KR100560122B1 - The screening method and the screening kit for inhibiting or enhancing agent of signal transduction in G-protein coupled receptorGPCR - Google Patents

Abstract

The present invention relates to a search method and a search kit for signaling inhibitors or promoters of G-protein coupled receptors (hereinafter referred to as GPCRs), and promote specific binding between G-proteins and GPCRs. The present invention is a screening method for screening a substance to be inhibited by surface plasmon resonance (SPR) or an immunoassay and an invention thereof. Specific application examples of the present invention include a fusion protein comprising an intracellular loop 3 (iL3), a third loop in a cell of serotonin receptor 6, a type of GPCR, and an α subunit (Gα _s ) of a Gs protein interacting with the receptor. And a method for searching for a signaling inhibitor or promoter of GPCR through measurement of the interaction between the fusion protein and the two proteins, and a search kit using the same. The search method and search kit of the present invention can be useful for the search for new signaling inhibitors or promoters acting on GPCR. In particular, the drug candidates for antipsychotics or antidepressants targeting serotonin receptors can be easily and at low cost. It can be used to search quickly.

GPCR, Serotonin Receptor 6, Search Method, Search Kit

Description

The screening method and the screening kit for inhibiting or enhancing agent of signal transduction in G-protein coupled receptor (GPCR)}             

Figure 1a is a schematic diagram showing the interaction between the intracellular loop 3 (iL3) and the α subunit (Gα _s ) of the Gs protein complex, a loop connecting the fifth and sixth transmembrane sites of serotonin receptor 6,

1B is a schematic diagram showing the structure of GST (Glutathione-S-transferase) -iL3, which is a fusion protein containing the iL3 region of serotonin receptor 6,

Figure 2a is a schematic diagram showing the primary structure of the GST-iL3 protein consisting of the GST site and the iL3 site of serotonin receptor 6,

FIG. 2B is a schematic diagram showing the primary structure of His-Gα _s protein in which six repetitive histidine amino acids are linked to the N-terminus of the α subunit (Gα _s ) of the Gs protein. FIG.

3A is a cleavage map of pGST-ApLZ-iL3,

3B is a cleavage map of pGST-iL3, which is an expression vector of GST-iL3 protein,

3C is a cleavage map of pH-Gα _{s that} is an expression vector of His-Gα _s protein,

4 is a diagram illustrating a process for measuring the interaction between GST-iL3 protein and His-Gα _s protein by enzyme immunoassay,

5 is a graph showing the binding specificity of GST-iL3 and His-Gα _s by enzyme immunoassay.

Figure 6 is a graph showing the binding specificity of GST-iL3 and His-Gα _s by surface plasmon resonance method, GST-iL3 in the concentration of 15, 10, 5, 2.5, 1 μM immobilized His-Gα _s The sensorgram obtained when processed was displayed sequentially from the top of the graph.

The present invention relates to a method and a search kit for detecting a signaling inhibitor or promoter of a G-protein coupled receptor (GPCR), and more specifically, a third in a cell of serotonin receptor 6, a type of GPCR. Fusion protein comprising an intracellular loop 3 (iL3) loop, a fusion protein comprising an α subunit (Gα _s ) of Gs protein interacting with the receptor, and a signal from the GPCR through measurement of the interaction between the two proteins A method of searching for a delivery inhibitor or promoter and a search kit using the same.

GPCR is a receptor that is involved in various signaling in vivo, and is particularly present in the neurotransmitter, and is a receptor that contains neurotransmitters such as adrenergic, dopamine or serotonin as ligands. It is estimated that about 2,000 GPCRs exist in humans, and they bind to various ligands to recognize signals outside the cell and interact with G-proteins in the cell to transmit signals into the cell. In addition, GPCRs have seven transmembrane helixes (7TM) and have common characteristics of transmitting signals via G-proteins. In most cases, ligands in GPCRs bind within the cell membrane permeation helix and some ligands bind to extracellular loops outside the cell membrane. The binding of the ligand induces the signaling inside the cell by inducing the binding of the intracellular loop of the GPCR to the G-protein, in particular the alpha subunit (Gα).

A type of GPCR, a serotonin receptor known to interact with the stimulatory G-protein (Gs), a type of G-protein, is a serotonin ligand and regulates sleep, eating, pain awareness, temperature and blood pressure, depression, anxiety, and mental Involved in central nervous system diseases related to schizophrenia and other bodies. In addition, serotonin and serotonin receptors have been shown to modulate various contraction, secretion, and electrophysiological effects in peripheral nervous systems such as the gastrointestinal system. Due to this broad role, drugs that affect the neurotransmitter system in which serotonin receptors are involved in recent years are of great interest. It becomes the target of. In particular, receptor specific agents and antagonists can be usefully used in the treatment of a wide range of disorders including anxiety, depression, hypertension, migraine, obesity, obsessive compulsive disorder, schizophrenic emesis.

