JP2003342192A - Method for screening diabetes-treating agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diabetes-treating pharmaceutical composition, by using a simple screening tool and procedure for obtaining a diabetes-treating agent, and to provide a method for producing the composition. <P>SOLUTION: This diabetes-treating pharmaceutical composition is produced by using the screening tool, wherein the tool comprises a G protein conjugate- type receptor which accelerates insulin secretion by being activated in a high glucose concentration, a functionally equivalent transformant thereof, a homogeneous polypeptide, or a cell which is transformed by an expression vector containing a polynucleotide coding for the polypeptide and expresses the polypeptide. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for screening a therapeutic agent for diabetes.

[0002]

2. Description of the Related Art Diabetes is a disease associated with persistent hyperglycemia and is said to occur as a result of the action of many environmental and genetic factors. Insulin is the major regulator of blood glucose, and hyperglycemia is a factor that impairs insulin deficiency or its action (eg, genetic predisposition,
Excessive exercise, obesity, stress, etc.) are known to occur. There are mainly two types of diabetes, and insulin-dependent diabetes mellitus (IDD) caused by a decrease in pancreatic insulin secretory function due to autoimmune diseases and the like.
M) and non-insulin-dependent diabetes mellitus (NIDDM) caused by a decrease in pancreatic insulin secretory function due to pancreatic exhaustion associated with persistently high insulin secretion. More than 95% of Japanese diabetic patients are said to have NIDDM, and an increase in the number of patients has become a problem due to changes in lifestyle.

In the treatment of diabetes, in mild cases, diet therapy, exercise therapy, improvement of obesity, etc. are mainly carried out, and when further progressed, oral diabetes drugs (for example, insulin secretagogues such as sulfonylurea drugs) are administered. If more severe, insulin preparations are being administered [edited by Ryuzo Abe and Masato Kasuga, "Evidence-B"
Diabetes Treatment Aiming for "Assured Medicine," Nankodo, 1997; Richard A. et al. Harrig
an et al., Annals of Energy M
edicine, 38 (1), 68-78, 2001;
In addition, “The Diabetes Treatment Guide 200” edited by Japan Diabetes Society
0 ", Bunkodo, 2000].

[0004] Sulfonylurea agents stimulate pancreatic β cells and promote endogenous insulin secretion, but the timing and amount of insulin secretion are determined by the timing and amount of drug administration, regardless of blood glucose level. Therefore, as a side effect, hypoglycemia due to the continued action of the drug may be exhibited. In addition, digestive symptoms such as anorexia appear. Furthermore, it is contraindicated in patients with severe ketosis or liver or renal dysfunction [Richard A. Har
Rigan et al., Annals of Emergenc.
y Medicine, 38 (1), 68-78, 20
01]. Insulin preparations certainly lower blood glucose, but they must be administered by injection and can lead to hypoglycemia [McCrimmon RJ et al., Di.
abete. Metab. , 20 (6), 503-51
2, 1994].

As described above, the insulin secretagogues and insulin preparations conventionally used have the above-mentioned problems. Therefore, a drug capable of more advanced blood sugar control, that is, a drug capable of controlling blood sugar within a normal range, has been earnestly desired, rather than a drug merely lowering blood sugar.

On the other hand, GLP-1 (Glucagon-1)
IKE Peptide-1), PACAP (Pitu
itary adenylate cycle a
activating polypeptide) and GIP (Gastric Inhibitory Po)
It is known that each of them transmits a signal into cells via a specific G protein-coupled receptor to promote insulin secretion. Each of these G protein-coupled receptors is a type of receptor that is coupled to the Gs protein, activates adenylate cyclase, and increases intracellular cAMP concentration. It is known that the GLP-1 receptor, PACAP receptor, and GIP receptor all promote insulin secretion by increasing intracellular cAMP concentration. However, although the expression distribution of these G protein-coupled receptors is distributed in the pancreas, it is not pancreatic-specific [Dunphy JL et al., Mol. Cell. En
docrinol. , 141 (1-2), 179-18.
6, 1998; Timothy James Kief
fer et al., Endocrine Reviews, 20.
(6), 876-913, 1999; David Va.
udry et al., Pharmacological Revi
ews, 52 (2), 269-324, 2000; Je
an Claude Reubi et al., Cancer R
essearch, 60, 3105-3112, 200
0; and Ted B.A. Usdin et al., Endocrin
LOGY, 133 (6), 2861-2870, 19
93], and when the GIP receptor is activated, NIDD
It is known that M has no effect (Michael
A. Nauck et al. Clin. Invest. , 9
1, 301-307, 1993).

The "polypeptide having the amino acid sequence represented by SEQ ID NO: 2" which can be used in the present invention
Various reports (WO00 / 22131, WO00 / 3125) on the nucleotide sequence encoding the same amino acid sequence as the above and the deduced amino acid sequence encoded by the nucleotide sequence.
8 and each pamphlet of WO00 / 50562). However, in these reports, the function of "the polypeptide having the amino acid sequence represented by SEQ ID NO: 2" in vivo was not clarified. For example, WO00 / 2
2131 and WO00 / 31258 each describe a human orphan G protein-coupled receptor, and WO00 / 50562 pamphlet describes “a polypeptide having an amino acid sequence represented by SEQ ID NO: 2”. As for the use of agonists and antagonists, a large number of diseases identical to both agonists and antagonists are listed, but neither agonist nor antagonist is proved to be useful for the treatment of those diseases. .

[0008]

An object of the present invention is to provide a polypeptide which is pancreatic-specific and which is activated at high glucose concentration to promote insulin secretion, and a polynucleotide encoding the same. A simple screening system for obtaining a substance provided and useful as a therapeutic agent for diabetes capable of controlling blood glucose within a normal range (particularly, insulin secretagogue, more preferably specific insulin secretagogue under high glucose concentration) The present invention also provides a therapeutic agent for diabetes containing the substance obtained by the screening system.

[0009]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention have found that a polyhedron having an amino acid sequence represented by SEQ ID NO: 2 is specifically expressed in the pancreas. Overexpression of "peptide" in pancreatic β-cell lines and activation under high glucose concentration increases insulin secretion, whereas when activated under low glucose concentration, insulin secretion changes. Acquiring the finding that the polypeptide and cells expressing the polypeptide have a specific insulin secretion promoting effect under high glucose concentration, and a screening tool for a diabetes therapeutic agent capable of controlling blood glucose within a normal range. I found that. Further, they have succeeded in providing a novel method for screening a therapeutic agent for diabetes using the above-mentioned screening tool. And, a known compound not known to have a therapeutic effect on diabetes, an activator obtained by subjecting it to the screening method, exhibits an action of increasing plasma insulin level in rats upon glucose load, and an action of lowering blood glucose. In addition, in a diabetic model rat, it was confirmed that an increase in blood glucose level was suppressed during glucose load, the utility of the screening method of the present invention was further clarified, and the diabetes treatment using the analysis of the polypeptide activation was performed. The present invention has been completed by establishing a method for producing a pharmaceutical composition.

That is, the present invention provides [1] (1) a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16, or (2) represented by SEQ ID NO: 2. In the amino acid sequence represented by SEQ ID NO: 16 or
It has an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added, and (a) is activated under high glucose concentration to promote insulin secretion from pancreatic β cells. Activity, and / or (b) a polypeptide showing an activity of increasing the intracellular cAMP amount by being activated; a diabetes therapeutic agent screening tool; [2] the amino acid sequence represented by SEQ ID NO: 2 Or containing the amino acid sequence represented by SEQ ID NO: 16 and (a) being activated under high glucose concentration to promote insulin secretion from pancreatic β cells, and / or (b) being activated. The polypeptide for showing the activity of increasing the intracellular cAMP amount by the above, a screening tool for a therapeutic agent for diabetes; [3] the amino acid represented by SEQ ID NO: 2 Insulin from pancreatic β-cells, which has an amino acid sequence having a homology of 90% or more with the sequence or the amino acid sequence represented by SEQ ID NO: 16 and (a) is activated under high glucose concentration. Diabetes therapeutic agent screening tool, which is a polypeptide showing an activity of promoting secretion and / or (b) an activity of increasing intracellular cAMP amount by being activated; [4] [1] to [3] ] The polypeptide described in (a) is activated under high glucose concentration to promote insulin secretion from pancreatic β cells, and (b) is activated to produce the intracellular cAMP amount. A therapeutic tool for treating diabetes mellitus showing an activity of increasing the activity of [5] an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence represented by SEQ ID NO: 16 A diabetic therapeutic agent screening tool which is a polypeptide comprising a no acid sequence (hereinafter, the diabetic therapeutic agent screening tools described in [1] to [5] are collectively referred to as a polypeptide-type diabetic therapeutic agent screening tool); [6] Transformation with an expression vector containing a polynucleotide encoding the polypeptide according to [1] to [5],
Diabetes therapeutic agent screening tool (hereinafter referred to as transformed cell type diabetes therapeutic agent screening tool) which is a cell expressing the polypeptide; [7] [6] The cell or cell membrane thereof, and a test compound And a step of analyzing whether the polypeptide described in [1] to [5] is activated,
A method for screening a therapeutic agent for diabetes; the cells according to [8] and [6], or the cell membrane thereof, or the polypeptide according to [1] to [5], and a test compound, according to [1] to [5]. A method for screening a therapeutic agent for diabetes, which comprises a step of contacting the polypeptide in the presence of a labeled agonist, and a step of analyzing a change in the amount of the labeled agonist bound to the cell or its cell membrane or the polypeptide; [9] The antidiabetic agent is an insulin secretagogue,
[7] or the screening method according to [8]; [10] A pharmaceutical composition for treating diabetes, which contains an activator of the polypeptide according to [1] to [5]; [8] A pharmaceutical composition for treating diabetes, which comprises a substance obtained by the method described in [8]. [12] A pharmaceutical composition for promoting insulin secretion,
The pharmaceutical composition for treating diabetes according to [10] or [11]; [13] the step of contacting the cell or cell membrane thereof with a test compound, and the polypeptide according to [1] to [5]. A method for producing a pharmaceutical composition for treating diabetes, which comprises a step of analyzing whether or not is activated, and a step of formulating the analyzed substance; [14] [6] The cell or the cell membrane thereof; Contacting with a test compound in the presence of a labeled agonist of the polypeptide according to [1] to [5], a step of analyzing a change in the amount of the labeled agonist bound to the cell or its cell membrane, and an analyzed substance [15] A method for producing a pharmaceutical composition for treating diabetes, which comprises a step of: [15] A method for producing [13] or [14], wherein the pharmaceutical composition for treating diabetes is a pharmaceutical composition for promoting insulin secretion; [16] [1 ]-[5] The polypeptide activator which comprises administering the activator of the polypeptide of [5] to a subject in need of diabetes treatment in an effective amount; [17] The polypeptide of [1]-[5] Of the activator of the polypeptide according to [18] [1] to [5], which comprises administering an effective amount of the activator of [18] to a subject in need of promoting insulin secretion; It relates to use for producing a pharmaceutical composition for treating diabetes and / or a pharmaceutical composition for promoting insulin secretion.

As used herein, the term "diabetic therapeutic agent" and "medical composition for treating diabetes" include not only pharmaceuticals used to cure diabetic patients but also pharmaceuticals used to prevent progression of diabetes and the like. . Further, in the present specification,
The “screening tool” refers to an object used for screening (specifically, a polypeptide or a cell expressing the polypeptide used for screening. The “diabetes therapeutic agent screening tool” refers to diabetes treatment. A cell or polypeptide to be contacted with a test compound in the method for screening a therapeutic agent for diabetes of the present invention for screening an agent, the polypeptide according to [1] to [5], or [6]. The use of the described cells for screening anti-diabetic agents is also included in the present invention.

Regarding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, in EP1092727 published after the priority date of the present application, a polypeptide consisting of the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 is disclosed. Although the DNA sequence encoding the amino acid sequence of the peptide (PFI-007) and the deduced amino acid sequence encoded by the DNA sequence are described, the above-mentioned polypeptide PFI-00 is described.
There is no description that 7 was acquired. And the polypeptide P
Uses of agents that modulate FI-007 include treatment of a number of diseases, including the polypeptide PFI-007.
There is a claim for the treatment of diabetes which consists of administering a substance which modulates (antagonist or agonist). However, there is no evidence that an agonist of the polypeptide PFI-007 has a therapeutic effect on diabetes, and it is described that an antagonist also has a therapeutic effect on diabetes. Therefore, it was found that the agonist has a therapeutic effect on diabetes. It is obvious that the present inventor is the first to do so. In addition, when the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is activated, it promotes insulin secretion and is activated in pancreatic β cells under high glucose concentration to It has not been described at all that it has the activity of promoting insulin secretion (hereinafter sometimes referred to as high glucose-dependent insulin secretion promoting activity).

As described above, the antidiabetic agent (particularly insulin secretagogue) screening tool described in the present application, the antidiabetic agent (particularly insulin secretagogue) screening method, the pharmaceutical composition for treating diabetes (particularly insulin secretagogue), The method for producing the pharmaceutical composition and the method for producing the pharmaceutical composition are the first inventions made by the present inventors.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. (1) Diabetes therapeutic agent screening tool The diabetes therapeutic agent screening tool of the present invention includes a polypeptide type diabetes therapeutic agent screening tool and a transformed cell type diabetes therapeutic agent screening tool. 1) Polypeptide type diabetes therapeutic agent screening tool As a polypeptide that can be used as the polypeptide type diabetes therapeutic agent screening tool of the present invention,
For example, (1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16; (2) the amino acid sequence represented by SEQ ID NO: 2 or the amino acid represented by SEQ ID NO: 16 The sequence has an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added, and (a) under high glucose concentration, pancreatic β
The activity of promoting insulin secretion from the pancreatic β cell by being activated in a cell, and / or (b)
When activated in a cell, the intracellular cA
A polypeptide showing an activity of increasing MP amount (hereinafter referred to as a functionally equivalent variant); and (3) homology with the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16 It has an amino acid sequence of 90% or more, and (a) is activated in pancreatic β cells under high glucose concentration to promote insulin secretion from the pancreatic β cells, and / or (b) ) A polypeptide that exhibits an activity of increasing the intracellular cAMP amount when activated in a cell (hereinafter referred to as a homologous polypeptide) can be mentioned. Hereinafter, these various polypeptides that can be used as a screening tool for the polypeptide-type diabetes therapeutic agent of the present invention are generically referred to as screening tool polypeptides.

