in a wild-type platypus V2R consisting of the amino acid sequence of SEQ ID NO: 1, extracellular loop region 3 (ECL3) can be replaced with ECL3 of human V2R.

[0020] The sequence listing shows, as SEQ ID NO: 17, the amino acid sequence of the platypus V2 receptor in which a predetermined portion has been replaced with hECL3. The sequence listing also shows, as SEQ ID NO: 18, the amino acid sequence of the platypus V2 receptor in which a predetermined portion has been replaced with hECL3 and which further has the mutations D126Y, Q129A, and M205N. The sequence listing also shows, as SEQ ID NO: 19, the amino acid sequence (PEAPLE) of the extracellular loop region 3 (hECL3) of the human V2 receptor.

[0021] In one embodiment, an amino acid mutation is made in a region of wild-type platypus V2R consisting of the amino acid sequence of SEQ ID NO: 1 that directly or indirectly interacts with a ligand. For example, an amino acid substitution can be made in extracellular loop region 2 (ECL2) of wild-type platypus V2R consisting of the amino acid sequence of SEQ ID NO: 1. ECL2 has been reported to not only directly interact with AVP but also stabilize the structure of extracellular loop region 1 (ECL1) (Zhou, F., Ye, C., Ma, X. et al. Molecular basis of ligand recognition and activation of human V2 vasopressin receptor. Cell Res 31, 929-931 (2021). https: / / doi.org / 10.1038 / s41422-021-00480-2). From the viewpoint of reducing cross-reactivity to DDAVP while maintaining reactivity to AVP, at least one mutation selected from the group consisting of an amino acid other than A at position 214 and an amino acid other than glutamic acid (E) at position 218 in the amino acid sequence of SEQ ID NO: 1 is made. Examples of amino acids other than A include valine (V) and similar amino acids whose side chains have a relatively small effect. Examples of amino acids other than E include A and serine (S).

[0022] When a V2R comprises a specific amino acid sequence, any amino acid in the amino acid sequence may be chemically modified. Even in such cases, the V2R according to the embodiment of the present invention can be said to comprise a specific amino acid sequence. Generally, known chemical modifications of amino acids contained in proteins in vivo include, for example, N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, glycosylation, etc.).

[0023] The cultured cells may be any cells that transiently or stably express the V2 receptor, and are preferably animal cells. Among these, human embryonic kidney cell-derived cell lines (HEK293 cells, HEK293T cells, HEK293A cells, etc.), Chinese hamster ovary-derived cell lines (CHO cells), and human osteosarcoma cell lines (U2OS cells) may be used in terms of gene transfer efficiency and stable growth.

[0024] Mammalian cells expressing the V2 receptor can be prepared by genetic engineering techniques commonly used in this field. For example, they can be obtained by transfecting a vector (e.g., pcDNA3.1(+), pcDM8, pAGE107, pAS3-3, pCDM8) containing a promoter (e.g., the cytomegalovirus [CMV] IE [immediate early] gene promoter, SV40 [Simian virus 40] early promoter, retroviral promoter, metallothionein promoter, heat shock promoter, SRα promoter, NFAT promoter, or HIF promoter) and a gene encoding the V2 receptor into cultured cells using methods such as electroporation, calcium phosphate transfection, lipofection, DEAE (Diethylaminoethyl) dextran transfection, or viral infection. For use in measuring AVP concentrations, genes other than the V2R-encoding gene may also be introduced, as appropriate. For example, a cAMP biosensor may be introduced in the method described in Non-Patent Document 2, which uses a cAMP biosensor.

[0025] In this specification, examples of cultured cells include cells derived from rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees. Of these, preferred examples include cells derived from mice, pigs, or humans.

[0026] The V2R mutant provided in one embodiment has the above mutation, and therefore has an improved AVP / DDAVP ratio. Because the V2R mutant has an improved AVP / DDAVP ratio, it can be suitably used for measuring AVP concentration. The method for measuring AVP concentration that can be applied is not particularly limited, and examples thereof include the method described in Patent Document 1 and the method described in Non-Patent Document 2.

[0027] (protein) One aspect of the present invention is the above-mentioned V2R mutant protein. (2) a protein having an amino acid sequence of SEQ ID NO: 1, which has at least one mutation selected from the group consisting of phenylalanine (F) corresponding to position 126, asparagine (N) corresponding to position 126, tyrosine (Y) corresponding to position 126, alanine (A) corresponding to position 129, and N corresponding to position 205; (3') A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of the protein set forth in SEQ ID NO: 1 or (2) (excluding the amino acid sequence of SEQ ID NO: 1), and having AVP receptor activity. Preferably, the protein consists of an amino acid sequence having at least 90% identity with the amino acid sequence of the protein set forth in SEQ ID NO: 1 or (2) (excluding the amino acid sequence of SEQ ID NO: 1), with the proviso that in the amino acid sequence of SEQ ID NO: 1, the protein has at least one mutation selected from the group consisting of phenylalanine (F) at the position corresponding to position 126, asparagine (N) at the position corresponding to position 126, tyrosine (Y) at the position corresponding to position 126, alanine (A) at the position corresponding to position 129, and N at the position corresponding to position 205, and has AVP receptor activity. (4') A protein having an amino acid sequence having at least 75% identity with the amino acid sequence of the protein set forth in SEQ ID NO: 1 or (2) (excluding the amino acid sequence of SEQ ID NO: 1), with the proviso that the portion corresponding to positions 219 to 333 has at least 95% identity, and having AVP receptor activity. Preferably, a protein having an amino acid sequence having at least 75% identity with the amino acid sequence of the protein set forth in SEQ ID NO: 1 or (2) (excluding the amino acid sequence of SEQ ID NO: 1), with the proviso that the portion corresponding to positions 219 to 333 has at least 95% identity, with the proviso that in the amino acid sequence of SEQ ID NO: 1, the protein has at least one mutation selected from the group consisting of phenylalanine (F) corresponding to position 126, asparagine (N) corresponding to position 126, tyrosine (Y) corresponding to position 126, alanine (A) corresponding to position 129, and N corresponding to position 205, and having AVP receptor activity; (5) A protein in which the portion corresponding to positions 334 to 339 in the amino acid sequence of the protein described in (2), (3'), and (4') is the amino acid sequence of SEQ ID NO: 19.