Serotonin receptors formally include 14-18 subspecies receptors. Dual serotonin receptor 6 was identified in 1993 (Monsma et al ., Mol. Pharmacol. 43, 320-327, 1993) and reported to be associated with stress and anxiety conditions (Yoshioka et al ., Life Science 17/18, 1473-1477, 1998). The human serotonin receptor 6 consists of 440 amino acid sequences and has a molecular weight of about 47 kDa, which consists of seven transmembrane domains, four extracellular loops (eLs), and four cells. It consists of an intracellular loop (iL). When the ligand, serotonin, binds to a binding site located at the transmembrane site of the serotonin receptor, the structural changes of the serotonin receptors are transferred to loops (iLs) located inside the cell membrane, and these structural changes are recognized by the G-protein and thus G-protein. Combined with. The intracellular loop 3 (iL3) region of the serotonin receptor is expected to play an important role in the interaction with G-protein. There are two kinds of Gs proteins known as G-proteins that interact with serotonin receptor 6 to transmit a signal, and have a molecular weight of 45.7 kDa and 44.3 kDa. The latter protein is composed of 380 amino acid sequences. The Gs protein is composed of three subunits of α, β, and γ, and it is known that the α subunit (Gα _s ) participates in the interaction with serotonin receptor 6 to increase the cAMP concentration in cells (Kohen et. al ., J. Neurochem. 66, 47-56, 1996; Ruat et al., Biochem. Biophys.Res.Commun . 193, 268-276, 1993).

Serotonin receptor 6 is expressed in the brain, especially olfactory tubercle, cerebral cortex, striatum, nucleus accumbens and hippocampus, and weakly in the thalamus. Expressed. Little is known about the functions related to serotonin receptor 6, but representative antidepressants such as clomipramine, amitriptyline, doxepin and nortryptyline and clozapine The attention of the pharmaceutical industry is increasing, as it is known that antipsychotics such as olanzapine, fluperlapine and seroquel bind strongly to these receptors. It is estimated to be very large.

Conventional methods of screening for new drug candidates through GPCRs mainly aim to search for substances that bind to GPCR ligand binding sites and modulate the signaling activity of GPCRs. When there is a need for subtypes of GPCRs having the same type of ligand as serotonin receptors, there is a problem that signaling regulatory substances targeting ligand binding sites may have low selectivity for each subtype GPCR.

Accordingly, the present inventors have studied to find a method for easily and quickly searching for candidate substances that can modulate (inhibit or promote) the signaling activity of GPCR. As a result, the intracellular loop of GPCR, which plays an important role in GPCR signal transmission, has been studied. We have devised a method for measuring in vitro the interaction between intracellular loop sites and α-subunits of G-proteins. In other words, we devised a fusion protein containing iL3 and Gα _s of serotonin receptor 6, which is a type of GPCR, and a method for measuring the interaction between them. By this, the present invention was completed.

An object of the present invention is to devise a fusion protein comprising an intracellular loop domain of GPCR and a fusion protein comprising an α-subunit (Gα) of G-protein interacting therewith and a method for measuring the interaction between these proteins. By providing a method for in vitro detection of signaling inhibitors or promoters targeting GPCR.

In order to achieve the above object, the present invention provides a fusion protein comprising a intracellular loop site of GPCR and a fusion protein including Gα and a signaling inhibitor or GP of the GPCR through the measurement of the interaction between the fusion protein Provides a way to search for authenticity.

The present invention also provides a search kit for a GPCR signaling inhibitor or promoter comprising a fusion protein comprising Gα and a substrate solution containing a fusion protein, a label antibody conjugate, and a coloring agent comprising an intracellular loop portion of GPCR. .

Hereinafter, the present invention will be described in detail.

The present invention provides a method for searching for a signaling inhibitor or promoter for GPCR using a fusion protein comprising an intracellular loop site of GPCR, a fusion protein including Gα, and measurement of the interaction between the fusion proteins.

GPCRs are structural specifics with seven transmembrane helixes (7TM) as receptors present in the cell membrane, and it is estimated that about 2,000 GPCRs exist in humans. These GPCRs bind to a variety of ligands to recognize signals outside the cell and interact with the G-proteins in the cell to deliver signals intracellularly. In particular, GPCRs have common properties through seven cell membrane helix sites and via G-proteins in signaling. In most cases, ligands in GPCRs bind within the cell membrane penetrating helix and some ligands bind to loops outside the cell membrane. The binding of these ligands induces the binding between the loops in the cell membrane of the GPCR and the G-protein, in particular the alpha monomer (Gα), thereby allowing the signal to be transferred into the cell.