Polypeptide for screening tools 1
The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is a human-derived G protein-coupled receptor consisting of 335 amino acid residues. In addition, one of the polypeptides for screening tools, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16, is:
It is a rat-derived G protein-coupled receptor consisting of 335 amino acid residues. A human-derived polypeptide having the amino acid sequence represented by SEQ ID NO: 2 and SEQ ID NO: 1
It shows 80.6% homology in the amino acid sequence comparison with the rat-derived polypeptide having the amino acid sequence represented by 6. The "homology" in the present specification
And BLAST (Basic local alig
nment search tool; Altsch
l, S. F. Et al., J. Mol. Biol. , 215, 4
03-410, 1990),
Amino acid sequence homology can be determined using the BLAST search algorithm. Specifically, BLAS
T package (sgi32bit version, version 2.
0.12; bl2seq program (obtained from NCBI) (Tatiana A. Tatusova and Thom)
as L.L. Madden, FEMS Microbio
l. Lett. , 174, 247-250, 1999).
Can be calculated according to the default parameters. Use the program name "blastp" as the pairwise alignment parameter
The p insertion cost value is “0”, the Gap expansion cost value is “0”, and “SE” is set as the filter of the Query array.
G ”and“ BLOSUM62 ”as Matrix, respectively.

These polypeptides consisting of the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16 are (a) high glucose-dependent insulin secretagogue activity, and (b) activated in cells. As a result, the activity of increasing the intracellular cAMP amount (hereinafter, sometimes referred to as intracellular cAMP increasing activity) is exhibited. In addition, these polypeptides consisting of the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16 are activated by being overexpressed in pancreatic β cells under low glucose concentration, It does not promote insulin secretion from the pancreatic β cells. In the present specification, “under high glucose concentration” means, for example, a state in which the glucose concentration in the blood or the environment surrounding the cells exceeds the glucose concentration range in a normal state, and in particular, 16 It is 0.8 mmol / L.
Further, “under a low glucose concentration” means, for example, a state in which the glucose concentration is lower than the glucose concentration range in a normal state, and is particularly 3.3 mmol / L or less.

As used herein, a certain polypeptide "is activated in pancreatic β cells, thereby
Whether or not it exhibits "activity for promoting insulin secretion from cells" is not particularly limited, but it can be confirmed by, for example, the following method (preferably the method described in Example 5 below). it can. That is, an expression vector containing a polynucleotide encoding the polypeptide,
Pancreatic β cells were each transformed with the control expression vector containing no polynucleotide, and after a lapse of a predetermined number of days (for example, 2 or 3 days), the cells were replaced with a buffer solution containing a predetermined concentration of glucose. Then, the cells are further incubated for a predetermined time (for example, several hours), and the insulin secretion amount in the buffer solution (that is, the culture supernatant) is measured. Compared to the amount of insulin secreted in the culture supernatant of the cells transformed with the control expression vector (control cells), the culture in the cells transformed with the expression vector containing the polynucleotide encoding the polypeptide (test cells) If the amount of insulin secretion in the serum is increased, it can be determined that the polypeptide exhibits “activity of promoting insulin secretion from pancreatic β cells by being activated in pancreatic β cells”. . Whether the test cells have a significantly increased insulin secretion amount relative to the control cells is determined by Student's t-test. Insulin secretion was increased in the test cells, and the significant difference from the control cells was p <0.0.
When 5, preferably p <0.01, it is judged that the insulin secretion amount is significantly increased.

[0018] In the present specification, whether or not a certain polypeptide exhibits "activity of increasing the intracellular cAMP amount by being activated in cells" is not particularly limited. For example, it can be confirmed by the following method (preferably, the method described in Example 4 below). That is, each of the expression vector containing the polynucleotide encoding the polypeptide and the control expression vector not containing the polynucleotide is transformed into cells for a predetermined time (for example,
After 20 hours), a phosphodiesterase inhibitor [eg, IBMX (3-isobutyl-1-me
[thylxanthine)] and the medium is further incubated for a predetermined time (for example, 40 minutes) to measure the amount of cAMP in each cell. CAMP in cells transformed with an expression vector containing a polynucleotide encoding the polypeptide as compared to cells transformed with a control expression vector
If the amount is increased, it can be determined that the polypeptide exhibits "activity that increases the intracellular cAMP amount by being activated in cells".

In the present specification, a state in which a polypeptide for a screening tool which is a G protein-coupled receptor is "activated" means that the polypeptide downstream of the G protein-coupled receptor is irrespective of the presence or absence of binding with a ligand. Means that the signal is transmitted to. These polypeptides are activated when the absolute amount of activated G protein-coupled receptors exceeds a certain amount. The G protein-coupled receptor is in an equilibrium state between the inactive form and the active form, and by binding the ligand to the G protein-coupled receptor,
The equilibrium shifts to the active form. Even when the G protein-coupled receptor is overexpressed, the absolute amount of the activated G protein-coupled receptor increases, so that it is activated and a signal is transmitted downstream even in the absence of a ligand. (Milano, CA et al., Scie).
No. 264, 582-586, 1994). That is, the G protein-coupled receptor can detect a signal from the G protein-coupled receptor by overexpressing the G protein-coupled receptor in cells even if the ligand is not specified. There are cases. In each of the experiments described below in Examples 4 and 5, even in the absence of a ligand for the screening tool polypeptide,
When these polypeptides are overexpressed, they are activated in the same state as activation by binding with an agonist.

A functionally equivalent variant which can be used as a screening tool for a polypeptide type diabetes therapeutic agent of the present invention is one or more of the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16. 1 or more (preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5), for example, 1 to several amino acids are deleted, substituted, and / or added at a position. And having (a) high glucose-dependent insulin secretagogue activity and / or (b) intracellular cAMP increasing activity [preferably (a) high glucose-dependent insulin secretagogue activity. , And (b) both show intracellular cAMP increasing activity, and more preferably, in addition to these activities, in (c) low glucose concentration Be activated by overexpression, unless the do not promote insulin secretion from pancreatic β cells] be a polypeptide capable, not limited in particular, its origin is not limited to a human or rat.

For example, it includes not only a human variant of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a rat variant of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16. , Functionally equivalent variants derived from organisms other than humans and rats (eg, mouse, hamster, or dog). Furthermore, their natural polypeptides (ie, variants derived from human or rat, or functional equivalent variants derived from organisms other than human and rat), or the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: A polypeptide in which the amino acid sequence of the amino acid sequence represented by 16 is artificially modified by genetic engineering is included. In the present specification, "variant" (va
(riation) means the individual difference found in the same polypeptide within the same species, or the difference found in homologous polypeptides between several species.

A human variant of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a rat variant of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 16, or a human or non-rat organism Those skilled in the art will appreciate that the functionally equivalent variant of
Or represented by SEQ ID NO: 16 based on the information on the nucleotide sequence of the polynucleotide encoding the polypeptide consisting of the amino acid sequence represented by (for example, the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing) A nucleotide sequence of a polynucleotide encoding a polypeptide consisting of an amino acid sequence (for example, the nucleotide sequence represented by SEQ ID NO: 15 in the sequence listing)
It can be obtained based on the information of. Regarding the gene recombination technique, unless otherwise specified, known methods (eg, Maniatis, T. et al., “Mole”) are used.
color Cloning-A Laboratory
Manual ", Cold Spring Harb
or Laboratory, NY, 1982).

For example, an appropriate primer or probe is designed based on the information on the nucleotide sequence of the polynucleotide encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16. , The primer or probe, and an organism of interest [eg, mammals (eg, human, mouse, rat,
Hamster, or dog)] sample (eg total RN)
A or mRNA fraction, cDNA library, or phage library) and polymerase chain reaction (PCR) method (Saiki, RK, et al., Science).
ce, 239, 487-491, 1988) or a hybridization method to obtain a polynucleotide encoding the polypeptide, and express the polynucleotide using an appropriate expression system. For example, by the method described in Example 5, it is confirmed that it exhibits high glucose-dependent insulin secretagogue activity, or by the method described in Example 4, it is confirmed that it exhibits intracellular cAMP increasing activity. As a result, the desired polypeptide can be obtained.

The above-mentioned polypeptide artificially modified by genetic engineering can be prepared by a conventional method, for example, a site-specific mutagenesis method (site-specific mutagenesis).
Mark; Mark, D .; F. Et al., Proc. Nat
l. Acad. Sci. USA, 81,5662-56.
66, 1984), a polynucleotide encoding the polypeptide is obtained, and the polynucleotide is expressed using an appropriate expression system.
For example, is it confirmed by the method described in Example 5 that it exhibits high glucose-dependent insulin secretagogue activity,
Alternatively, intracellular cAM can be obtained by the method described in Example 4.
A desired polypeptide can be obtained by confirming that it exhibits P-increasing activity.

The functionally equivalent variant is SEQ ID NO: 2
Containing the amino acid sequence represented by or the amino acid sequence represented by SEQ ID NO: 16, and (a) being activated under high glucose concentration, promoting the secretion of insulin from pancreatic β cells, and / Alternatively, (b) a polypeptide having an activity of increasing the intracellular cAMP amount when activated is included, and, for example, from the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16. A polypeptide obtained by adding an appropriate marker sequence or the like to the N-terminal and / or C-terminal of the polypeptide (that is, a fusion polypeptide) also has (a) high glucose-dependent insulin secretagogue activity, and / or (b) It is included as long as it shows intracellular cAMP increasing activity. As the marker sequence, a sequence that facilitates confirmation of polypeptide expression, intracellular localization, or purification can be used, and examples thereof include FLAG epitope, hexa-histidine tag, and hemagglutinin. Tag or m
A yc epitope etc. can be mentioned.

The homologous polypeptide which can be used as a screening tool for the polypeptide type diabetes therapeutic agent of the present invention has 90% homology with the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16. It has the above amino acid sequence, and (a) high glucose-dependent insulin secretagogue activity, and / or (b)
The polypeptide having intracellular cAMP increasing activity is not particularly limited, but is preferably 95% or more, more preferably 95% or more with respect to the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16. It may have an amino acid sequence having a homology of preferably 98% or more, more preferably 99% or more.

The polypeptide for a screening tool, which can be used as a screening tool for the polypeptide type diabetes therapeutic agent of the present invention, can be obtained by various known methods. For example, it is known by using a polynucleotide encoding a target protein. It can be prepared by the genetic engineering method of. More specifically, a transformant cell for a screening tool described later (that is, a transformant cell transformed with an expression vector containing a DNA encoding a polypeptide for the screening tool and expressing the polypeptide) is screened. It can be prepared by culturing under conditions that allow expression of the polypeptide for the tool, and separating and purifying the target protein from the culture by a method generally used for separating and purifying the receptor protein.

Examples of the polynucleotide encoding the polypeptide for screening tool include, for example, the polynucleotide encoding the polypeptide having the amino acid sequence represented by SEQ ID NO: 2 and the polypeptide having the amino acid sequence represented by SEQ ID NO: 16. A polynucleotide that encodes a polynucleotide that encodes a functional variant,
And a polynucleotide encoding a homologous polypeptide. The term "polynucleotide" as used herein includes both DNA and RNA.

The method for producing a polynucleotide encoding a polypeptide for a screening tool is not particularly limited, but for example, (1) a method using PCR, (2) a conventional genetic engineering method (ie, , CDN
Examples of the method include a method of selecting a transformant containing a desired cDNA from a transformant transformed with the A library), or (3) a chemical synthesis method.
Hereinafter, each manufacturing method will be sequentially described.

In the method using PCR, a polynucleotide encoding a polypeptide for a screening tool can be produced, for example, by the following procedure. That is, mRNA is extracted from a human cell or tissue capable of producing a polypeptide for a screening tool. Then, based on the nucleotide sequence of the polynucleotide encoding the screening tool polypeptide, a pair of two primer sets capable of sandwiching the entire length of mRNA corresponding to the screening tool polypeptide,
Alternatively, a set of two primers that can sandwich the part of the mRNA region is prepared. Reverse transcriptase-
By carrying out polymerase chain reaction (RT-PCR), the full-length cDNA of the polypeptide for screening tools
NA or part thereof can be obtained.

More specifically, first, total RNA including mRNA encoding a polypeptide for a screening tool is extracted by a known method from a cell or tissue (for example, pancreas) capable of producing the polypeptide for a screening tool. . Examples of the extraction method include guanidine
Thiocyanate hot phenol method, guanidine
The thiocyanate-guanidine / hydrochloric acid method or the guanidine / thiocyanate cesium chloride method can be mentioned, but the guanidine / thiocyanate cesium chloride method is preferably used. The cell or tissue having the ability to produce a polypeptide for a screening tool is, for example, a Northern blotting method using a polynucleotide encoding the polypeptide for a screening tool or a part thereof, or an antibody specific to the polypeptide for a screening tool. Can be specified by the Western blotting method using

Subsequently, the extracted mRNA is purified. m
Purification of RNA may be carried out by a conventional method, for example, mRNA can be purified by adsorbing it on an oligo (dT) cellulose column and then eluting it. If desired, mRNA can be further fractionated by a sucrose density gradient centrifugation method or the like. Alternatively, commercially available extracted and purified mRNA can be used without extracting the mRNA. Next, the purified mRNA is subjected to a reverse transcriptase reaction in the presence of, for example, a random primer, an oligo dT primer, and / or a custom-synthesized primer to synthesize a first-strand cDNA. This synthesis can be performed by a conventional method. Using the obtained first-strand cDNA, PCR is carried out using the full-length target polynucleotide or two kinds of primers sandwiching a part of the region, and the desired c
The DNA can be amplified. The obtained DNA is fractionated by agarose gel electrophoresis or the like. If desired, the DNA fragment of interest can be obtained by cutting the DNA with a restriction enzyme or the like and connecting them. In addition, a target DNA fragment can be obtained from genomic DNA.

In the method using a conventional genetic engineering technique, a polynucleotide encoding a polypeptide for a screening tool can be produced, for example, by the following procedure. First, single-stranded cDNA is synthesized using reverse transcriptase using the mRNA prepared by the above-mentioned method using PCR as a template, and then double-stranded cDNA is synthesized from this single-stranded cDNA. As the method, for example, S
1 nuclease method (Efstradiadis, A .;
Et al., Cell, 7, 279-288, 1976), La.
nd method (Land, H. et al., Nucleic Acid
s Res. , 9, 2251-2266, 1981),
O. Joon Yoo method (Yoo, OJ, et al., Pro
c. Natl. Acad. Sci. USA, 79, 10
49-1053, 1983), or Okayama-B.
erg method (Okayama, H. and Berg, P.,
Mol. Cell. Biol. , 2, 161-170,
1982) and the like.

Next, after preparing a recombinant plasmid containing the double-stranded cDNA, E. coli (eg, DH5α strain)
Then, the recombinant is selected by using, for example, drug resistance to tetracycline or ampicillin as an index. Transformation of a host cell is carried out by the method of Hanahan (Hana, for example, when the host cell is E. coli).
han, D.H. J. , Mol. Biol. , 166,55
7-580, 1983), that is, CaCl ₂ , Mg.
It can be carried out by a method of adding the above recombinant DNA to a competent cell prepared in the coexistence of Cl ₂ or RbCl. In addition to the plasmid, a phage vector such as lambda system can be used as the vector.