[0028] (Polynucleotides, Vectors) One aspect of the present invention is a polynucleotide encoding the above-mentioned V2R. Specifically, it is any one of the following:

[0029] (2) a polynucleotide encoding a protein having an amino acid sequence of SEQ ID NO: 1, which has at least one mutation selected from the group consisting of phenylalanine (F) corresponding to position 126, asparagine (N) corresponding to position 126, tyrosine (Y) corresponding to position 126, alanine (A) corresponding to position 129, and N corresponding to position 205; (3') a polynucleotide encoding a protein having an amino acid sequence having at least 90% identity with the amino acid sequence of the protein set forth in SEQ ID NO: 1 or (2) (excluding the amino acid sequence of SEQ ID NO: 1) and having AVP receptor activity; (4') a polynucleotide encoding a protein having an amino acid sequence having at least 75% identity with the amino acid sequence of the protein set forth in SEQ ID NO: 1 or (2) (excluding the amino acid sequence of SEQ ID NO: 1), with the proviso that the portion corresponding to residues 219 to 333 has at least 95% identity, and having AVP receptor activity; (5) A polynucleotide encoding a protein in which the portion corresponding to positions 334 to 339 in the amino acid sequence of the protein according to (2), (3'), and (4') is the amino acid sequence of SEQ ID NO: 19.

[0030] Polynucleotides can be synthesized using a DNA / RNA synthesizer. Alternatively, they can be purchased from a contract manufacturer that synthesizes DNA or RNA bases (e.g., Invitrogen Corporation, Takara Bio Inc., etc.). The polynucleotide sequence of the present invention can be appropriately designed based on the amino acid sequence of the V2R mutant of the present invention and the genetic code table.

[0031] One aspect of the present invention is a vector containing the above-described polynucleotide. Such a vector can be prepared by commonly known molecular biology techniques, such as TA cloning, blunt-end cloning, ligation, in-fusion cloning, Gateway cloning, and assembly.

[0032] Examples of vectors that can be used to introduce the polynucleotide include E. coli-derived plasmids (e.g., pBR322, pUC12, pET-Blue-2), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5), yeast-derived plasmids (e.g., pSH19, pSH15), animal cell expression plasmids (e.g., pA1-11, pcDNAI / Neo), bacteriophage vectors such as λ phage, and vectors derived from viruses such as adenovirus, retrovirus, and baculovirus. These vectors may contain components necessary for protein expression, such as a promoter, a replication origin, or an antibiotic resistance gene. The vector may also be an expression vector.

[0033] (cAMP biosensor) One aspect of the present invention is a cultured cell that expresses the above-mentioned V2R mutant and further expresses a cAMP biosensor.

[0034] In the description of the present invention, the term "cAMP biosensor" refers to a protein whose own indicator (e.g., enzyme activity level, color level, luminescence [fluorescence] level) that can be visualized (imaged) and / or quantified changes depending on the amount and / or concentration of cAMP produced in mammalian cells. The cAMP biosensor typically has a cAMP-binding domain, and upon binding of cAMP to the cAMP-binding domain, the three-dimensional structure of the cAMP biosensor changes, resulting in an allosteric effect such as a change from an inactivated state to an activated state or from an invisible state to a visible state.

[0035] Examples of cAMP biosensors that can be used include reporter proteins (e.g., horseradish peroxidase [HRP]; alkaline phosphatase; β-D-galactosidase; luciferases such as green luciferase [SLG], orange luciferase [SLO], and red luciferase [SLR]; and fluorescent proteins such as green fluorescent protein [GFP], red fluorescent protein [DsRed], and cyan fluorescent protein [CFP]) that contain a cAMP-binding domain (e.g., a cAMP-binding domain derived from the regulatory subunit of protein kinase A [PKA] or a cAMP-binding domain derived from Epac1). Specific examples include GloSensor cAMP (Promega), a luciferase that contains a cAMP-binding domain derived from the regulatory subunit of PKA, and Pink Flamindo (Pink Fluorescent cAMP indicator), a red fluorescent protein that contains a cAMP-binding domain derived from Epac1 (Harada K., et al., Sci Rep. 2017 Aug 4;7(1):7351). doi: 10.1038 / s41598-017-07820-6.), and GloSensor cAMP (Promega) is preferred because its effectiveness has been demonstrated in this example.