GPCRs have seven transmembrane helixes (7TM) and have the common characteristics of transmitting signals via G-proteins. Thus, in a preferred embodiment of the present invention, serotonin receptor 6 is a type of GPCR. A fusion protein comprising an intracellular loop 3 (iL3) and a fusion protein comprising Gα _s (stimulatory Gα protein), which is a kind of Gα, were prepared.

The fusion protein comprising iL3 of serotonin receptor 6 is composed of a site for facilitating purification of the protein and iL3, and preferably may be composed of GST (Glutathione-S-transferase) and iL3. The fusion protein has an amino acid sequence of SEQ ID NO: 12 encoded by the nucleotide sequence described in SEQ ID NO: 11 , wherein the nucleotide sequence encoding the amino acid sequence of iL3 uses a base sequence consisting of codons used at high frequencies in E. coli It is desirable to.

In addition, the fusion protein comprising the α subunit (Gα _s ) of the Gs protein is composed of a site and Gα _s to facilitate purification, and preferably 6 consecutive histidine amino acid residues at the N-terminus of Gα _s It can be configured as attached. The fusion protein has the amino acid sequence of SEQ ID NO: 14 encoded by the nucleotide sequence described in SEQ ID NO: 13.

Expression vector comprising the gene of the fusion protein to express the fusion protein comprising the intracellular loop 3 (iL3) of the serotonin receptor 6 and the fusion protein comprising the α subunit (Gα _s ) of the Gs protein in E. coli pGST-iL3 and pH-Gα _s were prepared. The degree of interaction between the two fusion proteins prepared as described above was measured by the following enzyme immunoassay method and surface plasmon resonance method. As a result, the degree of dissociation constant (KD) of 600 nM was shown to detect the inhibitor or promoter of GPCR. It was found that it can be used in the method.

Method for screening the inhibitor or promoter of GPCR of the present invention using enzyme immunoassay comprises the steps of: i) adsorbing a fusion protein comprising Gα to a vessel; Ii) treating the GPCR signaling inhibitor or promoter candidate; Iii) adding a fusion protein comprising an intracellular loop of GPCR; Iii) binding the labeling antibody conjugate to a fusion protein comprising the intracellular loop of GPCR in step iii); Iii) color development using bound label antibody conjugates; And iii) comparing the degree of color development.

In addition, a method of searching for a signaling inhibitor or promoter of a serotonin receptor using a surface plasmon resonance method may include: i) adsorbing a fusion protein containing Gα to a sensor chip; Ii) adding a fusion protein comprising a GPCR signaling inhibitor or promoter candidate to the fusion protein comprising adsorbed Gα and an intracellular loop of GPCR; Iii) comparing the combined degree.

In addition, the present invention provides a fusion protein comprising iii) a fusion protein comprising Gα and an intracellular loop site of GPCR; Ii) a search kit for signaling inhibitors or promoters of GPCRs comprising a label antibody conjugate and iii) a substrate solution containing a chromophore, preferably iii) a fusion protein comprising Gα _s , a type of Gα, and A fusion protein comprising an intracellular loop 3 region of serotonin receptor 6, a type of GPCR; Ii) labeling antibody conjugates; Iii) Provides a kit for screening inhibitors or promoters of GPCRs comprising a substrate solution containing a colorant.

In the search kit, the conjugate of the labeled antibody may be an antibody labeled with a horse reddish peroxydase (HRP), an alkaline phosphatase, a fluorescein or a dye, and the like. Anti GST Ab-HRP conjugates can be used.

As described above, a method and a search kit of a GPCR signaling inhibitor or promoter provided in the present invention are useful for searching for a novel substance that modulates signaling specifically made between GPCR and G-protein, such as serotonin receptor 6. Can be used.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of GST-iL3 Protein

<1-1> Expression vector preparation of GST-iL3 gene

Since GPCR is a membrane protein, there are many problems to apply it as it is under laboratory conditions. Therefore, the inventors have devised a gene structure encoding the amino acid sequence of the iL3 region of human ( Homo sapiens ) serotonin receptor 6, a type of GPCR . 2a ) was expressed in E. coli. That is, a gene of SEQ ID NO: 11 having a GST gene sequence that facilitates expression and purification in Escherichia coli and an amino acid sequence identical to iL3 but consisting of codons that are frequently used in Escherichia coils is designed. From this, GST-iL3 protein was expressed.