From the transformant thus obtained,
Examples of the method for selecting a transformant having the desired cDNA include (1) a screening method using a synthetic oligonucleotide probe, (2) a screening method using a probe prepared by PCR, and
(3) A method of producing a target polypeptide in another animal cell for screening, (4) a method of selecting using an antibody against the polypeptide for a screening tool, or (5) a selective hybridization translation system The method can be adopted.

In the screening method using a synthetic oligonucleotide probe, a transformant having the desired cDNA can be selected by the following procedure, for example. That is, synthesize an oligonucleotide corresponding to all or part of the screening tool polypeptide,
Using this as a probe (labeled with ³² P or ³³ P), the DNA of the transformant is hybridized with a denaturing and immobilized nitrocellulose filter, and the obtained positive strain is searched and selected. When synthesizing an oligonucleotide for a probe, it may be a nucleotide sequence derived by using the frequency of codon usage, or may be a plurality of nucleotide sequences combining possible nucleotide sequences. . In the latter case,
Inosine can be included to reduce its variety.

In the screening method using the probe prepared by PCR, for example, the transformant having the desired cDNA can be selected by the following procedure. That is, each oligonucleotide of a sense primer and an antisense primer corresponding to a part of a polypeptide for a screening tool is synthesized, PCR is performed by combining these oligonucleotides, and a DNA fragment encoding all or part of the target polypeptide is amplified. To do. As the template DNA used here, cDNA synthesized by reverse transcription reaction from mRNA of cells producing the screening tool polypeptide or genomic DNA can be used. The DNA fragment prepared in this manner is used, for example, for ³² P or
A transformant having the desired cDNA is selected by labeling with ³³ P and using this as a probe for colony hybridization or plaque hybridization.

In the method of producing a desired polypeptide in other animal cells and screening it, for example, a transformant having the desired cDNA can be selected by the following procedure. That is, the transformant is cultured, the polynucleotide is amplified, the polynucleotide is transfected into an animal cell, and the polypeptide encoded by the polynucleotide is produced on the cell surface. In this case, either a plasmid capable of self-replication and containing a transcription promoter region, or a plasmid which can be integrated into the chromosome of an animal cell can be used. An antibody against the polypeptide for the screening tool is used to detect the polypeptide for the screening tool to select a transformant having the desired cDNA from the original transformants.

In the selection method using an antibody against a polypeptide for a screening tool, for example, a transformant having the desired cDNA can be selected by the following procedure. That is, cDNA is previously incorporated into an expression vector to produce a polypeptide on the cell surface of a transformant, and a desired polypeptide-producing strain is detected using an antibody against the polypeptide for a screening tool and a secondary antibody against the antibody. Then, a transformant having the desired cDNA is selected.

Selective hybridization
In the method using the translation system, for example, the transformant having the desired cDNA can be selected by the following procedure. That is, cDNA obtained from the transformant is blotted on a nitrocellulose filter or the like, and mRNA separately prepared from cells having the ability to produce a polypeptide for a screening tool is hybridized, and then the mRNA bound to the cDNA is dissociated. ,
to recover. The recovered mRNA is translated into a polypeptide using an appropriate polypeptide translation system, for example, injection into Xenopus oocytes or a cell-free system such as rabbit reticulocyte lysate or wheat germ. A transformant having the cDNA of interest is selected by detection using an antibody against the polypeptide for a screening tool.

A method for collecting a polynucleotide encoding a polypeptide for a screening tool from the obtained transformant of interest is a known method (for example, Mania).
tis, T .; Et al., “Molecular Clonin
g-A Laboratory Manual ", Co
ld Spring Harbor Laborato
ry, NY, 1982). For example, it can be carried out by separating a fraction corresponding to the plasmid DNA from the cells and cutting out the cDNA region from the obtained plasmid DNA.

In the method using the chemical synthesis method, for example, a polynucleotide encoding a polypeptide for a screening tool can be produced by ligating a DNA fragment produced by the chemical synthesis method. Each DN
A is a DNA synthesizer [eg, Oligo 1000M
DNA Synthesizer (manufactured by Beckman) or 394 DNA / RNA Synthesi
zer (manufactured by Applied Biosystems)
Etc.] can be used for the synthesis. In addition, a polynucleotide encoding a polypeptide for a screening tool can be synthesized, for example, by the phosphite triester method (H
unkapiller, M .; Et al, Nature, 10,
105-111, 1984) and the like, and can also be produced by chemical synthesis of nucleic acid. The codon for the desired amino acid is known per se, and its selection may be arbitrary. For example, it can be determined according to a conventional method in consideration of the frequency of codon usage of the host to be used (Cra).
ntham, R.N. Et al. Nucleic Acids R
es. , 9, r43-r74, 1981). Furthermore, partial modification of the codons of these base sequences is carried out according to a conventional method by a site-directed mutagenesis method (site) using a primer composed of a synthetic oligonucleotide encoding the desired modification.
specific mutagenesis) (Ma
rk, D.I. F. Et al., Proc. Natl. Acad. S
ci. USA, 81, 5662-5666, 1984).
Etc. can be implemented.

Sequencing of the DNA obtained by the various methods described above can be performed, for example, by the Maxam-Gilbert chemical modification method (Maxam, AM and Gilber).
t, W. , "Methods in Enzymolo
gy ", 65, 499-559, 1980) and dideoxynucleotide chain termination method (Messing, J. and V.
ieira, J .; , Gene, 19, 269-276,
1982) and the like.

A host cell (preferably a eukaryote, particularly preferably a 293-EBNA cell) is transformed by reintegrating the isolated polynucleotide encoding the polypeptide for a screening tool into an appropriate vector DNA. be able to.

By culturing the transformed cells,
The polypeptide for a screening tool produced on the cell surface of the cells can be separated and purified by various known separation procedures utilizing the physical properties and biochemical properties of the polypeptide. Specifically, for example,
A cell membrane fraction containing the polypeptide for a screening tool can be obtained by culturing cells expressing the polypeptide for a screening tool on the surface, suspending them in a buffer, homogenizing and centrifuging. After solubilizing the obtained cell membrane fraction, treatment with a usual protein precipitant, ultrafiltration, various liquid chromatography [eg, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, Affinity chromatography or high performance liquid chromatography (HPLC)
Etc.], or by a dialysis method or a combination thereof,
Polypeptides for screening tools can be purified. When solubilizing the cell membrane fraction, a solubilizing agent (eg, CHAPS, Trito) that is as mild as possible is used.
n X-100, dichitonin, etc.) can retain the properties of the receptor even after solubilization.

Polypeptides for screening tools are
The expression in fusion with the marker sequence in frame facilitates confirmation of expression of the polypeptide for screening tools, confirmation of intracellular localization, purification, and the like. Examples of the marker sequence include FLAG epitope, hexa-histidine tag, hemagglutinin tag, myc epitope, and the like.
In addition, by inserting a specific amino acid sequence recognized by a protease (for example, enterokinase, factor Xa, thrombin, etc.) between the marker sequence and the polypeptide for a screening tool, the marker sequence portion is inserted into these proteases. Can be removed by cutting. For example, there is a report in which a muscarinic acetylcholine receptor and a hexahistidine tag are linked with a thrombin recognition sequence (Hayashi, M.K. and Haga, T., J. Biochem., 120, 1).
232-1238, 1996).

2) Transformed Cell Type Diabetes Therapeutic Agent Screening Tool Transformed cells that can be used as the transformed cell type antidiabetic agent screening tool of the present invention (hereinafter referred to as transforming cells for screening tools) are, for example, (I) a trait that is transformed with an expression vector containing a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16 and expressing the polypeptide A transformed cell; (ii) a transformed cell which is transformed with an expression vector containing a polynucleotide encoding a functionally equivalent variant and expresses the polypeptide; and (iii) a polynucleotide encoding a homologous protein. Is transformed with an expression vector and expresses said polypeptide Transformed cells can be mentioned.

The transformed cells for the screening tool are
For example, a polynucleotide encoding a screening tool polypeptide isolated by the above-mentioned method is reintegrated into an appropriate vector DNA to give a host cell (preferably eukaryote, particularly preferably 293-).
It can be obtained by transforming EBNA cells). Also, by introducing an appropriate promoter and a sequence involved in expression into these vectors, it is possible to express the polynucleotide in each host cell.

The present inventor has made it possible to overexpress the screening tool polypeptide on the cell membrane by using an expression vector capable of adding a signal sequence to the N-terminus of the screening tool polypeptide. The expression vector is not particularly limited as long as it includes a polynucleotide encoding a polypeptide for a screening tool, and for example, a known expression vector appropriately selected according to the host cell to be used is inserted with the polynucleotide. The expression vector obtained by this can be mentioned.

The present inventor has also made it possible to overexpress the screening tool polypeptide on the cell membrane by using 293-EBNA cells. A transformant cell for a screening tool that can be used as a transformant cell type diabetes therapeutic agent screening tool of the present invention is a transformant cell type transformed with the expression vector and containing a polynucleotide encoding a polypeptide for the screening tool. It is not particularly limited as long as it expresses the above polypeptide when used as a screening tool for therapeutic agent for diabetes, for example, a polynucleotide encoding a polypeptide for a screening tool is a cell integrated into the chromosome of a host cell. Or it can be a cell containing in the form of an expression vector containing a polynucleotide encoding a polypeptide for a screening tool. A transformant cell for a screening tool can be obtained by transforming a desired host cell with an expression vector containing a polynucleotide encoding a polypeptide for a screening tool, for example.

For example, eukaryotic host cells include cells of vertebrates, insects, yeasts and the like. Examples of vertebrate cells include monkey cells such as COS cells (Glu).
zman, Y. , Cell, 23, 175-182, 1
981), Chinese hamster ovary cells (CH
O) dihydrofolate reductase-deficient strain (Urlau)
b, G.I. And Chasin, L .; A. , Proc. Na
tl. Acad. Sci. USA, 77, 4216-4
220, 1980), human embryonic kidney-derived HEK293 cells, and 293-EBN into which the EBNA-1 gene of Epstein-Barr virus has been introduced into the HEK293 cells.
A cell (Invitrogen) etc. can be mentioned.

As an expression vector for vertebrate cells, one having a promoter located upstream of the polynucleotide to be normally expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence, etc. can be used. Further, if necessary, it may have an origin of replication. Examples of the expression vector include S
PSV2dhfr with V40 early promoter
(Subramani, S. et al., Mol. Cell. B
iol. , 1,854-864, 1981), pEF-BOS (Mizu) having a human elongation factor promoter.
Shima, S .; And Nagata, S .; , Nucle
ic Acids Res. , 18,5322,199
0), pC with cytomegalovirus promoter
EP4 (Invitrogen) and the like can be mentioned. In addition, upstream of the polypeptide to be expressed,
It is also possible to use an expression vector designed so that an extracellular secretion signal sequence (signal sequence), for example, an influenza hemagglutinin signal sequence can be fused in frame (J. Biol. Chem., 267, 21995-). Two
1998, 1992). As such an expression vector, for example, a plasmid (pEF-BOS signal seq) in which a sequence encoding a signal sequence and a FLAG epitope is introduced into the above pEF-BOS.
uence flag plasmid) can be used.

When 293-EBNA cells are used as the host cells, pCEP4 (Invitrogen) having an Epstein-Barr virus origin of replication as an expression vector and capable of self-propagation in 293-EBNA cells.
Company) can be used.

When a COS cell is used as a host cell, it has an SV40 origin of replication as an expression vector and is capable of autonomous growth in a COS cell.
Those provided with a transcription promoter, transcription termination signal, and RNA splice site can be used, and for example,
pME18S (Maruyama, K. and Takeb)
e, Y. , Med. Immunol. , 20, 27-3
2, 1990), pEF-BOS (Mizushim)
a, S. And Nagata, S .; , Nucleic A
cids Res. , 18, 5322, 1990), or pCDM8 (Seed, B., Nature, 32).
9, 840-842, 1987) and the like.

The expression vector is, for example, DEAE-
Dextran method (Luthman, H. and Magnu)
Sson, G.S. , Nucleic Acids Re
s. , 11, 1295-1308, 1983), calcium phosphate-DNA coprecipitation method (Graham, FL.
And van der Ed, A .; J. , Virolog
y, 52, 456-457, 1973), commercially available transfection reagents (eg, FuGENE ^™ 6 Tr).
protection Reagent; Roche
Diagnostics) or electric pulse drilling (Neumann, E. et al., EM)
BO J. , 1, 841-845, 1982) and the like, and can be incorporated into COS cells.

Furthermore, when CHO cells are used as host cells, G cells are used together with an expression vector containing a polynucleotide encoding a polypeptide for a screening tool.
A vector capable of expressing the neo gene that functions as a 418 resistance marker, for example, pRSVneo
(Sambrook, J. et al., "Molecular C"
longing-A Laboratory Manual
l ", Cold Spring Harbor Lab
orory, NY, 1989) or pSV2-ne.
o (Southern, PJ and Berg, P.,
J. Mol. Appl. Genet. , 1,327-3
41, 1982) and the like, and G41
By selecting 8-resistant colonies, transformed cells that stably produce the screening tool polypeptide can be obtained.

The transformed cells for the screening tool are
It can be cultured according to a conventional method, and the polypeptide for a screening tool is produced on the cell surface by the culture. As the medium that can be used for the culture, various commonly used mediums can be appropriately selected depending on the host cell used. For example, in the case of COS cells, for example, a medium such as RPMI-1640 medium or Dulbecco's modified Eagle minimum essential medium (DMEM) to which a serum component such as fetal bovine serum (FBS) is added is used. can do. Further, in the case of 293-EBNA cells, a medium obtained by adding G418 to a medium such as Dulbecco's modified Eagle minimum essential medium (DMEM) supplemented with serum components such as fetal bovine serum (FBS) can be used.

(2) Method for screening anti-diabetic agent When a polypeptide for a screening tool or a transformed cell for a screening tool is used, a substance capable of controlling the activity of the polypeptide for a screening tool, particularly,
A substance that activates the polypeptide for a screening tool (that is, an agonist) can be screened. As described above, the polypeptide for a screening tool has an activity of promoting insulin secretion from the pancreatic β cell when activated in the pancreatic β cell under a high glucose concentration. Therefore, a substance that activates a polypeptide for a screening tool can be used as an active ingredient of an insulin secretagogue capable of promoting insulin secretion from pancreatic β cells under high glucose concentration, and further, as an effective therapeutic agent for diabetes. It is useful as an ingredient. Therefore, the polypeptide itself for the screening tool or the transformed cell itself for the screening tool is used as a therapeutic agent for diabetes (in particular, an insulin secretagogue, more specifically, a specific insulin secretagogue under a high glucose concentration). Can be used as a screening tool.