[0036] (kit) One aspect of the present invention is a kit for measuring AVP, which comprises animal cells expressing the above-mentioned V2R mutant.

[0037] In one embodiment, the AVP measurement kit can be used for testing and diagnosing diseases or conditions associated with increased or decreased AVP levels in blood, such as central diabetes insipidus.

[0038] The kit can be made of commonly used reagents. For example, the kit may contain an animal cell solution, a luminescent substrate solution, a reaction plate, a standard solution, and a diluent. The kit may be an in vitro diagnostic drug.

[0039] (Method of analyzing AVP levels) One aspect of the present invention is a method for analyzing AVP levels in a sample, comprising the steps of: (a) adding a sample to cultured cells expressing AVP receptors (more specifically, V2 receptors) and incubating; In this case, the AVP receptor is selectively activated by AVP, while its activation by 1-deamino-8-D-arginine vasopressin (DDAVP) is inhibited; (b) analyzing the activation level of AVP receptors (more specifically, V2R activation level) after incubation; (c) Based on the obtained analysis results, the effect of DDAVP on the reduced AVP level is analyzed.

[0040] The activation level of the AVP receptor can be analyzed as cAMP level, which means the amount of cAMP produced in mammalian cells due to V2R, and can be defined as RLU when measured by the method described below.

[0041] [Animal cells used] Human fetal kidney cell-derived cell line (293A cells) (obtained from Thermo Fischer Scientific, Cat. no. R70507).

[0042] [Vector used] A plasmid vector (pGloSensor-22F, manufactured by Promega) expressing GloSensor cAMP, a protein containing amino acid residues 359 to 544 of firefly luciferase, the cAMP-binding domain of the regulatory subunit of protein kinase A (PKA), and amino acid residues 4 to 355 of firefly luciferase, and pCAGGS (pCAGGS_V2R) expressing V2R. In the method described below, pCAGGS expressing a V2R mutant may be used instead of pCAGGS_V2R.

[0043] [Cell preparation method] [1] 4.0 x 10 6 293A cells are seeded in D-MEM medium containing 10% FBS and cultured for 24 hours. [2] pGloSenso-22F and pCAGGS_V2R are transfected into 293A cells using Polyethyleneimine MAX (Polyscience) according to the protocol attached to the GloSensor cAMP Assay (Promega). [3] After culturing the transfected cells for 24 hours, cells that show an increase in luminescence intensity upon addition of a V2R stimulator (e.g., AVP or DDAVP) are selected.

[0044] [Activity measurement conditions] [1] After culturing the cells for 24 hours, the incubation medium is replaced with an incubation medium containing 2% (v / v) D-Luciferin Potassium Salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and equilibration is carried out at room temperature for 2 hours. [2] After adding a V2R stimulant and incubating for 20 minutes, RLU was measured using a luminometer. The measured RLU was defined as the cAMP level.

[0045] The method for measuring cAMP levels herein can be modified as appropriate within the scope that ensures equivalence to the above-mentioned measurement method.

[0046] In the description of the present invention, the term "V2R activation level" refers to the responsiveness of V2R to a V2R stimulant, and is defined by the ratio (fold change) of the cAMP level in the presence of a V2R stimulant to the constitutive cAMP level in mammalian cells.

[0047] In the description of the present invention, the term "constitutive cAMP level" refers to the cAMP level observed in the absence of a V2R stimulant. In this specification, the constitutive cAMP level can be measured under the activity measurement conditions [2] of the cAMP level measurement method described above, without adding a V2R stimulant.

[0048] In the description of the present invention, the term "V2R stimulator" refers to a ligand that stimulates (activates) V2R, and examples thereof include AVP and DDAVP.

[0049] The AVP receptor used, more specifically V2R, is one that is inhibited from being activated by DDAVP and selectively activated by AVP. In a particularly preferred embodiment, the above-mentioned mutant is used as the AVP receptor. The mutant has an improved AVP / DDAVP ratio compared to the wild-type.

[0050] In the description of the present invention, the "AVP / DDAVP ratio" refers to the pEC50 (-log 10 pEC50 refers to the ratio of pEC50 (units) when AVP was added to pEC50 (units) when DDAVP was added, specifically, it refers to the value obtained by subtracting pEC50 (units) when DDAVP was added. The method for measuring pEC50 is as described in the Examples below.

[0051] As can be understood from the above definition, the "AVP / DDAVP ratio" is an index showing the relationship between the affinity of expressed V2R for AVP and the affinity for DDAVP.

[0052] In the description of the present invention, the term "high (low) AVP / DDAVP ratio" refers to the magnitude of the aforementioned AVP / DDAVP ratio. As can be understood from this definition, a high AVP / DDAVP ratio means that the expressed V2R has a higher affinity for AVP than for DDAVP. A low AVP / DDAVP ratio means that the expressed V2R has a higher affinity for DDAVP than for AVP.

[0053] In addition to the contents described in this specification, for details of the cAMP level measurement method, animal cell preparation method, and AVP concentration measurement kit related to the present invention, the literature described in this specification, particularly Patent Publication No. 2021-103947, and the contents of the literature described therein can be appropriately referred to.