First, using the approximately 100 nucleotides in SEQ ID NO: 1, 2, 3, and 4, a nucleotide oligonucleotide synthesis, and Pfu polymerase with the nucleotides in the oligonucleotide consisting of the template to obtain a gene sequence of iL3 1 min at 94 ℃; 1 minute at 55 ° C .; The polymerase chain reaction (PCR) was performed by repeating 10 times at 72 ° C. for 1 minute period. Subsequently, using a primer of SEQ ID NOs: 5 and 6 as a template using the DNA synthesized in the PCR and containing a restriction enzyme recognition sequence at 94 ° C. for 1 minute; 1 minute at 55 ° C .; The second PCR was carried out by repeating 25 times at 72 DEG C for 1 minute. The DNA amplification where the sequence number by inserting by using T4 DNA ligase (ligase) enzyme to the BamH I and Xho I restriction enzymes, one then the pGEX4T-1 plasmid DNA (Amersham Biosciences, Sweden) digested with the same restriction enzyme digestion with A pGST-ApLZ-iL3 vector containing 15 GST-ApLZ-iL3 genes was prepared ( FIG. 3A ). 1 minute at 94 ° C. using SEQ ID NOs: 7 and 8 primers as pGST-ApLZ-iL3 template to make expression vectors producing GST-iL3 protein; 1 minute at 55 ° C .; PCR was repeated 25 times at 1 min intervals at 72 ° C. The amplified DNA was digested with BamH I and Xho I restriction enzymes, and then inserted into pGEX4T-1 plasmid DNA (Amersham Biosciences, Sweden) using T4 DNA ligase enzymes digested with the same restriction enzymes. PGST-iL3, a protein-producing expression vector, was prepared ( FIG. 3B ). DNA sequence analysis confirmed that the synthesized DNA sequence and the DNA was properly inserted into the expression vector.

<1-2> Expression and Purification of GST-iL3 Protein

For expression and purification of GST-iL3 protein, first, the expression vector pGST-iL3 prepared in Example <1-1> was introduced into E. coli DH5α (Invitrogen, USA) to prepare a transformant, and then the transformant. Was cultured at 37 ° C. using an LB culture medium containing 100 mg / ml of antibiotics ampicillin (Ampicilline). When the absorbance at 600 nm reached 0.7, Isopropyl-β-D-Thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM to induce the expression of the protein and further incubated at 18 ° C. for 16 hours, E. coli transformants were harvested by centrifugation. The collected E. coli transformants were suspended in a buffer solution (50 mM Na phosphate, pH 6.0) and passed through a French press at a pressure of 12,000 psi to break the cell wall and centrifuged (10,000 × g, 30 minutes). Insoluble materials such as cell walls were removed. The expressed protein was passed through a GST adsorption column, extracted with a buffer containing 10 mM reduced glutatione, and the extracted fraction was purified once again using gel filtration chromatography. GST-iL3 protein expressed and purified by the above method has a purity of about 90%.

Example 2 His-Gα _s  Preparation of Protein

<2-1> His-Gα _s Preparation of Gene Expression Vector

For easy purification of the Gα _s protein, a gene of SEQ ID NO: 13 with six consecutive histidine residues attached to the N-terminus was designed and an expression vector comprising the same was prepared. Specifically, 1 minute at 94 ° C using primers of SEQ ID NOs: 9 and 10 as templates for cDNA derived from human brain tissue; 1 minute at 55 ° C .; PCR was performed 25 times at a cycle of 1 minute at 72 ° C. The amplified DNA is thereby inserted using T4 DNA ligase enzyme to Nde I and Sac I Two kinds of DNA restriction enzymes, one then the pET28a plasmid DNA (Novagen, USA) digested with the same restriction enzyme digestion to, His-Gα _s PH-Gα _s , an expression vector that produces proteins, was prepared ( FIG. 3C ). Analysis of the DNA sequence confirmed that the synthesized DNA sequence and the DNA were properly inserted into the expression vector.

<2-2> His-Gα _s  Expression and Purification of Proteins

For expression and purification of His-Gα _s protein, the expression vector pH-Gα _s was introduced into Escherichia coli BL21 (DE3) RP codon plus (Stratagen, USA) to prepare transformants and the transformants were kanamycine. The culture was shaken at 37 ° C. in an LB culture medium containing 100 μg / ml antibiotic. When the absorbance of the culture reached 0.4 at 600 nm, IPTG was added to a final concentration of 30 μM to induce the expression of the protein, and after further incubation at 18 ° C. for 14 hours, E. coli transformants were collected by centrifugation. The collected E. coli transformants were suspended in a buffer solution (50 mM Tris, pH 8.0) and passed through a French Press at a pressure of 12,000 psi to break the cell wall and centrifuged (10,000 × g, 30 minutes). Insoluble substances such as cell walls were removed. The expressed protein was passed through a Ni-chelating column, and then the adsorbed protein was extracted with a buffer containing 200 mM of imidazole. The extracted protein was once again purified using anion exchange column. His-Gα _s protein expressed and purified by the above method has a purity of about 95%.