In the present specification, "specific insulin secretion promotion under high glucose concentration" means a test in which the insulin secretion amount is significantly increased with respect to the control group under the high glucose concentration, and This is the case where the increase in insulin secretion under high glucose concentration in the compound-treated group is 1.5 times or more, preferably 3 times or more, the increase in insulin secretion under low glucose concentration, and more preferably under low glucose concentration. Indicates the case where the amount of insulin secretion is not significantly increased in the test compound-treated group with respect to the control group. Whether or not the amount of insulin secretion in the test compound-treated group is significantly higher than that in the control group can be determined by, for example, a Student's t-test by conducting an experiment under the conditions shown in Example 8 or 12. Insulin secretion was increased in the test compound treatment group, and the significant difference from the control group was p <
When 0.05, preferably p <0.01, it is judged that the amount of insulin secretion is significantly increased.

The test compound that can be used for screening using the antidiabetic agent screening tool of the present invention is not particularly limited, but for example, various known compounds (peptides Including), combinatorial chemistry technology (Terrett, NK et al., Tetrahhe)
dron, 51, 8135-8137, 1995) or the phage display method (Felici, F. et al., J. Mol. Bio).
l. , 222, 301-310, 1991) and the like, and a random peptide group created by applying the same can be used. In addition, culture supernatants of microorganisms, natural components derived from plants or marine organisms, extracts from animal tissues, and the like can also be used as test compounds for screening. Furthermore,
A compound (including peptide) chemically or biologically modified with a compound (including peptide) selected by the antidiabetic agent screening tool of the present invention can be used.

The method for screening a therapeutic agent for diabetes (in particular, an insulin secretagogue, more preferably a specific insulin secretagogue under a high glucose concentration) of the present invention expresses a polypeptide for a screening tool so as to function as a receptor. It is not particularly limited as long as it includes the step of contacting the transformed cell for the screening tool or the cell membrane thereof with a test compound, and the step of analyzing whether or not the polypeptide is activated. Based on the difference in the method used to analyze the activation of the polypeptide, for example, 1) a screening method using the fluctuation of intracellular cAMP concentration as an index (hereinafter referred to as a cAMP type screening method), 2) a GTPγS binding method (Hereinafter, referred to as GTPγS-binding screening method and To), and 3) a screening method using a ligand-binding assay (hereinafter, referred to as ligand-binding-type screening method) can be exemplified.

1) Screening method for cAMP type Useful as an active ingredient of a therapeutic agent for diabetes (in particular, an insulin secretagogue, more preferably a specific insulin secretagogue under high glucose concentration), using the fluctuation of intracellular cAMP concentration as an index. When screening a substance that activates a polypeptide for a screening tool (that is, an agonist), the transformed cell for a screening tool is contacted with a test compound, and the intracellular cAM
Whether the polypeptide is activated is analyzed by directly (or indirectly) analyzing (ie, measuring or detecting) the change in P concentration. That is, intracellular cAMP
The cAMP-type screening method of the present invention in which the fluctuation of the concentration is used as an index includes a step of contacting a transformant cell for a screening tool with a test compound, and a step of analyzing a change in the cAMP concentration in the cell. More specifically, it is preferably carried out by each method described in Examples 6, 7, 10 or 11 described later. For example, the test compound is allowed to act for a certain period of time, and the EC ₅₀ thereof is 10 μM or less (more preferably 1%) using the increase in intracellular cAMP concentration as an index.
A test compound (μM or less) can be selected as a substance having an agonist activity. The change in cAMP concentration is
For example, as shown in Example 6 or 11 described later, a commercially available cAMP measurement kit (Amersham, etc.) can be used to directly analyze the change in cAMP concentration, or Example 7 described below. Alternatively, as shown in 10, by analyzing the transcription activity of a gene whose transcription level is regulated depending on the cAMP concentration [for example, a gene in which a cAMP response element (CRE) is inserted upstream of the luciferase gene], It is also possible to analyze changes in cAMP concentration.

When a transformant for a screening tool and a test compound are contacted with each other, the intracellular cAMP
When the concentration increases, the test compound can be determined to be an agonist for the screening tool polypeptide. As a control, instead of the transformant cell for the screening tool, a host cell in which the polypeptide for the screening tool is not expressed, or a transformant cell transformed with an empty vector is subjected to the same operation, and the test described above is performed. It is preferable to confirm that the compound does not increase the cAMP concentration in these control cells.

Commercially available cAMP measurement kit (Amersh
In the case of screening a substance that activates the polypeptide for a screening tool by directly analyzing the change in the cAMP concentration using Am, etc.), for example, as shown in Example 6 described later, It can be carried out by the following procedure. That is, cells into which a gene encoding a polypeptide for a screening tool has been introduced are cultured for 20 hours after the gene introduction, and then the medium is aspirated, followed by 1 mmol / L-IBMX (3-iso.
butyl-1-methylxanthine) / D
Add 400 μL of MEM and incubate at 37 ° C. for 10 minutes in the presence of 5% CO ₂ . Furthermore, 1 mmol / L-
Add a test compound (eg, compound, peptide, antibody, etc.) diluted in 100 μl of IBMX / DMEM,
Incubate for another 30 minutes. The medium was aspirated, and the amount of cAMP in the obtained cells was measured using a commercially available cAMP measurement kit [eg, cAMP enzyme immunoassay system (cAMP
enzyme immunoassay system
m; Amersham pharmacia biot
ech company)]. A test compound in which a specific increase in the amount of cAMP in the presence of the test compound is observed can be screened as a substance that activates the polypeptide for a screening tool, that is, a therapeutic agent for diabetes.

When screening a substance that activates a polypeptide for a screening tool by analyzing the transcriptional activity of a gene whose transcription amount is regulated depending on the cAMP concentration and indirectly analyzing the change in the cAMP concentration For example, it can be carried out by the following procedure, as shown in Example 7 described later. That is, a gene encoding a polypeptide for a screening tool and cA
A gene whose transcription level is regulated depending on MP concentration [for example, a gene in which a cAMP response element (CRE) is inserted upstream of a luciferase gene; for example, pCRE-Lu
c vector (CLONTECH)] was introduced into the cells, and then the cells were cultured for 18 to 20 hours, and the test compound diluted with the medium was added to the cells at 37 ° C. in the presence of 5% CO _2.
Incubate for 5-6 hours. After aspirating the medium and lysing with a cell lysate, its luciferase activity is measured. A substance or the like in which a specific increase in reporter activity in the presence of a test compound is observed can be screened as a substance that activates a polypeptide for a screening tool, that is, a therapeutic agent for diabetes.

2) GTPγS binding type screening method GTPγS binding method (Lazareno, S. and Bir)
dsall, N .; J. M. , Br. J. Pharmac
ol. , 109, 1120-1127, 1993), and a therapeutic agent for diabetes (in particular, an insulin secretagogue,
More preferably, when screening a substance that activates a polypeptide for a screening tool (that is, an agonist) that is useful as an active ingredient of a specific insulin secretion promoter under high glucose concentration, for example, the following procedure is used. It can be carried out. That is, the cell membrane expressing the polypeptide for the screening tool was treated with 20 mmol / L-HEPES (pH 7.4),
100 mmol / L-NaCl, 10 mmol / L-M
GTPγS labeled with ³⁵ S (400 pmol / gCl ₂ and 50 mmol / L-GDP mixed solution)
L). After incubation in the presence of the test compound and in the absence of the test compound, the reaction solution is filtered with a glass filter or the like, and the radioactivity of GTPγS remaining on the filter is measured with a liquid scintillation counter or the like. An agonist to the polypeptide for a screening tool, that is, a therapeutic agent for diabetes can be screened by using as an index a specific increase in GTPγS binding in the presence of a test compound.

GT of the Invention Utilizing the GTPγS Binding Method
In the PγS-binding screening method, in the presence of GTPγS labeled with ³⁵ S, a step of bringing a cell membrane of a transformant cell for a screening tool into contact with a test compound, and GTPγS bound to the cell membrane and unbound GTPγS The step of separating and analyzing the radioactivity contained in either separated is included.

3) Ligand binding type screening method Utilizing a ligand binding assay method, it is useful as an active ingredient of a therapeutic agent for diabetes (in particular, an insulin secretagogue, more preferably a specific insulin secretagogue under high glucose concentration). When screening a substance that binds to a polypeptide for a screening tool, for example, it can be carried out by the following procedure. That is, a transformed cell for a screening tool expressing a polypeptide for a screening tool, a cell membrane thereof, or a polypeptide for a screening tool (preferably a purified sample thereof) is prepared. Optimized assay conditions such as buffer, ions, and / or pH, and transformed cells or cell membranes thereof expressing said polypeptide in said optimized buffer or said polypeptide and, for example, said cAMP-type Screening method and / or GT
Substances that can be obtained by the PγS-binding screening method [ie, agonists; for example, 2- (pyridin-4-yl) ethyl thiobenzoate or L-α-
The lysophosphatidylcholine oleoyl] label is incubated with the test compound for a period of time. After the reaction, the mixture is filtered with a glass filter or the like, washed with an appropriate amount of buffer, and the radioactivity remaining on the filter is measured with a liquid scintillation counter or the like. The ligand of the polypeptide for a screening tool can be selected using the obtained binding inhibition of the labeled substance as an index. Whether this ligand is an agonist or an antagonist can be confirmed by the above-mentioned cAMP type screening method and / or GTPγS binding type screening method and the like.

(3) Pharmaceutical composition for treating diabetes In the present invention, for example, a substance that activates a polypeptide for a screening tool that can be selected by the screening method of the present invention [for example, DNA, protein (antibody or antibody (Including antibody fragments), peptides, or other compounds] as an active ingredient.
The pharmaceutical composition of the present invention is preferably a pharmaceutical composition for preventing and / or treating diabetes (a preventive and / or therapeutic agent for diabetes; particularly insulin secretion), which contains a substance that activates a polypeptide for a screening tool as an active ingredient. A pharmaceutical composition for promoting, more preferably a pharmaceutical composition for promoting specific insulin secretion under high glucose concentration).

Further, in the confirmation test of the quality standard of the pharmaceutical composition for treating diabetes, (1) the step of contacting the cell for screening tool or its cell membrane with a test compound, and the polypeptide for screening screen is activated. Or (2) contacting the cell for screening tool or its cell membrane with a test compound in the presence of a labeled agonist of the polypeptide for screening tool, and the cell or The present invention also includes a method for producing a pharmaceutical composition for treating diabetes, which comprises performing an analysis comprising a step of analyzing a change in the amount of labeled agonist bound to the cell membrane and then formulating the analyzed substance. The present invention also includes a method for producing a pharmaceutical composition for treating diabetes, which comprises formulating the substance obtained by the screening method of the present invention including the analysis by the above steps.

As the active ingredient in the pharmaceutical composition of the present invention, a substance that activates the polypeptide for a screening tool can be used, and the activation substance can be selected, for example, by the screening method of the present invention. . Examples of the substance that activates the polypeptide for a screening tool include 2- (pyridin-4-yl) ethyl thiobenzoate (see Example 7 described later) or L-α-lyso selected by the screening method of the present invention. Examples thereof include phosphatidylcholine oleoyl (see Example 10), and 2- (pyridin-4-yl) ethyl thiobenzoate is preferable. The pharmaceutical composition of the present invention is not limited to a pharmaceutical composition containing a substance obtained by the screening method of the present invention as an active ingredient, and a pharmaceutical composition for treating diabetes containing a substance that activates a polypeptide for a screening tool as an active ingredient. All compositions are included, and a pharmaceutical composition for promoting insulin secretion is preferable, and a pharmaceutical composition for promoting specific insulin secretion under high glucose concentration is more preferable. The confirmation of the therapeutic effect on diabetes can be carried out by using a method known to those skilled in the art or a method improved from it. For example, confirmation of the insulin secretory action can be performed, for example, by the method described in Example 8 described later, and confirmation of the action of increasing plasma insulin amount or confirmation of the blood glucose lowering action can be performed, for example, in the Example described later. The method described in 9 can be used.

Substances that activate the polypeptide for screening tools [eg, DNA, protein (including antibody or antibody fragment), peptide, or other compound]
The formulation containing the active ingredient is, depending on the type of the active ingredient, a pharmacologically acceptable carrier, an excipient, and / or other additives which are usually used in the formulation thereof,
It can be prepared as a pharmaceutical composition. The pharmaceutical composition of the present invention is preferably a pharmaceutical composition for preventing and / or treating diabetes which comprises a substance that activates a polypeptide for a screening tool and promotes insulin secretion as an active ingredient, and more preferably, screening A pharmaceutical composition for preventing and / or treating diabetes, which comprises a substance that activates a polypeptide for a tool and specifically promotes insulin secretion under a high glucose concentration as an active ingredient. Alternatively,
The present invention includes the step of administering a substance that activates a polypeptide for a screening tool, and preventing and / or treating diabetes.
Or it is a treatment method.

As the administration, for example, oral administration such as tablets, pills, capsules, granules, fine granules, powders, and oral liquids, or injections such as intravenous injection or intramuscular injection, suppositories, Parenteral administration such as transdermal administration or transmucosal administration can be mentioned. Particularly for peptides that are digested in the stomach, parenteral administration such as intravenous injection is preferable.

In solid compositions for oral administration,
Mixing one or more active substances with at least one inert diluent such as lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropyl cellulose, starch, or polyvinylpyrrolidone, magnesium aluminometasilicate, etc. You can According to a conventional method, the composition may contain an additive other than an inert diluent, for example, a lubricant, a disintegrating agent, a stabilizing agent, a solubilizing agent or a solubilizing agent. If necessary, the tablets or pills may be coated with a sugar coating or a film of a gastric or enteric substance. Liquid compositions for oral use may contain, for example, emulsions, solutions, suspensions, syrups, or elixirs, and generally used inert diluents such as purified water or It can include ethanol. The composition may contain additives other than inert diluents, such as wetting agents, suspending agents, sweetening agents, flavoring agents or preservatives.

The injection for parenteral administration may include a sterile aqueous or non-aqueous solution, suspension or emulsion. The water-soluble solution or suspension may contain, for example, distilled water for injection or physiological saline as a diluent. Examples of the diluent for a water-insoluble solution or suspension include propylene glycol, polyethylene glycol, vegetable oil (eg olive oil), alcohols (eg ethanol), polysorbate 80 and the like. The composition may further contain a wetting agent, an emulsifying agent, a dispersing agent, a stabilizing agent, a solubilizing or solubilizing agent, an antiseptic agent and the like. The composition can be sterilized by, for example, filtration through a bacteria-retaining filter, addition of a bactericide, or irradiation. In addition, a sterile solid composition can be produced and used by dissolving it in sterile water or other sterile injectable medium before use.