[0054] (Other terms) In the description of the present invention, with regard to amino acids or amino acid residues, unless otherwise specified, A represents alanine, C represents cysteine, D represents aspartic acid, E represents glutamic acid, F represents phenylalanine, G represents glycine, H represents histidine, I represents isoleucine, K represents lysine, L represents leucine, M represents methionine, N represents asparagine, P represents proline, Q represents glutamine, R represents arginine, S represents serine, T represents threonine, U represents selenocysteine, V represents valine, W represents tryptophan, and Y represents tyrosine.

[0055] In the description of the present invention, the term "polynucleotide" includes those composed of multiple nucleotides or bases, or their equivalents, linked together. The nucleotides and bases include DNA bases or RNA bases. The above equivalents include, for example, DNA bases or RNA bases that have been chemically modified, such as by methylation, or nucleotide analogs. Nucleotide analogs include unnatural nucleotides.

[0056] In the description of the present invention, "wild-type V2R" refers to a V2R that has not been mutated. The amino acid sequence of a V2R that has not been mutated is the V2R amino acid sequence registered in the NCBI database (http: / / www.ncbi.nlm.nih.gov / guide / ). The wild-type V2R may also be abbreviated as "WT."

[0057] In the description of the present invention, when a mutant is represented by a character string consisting of one letter and a number followed by another letter, the leftmost letter indicates the amino acid before mutation, the central number indicates the position of the amino acid residue, and the rightmost letter indicates the amino acid after mutation, and the leftmost amino acid indicates a mutant in which a point mutation has been introduced from the leftmost amino acid to the rightmost amino acid. For example, "D126Y" indicates a mutant in which a point mutation has been introduced, substituting aspartic acid at position 126 with tyrosine.

[0058] In the description of the present invention, when a protein or amino acid sequence is referred to as "an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added," the number of amino acids to be substituted, etc. is not particularly limited, unless otherwise specified, for any protein, as long as the protein consisting of that amino acid sequence has the desired function, but may be approximately 1-750, 1-500, 1-250, 1-200, 1-150, 1-100, 1-50, 1-40, 1-30, 1-20, 1-15, 1-9, or 1-4 amino acids, or even greater numbers of substitutions, etc., as long as the substitutions are with amino acids with similar properties. Means for preparing polynucleotides or proteins with such amino acid sequences are well known to those skilled in the art.

[0059] In the present invention, when referring to a base sequence (sometimes referred to as a nucleotide sequence) or an amino acid sequence, "identity" is defined as a value measured by BLAST of NCBI (http: / / www.ncbi.nlm.nih.gov / ), unless otherwise specified. When comparing amino acid sequences with BLAST, Blastp can be used with the default settings as the algorithm. The measurement results are quantified as positives or identities.

[0060] In the present invention, when referring to a base sequence or amino acid sequence, identity refers to a sequence identity of at least 50%, for example, 60% or more, 70% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97.5% or more, and even more preferably 99% or more, unless otherwise specified.

[0061] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Example]

[0062] [Example 1. Screening using various animal V2Rs] According to the method described below, highly sensitive receptors were searched for using pEC50 as an indicator, using V2Rs derived from 11 animal species: human (Human, SEQ ID NO: 7), platypus (Platypus, SEQ ID NO: 1), marmoset (Marmoset, SEQ ID NO: 8), green monkey (Vervet, SEQ ID NO: 9), naked mole rat (Naked mole rat, SEQ ID NO: 10), rat (Rat, SEQ ID NO: 11), megabat (Megabat, SEQ ID NO: 12), pig (Pig, SEQ ID NO: 13), horse (Horse, SEQ ID NO: 14), cow (Cow, SEQ ID NO: 15), and rabbit (Rat, SEQ ID NO: 16).

[0063] 1-1. Preparation of expression vectors for various animal V2Rs The DNA sequences of various animal V2Rs were obtained by requesting artificial gene synthesis from GenScript, Inc., with reference to NCBI (National Center for Biotechnology Information). The obtained V2R genes were subcloned into the mammalian expression vector pCAGGS to prepare expression vectors for various animal V2Rs.

[0064] 1-2. Obtaining animal V2R transiently expressing cell lines 4.0×10 6Human fetal kidney cell-derived cell line (293A cells) (obtained from Thermo Fischer Scientific, Cat. no. R70507) were seeded onto a 6-well multiplate together with D-MEM medium containing 10% FBS and cultured for 24 hours.

[0065] 293A cells were transfected with 0.02 μg of the animal V2R expression vector obtained in 1-1 and 1 μg of a plasmid vector (pGloSenso-22F, Promega) expressing a protein containing amino acid residues 359 to 544 of firefly luciferase, the cAMP-binding domain of the regulatory subunit of protein kinase A (PKA), and amino acid residues 4 to 355 of firefly luciferase, using Polyethylenimine Max (Polyscience). 293A cells transiently expressing various animal V2Rs were obtained by the above procedure.

[0066] [Example 2. Measurement of activity of animal V2R against AVP] AVP (Peptide Institute) was added to the 293A cells transiently expressing various animal V2Rs obtained in Example 1, and the activity of various animal V2Rs against AVP was measured.