Example 3 Development of Search Method Using Enzyme Immunoassay

<3-1> GST-iL3 Protein and His-Gα _s Enzyme Immunoassay Using Protein

0.1 ml of 5 μg / ml of His-Gα _s protein (20 mM Na phosphate, 150 mM Na chloride, pH 7.4) was added to a 96 well cell culture medium with medium binding capacity, followed by a plastic surface at 4 ° C. for 14 hours. It was made to adsorb | suck to. After washing four times with 200 μl of wash buffer (20 mM Na phosphate, 150 mM Na chloride, 0.5% Triton X-100, 0.001% antifoam 289 (Sigma, USA), pH 7.4) to remove the aqueous protein solution, Buffer solution containing 0.3 ml of 5% skim milk powder (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) was allowed to stand at room temperature for 1 hour, washed again 6 times with washing buffer and then buffered ( 100 μl of GST-iL3 protein diluted to 100 nM in sodium phosphate buffer pH 7.4) was added and reacted at room temperature for 1 hour. After removing the aqueous protein solution, the solution was washed 6 times with a washing buffer, and 100 μl of HRP-binding antibody (HRP labeled Anti-GST from Goat, 1 / 20,000 dilution) was added for 1 hour at room temperature. I was. After removing the aqueous solution of the antibody and washing it six times with a washing buffer, 100 μl of an OPD coloring solution containing 1 mg / ml of OPD (o-phenylenediamine Dihydrochloride) was added to 10 × substrate buffer, followed by reaction for 3 to 5 minutes. 100 μl of 2.5 M sulfuric acid (H ₂ SO ₄ ) was added thereto to stop the reaction, and the absorbance of the aqueous solution was measured at 490 nm. As a result, it was confirmed that both proteins bind with affinity and their binding degree was 600 nM dissociation constant (KD).

<3-2> GST-iL3 Protein and His-Gα by Enzyme Immunoassay _s Measure Interaction Between Proteins

In order to confirm whether the enzyme immunoassay method of Example <3-1> can selectively measure the binding of the His-Gα _s protein to the iL3 loop domain of the GST-iL3 protein, the adsorbed His-Gα _s protein When the protein was not treated with any of the proteins, the GST protein was treated, and the GST-iL3 protein was treated with the anti-GST antibody-HRP conjugate, respectively. As a result, as shown in FIG. 5 , when the GST-iL3 protein was treated, the color reaction occurred strongly, and when only the GST protein was present, the color was developed very weakly. These results show that the color reaction of the enzyme immunoassay is due to the interaction between the iL3 domain of serotonin receptor 6 and His-Gα _s protein. Subtracting the binding force of the GST protein from the total binding strength of the GST-iL3 protein can determine the specific binding force between the iL3 domain and His-Gα _s protein. Therefore, it is possible to search for new drug candidates that can promote or inhibit signaling of serotonin receptors using the enzyme immunoassay of the present invention.

Example 4 Development of Search Method Using Surface Plasmon Resonance Measurement

<4-1> GST-iL3 Protein and His-Gα _s Surface Plasmon Resonance Measurement Using Protein

A BIAcore 3000 analyzer (BIACORE, Uppsala, Sweden) was used to measure the mutual binding between GST-iL3 and His-Gα _s by surface plasmon resonance. Biosensor analysis of the EDC / NHS (N '- ( 3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysuccinimide) a CM5 sensor chip activated (BIACORE, Uppsala, Sweden) His -Gα s protein at a flow rate of 5 ㎕ / min to ( 5 μg / ml in 20 mM Na phosphate, 150 mM Na chloride, pH 7.4) was fixed for 7 minutes by flowing. The active part remaining on the surface of the sensor chip was deactivated by adding 1.0 M ethanolamine. GST-iL3 was diluted in buffer solution for each concentration (10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ , 2.7 mM KCl, 140 mM NaCl pH 7.4), and flowed at a flow rate of 30 μl / min. Gram was confirmed and dissociation sensorgram was confirmed by flowing buffer (10 mM Na ₂ HPO ₄ , 1.8 mM K ₂ HPO ₄ , 2.7 mM KCl, 140 mM NaCl pH 7.4). In order to correct the sensorgram generated by nonspecific binding with the sensor chip, a cell completely deactivated with ethanolamine was used and the nonspecific binding was corrected by subtracting the sensorgram of ethanolamine from the sensorgram of GST-iL3. The surface of the sensor chip was regenerated by flowing regeneration buffer (80 mM NaCl, 20 mM NaOH).