The dosage depends on the strength of activity of active ingredient, symptoms,
It can be appropriately determined in consideration of the age, sex, etc. of the administration subject. For example, in the case of oral administration, the dose is usually about 0.1 to 100 mg, preferably 0.1 to 50 per day in an adult (body weight is 60 kg).
mg. In the case of parenteral administration, in the form of injection, 0.01 to 50 mg per day, preferably 0.01 to 10 mg
mg.

The polynucleotide encoding the polypeptide for the screening tool is as described above,
It can be used for manufacturing the screening tool of the present invention, but is also useful for other purposes, such as gene therapy. For example, in gene therapy using a polynucleotide encoding a polypeptide for a screening tool, the polynucleotide is introduced into the pancreas specifically,
When a polypeptide for a screening tool is overexpressed in a pancreas-specific manner, it spontaneously activates even in the absence of a ligand and promotes insulin secretion at high glucose concentration, which is useful for the treatment of diabetes. Gene therapy is described in, for example, “Gene Therapy Development Research Handbook” edited by Japan Gene Therapy Society, NTS Co., Ltd., 1999 or Tadashi Ariga and Y.
ukio Sakiyama (Tadashi Ariga and Yukio Sakiyama), Protein Nucleic Acid Enzyme, 40 (17), 2772-
It can be performed according to the method described in 2780, 1995 and the like.

A substance that promotes the expression of a polypeptide for a screening tool (particularly, a natural polypeptide, for example, a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16) (For example, a substance that promotes the transcription activity of a polynucleotide encoding a polypeptide for a screening tool)
Is capable of promoting the secretion of insulin under high glucose concentration by overexpressing a polypeptide for a screening tool. Therefore, a therapeutic agent for diabetes (in particular, an insulin secretagogue, more preferably under high glucose concentration) It is useful as an active ingredient of a specific insulin secretagogue. The expression promoting substance is, for example,
A promoter region of a polynucleotide encoding a screening tool polypeptide was ligated upstream of an appropriate reporter gene (for example, a luciferase gene) to prepare an expression vector, and cells transformed with this expression vector and a test compound were combined. It can be selected by contacting and analyzing the change in the expression of the reporter gene.

[0079]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Unless otherwise specified, known methods (Mania)
tis, T .; Et al., “Molecular Clonin
g-A Laboratory Manual ", Co
ld Spring Harbor Laborato
ry, NY, 1982).

Example 1: Amino acid represented by SEQ ID NO: 2
Polynucleotide encoding a polypeptide consisting of a sequence
The full-length cDNA encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is human genomic DNA (h
human genomic DNA; TOYOBO)
Reverse transcriptase-polymerase chain reaction (RT
-PCR) was obtained by the following procedure. An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 3 was used as the forward primer, and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4 was used as the reverse primer. An XbaI recognition sequence was added to the 5'end of each of the above primers. RT-PCR is a DNA polymerase (Pyro
best DNA polymerase; Takara Shuzo)
In the presence of 5% dimethylsulfoxide (DMSO) at 98 ° C. (10 seconds), 58 ° C. (30 seconds) and 72 ° C.
It was carried out by repeating a cycle consisting of ° C (2 minutes) 34 times. As a result, a DNA fragment of about 1.0 kbp was amplified.

The obtained fragment was digested with the restriction enzyme XbaI, and then the pEF-BOS plasmid and pEF-BO were digested.
S signal sequence flag plasmid (pEF-
BOS signal sequence flag
plasmid; Mizushima, S .; And Nag
ata, S .; , Nucleic Acids Re
s. , 18, 5322, 1990). The nucleotide sequence of the obtained clone is based on the dideoxy terminator.
DNA sequencer (ABI377)
DNA Sequencer; Applied Bio
system) and the base sequence represented by SEQ ID NO: 1 was obtained.

Next, in order to determine the 5'end and 3'end of the cDNA encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, cDNA derived from human pancreas
(Human Pancreas Marathon-
Ready cDNA; Clontech) was used as a template and RACE (rapid amplicica) was used.
(ion of cDNA ends) method was performed.
The specific operation was according to the manual attached to the cDNA.

In 5'-RACE, for the first PCR, an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 5 and AP1 primer (attached to the above cDNA)
And the AP2 primer (attached to the above-mentioned cDNA) and the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6 were used for the second PCR. Also,
For 3'-RACE, SEQ ID NO: 7 was used for the first PCR.
Using the oligonucleotide consisting of the nucleotide sequence represented by and the AP1 primer, the second PCR,
An oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 8 and the AP2 primer were used.

The first PCR of 5'-RACE and 3'-RACE was performed using Taq polymerase (LA Taq; C
64 ° C (20 seconds) at 64 ° C using Lontech
It was carried out by repeating a cycle consisting of ° C (30 seconds) and 72 ° C (3 minutes) 27 times. Second PCR
Is Taq polymerase (LA Taq; Clonte
ch company) was carried out by repeating a cycle consisting of 98 ° C. (20 seconds), 64 ° C. (30 seconds) and 72 ° C. (3 minutes) 34 times. The nucleotide sequences of the obtained PCR products were respectively determined by the dideoxy terminator method to be used as a DNA sequencer (ABI377 DNA Seq).
uencer; Applied Biosystems
Company).

By 5'-RACE, the nucleotide sequence represented by SEQ ID NO: 9 was obtained as the 5'-terminal sequence, and 3'-
According to RACE, the sequence at the 3'end is SEQ ID NO: 10
A base sequence represented by In the base sequence represented by SEQ ID NO: 9, the putative initiation codon (atg;
A stop codon (tag; immediately upstream from 0 to 202);
Nos. 161 to 163) exist, and there is no sequence that can be a start codon in frame between the stop codon and the start codon. In the base sequence represented by SEQ ID NO: 10, a stop codon (ta
a; Nos. 217 to 219) existed. Therefore, it was revealed that the base sequence represented by SEQ ID NO: 1 is a base sequence encoding a full-length amino acid sequence including a start codon and a stop codon. The amino acid sequence (335 amino acids) predicted from this sequence was the amino acid sequence represented by SEQ ID NO: 2. Since the predicted amino acid sequence has seven hydrophobic regions, which are characteristic of G protein-coupled receptors and are considered to be transmembrane domains, the nucleotide sequence represented by SEQ ID NO: 1 represents G protein-coupled receptors. Turned out to code.

Example 2: Amino acid represented by SEQ ID NO: 2
Confirmation of expression distribution of mRNA of polypeptide consisting of sequence The expression distribution of the polynucleotide encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was analyzed by the RT-PCR method according to the following procedure. First, human organs [brain (amygdala, caudate, hippocampus, corpus callosum, substantia nigra, and cerebellum), spinal cord, pituitary gland, heart, placenta, lung, trachea, liver,
Kidney A, pancreas, small intestine, stomach, spleen, bone marrow, thymus, thyroid, salivary gland, adrenal gland, mammary gland, prostate, testis, and ovary].
The reaction was carried out at 37 ° C. for 15 minutes using ASE (DNase; Nippon Gene). Of the poly A + RNA treated with DNase, 4 μg was used for reverse transcriptase (MMLV Reverse Transcripta).
se; Clontech) at 42 ° C. for 60 minutes,
And were sequentially reacted at 94 ° C. for 5 minutes to synthesize cDNA. The synthesized cDNA was dissolved in 800 μl of sterilized water.

The distribution of mRNA expression of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was obtained by using the cDNA of each human organ thus obtained as a template and SEQ ID NO: 11
Was analyzed by PCR using an oligonucleotide consisting of the nucleotide sequence represented by and an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12 as a primer set. The PCR was performed using Taq polymerase (Ex T
aq; Takara Shuzo) in the presence of 5% DMSO
It was carried out by repeating a cycle of 30 ° C. (30 seconds), 50 ° C. (30 seconds) and 72 ° C. (1 minute) 30 times. As an internal standard, the human G3PDH control amplification set (Human G3PDH Control A) was prepared using the cDNA of each human organ as a template.
The gene for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was amplified by PCR under the same conditions using mplimer Set; Clontech). The PCR reaction product was analyzed by electrophoresis on a 1% agarose gel. Approximately 500b derived from mRNA of polypeptide consisting of amino acid sequence represented by SEQ ID NO: 2
The amplification product of p was detected only in the pancreas.

Example 3: Amino acid represented by SEQ ID NO: 2
Polynucleotide encoding a polypeptide consisting of a sequence
Isolation and development of rat-derived polynucleotide corresponding to
Confirmation of current distribution The rat-derived polynucleotide corresponding to the human-derived polynucleotide encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 isolated in Example 1 above was obtained by the following procedure. First, rat genome DN
A (rat genomic DNA; Clontec
h company) as a template and SEQ ID NO: 1 used in Example 2 above.
PCR was performed using the oligonucleotide consisting of the nucleotide sequence represented by 1 and the oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12 as a primer set. The PCR is a DNA polymerase (Pyrobe).
st DNA polymerase (Takara Shuzo Co., Ltd.) in the presence of 5% DMSO at 98 ° C. (10 seconds) and 5
A cycle consisting of 7 ° C. (30 seconds) and 72 ° C. (1 minute) was repeated 34 times, and the PCR product was used as a template for DNA polymerase (Pyrobest DNA).
5% DM using polymerase (Takara Shuzo)
It was carried out by repeating a cycle consisting of 98 ° C. (10 seconds), 55 ° C. (30 seconds) and 72 ° C. (1 minute) 34 times in the presence of SO. Partial sequence analysis of the obtained PCR fragment was performed, and 4 types of oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 21 to 24 were designed as primers used in the RACE method.

In the following RACE method, rat brain cDNA (Marathon-Ready cDNA) was used as a template.
A; Clontech) was used. 5'-RACE
Then, in the first PCR, the oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 21 and the AP1 primer (attached to the cDNA) were used, and the second PCR
For this, an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 22 and an AP2 primer (attached to the cDNA) were used. Further, in 3′-RACE, the oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 23 and the AP1 primer were used for the first PCR, and the oligonucleotide represented by SEQ ID NO: 24 was used for the second PCR. The above-mentioned AP2 primer was used with an oligonucleotide having a nucleotide sequence of

5'-RACE first and second PC
R is Taq polymerase (LA Taq;
Clontech) and 98 ° C (20 seconds) and 6
It was carried out by repeating a cycle consisting of 5 ° C. (30 seconds) and 72 ° C. (3 minutes) 34 times. Also, 3'-
The first and second PCRs of RACE were performed using T
aq polymerase (LA Taq; Clontech
Company), 98 ° C (20 seconds) and 65 ° C (30 seconds)
And 72 ° C. (5 minutes) were repeated 34 times. 5'-RACE and 3'-RA
The nucleotide sequences of the PCR products obtained by CE were analyzed.

Based on the determined base sequence, an oligonucleotide having the base sequence represented by SEQ ID NO: 25 and an oligonucleotide having the base sequence represented by SEQ ID NO: 26 were designed. PCR using the rat brain cDNA as a template was carried out using this primer set. The PCR is a DNA polymerase (pfu turbo).
DNA polymerase; STRATAGEN
12 cycles of 98 ° C. (20 seconds), 64 ° C. (30 seconds) and 74 ° C. (2 minutes).
98 ° C (20 seconds), 61 ° C (30 seconds) and 74 ° C (2 seconds)
For 12 minutes and 98 ° C
(20 seconds), 58 ° C (30 seconds) and 74 ° C (2 minutes)
The cycle consisting of and was repeated 16 times. Furthermore, this P
Using the CR product as a template, DNA polymerase (pfu t
urbo DNA polymerase; STRAT
Using AGENE), 98 ° C (20 seconds) and 64 ° C
12 cycles of (30 seconds) and 74 ° C (2 minutes 30 seconds), 1 cycle of 98 ° C (20 seconds), 61 ° C (30 seconds) and 74 ° C (2 minutes 30 seconds)
Two cycles, one cycle consisting of 98 ° C (20 seconds), 58 ° C (30 seconds) and 74 ° C (2 minutes 30 seconds)
Repeated 6 times. This PCR product (hereinafter referred to as PCR product A
(Hereinafter referred to as ")" was subcloned and the nucleotide sequence was confirmed.

Then, based on the determined nucleotide sequence,
An oligonucleotide having a base sequence represented by SEQ ID NO: 13 (forward primer) and an oligonucleotide having a base sequence represented by SEQ ID NO: 14 (reverse primer) were designed. An XbaI recognition sequence was added to the 5'end of each of the above primers. Using this primer set, PCR was performed using the PCR product A previously subcloned as a template. The PCR is a DNA polymerase (pfu t
urbo DNA polymerase; STRAT
Using AGENE), 98 ° C (20 seconds) and 59 ° C
It was carried out by repeating a cycle consisting of (30 seconds) and 74 ° C. (1 minute 30 seconds) 25 times. as a result,
A DNA fragment of about 1.0 kbp was amplified. This fragment was digested with the restriction enzyme XbaI and then cloned using the pEF-BOS plasmid. The nucleotide sequence of the obtained clone was analyzed by the dideoxy terminator method to obtain a DNA sequencer (ABI377DNA Sequence).
r; Applied Biosystems), and the base sequence represented by SEQ ID NO: 15 was obtained. The amino acid sequence predicted from this sequence was the amino acid sequence represented by SEQ ID NO: 16.

Next, an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 17 and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 18 designed based on the obtained base sequence was used as a primer set, Rat pancreatic β cell line RIN-5F (ATCC: CRL-2058)
PCR was performed using the derived cDNA as a template. In addition,
The cDNA was synthesized by preparing total RNA using a total RNA purification reagent (ISOGEN; Nippon Gene) and then performing a reverse transcriptase reaction. In addition, the P
For CR, Taq polymerase (rTaq; Takara Shuzo Co., Ltd.) was used and 94 ° C. (30 seconds) and 5 were used in the presence of 5% DMSO.
It was carried out by repeating a cycle consisting of 7 ° C. (30 seconds) and 72 ° C. (1 minute) 34 times. The PCR reaction product was analyzed by electrophoresis on a 1% agarose gel.
As a result, in the rat pancreatic β cell line, mRNA of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16
Was confirmed to be expressed.

Further, cDNA prepared from mouse pancreatic β cell line NIT-1 cells (ATCC: CRL-2055)
[In the same manner as in the rat pancreatic β cell line RIN-5F,
Total RNA was prepared using a total RNA purification reagent (ISOGEN; Nippon Gene), and then synthesized by performing a reverse transcriptase reaction.] For the nucleotide sequence of the rat-derived polynucleotide obtained above (ie, SEQ ID NO: Except that the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 19 and the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 20 were used as a primer set. ,
As a result of performing PCR under the same conditions as in the rat pancreatic β cell line RIN-5F, a DNA fragment of about 400 bp was detected. The DNA fragment is a human-derived polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 1
It is considered to be derived from a mouse-derived polynucleotide corresponding to the rat-derived polynucleotide consisting of the nucleotide sequence represented by 5, and the mouse pancreatic β-cell line also comprises a polynucleotide consisting of the amino acid sequence represented by SEQ ID NO: 2 or 16. It was confirmed that the mRNA of the mouse-derived polynucleotide corresponding to the polynucleotide encoding the peptide was expressed.