[0067] The measurement method is as follows. (Measurement conditions) 293A cells transiently expressing animal V2R were cultured for 24 hours, then harvested and suspended in Hank's balanced buffer containing 10 mM D-luciferin potassium salt (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.01% (w / v) bovine serum albumin. AVP was added at 0, 1, 10, 100, 1,000, 10,000, or 100,000 pM, and forskolin was added at 10 μM as a positive control. After 20 minutes of incubation, the RLU values ​​were measured using a 96-well multiplate luminometer (Molecular Devices). The RLU values ​​were divided by the RLU value at 0 pM (fold change) and normalized to 100% forskolin-treated wells (cAMP accumulation value). The obtained cAMP accumulation values ​​were plotted on a semi-logarithmic graph, with the cAMP accumulation value on the vertical axis and the added ligand concentration on the horizontal axis, to obtain a concentration-response curve. The 50% effective concentration (EC 50 ) was calculated and the obtained EC 50 From pEC50(-log 10 EC50) was obtained.

[0068] The pEC50 values ​​for each animal's V2R upon addition of AVP are shown in Figure 1. The pEC50 values ​​varied depending on the animal species, and it can be seen that the receptor with the highest pEC50 was the platypus V2R, suggesting that the platypus V2R is the most sensitive receptor.

[0069] [Example 3. Construction of platypus V2R mutant] A platypus V2R mutant in which one amino acid was substituted from the wild-type platypus V2R was prepared according to the method described below.

[0070] 3-1. Design of platypus V2R mutant The following mutations were investigated as mutations that are suspected to affect V2R activity (Fig. 2). D126, M205

[0071] 3-2. Preparation of expression vector for platypus V2R mutant To correspond to the mutations described in 3-1, 20-30mer artificial primers were designed on the 3' and 5' sides, flanking the nucleotides corresponding to the mutation sites. Point mutations were introduced into the V2R sequence by inverse PCR using the wild-type platypus V2R 3' primer and a 5' primer with 1-3 additional bases added for mutation introduction, along with the wild-type platypus V2R expression vector pCAGGS_pV2R, using Toyobo KOD-One (Code No. KMM-101). Plasmids were purified from single colonies of E. coli transformed with the point mutation-introduced plasmid, and the introduction of the point mutations was confirmed by sequence analysis.

[0072] 3-3. Obtaining cell lines transiently expressing platypus V2R mutants 4.0×10 6 Human fetal kidney cell-derived cell line (293A cells) (obtained from Thermo Fischer Scientific, Cat. no. R70507) were seeded onto a 6-well multiplate together with D-MEM medium containing 10% FBS and cultured for 24 hours.

[0073] 293A cells were transfected with 0.02 μg of the platypus V2R mutant expression vector obtained in 3-2 and 1 μg of a plasmid vector (pGloSenso-22F, Promega) expressing a protein containing amino acid residues 359 to 544 of firefly luciferase, the cAMP-binding domain of the regulatory subunit of protein kinase A (PKA), and amino acid residues 4 to 355 of firefly luciferase, using Polyethylenimine Max (Polyscience). 293A cells transiently expressing various platypus V2R mutants were obtained by the above procedure.

[0074] [Example 4. Measurement of activity of platypus V2R mutants against AVP or DDAVP] AVP or DDAVP (Peptide Institute) was added to 293A cells transiently expressing the platypus V2R mutant obtained in Example 3, and the activity of the platypus V2R mutant against AVP or DDAVP was measured.

[0075] The measurement method is as follows. (Measurement conditions) 293A cells transiently expressing the platypus V2R mutant were cultured for 24 hours, then harvested and suspended in Hank's balanced buffer containing 10 mM D-luciferin potassium salt (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.01% (w / v) bovine serum albumin. AVP or DDAVP was added at 0, 1, 10, 100, 1,000, 10,000, or 100,000 pM. Forskolin was also added at 10 μM as a positive control. After 20 minutes of incubation, the RLU values ​​were measured using a 96-well multiplate luminometer (Molecular Devices). The RLU values ​​were divided by the RLU value at 0 pM (fold change) and normalized to 100% forskolin-treated wells (cAMP accumulation value). The cAMP accumulation values ​​were plotted on a semi-logarithmic graph, with the cAMP accumulation value on the vertical axis and the ligand concentration on the horizontal axis to obtain a concentration-response curve, from which the half effective concentration (EC50) was calculated.

[0076] The concentration-response curves for the platypus V2R D126 mutant and M205 mutant upon addition of AVP or DDAVP are shown in Figures 3 and 4, respectively. While the concentration-response curves for wild-type platypus V2R upon addition of AVP and DDAVP are nearly identical, the concentration-response curves for the platypus V2R mutant do not match, indicating that AVP is the dominant factor in increasing intracellular cAMP levels.

[0077] The AVP / DDAVP ratio values ​​for each platypus V2R mutant are shown in the table below. Each row represents an introduced mutation, and WT indicates the wild type.

[0078] [Table 1]

[0079] [Table 2]

[0080] The above results show that the D126F, D126N, and D126Y mutants have reactivity to AVP equivalent to that of the wild type, but have significantly reduced reactivity to DDAVP.