<4-2> GST-iL3 Protein and His-Gα by Surface Plasmon Resonance Assay _s Protein Interaction Measurement

In order to confirm whether the surface plasmon resonance method of Example <4-1> can selectively measure the binding between the iL3 loop domain of the GST-iL3 protein and the His-Gα _s protein, it is immobilized on a CM5 sensor chip. GST-iL3 protein was flowed into His-Gα _s protein at concentrations of 15, 10, 5, 2.5, and 1 μM, and binding sensorgrams were examined. As a result, as shown in FIG. 6 , the GST-iL3 protein shows a sensorgram that specifically binds to His-Gα _s protein, and the increase in RU value according to the amount of GST-iL3 bound is increased in the concentration of GST-iL3. It can be seen that the proportion increases. These results show that the increase curve of the binding sensorgram of the surface plasmon resonance measurement is due to the interaction between the iL3 domain of serotonin receptor 6 and His-Gα _s protein. Therefore, it is possible to search for drug candidates that can promote or inhibit signaling of serotonin receptors using the surface plasmon resonance method of the present invention.

The method of screening a GPCR signaling inhibitor or promoter using a fusion protein comprising an intracellular loop and a Gα protein of the GPCR of the present invention has the following characteristics. First, the conventional screening method is mainly to search for a substance that binds to the ligand binding site of GPCR to regulate the signaling activity of GPCR, but the screening method of the present invention is a substance that binds to the interaction site between GPCR and G-protein. Aim for search. Secondly, since the interaction site between GPCR and G-protein is expressed in large quantities as a water-soluble protein to measure the interaction in vitro, a large-scale search can be performed at low cost since no expensive cell culture step is required. Third, in the presence of subspecies GPCRs with the same type of ligand, such as serotonin receptors, modulators targeting the ligand binding site may have low selectivity for each subspecies GPCR. In the case of subspecies GPCRs, the sequence of loop sites in cells is highly different, so the interactions between these GPCRs and G-proteins are different. Therefore, the modulators detected for the interaction sites of GPCRs and G-proteins are separated from the subspecies GPCRs. Since there is an advantage of having a high selectivity, a method for screening a GPCR signaling inhibitor or promoter using a fusion protein comprising an intracellular loop and a Gα protein of the GPCR of the present invention can be used to search for drug candidates for GPCR. It can be usefully used.