From the results of Example 2 and this Example, the polynucleotide encoding the polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or the mRNA of rat or mouse polynucleotide corresponding thereto
Was found to be specifically expressed in the pancreas, which is an organ greatly related to diabetes. It was also revealed that it was also expressed in the pancreatic β cell line. Therefore, a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a rat or mouse polynucleotide corresponding thereto is introduced into various cell lines without expressing the target polypeptide. Also, using a pancreatic β cell line or pancreatic cells, it is possible to select a substance that activates the polypeptide or the like consisting of the amino acid sequence represented by SEQ ID NO: 2.

Example 4: Amino acid represented by SEQ ID NO: 2
Of the polypeptide comprising the sequence in 293-EBNA cells
Change in intracellular cAMP concentration due to expression and overexpression The clone prepared in Example 1 in order to express the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, that is, the amino acid represented by SEQ ID NO: 2 A pEF-BOS signal sequence flag plasmid (hereinafter referred to as plasmid pEF-BOSSSF-NA) into which a full-length cDNA encoding a polypeptide consisting of a sequence was introduced was used. This is the N of the polypeptide of interest.
This is because the target polypeptide can be expressed at high frequency in the cell membrane by using an expression vector capable of adding a signal sequence to the end.

First, 293-EBNA cells (7 × 10 ⁴ cells / well) were placed on a 24-well plate coated with collagen.
Was seeded and cultured for 24 hours, and then the transfection reagent (FuGENE6; Boeringer Mann
Heim) was used to transform the cells into plasmid pEF-.
BOS SSF-NA or plasmid pEF-BOS
(Empty vector for control) was introduced into the gene. After gene transfer, the cells were further cultured for 20 hours, and then the medium was aspirated, followed by 1 mmol / L-IBMX (3-isob
utyl-1-methylxanthine) / DM
Add 500 μl of EM and add 4% at 37 ° C. in the presence of 5% CO _2.
Incubation was carried out for 0 minutes. In addition, the IBMX
Is a phosphodiesterase inhibitor. Aspirate the medium,
The obtained cells were subjected to cAMP amount measurement. The amount of cAMP can be measured by a commercially available cAMP enzyme immunoassay system (cAMP
enzyme immunoassay system
m; Amersham pharmacia biot
ech).

Plasmid pEF-BOS SSF-NA
It was found that the amount of intracellular cAMP was increased in a plasmid-introduced manner in the cells introduced with. On the other hand, plasmid p
In cells into which EF-BOS was introduced, there was no change in intracellular cAMP amount. By overexpressing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, cAM
Since the amount of P was increased, it was revealed that one of the second messengers in the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is cAMP.

Example 5: Amino acid represented by SEQ ID NO: 2
Mouse pancreatic β cell line NIT-of polypeptide consisting of sequence
Of insulin secretion by expression and overexpression by one cell
Changes The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or 16 is specifically expressed in the pancreas, and its expression has been confirmed in the pancreatic β cell line (see Examples 2 and 3 above). Therefore, the function of the polypeptide can be estimated by overexpressing the polypeptide in the pancreatic β cell line. The same plasmid pEF-BOS SS used in Example 4 was used to express a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
F-NA was used.

First, NIT-1 cells (4 × 10 ⁵ cells) were seeded on a 24-well plate and cultured for 24 hours, and then the transfection reagent (FuGENE6; Boeri) was used.
The plasmid pEF-BOS SSF-NA or the plasmid pEF-BOS (empty vector for control) was introduced into the cells using Nger Mannheim. After gene transfer, the cells are cultured for 2 or 3 days, and then the medium is aspirated, followed by phosphate buffer solution (PB
S) wash with 3.3 mmol / L glucose containing KRB
B (Krebs-Ringer bibonbonat
e buffer) 1 mL was added, and in the presence of 5% CO ₂ ,
Incubated at 37 ° C for 1-2 hours. The buffer was removed by suction, and K containing 3.3 mmol / L glucose was used.
Either 1 mL of RBB or 1 mL of 16.8 mmol / L glucose-containing KRBB was added, and the mixture was incubated at 37 ° C. for 2 hours in the presence of 5% CO ₂ . This supernatant was subjected to insulin secretion measurement. The insulin secretion amount was measured by a commercially available insulin radioimmunoassay kit (Fadeceph Insulin; Pharmacia Upjohn).

As a result, by overexpressing the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2,
The insulin secretion amount did not change in the presence of 3.3 mmol / L glucose, but the insulin secretion amount increased in the presence of 16.8 mmol / L glucose. From the above results, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is a G protein-coupled receptor that is specifically expressed in the pancreas as shown in Example 2, and this Example As shown in, it was shown to have a glucose concentration-dependent insulin secretagogue action. Therefore, it is considered possible to prevent and / or treat pancreatic diseases, especially diabetes by activating the activity of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.

Example 6: Indication of changes in intracellular cAMP concentration
Consisting of the amino acid sequence represented by SEQ ID NO: 2
Screening for substances that modify the activity of polypeptides
(1) In order to express a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, the full length encoding the clone prepared in Example 1, that is, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 pEF-BOS plasmid into which cDNA has been introduced (hereinafter referred to as plasmid pEF-
BOS-NA) was used.

First, 293-EBNA cells (7 × 10 ⁴ cells / well) were placed on a 24-well plate coated with collagen.
After inoculation and culturing for 24 hours, the transfection reagent (FuGENE6; Boeringer Mannhe
plasmid) pEF-BO to the cells using
50 ng of S-NA or plasmid pEF-BOS (empty vector for control) was introduced into the gene. After gene transfer, the cells were further cultured for 20 hours, and then the medium was aspirated, followed by 1 mmol / L-IBMX (3-isobuty
1-1-methylxanthine) / DMEM4
00 μL was added, and the mixture was incubated at 37 ° C. for 10 minutes in the presence of 5% CO ₂ . Furthermore, 1 mmol / L-IBMX
/ Test compound diluted in 100 μl of DMEM (eg,
Compound, peptide, antibody, etc.) was added and incubated for an additional 30 minutes. The medium was aspirated and the obtained cells were subjected to cAMP amount measurement. The amount of cAMP is measured by a commercially available cAMP enzyme immunoassay system (cAMP enzyme).
immunoassay system; Amersha
mpharmacia biotech), and a test compound in which an increase in the amount of cAMP specific to cells expressing the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was observed was the amino acid represented by SEQ ID NO: 2. It can be selected as a substance that activates the polypeptide consisting of the sequence.

Example 7: Indication of changes in intracellular cAMP concentration
Consisting of the amino acid sequence represented by SEQ ID NO: 2
Screening for substances that modify the activity of polypeptides
(2) In this example, the same plasmid pEF-BOS-NA as that used in Example 6 was used. 293-EBNA cells (1 x 1
0 ⁴ cells / well were seeded and 10% fetal calf serum (FC
S) -containing Dulbecco's modified Eagle's medium (DMEM) after overnight culture, followed by transfection reagent (LIPOF).
ECTAMINE 2000; GIBCO BRL)
To the cells using the plasmid pEF-BOS-NA.
Alternatively, 0.01 ng of plasmid pEF-BOS (empty vector for control) and pCRE-Luc vector (C
5 ng of LONTECH) was gene-transferred. After gene transfer, the cells were further cultured for 18 to 20 hours, and a test compound (a known compound not known to have a therapeutic effect on diabetes) diluted with a medium was added to the mixture at 37 ° C. in the presence of 5% CO _2.
And incubated for 5-6 hours. After aspirating the medium and lysing with a cell lysate (cell lysate LCβ; Toyo Ink), its luciferase activity was measured using a commercially available measurement kit (Picagene luminescence kit; Toyo Ink) and measurement device (M
L3000 microtiter plate lu
minometer; Dynatech Labora
Tories).

A substance in which an increase in the reporter activity specific to cells expressing the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was observed was selected as a substance enhancing the activity of the polypeptide. , 2- (pyridin-4-yl) ethyl thiobenzoate (LT-1
Z 0059519; LaboTest), etc., and four different compounds could be obtained. The EC ₅₀ value of 2- (pyridin-4-yl) ethylthiobenzoate is
It was 3.2 μM.

Example 8: Mouse pancreatic β cell line MIN6 cells
(1) MIN6 cells (2 × 10 ⁵ cells) were seeded on a 24-well plate and cultured in DMEM containing 10% FCS for 2 days.
Then, after aspirating the medium, KRB-HEPES (14
0 mmol / L-NaCl, 3.6 mmol / L-KC
1, 0.5 mmol / L-NaH ₂ PO ₄ , 0.5 mmo
1 / L-MgSO ₄ , 1.5 mmol / L-CaCl ₂ ,
10 mmol / L-Hepes, 2 mmol / L-Na
Wash with HCO ₃ , 0.1% BSA, pH 7.4), 2.
8 mmol / L glucose-containing KRB-HEPES (1
ml) was added, and in the presence of 5% CO ₂ at 37 ° C., 30 to 60.
Incubated for minutes.

Of the four screening results obtained in Example 7, after removing the buffer by suction, 2-
(Pyridin-4-yl) ethyl thiobenzoate was diluted with 2.8 mmol / L or 16.8 mmol / L glucose-containing KRB-HEPES 2- (pyridin-).
0.5 ml of 4-yl) ethyl thiobenzoate solution was added and incubated at 37 ° C. for 20 minutes in the presence of 5% CO ₂ . This supernatant was subjected to insulin secretion measurement.
The insulin secretion amount was measured by a commercially available insulin / radioimmunoassay kit (Fadecef Insulin; Pharmacia Upjohn).

As a result, by stimulation with 2- (pyridin-4-yl) ethyl thiobenzoate, 2.8 mmol /
Insulin secretion was not increased in the presence of L glucose, but insulin secretion was increased in the presence of 16.8 mmol / L glucose. Therefore, it was found that 2- (pyridin-4-yl) ethyl thiobenzoate exhibits an insulin secretion promoting action only when stimulated with high concentration glucose. The remaining 3 obtained in Example 7 other than 2- (pyridin-4-yl) ethyl thiobenzoate
An experiment similar to that of 2- (pyridin-4-yl) ethyl thiobenzoate was carried out for one of the screening results. As a result, the amount of insulin secretion did not increase in the presence of 2.8 mmol / L glucose due to the stimulation of the compound, but 16.8 mmol
Insulin secretion was increased in the presence of / L glucose. Therefore, it was revealed that the above compound also exhibits an insulin secretagogue action only when stimulated with high-concentration glucose.

Example 9: Using SD rat and GK rat
The single oral glucose tolerance test SD rat (4 weeks old; CLEA Japan, Inc.) was fasted overnight and glucose 2 g / kg was orally administered. It should be noted that 2- (pyridin-4-yl) ethyl thiobenzoate (LT-1 Z 005951) was added 5 minutes before glucose loading.
9) 100 mg / kg was intraperitoneally administered (ip).
After the glucose load, 0 minutes, 30 minutes, 60 minutes, and 120 minutes later, an appropriate amount of blood was collected and used for measurement of blood glucose level and plasma insulin concentration.

Blood and 0.33 mol were used to measure blood glucose.
/ L perchloric acid (blood: 0.33 mol / L perchloric acid = 1:10), followed by centrifugation (3000 xg, 1
The supernatant was used for 0 minutes at 4 ° C. Blood plasma was centrifuged (3000 xg, 10
The supernatant was used for 4 minutes. Glucose C Test Wako (Wako) was used for measuring the blood glucose level. In addition, rat-
An insulin assay system (Amersham) was used.

The results are shown in FIGS. 1 and 2. FIG. 1 shows the plasma insulin concentration (unit = ng) after oral glucose loading.
/ ML), and FIG. 2 shows the time course of the blood glucose level (unit = mg / dL) after oral glucose loading.
The symbol “*” shown in FIGS. 1 and 2 indicates that the significant difference from the control group [ie, the 2- (pyridin-4-yl) ethyl thiobenzoate-free group] was p <0.05.
(Student's t test).

As shown in FIG. 1, 2- (pyridine-4-)
After administration of 100 mg / kg of (yl) ethyl thiobenzoate, a significant increase in plasma insulin concentration was observed 30 minutes after glucose loading. Also,
After 30 minutes from glucose loading, 100 mg of 2- (pyridin-4-yl) ethyl thiobenzoate
The increase in blood glucose level due to glucose load was significantly suppressed in the / kg administration group. Therefore, in sugar-loaded SD rats, 2-
It was confirmed that (pyridin-4-yl) ethyl thiobenzoate has an action of increasing the amount of insulin in plasma and an action of lowering blood glucose.

Next, GK (Goto-Kakizak)
i) Rat (insulin deficiency type 2 diabetes model, 7
Weekly; A single oral glucose tolerance test was performed using Charles River Japan. The GK rat was prepared by Professor Associate Professor Yoshio Goto, Tohoku University School of Medicine, in 1975.
It is a strain established by selective mating using poor glucose tolerance during an oral glucose tolerance test in r strain rats as an index.

The oral glucose tolerance test was conducted using 2- (pyridine-4-).
Orally) (p.yl) ethyl thiobenzoate (p.
o. ) Was performed in the same manner as in SD rats. Blood glucose level after oral glucose loading (unit = mg / d
The change with time of L) is shown in FIG. Symbol “*” shown in FIG.
Is a control group [ie, 2- (pyridine-4-
[Ill) ethyl thiobenzoate-free group] is p <0.05 (Student's t-test), and the symbol “**” indicates that the significant difference is p <0.05.
It means 0.01. As shown in FIG. 3, after 30 minutes and 60 minutes after the glucose load,
Administration of 2- (pyridin-4-yl) ethyl thiobenzoate 100 mg / kg significantly suppressed the increase in blood glucose level, and the efficacy of 2- (pyridin-4-yl) ethyl thiobenzoate was confirmed in diabetic model rats. It was

Example 10: Changes in intracellular cAMP concentration
It consists of the amino acid sequence represented by SEQ ID NO: 2 as an index.
For substances that modify the activity of polypeptides
(3) In this example, the same plasmid pEF-BOS-NA as that used in Example 6 was used. 293-EBNA cells (7 x 1
0 ⁴ cells / well) and seeded with 1% fetal calf serum (FC
S) -containing Dulbecco's modified Eagle's medium (DMEM) after overnight culture, followed by transfection reagent (LIPOF).
ECTAMINE 2000; GIBCO BRL)
To the cells using the plasmid pEF-BOS-NA.
Alternatively, 0.1 ng of plasmid pEF-BOS (empty vector for control) and pCRE-Luc vector (CL
ONTECH Co., Ltd.) 20 ng was introduced into the gene. After gene transfer, culture is continued for 18 to 20 hours and 0.1% BS
The test compound diluted with the A-containing medium was added, and the mixture was incubated at 37 ° C. for 6 hours in the presence of 5% CO ₂ . After aspirating the medium and lysing it with a cell lysate (cell lysate LCβ; Toyo Ink), its luciferase activity was measured using a commercially available measurement kit (Picagene luminescence kit; Toyo Ink) and measuring device (ML3000 microtiter plate).
luminometer; Dynatech Lab
oratories).