[0081] [Example 5. Construction of platypus V2R double mutant] According to the method described below, a platypus V2R double mutant was generated in which two single amino acid mutations were introduced into the wild-type platypus V2R.

[0082] 5-1. Design of the platypus V2R double mutant Based on the results of Example 4, the following two mutants were designed as mutants that may increase the intracellular cAMP concentration in a more AVP-dominant manner. D126Y / M205N, D126N / M205N

[0083] 5-2. Preparation of expression vector for platypus V2R double mutant To correspond to the mutations described in 5-1, 20-30mer artificial primers were designed on the 3' and 5' sides, flanking the nucleotide corresponding to the mutation site. Point mutations were introduced into the V2R sequence by inverse PCR using the platypus V2R M205N 3' primer and a 5' primer with 1-3 additional bases for mutation introduction, and the vector pCAGGS_pV2R M205N expressing platypus V2R M205N using Toyobo KOD-One (Code No. KMM-101). Plasmids were purified from single colonies of E. coli transformed with the point mutation-introduced plasmid, and the introduction of the point mutations was confirmed by sequence analysis.

[0084] 5-3. Obtaining a cell line transiently expressing the platypus V2R double mutant 4.0×10 6 Human fetal kidney cell-derived cell line (293A cells) (obtained from Thermo Fischer Scientific, Cat. no. R70507) were seeded onto a 6-well multiplate together with D-MEM medium containing 10% FBS and cultured for 24 hours.

[0085] 293A cells were transfected with 0.02 μg of the platypus V2R double mutant expression vector obtained in 5-2 and 1 μg of a plasmid vector (pGloSenso-22F, Promega) expressing a protein containing amino acid residues 359 to 544 of firefly luciferase, the cAMP-binding domain of the regulatory subunit of protein kinase A (PKA), and amino acid residues 4 to 355 of firefly luciferase, using Polyethylenimine Max (Polyscience). 293A cells transiently expressing the platypus V2R double mutant were obtained by the above procedure.

[0086] Example 6: Measurement of activity of platypus V2R double mutant against AVP or DDAVP AVP or DDAVP (Peptide Institute) was added to 293A cells transiently expressing the platypus V2R mutant obtained in Example 5, and the activity of the platypus V2R double mutant against AVP or DDAVP was measured.

[0087] The measurement method is as follows. (Measurement conditions) 293A cells transiently expressing the platypus V2R double mutant were cultured for 24 hours, then harvested and suspended in Hank's balanced buffer containing 10 mM D-luciferin potassium salt (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.01% (w / v) bovine serum albumin. AVP or DDAVP was added at 0, 1, 10, 100, 1,000, 10,000, or 100,000 pM. Forskolin was also added at 10 μM as a positive control. After 20 minutes of incubation, the RLU values ​​were measured using a 96-well multiplate luminometer (Molecular Devices). The RLU values ​​were divided by the RLU value at 0 pM (fold change) and normalized to 100% forskolin-treated wells (cAMP accumulation value). The cAMP accumulation values ​​were plotted on a semi-logarithmic graph, with the cAMP accumulation value on the vertical axis and the ligand concentration on the horizontal axis to obtain a concentration-response curve, from which the half effective concentration (EC50) was calculated.

[0088] The concentration-response curves for each platypus V2R double mutant upon addition of AVP or DDAVP are shown in Figures 5 and 6. While the concentration-response curves for wild-type platypus V2R upon addition of AVP and DDAVP are nearly identical, the concentration-response curves for the platypus V2R double mutant do not match, indicating that AVP is the dominant factor in increasing intracellular cAMP levels.

[0089] The AVP / DDAVP ratio values ​​for each platypus V2R mutant are shown in the table below. Each row represents an introduced mutation, and WT indicates the wild type.

[0090] [Table 3]

[0091] The above results show that D126Y / M205N has a higher AVP / DDAVP ratio than the mutants into which D126Y and M205N were introduced alone, and is able to suppress receptor activation by DDAVP.

[0092] [Example 7. Construction of human-platypus chimera V2R] According to the method described below, a human-platypus chimeric V2R was prepared in which a portion of the amino acid sequence of platypus V2R was replaced with an amino acid sequence derived from human V2R.

[0093] 7-1. Design of the human-platypus chimera V2R The amino acid sequences of platypus V2R (SEQ ID NO: 1) and human V2R (SEQ ID NO: 7) were obtained from NCBI, and multiple sequence alignment (MSA) was performed on each amino acid sequence using CLUSTALW (https: / / www.genome.jp / tools-bin / clustalw). Domain information for human V2R was also obtained from GPCRdb (https: / / gpcrdb.org / ), and the domain information for platypus V2R was determined by comparing it with the MSA results. The table below shows the domain information for human V2R and platypus V2R. In the table below, the domain indicated as NTD is the N-terminal domain, TM is the transmembrane domain, ECL is the extracellular loop region, ICL is the intracellular loop region, and CTD is the C-terminal domain. Figure 7 shows schematic diagrams of chimera V2R-1, chimera V2R-1.1, chimera V2R-1.2, chimera V2R-2, chimera V2R-2.1, and chimera V2R-2.2.