<110> Korea Institute of Science and Technology <120> The screening method and the screening kit for regulating agent         of G protein coupled receptor <130> 4p-01-06 <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> oligo1 <400> 1 aaaaagaact gcaggctaac aaaaaagaac tggctcagct gaaatgggaa 60 tgaaaaaaga actgtgccgt atcctgctgg ctgctcgta 109 <210> 2 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> oligo2 <400> 2 gcagggtttc ggaagcctgg gaagccatac cggtggtcag ggaagcaacc 60 gtttacgagc agccagcagg atacggca 98 <210> 3 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> oligo3 <400> 3 cctgcaggtt ccgcgtaccc cgcgtccggg tgttgaatcc gctgactccc 60 taccaaacac tcccgtaaag ctctgaaa 98 <210> 4 <211> 115 <212> DNA <213> Artificial Sequence <220> <223> oligo4 <400> 4 tttttttcca gagcctggtt tttccattcc agctgagcca gttttttttc 60 agtttttttt ccagctggca tttcagagct ttacgggagt gtttg 115 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer1 <400> 5 cctacgctct gaaaaaagaa ctgcag 36 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer2 <400> 6 agctgagcca gttttttttc cagagc 36 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer3 <400> 7 cctgccgtat cctgctggct gctcg 35 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer4 <400> 8 agtttcagag ctttacggga gtgtttg 37 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer5 <400> 9 tgggctgcct cgggaacagt aagacc 36 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer6 <400> 10 tcttagagca gctcgtactg acgaag 36 <210> 11 <211> 873 <212> DNA <213> Artificial Sequence <220> <223> GST-iL3 DNA <400> 11 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660 gtggatcctg ccgtatcctg ctggctgctc gtaaacaggc tgttcaggtt 720 ccaccggtat ggcttcccag gcttccgaaa ccctgcaggt tccgcgtacc 780 gtgttgaatc cgctgactcc cgtcgtctgg ctaccaaaca ctcccgtaaa 840 tcgagcggcc gcatcgtgac tga 873 <210> 12 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> GST-iL3 protein <400> 12 Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro  1 5 10 15 Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu             20 25 30 Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu         35 40 45 Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys     50 55 60 Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu                 85 90 95 Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser            100 105 110 Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu        115 120 125 Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn    130 135 140 Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp                 150 155 160 Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu                165 170 175 Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr            180 185 190 Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala        195 200 205 Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg    210 215 220 Ser Cys Arg Ile Leu Leu Ala Ala Arg Lys Gln Ala Val Gln Val                 230 235 240 Ser Leu Thr Thr Gly Met Ala Ser Gln Ala Ser Glu Thr Leu Gln                245 250 255 Pro Arg Thr Pro Arg Pro Gly Val Glu Ser Ala Asp Ser Arg Arg            260 265 270 Ala Thr Lys His Ser Arg Lys Ala Leu Lys Leu Glu Arg Pro His        275 280 285 Asp    290 <210> 13 <211> 1203 <212> DNA <213> Artificial Sequence <220> <223> His-Ga DNA <400> 13 gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 tcgggaacag taagaccgag gaccagcgca acgaggagaa ggcgcagcgt 120 aaaagatcga gaagcagctg cagaaggaca agcaggtcta ccgggccacg 180 tgctgctggg tgctggagaa tctggtaaaa gcaccattgt gaagcagatg 240 atgttaatgg gtttaatgga gacagtgaga aggcaaccaa agtgcaggac 300 acctgaaaga ggcgattgaa accattgtgg ccgccatgag caacctggtg 360 agctggccaa ccccgagaac cagttcagag tggactacat cctgagtgtg 420 ctgactttga cttccctccc gaattctatg agcatgccaa ggctctgtgg 480 gagtgcgtgc ctgctacgaa cgctccaacg agtaccagct gattgactgt 540 tcctggacaa gatcgacgtg atcaagcagg ctgactatgt gccgagcgat 600 ttcgctgccg tgtcctgact tctggaatct ttgagaccaa gttccaggtg 660 acttccacat gtttgacgtg ggtggccagc gcgatgaacg ccgcaagtgg 720 tcaacgatgt gactgccatc atcttcgtgg tggccagcag cagctacaac 780 gggaggacaa ccagaccaac cgcctgcagg aggctctgaa cctcttcaag 840 acaacagatg gctgcgcacc atctctgtga tcctgttcct caacaagcaa 900 ctgagaaagt ccttgctggg aaatcgaaga ttgaggacta ctttccagaa 960 acactactcc tgaggatgct actcccgagc ccggagagga cccacgcgtg 1020 agtacttcat tcgagatgag tttctgagga tcagcactgc cagtggagat 1080 actgctaccc tcatttcacc tgcgctgtgg acactgagaa catccgccgt 1140 actgccgtga catcattcag cgcatgcacc ttcgtcagta cgagctgctc 1200                                                                     1203 <210> 14 <211> 400 <212> PRT <213> Artificial Sequence <220> <223> His-Ga protein <400> 14 Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro  1 5 10 15 Gly Ser His Met Gly Cys Leu Gly Asn Ser Lys Thr Glu Asp Gln             20 25 30 Asn Glu Glu Lys Ala Gln Arg Glu Ala Asn Lys Lys Ile Glu Lys         35 40 45 Leu Gln Lys Asp Lys Gln Val Tyr Arg Ala Thr His Arg Leu Leu     50 55 60 Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gln Met 65 70 75 80 Ile Leu His Val Asn Gly Phe Asn Gly Asp Ser Glu Lys Ala Thr                 85 90 95 Val Gln Asp Ile Lys Asn Asn Leu Lys Glu Ala Ile Glu Thr Ile            100 105 110 Ala Ala Met Ser Asn Leu Val Pro Pro Val Glu Leu Ala Asn Pro        115 120 125 Asn Gln Phe Arg Val Asp Tyr Ile Leu Ser Val Met Asn Val Pro    130 135 140 Phe Asp Phe Pro Pro Glu Phe Tyr Glu His Ala Lys Ala Leu Trp                 150 155 160 Asp Glu Gly Val Arg Ala Cys Tyr Glu Arg Ser Asn Glu Tyr Gln                165 170 175 Ile Asp Cys Ala Gln Tyr Phe Leu Asp Lys Ile Asp Val Ile Lys            180 185 190 Ala Asp Tyr Val Pro Ser Asp Gln Asp Leu Leu Arg Cys Arg Val        195 200 205 Thr Ser Gly Ile Phe Glu Thr Lys Phe Gln Val Asp Lys Val Asn    210 215 220 His Met Phe Asp Val Gly Gly Gln Arg Asp Glu Arg Arg Lys Trp                 230 235 240 Gln Cys Phe Asn Asp Val Thr Ala Ile Ple Val Val Ala Ser                245 250 255 Ser Tyr Asn Met Val Ile Arg Glu Asp Asn Gln Thr Asn Arg Leu            260 265 270 Glu Ala Leu Asn Leu Phe Lys Ser Ile Trp Asn Asn Arg Trp Leu        275 280 285 Thr Ile Ser Val Ile Leu Phe Leu Asn Lys Gln Asp Leu Leu Ala    290 295 300 Lys Val Leu Ala Gly Lys Ser Lys Ile Glu Asp Tyr Phe Pro Glu                 310 315 320 Ala Arg Tyr Thr Thr Pro Glu Asp Ala Thr Pro Glu Pro Gly Glu                325 330 335 Pro Arg Val Thr Arg Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu            340 345 350 Ile Ser Thr Ala Ser Gly Asp Gly Arg His Tyr Cys Tyr Pro His        355 360 365 Thr Cys Ala Val Asp Thr Glu Asn Ile Arg Arg Val Phe Asn Asp    370 375 380 Arg Asp Ile Ile Gln Arg Met His Leu Arg Gln Tyr Glu Leu Leu                 390 395 400 <210> 15 <211> 1047 <212> DNA <213> Artificial Sequence <220> <223> GST-ApLZ-iL3 DNA <400> 15 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660 gtggatccta cgctctgaaa aaagaactgc aggctaacaa aaaagaactg 720 aatgggaact gcaggctctg aaaaaagaac tgtgccgtat cctgctggct 780 aggctgttca ggttgcttcc ctgaccaccg gtatggcttc ccaggcttcc 840 aggttccgcg taccccgcgt ccgggtgttg aatccgctga ctcccgtcgt 900 aacactcccg taaagctctg aaatgccagc tggaaaaaaa actgcaggct 960 aactggctca gctggaatgg aaaaaccagg ctctggaaaa aaaactggct 1020 ggccgcatcg tgactga 1047