When a substance or the like in which an increase in reporter activity specific to cells expressing the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was observed was selected as a substance enhancing the activity of the polypeptide. , L-α-lysophosphatidylcholine oleoyl (L1881; SIGMA), which is a biological component, could be obtained.

Example 11: Changes in intracellular cAMP concentration
It consists of the amino acid sequence represented by SEQ ID NO: 2 as an index.
For substances that modify the activity of polypeptides
(4) In this example, the same plasmid pEF-BOS-NA as that used in Example 6 was used. 293-EBNA cells (1 x 1
0 ⁴ cells / well) and seeded for 24 hours and then transfected with transfection reagent (LIPOFECTAMINE 2
000; GIBCO BRL) was used for the plasmid pEF-BOS-NA or the plasmid pEF.
-BOS (empty vector for control) 3 ng was introduced into the gene. After gene transfer, cultivate for another 20 hours,
Then, after aspirating the medium, 1 mmol / L-IBMX
(3-isobutyl-1-methylxanth
ine) /0.1% BSA / DMEM 80 μL,
Incubated at 37 ° C. for 10 minutes in the presence of 5% CO ₂ . Furthermore, 1 mmol / L-IBMX / 0.1% BSA
Test compounds diluted in 20 μl of / DMEM were added and incubated for a further 30 minutes. The medium was aspirated and the obtained cells were subjected to cAMP amount measurement. The amount of cAMP is measured by a commercially available cAMP enzyme immunoassay system (cyclic).
AMP kit; Nippon Schering Co., Ltd.). With the compound selected in Example 10 (L-α-lysophosphatidylcholine oleoyl), an increase in the amount of cAMP specific to cells expressing the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was observed, and therefore, This compound was selected as a substance that activates the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.

Example 12: Mouse pancreatic β cell line MIN6 cell
(2) Collagen-coated 24-well plate was seeded with MIN6 cells (2.5 × 10 ⁵ cells), and 10% FCS-containing D
MEM (Cat. No. 11995-065; GIBC
The cells were cultured for 2 days in O BRL. Then, after sucking the medium, DME containing 10% FCS and not containing glucose
M (Cat. No. 11966-025; GIBCO)
BRL) 0.4 mL was added, and in the presence of 5% CO ₂ , 37
Incubated for 2 hours at ° C. The medium was removed by suction, and 2.8 mmol / L or 16.8 mmol / L glucose-containing DMEM (Cat. No. 11966-02).
5; L-α-lysophosphatidylcholine oleoyl diluted with GIBCO BRL (that is, Example 1)
0.5 ml of the screening result solution obtained in 0) was added, and the mixture was incubated at 37 ° C. for 30 minutes in the presence of 5% CO ₂ . This supernatant was subjected to insulin secretion measurement.
The insulin secretion amount was measured by a commercially available insulin / radioimmunoassay kit (Fadecef Insulin; Pharmacia Upjohn). as a result,
In the presence of 16.8 mmol / L glucose, insulin secretion was increased by L-α-lysophosphatidylcholine oleoyl stimulation.

[0119]

Industrial Applicability The polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16, a functionally equivalent variant thereof, or a homologous polypeptide is pancreatic-specific and It has an activity of promoting insulin secretion under glucose concentration. Therefore,
According to the polypeptide, for obtaining a substance useful as a therapeutic agent for diabetes capable of controlling blood glucose within a normal range (particularly, insulin secretagogue, more preferably, a specific insulin secretagogue under high glucose concentration) A simple screening system can be constructed. Further, a pharmaceutical composition for treating diabetes (particularly insulin, which can be obtained by the screening tool of the present invention or the screening method of the present invention, and which has an activating substance of the polypeptide as an active ingredient and can control blood glucose within a normal range. A pharmaceutical composition for promoting secretion, more preferably, a pharmaceutical composition for promoting specific insulin secretion under high glucose concentration) can be produced. Further, the cell for a screening tool, which is one of the screening tools of the present invention, or the cell membrane thereof is used not only for screening a substance useful as a therapeutic agent for diabetes, but also in a test for confirming the quality standard of a pharmaceutical composition for treating diabetes. , Can also be used.

[0120]

[Sequence list free text] Numerical headings of the following sequence list <
223>, "Artificial Sequence"
"ce" is described. Specifically, the base sequences represented by the sequences of SEQ ID NOs: 3, 4, 13, and 14 in the sequence listing are artificially synthesized primer sequences.

[0121]

[Sequence list] <110> Yamanouchi Pharmaceutical Co., Ltd. <120> Method for screening agents for the treatment of diabetes <130> YAM033259P <150> JP 2000-367349 <151> 2000-12-01 <150> JP 2001-243841 <151> 2001-08-10 <160> 26 <210> 1 <211> 1008 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (1008) <400> 1 atg gaa tca tct ttc tca ttt gga gtg atc ctt gct gtc ctg gcc tcc 48 Met Glu Ser Ser Phe Ser Phe Gly Val Ile Leu Ala Val Leu Ala Ser   1 5 10 15 ctc atc att gct act aac aca cta gtg gct gtg gct gtg ctg ctg ttg 96 Leu Ile Ile Ala Thr Asn Thr Leu Val Ala Val Ala Val Leu Leu Leu              20 25 30 atc cac aag aat gat ggt gtc agt ctc tgc ttc acc ttg aat ctg gct 144 Ile His Lys Asn Asp Gly Val Ser Leu Cys Phe Thr Leu Asn Leu Ala          35 40 45 gtg gct gac acc ttg att ggt gtg gcc atc tct ggc cta ctc aca gac 192 Val Ala Asp Thr Leu Ile Gly Val Ala Ile Ser Gly Leu Leu Thr Asp      50 55 60 cag ctc tcc agc cct tct cgg ccc aca cag aag acc ctg tgc agc ctg 240 Gln Leu Ser Ser Pro Ser Arg Pro Thr Gln Lys Thr Leu Cys Ser Leu  65 70 75 80 cgg atg gca ttt gtc act tcc tcc gca gct gcc tct gtc ctc acg gtc 288 Arg Met Ala Phe Val Thr Ser Ser Ala Ala Ala Ser Val Leu Thr Val                  85 90 95 atg ctg atc acc ttt gac agg tac ctt gcc atc aag cag ccc ttc cgc 336 Met Leu Ile Thr Phe Asp Arg Tyr Leu Ala Ile Lys Gln Pro Phe Arg             100 105 110 tac ttg aag atc atg agt ggg ttc gtg gcc ggg gcc tgc att gcc ggg 384 Tyr Leu Lys Ile Met Ser Gly Phe Val Ala Gly Ala Cys Ile Ala Gly         115 120 125 ctg tgg tta gtg tct tac ctc att ggc ttc ctc cca ctc gga atc ccc 432 Leu Trp Leu Val Ser Tyr Leu Ile Gly Phe Leu Pro Leu Gly Ile Pro     130 135 140 atg ttc cag cag act gcc tac aaa ggg cag tgc agc ttc ttt gct gta 480 Met Phe Gln Gln Thr Ala Tyr Lys Gly Gln Cys Ser Phe Phe Ala Val 145 150 155 160 ttt cac cct cac ttc gtg ctg acc ctc tcc tgc gtt ggc ttc ttc cca 528 Phe His Pro His Phe Val Leu Thr Leu Ser Cys Val Gly Phe Phe Pro                 165 170 175 gcc atg ctc ctc ttt gtc ttc ttc tac tgc gac atg ctc aag att gcc 576 Ala Met Leu Leu Phe Val Phe Phe Tyr Cys Asp Met Leu Lys Ile Ala             180 185 190 tcc atg cac agc cag cag att cga aag atg gaa cat gca gga gcc atg 624 Ser Met His Ser Gln Gln Ile Arg Lys Met Glu His Ala Gly Ala Met         195 200 205 gct gga ggt tat cga tcc cca cgg act ccc agc gac ttc aaa gct ctc 672 Ala Gly Gly Tyr Arg Ser Pro Arg Thr Pro Ser Asp Phe Lys Ala Leu     210 215 220 cgt act gtg tct gtt ctc att ggg agc ttt gct cta tcc tgg acc ccc 720 Arg Thr Val Ser Val Leu Ile Gly Ser Phe Ala Leu Ser Trp Thr Pro 225 230 235 240 ttc ctt atc act ggc att gtg cag gtg gcc tgc cag gag tgt cac ctc 768 Phe Leu Ile Thr Gly Ile Val Gln Val Ala Cys Gln Glu Cys His Leu                 245 250 255 tac cta gtg ctg gaa cgg tac ctg tgg ctg ctc ggc gtg ggc aac tcc 816 Tyr Leu Val Leu Glu Arg Tyr Leu Trp Leu Leu Gly Val Gly Asn Ser             260 265 270 ctg ctc aac cca ctc atc tat gcc tat tgg cag aag gag gtg cga ctg 864 Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp Gln Lys Glu Val Arg Leu         275 280 285 cag ctc tac cac atg gcc cta gga gtg aag aag gtg ctc acc tca ttc 912 Gln Leu Tyr His Met Ala Leu Gly Val Lys Lys Val Leu Thr Ser Phe     290 295 300 ctc ctc ttt ctc tcg gcc agg aat tgt ggc cca gag agg ccc agg gaa 960 Leu Leu Phe Leu Ser Ala Arg Asn Cys Gly Pro Glu Arg Pro Arg Glu 305 310 315 320 agt tcc tgt cac atc gtc act atc tcc agc tca gag ttt gat ggc taa 1008 Ser Ser Cys His Ile Val Thr Ile Ser Ser Ser Glu Phe Asp Gly                 325 330 335 <210> 2 <211> 335 <212> PRT <213> Homo sapiens <400> 2 Met Glu Ser Ser Phe Ser Phe Gly Val Ile Leu Ala Val Leu Ala Ser   1 5 10 15 Leu Ile Ile Ala Thr Asn Thr Leu Val Ala Val Ala Val Leu Leu Leu              20 25 30 Ile His Lys Asn Asp Gly Val Ser Leu Cys Phe Thr Leu Asn Leu Ala          35 40 45 Val Ala Asp Thr Leu Ile Gly Val Ala Ile Ser Gly Leu Leu Thr Asp      50 55 60 Gln Leu Ser Ser Pro Ser Arg Pro Thr Gln Lys Thr Leu Cys Ser Leu  65 70 75 80 Arg Met Ala Phe Val Thr Ser Ser Ala Ala Ala Ser Val Leu Thr Val                  85 90 95 Met Leu Ile Thr Phe Asp Arg Tyr Leu Ala Ile Lys Gln Pro Phe Arg             100 105 110 Tyr Leu Lys Ile Met Ser Gly Phe Val Ala Gly Ala Cys Ile Ala Gly         115 120 125 Leu Trp Leu Val Ser Tyr Leu Ile Gly Phe Leu Pro Leu Gly Ile Pro     130 135 140 Met Phe Gln Gln Thr Ala Tyr Lys Gly Gln Cys Ser Phe Phe Ala Val 145 150 155 160 Phe His Pro His Phe Val Leu Thr Leu Ser Cys Val Gly Phe Phe Pro                 165 170 175 Ala Met Leu Leu Phe Val Phe Phe Tyr Cys Asp Met Leu Lys Ile Ala             180 185 190 Ser Met His Ser Gln Gln Ile Arg Lys Met Glu His Ala Gly Ala Met         195 200 205 Ala Gly Gly Tyr Arg Ser Pro Arg Thr Pro Ser Asp Phe Lys Ala Leu     210 215 220 Arg Thr Val Ser Val Leu Ile Gly Ser Phe Ala Leu Ser Trp Thr Pro 225 230 235 240 Phe Leu Ile Thr Gly Ile Val Gln Val Ala Cys Gln Glu Cys His Leu                 245 250 255 Tyr Leu Val Leu Glu Arg Tyr Leu Trp Leu Leu Gly Val Gly Asn Ser             260 265 270 Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp Gln Lys Glu Val Arg Leu         275 280 285 Gln Leu Tyr His Met Ala Leu Gly Val Lys Lys Val Leu Thr Ser Phe     290 295 300 Leu Leu Phe Leu Ser Ala Arg Asn Cys Gly Pro Glu Arg Pro Arg Glu 305 310 315 320 Ser Ser Cys His Ile Val Thr Ile Ser Ser Ser Glu Phe Asp Gly                 325 330 335 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 3 aaaatctaga atggaatcat ctttctcatt tg 32 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 4 cggctctaga ttagccatca aactctgagc tgg 33 <210> 5 <211> 23 <212> DNA <213> Homo sapiens <400> 5 gggctgcttg atggcaaggt acc 23 <210> 6 <211> 23 <212> DNA <213> Homo sapiens <400> 6 ctgcggagga agtgacaaat gcc 23 <210> 7 <211> 23 <212> DNA <213> Homo sapiens <400> 7 ggagctttgc tctatcctgg acc 23 <210> 8 <211> 23 <212> DNA <213> Homo sapiens <400> 8 acctctacct agtgctggaa cgg 23 <210> 9 <211> 390 <212> DNA <213> Homo sapiens <400> 9 ctgaggactg aaaagagagg gtgagtaatt cttcatgacc tgtaggatcc caaagatggc 60 gacctgccag cctggactgc cagcgaaggc cagaatcgtg ctgtagctct gaacccacag 120 ctcctctgcc cctggcccat gagaatttca gctggagaga tagcatgccc tggtaagtga 180 agtcctgcca cttcgagaca tggaatcatc tttctcattt ggagtgatcc ttgctgtcct 240 ggcctccctc atcattgcta ctaacacact agtggctgtg gctgtgctgc tgttgatcca 300 caagaatgat ggtgtcagtc tctgcttcac cttgaatctg gctgtggctg acaccttgat 360 tggtgtggcc atctctggcc tactcacaga 390 <210> 10 <211> 223 <212> DNA <213> Homo sapiens <400> 10 ctgtggctgc tcggcgtggg caactccctg ctcaacccac tcatctatgc ctattggcag 60 aaggaggtgc gactgcagct ctaccacatg gccctaggag tgaagaaggt gctcacctca 120 ttcctcctct ttctctcggc caggaattgt ggcccagaga ggcccaggga aagttcctgt 180 cacatcgtca ctatctccag ctcagagttt gatggctaag acg 223 <210> 11 <211> 21 <212> DNA <213> Homo sapiens <400> 11 gttgatccac aagaatgatg g 21 <210> 12 <211> 21 <212> DNA <213> Homo sapiens <400> 12 gaggcaatct tgagcatgtc g 21 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 13 aaaatctaga atggagtcat ctttctcatt tgg 33 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 14 aaaatctaga ctagccatcg agctccggc 29 <210> 15 <211> 1008 <212> DNA <213> Rattus sp. <220> <221> CDS <222> (1) .. (1008) <400> 15 atg gag tca tct ttc tca ttt gga gtg atc ctt gct gtc ctg acc atc 48 Met Glu Ser Ser Phe Ser Phe Gly Val Ile Leu Ala Val Leu Thr Ile   1 5 10 15 ctt atc att gct gtt aat gcg ctg gtg gtt gtg gct atg ctg cta tca 96 Leu Ile Ile Ala Val Asn Ala Leu Val Val Val Ala Met Leu Leu Ser              20 25 30 atc tac aag aat gat ggt gtt ggc ctt tgc ttc acc tta aat ctg gcc 144 Ile Tyr Lys Asn Asp Gly Val Gly Leu Cys Phe Thr Leu Asn Leu Ala          35 40 45 gtg gct gat acc ttg att ggc gtg gct att tct ggg cta gtt aca gac 192 Val Ala Asp Thr Leu Ile Gly Val Ala Ile Ser Gly Leu Val Thr Asp      50 55 60 cag ctc tcc agc tct gct cag cac aca cag aag acc ttg tgt agc ctt 240 Gln Leu Ser Ser Ser Ala Gln His Thr Gln Lys Thr Leu Cys Ser Leu  65 70 75 80 cgg atg gca ttc gtc act tct tct gca gcc gcc tct gtc ctc acg gtc 288 Arg Met Ala Phe Val Thr Ser Ser Ala Ala Ala Ser Val Leu Thr Val                  85 90 95 atg ctg att gcc ttt gac agg tac ctg gcc att aag cag ccc ctc cgt 336 Met Leu Ile Ala Phe Asp Arg Tyr Leu Ala Ile Lys Gln Pro Leu Arg             100 105 110 tac ttc cag atc atg aat ggg ctt gta gcc gga gga tgc att gca ggg 384 Tyr Phe Gln Ile Met Asn Gly Leu Val Ala Gly Gly Cys Ile Ala Gly         115 120 125 ctg tgg ttg ata tct tac ctt atc ggc ttc ctc cca ctt gga gtc tcc 432 Leu Trp Leu Ile Ser Tyr Leu Ile Gly Phe Leu Pro Leu Gly Val Ser     130 135 140 ata ttc cag cag acc acc tac cat ggg ccc tgc acc ttc ttt gct gtg 480 Ile Phe Gln Gln Thr Thr Tyr His Gly Pro Cys Thr Phe Phe Ala Val 145 150 155 160 ttt cac cca agg ttt gtg ctg acc ctc tcc tgt gct ggc ttc ttc cca 528 Phe His Pro Arg Phe Val Leu Thr Leu Ser Cys Ala Gly Phe Phe Pro                 165 170 175 gct gtg ctc ctc ttt gtc ttc ttc tac tgt gac atg ctc aag att gcc 576 Ala Val Leu Leu Phe Val Phe Phe Tyr Cys Asp Met Leu Lys Ile Ala             180 185 190 tct gtg cac agc cag cac atc cgg aag atg gaa cat gca gga gcc atg 624 Ser Val His Ser Gln His Ile Arg Lys Met Glu His Ala Gly Ala Met         195 200 205 gtt gga gct tgc cgg ccc cca cgg cct gtc aat gac ttc aag gct gtc 672 Val Gly Ala Cys Arg Pro Pro Arg Pro Val Asn Asp Phe Lys Ala Val     210 215 220 cgg act gta tct gtc ctt att ggg agc ttc acc ctg tcc tgg tct ccg 720 Arg Thr Val Ser Val Leu Ile Gly Ser Phe Thr Leu Ser Trp Ser Pro 225 230 235 240 ttt ctc atc act agc att gtg cag gtg gcc tgc cac aaa tgc tgc ctc 768 Phe Leu Ile Thr Ser Ile Val Gln Val Ala Cys His Lys Cys Cys Leu                 245 250 255 tac caa gtg ctg gaa aaa tac ctc tgg ctc ctt gga gtt ggc aac tcc 816 Tyr Gln Val Leu Glu Lys Tyr Leu Trp Leu Leu Gly Val Gly Asn Ser             260 265 270 ctg ctc aac cca ctc atc tat gcc tat tgg cag agg gag gtt cgg cag 864 Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp Gln Arg Glu Val Arg Gln         275 280 285 cag ctc tgc cac atg gcc ctg ggg gtg aag aag ttc ttt act tca atc 912 Gln Leu Cys His Met Ala Leu Gly Val Lys Lys Phe Phe Thr Ser Ile     290 295 300 ttc ctc ctt ctc tcg gcc agg aat cgt ggt cca cag agg acc cga gaa 960 Phe Leu Leu Leu Ser Ala Arg Asn Arg Gly Pro Gln Arg Thr Arg Glu 305 310 315 320 agc tcc tat cac atc gtc act atc agc cag ccg gag ctc gat ggc tag 1008 Ser Ser Tyr His Ile Val Thr Ile Ser Gln Pro Glu Leu Asp Gly                 325 330 335 <210> 16 <211> 335 <212> PRT <213> Rattus sp. <400> 16 Met Glu Ser Ser Phe Ser Phe Gly Val Ile Leu Ala Val Leu Thr Ile   1 5 10 15 Leu Ile Ile Ala Val Asn Ala Leu Val Val Val Ala Met Leu Leu Ser              20 25 30 Ile Tyr Lys Asn Asp Gly Val Gly Leu Cys Phe Thr Leu Asn Leu Ala          35 40 45 Val Ala Asp Thr Leu Ile Gly Val Ala Ile Ser Gly Leu Val Thr Asp      50 55 60 Gln Leu Ser Ser Ser Ala Gln His Thr Gln Lys Thr Leu Cys Ser Leu  65 70 75 80 Arg Met Ala Phe Val Thr Ser Ser Ala Ala Ala Ser Val Leu Thr Val                  85 90 95 Met Leu Ile Ala Phe Asp Arg Tyr Leu Ala Ile Lys Gln Pro Leu Arg             100 105 110 Tyr Phe Gln Ile Met Asn Gly Leu Val Ala Gly Gly Cys Ile Ala Gly         115 120 125 Leu Trp Leu Ile Ser Tyr Leu Ile Gly Phe Leu Pro Leu Gly Val Ser     130 135 140 Ile Phe Gln Gln Thr Thr Tyr His Gly Pro Cys Thr Phe Phe Ala Val 145 150 155 160 Phe His Pro Arg Phe Val Leu Thr Leu Ser Cys Ala Gly Phe Phe Pro                 165 170 175 Ala Val Leu Leu Phe Val Phe Phe Tyr Cys Asp Met Leu Lys Ile Ala             180 185 190 Ser Val His Ser Gln His Ile Arg Lys Met Glu His Ala Gly Ala Met         195 200 205 Val Gly Ala Cys Arg Pro Pro Arg Pro Val Asn Asp Phe Lys Ala Val     210 215 220 Arg Thr Val Ser Val Leu Ile Gly Ser Phe Thr Leu Ser Trp Ser Pro 225 230 235 240 Phe Leu Ile Thr Ser Ile Val Gln Val Ala Cys His Lys Cys Cys Leu                 245 250 255 Tyr Gln Val Leu Glu Lys Tyr Leu Trp Leu Leu Gly Val Gly Asn Ser             260 265 270 Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp Gln Arg Glu Val Arg Gln         275 280 285 Gln Leu Cys His Met Ala Leu Gly Val Lys Lys Phe Phe Thr Ser Ile     290 295 300 Phe Leu Leu Leu Ser Ala Arg Asn Arg Gly Pro Gln Arg Thr Arg Glu 305 310 315 320 Ser Ser Tyr His Ile Val Thr Ile Ser Gln Pro Glu Leu Asp Gly                 325 330 335 <210> 17 <211> 20 <212> DNA <213> Rattus sp. <400> 17 gtggctgata ccttgattgg 20 <210> 18 <211> 20 <212> DNA <213> Rattus sp. <400> 18 gcacagctgg gaagaagcca 20 <210> 19 <211> 20 <212> DNA <213> Rattus sp. <400> 19 gattggcgtg gctatttctg 20 <210> 20 <211> 20 <212> DNA <213> Rattus sp. <400> 20 ggaagaagcc agcacaggag 20 <210> 21 <211> 24 <212> DNA <213> Rattus sp. <400> 21 taagatatca accacagccc tgca 24 <210> 22 <211> 24 <212> DNA <213> Rattus sp. <400> 22 ctacaagccc attcatgatc tgga 24 <210> 23 <211> 21 <212> DNA <213> Rattus sp. <400> 23 ccgcctctgt cctcacggtc a 21 <210> 24 <211> 23 <212> DNA <213> Rattus sp. <400> 24 acaggtacct ggccattaag cag 23 <210> 25 <211> 22 <212> DNA <213> Rattus sp. <400> 25 tagagcacat ctaatcctgt cc 22 <210> 26 <211> 25 <212> DNA <213> Rattus sp. <400> 26 ttagagatga aagtcaggat ccagc 25