[0094] [Table 4]

[0095] 7-2. Preparation of expression vector for human-platypus chimeric V2R To match the chimeric receptors described in 7-1, 20-30mer artificial primers were designed on the 3' and 5' ends, flanking the nucleotides corresponding to each domain of human and platypus. Using the 3' and 5' primers for platypus V2R or human V2R and the vectors pCAGGS_pV2R and pCAGGS_hV2R expressing platypus V2R, the domain sequences were amplified by PCR using Takara Bio Primestar MAX DNA Polymerase (product number R045A). pCAGGS was then digested with two restriction enzymes (KpnI and XhoI) to obtain linearized vectors. Plasmids were prepared using the amplified domain sequences and linearized vectors with NEBuilder HiFi DNA Assembly Master Mix (product number E2621S). Plasmids were purified from single colonies of E. coli transformed with the constructed plasmids, and the nucleotide sequences of each chimeric V2R were confirmed by sequence analysis.

[0096] 7-3. Obtaining a cell line transiently expressing human-platypus chimeric V2R 4.0×10 6 Human fetal kidney cell-derived cell line (293A cells) (obtained from Thermo Fischer Scientific, Cat. no. R70507) were seeded onto a 6-well multiplate together with D-MEM medium containing 10% FBS and cultured for 24 hours.

[0097] 293A cells were transfected with 0.02 μg of the human-platypus chimeric V2R expression vector obtained in 7-2 and 1 μg of a plasmid vector (pGloSenso-22F, Promega) expressing a protein containing amino acid residues 359 to 544 of firefly luciferase, the cAMP-binding domain of the regulatory subunit of protein kinase A (PKA), and amino acid residues 4 to 355 of firefly luciferase, using Polyethylenimine Max (Polyscience). 293A cells transiently expressing each human-platypus chimeric V2R were obtained by the above procedure.

[0098] [Example 8. Measurement of activity of human-platypus chimera V2R against AVP] AVP (Peptide Institute) was added to 293A cells transiently expressing the human-platypus chimeric V2R obtained in Example 7, and the activity of the human-platypus chimeric V2R against AVP was measured.

[0099] The measurement method is as follows. (Measurement conditions) 293A cells transiently expressing the human-platypus chimeric V2R were cultured for 24 hours, then harvested and suspended in Hank's balanced buffer containing 10 mM D-luciferin potassium salt (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.01% (w / v) bovine serum albumin. AVP or DDAVP was added at 0, 1, 10, 100, 1,000, 10,000, or 100,000 pM. Forskolin was also added at 10 μM as a positive control. After 20 minutes of incubation, the RLU values ​​were measured using a 96-well multiplate luminometer (Molecular Devices). The RLU values ​​were divided by the RLU value at 0 pM (fold change) and normalized to 100% forskolin-treated wells (cAMP accumulation value). The cAMP accumulation values ​​were plotted on a semi-logarithmic graph, with the cAMP accumulation value on the vertical axis and the ligand concentration on the horizontal axis to obtain a concentration-response curve, from which the half effective concentration (EC50) was calculated.

[0100] The concentration-response curves for each human-platypus chimeric V2R upon addition of AVP are shown in Figure 8. The concentration-response curve for chimeric V2R-2.1 is shifted to lower concentrations than that for human V2R, indicating that the amino acid sequence from amino acid numbers 219 to 333 of platypus V2R is an important amino acid sequence for sensitive measurement of AVP concentration using cultured cells expressing platypus V2R.

[0101] [Example 9. Construction of chimeric V2R (ECL3 swap) and activity measurement] Using the same method as in Examples 7 and 8, human V2R and platypus V2R were prepared by exchanging extracellular loop regions (ECLs) 1 to 3, and their activities were measured.

[0102] As a result, introduction of ECL3 of platypus V2R (SEQ ID NO: 17) into human V2R reduced pEC50 (FIG. 9).

[0103] On the other hand, swapping the ECL3 between platypus V2R and human V2R slightly increases the pEC50 (Figure 10). Because the concentration to be measured is near the lower limit of the set concentration, evaluation using pEC50 is not considered appropriate. When the above data was evaluated using fold change, the results are as shown in the table below in Figure 10. Chimera V2R (hECL3) (SEQ ID NO: 17), which has swapped ECL3, is considered to be a more sensitive receptor than platypus V2R.

[0104] Example 10: Examination of the Q129X mutant Platypus V2R containing the Q129A mutation was constructed and its activity was measured. The Q129X mutation was evaluated in combination with Chimera V2R (hECL3) containing D126Y and M205N mutations (SEQ ID NO: 18).

[0105] The results are shown in the table below and in Figure 11. Introduction of Q129A reduced cross-reactivity to DDAVP. Substitution of Q129 with an amino acid other than A was also investigated, but Q129A was found to be the most suitable mutant for maintaining or increasing the pEC50 to AVP and reducing the pEC50 to DDAVP.

[0106] [Table 5]

[0107] Example 11: Study of ECL2 mutants We focused on the ECL2 domain, which has been reported to interact with many ligands. Comparing the amino acid sequences of ECL2 from human V2R and platypus V2R, we found that there was little amino acid identity, especially in the N-terminus.