Claims (17)

One containing the intracellular loop region of the G-protein coupled receptor (GPCR) and the other containing the α subunit Gα of the G-protein. A method of screening a GPCR signaling inhibitor or promoter for selecting candidate substances by measuring the specific binding degree between the fusion proteins by surface plasmon resonance (SPR) or immunoassay (immunoassay). The method of claim 1, wherein the fusion protein comprising the intracellular loop portion of the GPCR and the fusion protein comprising Gα each comprises a fusion protein comprising an intracellular loop 3 (iL3) region of serotonin receptor 6, and stimulatory A method of searching for a GPCR signaling inhibitor or promoter comprising a fusion protein comprising an α subunit of G _s (G α _s ). The method of claim 2, wherein the fusion protein comprising the iL3 site of serotonin receptor 6 is composed of a site for facilitating purification of the protein and an iL3 site. The method of claim 3, wherein the site for facilitating purification of the fusion protein comprising the iL3 site is GST (Glutathione-S-Transferase). The method of claim 2, wherein the fusion protein comprising Gα _s comprises a site for facilitating purification and Gα _s . The method of claim 5, wherein the site for facilitating purification of the fusion protein comprising Gα _s is six histidine tags at the N-terminus. Iii) adsorbing a fusion protein comprising Gα to the vessel; Ii) treating the GPCR signaling inhibitor or promoter candidate; Iii) adding a fusion protein comprising the intracellular loop portion of GPCR; Iii) binding the labeling antibody conjugate to the fusion protein comprising the intracellular loop site to the mixture of step iii); Iii) color development using the conjugated labeled antibody conjugates, and Iii) comparing the degree of color development by the binding between the two fusion proteins when the degree of color development of step v) and the candidate substance are not added to determine the degree of promotion or inhibition of GPCR binding (at which time no candidate substance is added) In this case, steps i) to v) are applied under the same conditions except for step ii). Search method of GPCR signaling inhibitors or promoters. Iii) adsorbing a fusion protein comprising Gα to a sensor chip for measuring SPR; Ii) adding a fusion protein comprising a GPCR signaling inhibitor or promoter candidate and an intracellular loop site of GPCR to a fusion protein comprising adsorbed Gα; iii) irradiating the sensor with an SPR measuring device to obtain a surface plasmon resonance signal thereof; and iv) comparing the resonance signal of iii) and the resonance signal by binding between the two fusion proteins when the candidate material is not added to determine the degree of promotion or inhibition of GPCR binding (when no candidate material is added) Is applied to steps i) to iv) under the same conditions, except for the addition of the candidate substance in step ii)). i) a fusion protein comprising Gα and a fusion protein comprising an intracellular loop region of GPCR; ii) labeling antibody conjugates; And, iii) a search kit for signaling inhibitors or promoters of GPCRs, including a substrate solution containing a colorant. 10. The kit according to claim 9, wherein Gα in step i) is Gα _s and the intracellular loop site of GPCR is iL3 of serotonin receptor 6. 10. The kit according to claim 9, wherein the labeling antibody conjugate of step ii) is an anti-GST Ab-HRP-conjugate. GST-iL3 fusion protein having the amino acid sequence set forth in SEQ ID NO: 12 . Article 12 Expression vector pGST-iL3 comprising a gene of SEQ ID NO: 11 encoding a GST-iL3 fusion protein. His-Gα _s fusion protein having the amino acid sequence set forth in SEQ ID NO: 14 . Expression of claim 14 comprising a gene of SEQ ID NO: 13 encoding the fusion protein vector His-Gα _s pH-Gα _s. An expression vector pGST-ApLZ-iL3 comprising a GST-ApLZ-iL3 gene having the nucleotide sequence set forth in SEQ ID NO: 15 . The method of claim 8, wherein the SPR measuring device of step iii) is a Biacore SPR measuring system.

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