[Brief description of drawings]

FIG. 1 is a graph showing a time course of plasma insulin concentration after oral glucose loading in SD rats intraperitoneally administered with 2- (pyridin-4-yl) ethyl thiobenzoate (LT-1 Z 0059519).

FIG. 2 is a graph showing the time course of blood glucose level after oral glucose loading in SD rats intraperitoneally administered with 2- (pyridin-4-yl) ethyl thiobenzoate (LT-1 Z 0059519).

FIG. 3 is a graph showing a time course of blood glucose level after oral glucose loading in GK rats orally administered with 2- (pyridin-4-yl) ethyl thiobenzoate (LT-1 Z 0059519).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/50 A61K 31/4436 // A61K 31/4436 C07K 14/705 C07K 14/705 A61K 37/02 ZNA (72) Inventor Mitsuyuki Matsumoto 21 Yamanouchi Pharmaceutical Co., Ltd., Miyukigaoka Tsukuba, Ibaraki Prefecture (72) Inventor Satoshi Saito 21 Miyukigaoka Tsukuba, Ibaraki Yamanouchi Pharmaceutical Co., Ltd. (72) Inventor Masazumi Kamabahara 21 Miyukigaoka, Tsukuba-shi, Ibaraki Yamanouchi Pharmaceutical Co., Ltd. (72) Inventor Takatoshi Soga Miyukigaoka, Tsukuba-shi, Ibaraki 21 Inventor Yamanouchi Pharmaceutical Co., Ltd. (72) Shigeru Yoshida Miyukigaoka, Tsukuba-shi, Ibaraki 21 Yamanouchi Pharmaceutical Co., Ltd. (72) Inventor Yoshitaka Ueda Miyukigaoka, Tsukuba, Ibaraki 21 Yamanouchi Pharmaceutical Co., Ltd. F Term (reference) 2G045 AA34 AA35 BB01 BB4 1 BB50 CB01 DA13 DA36 FB01 FB02 FB03 4C084 AA01 AA17 BA01 BA02 BA08 BA22 MA35 MA37 MA41 MA43 MA52 MA55 MA56 MA63 MA66 NA14 ZC032 ZC352 4C086 AA01 AA02 AA03 BC17 GA15 MA56 MA56 MA56 MA14 MA56 MA14 MA14 MA41 MA41 MA43 MA41 MA41 MA43 MA41 MA43 CA40 DA50 EA20 FA74

Claims (7)

[Claims] 1. A polypeptide having (1) the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16, and (2) the amino acid sequence represented by SEQ ID NO: 2 or represented by SEQ ID NO: 16. 1 in the amino acid sequence
It has an amino acid sequence in which 10 to 10 amino acids are deleted, substituted, and / or added, and (a) is activated under high glucose concentration to promote insulin secretion from pancreatic β cells. Activity, and / or (b) a polypeptide having an activity of increasing the intracellular cAMP amount by being activated, (3) the amino acid sequence represented by SEQ ID NO: 2 or the amino acid represented by SEQ ID NO: 16 Intracellular cAMP containing a sequence and (a) being activated under high glucose concentration to promote insulin secretion from pancreatic β-cells and / or (b) being activated. A polypeptide showing an activity of increasing the amount, (4) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16. And (a) is activated under high glucose concentration to promote insulin secretion from pancreatic β-cells, and / or (b) is activated to produce intracellular cAMP. Containing a polypeptide showing an activity of increasing the amount, or (5) an activator of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 16 A pharmaceutical composition for treating diabetes. 2. (1) Contacting a test compound with a cell transformed with an expression vector containing a polynucleotide encoding the polypeptide according to claim 1 and expressing the polypeptide, or a cell membrane thereof. A method for screening a therapeutic agent for diabetes mellitus, comprising the steps of: and a step of analyzing whether or not the polypeptide of claim 1 is activated, or (2) the cell, or a cell membrane thereof, or The method of contacting the polypeptide according to 1 with a test compound in the presence of the labeled agonist of the polypeptide according to claim 1, and the binding amount of the labeled agonist to the cell or its cell membrane or the polypeptide. Pharmaceutical composition for treating diabetes, comprising a substance obtained by a method for screening a therapeutic agent for diabetes, which comprises a step of analyzing changes . 3. The pharmaceutical composition for treating diabetes according to claim 1, which is a pharmaceutical composition for promoting insulin secretion. 4. A step of contacting the cell according to claim 2 or a cell membrane thereof with a test compound, a step of analyzing whether or not the polypeptide according to claim 1 is activated, and an analysis. A method for producing a pharmaceutical composition for treating diabetes, which comprises the step of formulating a substance. 5. A step of contacting the cell according to claim 2 or a cell membrane thereof with a test compound in the presence of the labeling agonist of the polypeptide according to claim 1, labeling the cell or a cell membrane thereof. Comprising the step of analyzing the change in the amount of binding of the agonist, and the step of formulating the analyzed substance,
A method for producing a pharmaceutical composition for treating diabetes. 6. The production method according to claim 4, wherein the pharmaceutical composition for treating diabetes is a pharmaceutical composition for promoting insulin secretion. 7. Use of the activator of the polypeptide according to claim 1 for producing a pharmaceutical composition for treating diabetes and / or a pharmaceutical composition for promoting insulin secretion.