[0108] [Table 6]

[0109] The C-terminal sequence is common to AVP receptors, and interactions with AVP and ECL1 have been reported. Because the amino acid sequences at the N-terminal end differ significantly between humans and platypus, it was thought difficult to predict important amino acids based on existing reports on human V2R and structural data on human V2R-AVP. Therefore, alanine scanning mutagenesis was performed on ECL2, and activity was measured using the same method as in Examples 7 and 8.

[0110] The results are shown in Figure 12. In the wild type (WT), the pEC50 values ​​of AVP and DDAVP were almost the same, while the pEC50 values ​​changed in some mutants.

[0111] W213 and A214 (in human V2R) have been previously reported to interact with AVP. D211 is predicted to interact with ECL1, and C212 is predicted to form a disulfide bond with a cysteine ​​in TM4. The remaining residues, F200, F216, and E218, have not been reported to interact with AVP, but they may be involved in ligand interactions that are not revealed in previously reported structural snapshots, such as cryo-EM structural data.

[0112] Since the purpose of this example is to reduce the reactivity to DDAVP while maintaining AVP reactivity close to that of the wild type, A214 and E218 are thought to be important target residues. [Industrial Applicability]

[0113] The present invention contributes to the diagnosis and treatment of central diabetes insipidus.

[0114] [Sequence listed in the sequence listing] SEQ ID NO: 1 Platypus V2R SEQ ID NO: 2 Platypus V2R D126Y SEQ ID NO: 3 Platypus V2R D126N SEQ ID NO: 4 Platypus V2R M205N SEQ ID NO: 5 Platypus V2R D126Y / M205N SEQ ID NO: 6 Platypus V2R D126N / M205N SEQ ID NO: 7 Human V2R SEQ ID NO: 8 Marmoset V2R SEQ ID NO: 9 Vervet-AGM V2R SEQ ID NO:10 Naked-mole-rat V2R SEQ ID NO:11 Rat V2R SEQ ID NO:12 Megabat V2R SEQ ID NO:13 Pig V2R SEQ ID NO:14 Horse V2R SEQ ID NO:15 Cow V2R SEQ ID NO:16 Rabbit V2R SEQ ID NO:17 Platypus V2R (hECL3 swap) SEQ ID NO:18 Platypus V2R (hECL3 swap) D126Y / Q129A / M205N SEQ ID NO:19 hECL3 SEQ ID NO:20 hECL2 SEQ ID NO:21 Platypus ECL2

Claims

1. A cultured cell expressing any one of the following proteins: (1) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (2) A protein having an amino acid sequence of SEQ ID NO: 1, which has at least one mutation selected from the group consisting of phenylalanine (F) corresponding to position 126, asparagine (N) corresponding to position 126, tyrosine (Y) corresponding to position 126, alanine (A) corresponding to position 129, N corresponding to position 205, an amino acid other than A corresponding to position 214, and an amino acid other than glutamic acid (E) corresponding to position 218; (3) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of the protein according to (1) or (2) and having AVP receptor activity; (4) A protein having an amino acid sequence having at least 75% identity with the amino acid sequence of the protein according to (1) or (2), with the proviso that the portion corresponding to residues 219 to 333 has at least 95% identity, and having AVP receptor activity; (5) A protein, wherein the portion corresponding to positions 334 to 339 in the amino acid sequence of the protein according to (1) to (4) is the amino acid sequence of SEQ ID NO:

19.

2. Any one of the following proteins: (2) A protein having an amino acid sequence of SEQ ID NO: 1, which has at least one mutation selected from the group consisting of phenylalanine (F) corresponding to position 126, asparagine (N) corresponding to position 126, tyrosine (Y) corresponding to position 126, alanine (A) corresponding to position 129, N corresponding to position 205, an amino acid other than A corresponding to position 214, and an amino acid other than glutamic acid (E) corresponding to position 218; (3') a protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of the protein set forth in SEQ ID NO: 1 or (2) (excluding the amino acid sequence of SEQ ID NO: 1), and having AVP receptor activity; (4') a protein having an amino acid sequence having at least 75% identity with the amino acid sequence of the protein set forth in SEQ ID NO: 1 or (2) (excluding the amino acid sequence of SEQ ID NO: 1), with the proviso that the portion corresponding to residues 219 to 333 has at least 95% identity, and having AVP receptor activity; (5) A protein in which the portion corresponding to positions 334 to 339 in the amino acid sequence of the protein described in (2), (3'), and (4') is the amino acid sequence of SEQ ID NO:

19.

3. The cultured cell of claim 1 , further expressing a cAMP biosensor.

4. A polynucleotide encoding the protein of claim 2.

5. A kit for measuring AVP, comprising the cultured cells according to claim 1 or 3.

6. The kit according to claim 5, which is an in vitro diagnostic pharmaceutical.

7. 1. A method for analyzing AVP levels in a sample, comprising the steps of: (a) adding a sample to cultured cells expressing AVP receptors and incubating the cells; In this case, the AVP receptor is one in which activation by 1-deamino-8-D-arginine vasopressin (DDAVP) is inhibited and the AVP receptor is selectively activated; (b) analyzing the activation level of AVP receptors after incubation; (c) analyzing the AVP level attenuated by the influence of DDAVP based on the obtained analysis results, 3. A method according to claim 1, wherein the AVP receptor is a protein as defined in claim 1 or a protein as described in claim 2.

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