JP3461852B2 - Human cholecystokinin B receptor - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cholecystokinin receptor derived from human brain cells; a cholecystokinin receptor gene encoding the receptor; an expression vector containing the gene; and a transformant having the expression vector. .

[0002]

Cholecystokinin (CCK) is a hormone which is present in cells of the digestive tract of mammals and also in cells of various regions of the brain. Of these, the central nervous system
CCK is speculated to be a neurotransmitter or neuromodulator (Williams, JA Biomed. Res. 3, 1
07-121 (1982)), especially the cerebral cortex, limbic system, striatum,
And in areas involved in emotional and cognitive processes such as motivation and anxiety. It is known that CCK has an anti-sedative effect, has a role of controlling pain and anesthesia tolerance such as inhibition of an analgesic effect of morphine, or a role of protecting cortical neurons from glutamate poison. Besides, CCK is thought to play various physiological and pathological roles in the human central nervous system, but the exact mechanism thereof has not yet been completely clarified.

CCK signals are transduced by the binding of CCK to specific cell surface membrane receptors. Such receptors include CCK-A (gastrointestinal type) and CCK
-There are at least two types of specific receptors called B (brain type) (Sakamoto, C. et al.,
Biochem. Biophys. Res. Commun. 124, 497-502 (198
Four)). The CCK of the central nervous system plays a role in enhancing satiety and regulating food intake. This activity is associated with CCK-A receptors present in afferent fibers of the gastric vagus nerve, the solitary nucleus and the posterior cortex, and CC present in the ventromedial paraventricular nucleus of the hypothalamus.
Mediated by both KB receptors (Smith, GP
, Am. J. Physiol. 249, R638-R641; Miceli, MO
Physiol. Behav. 38 855-860 (1986); and Douris.
h, CT, Science 245, 1509-1511 (1989)).

The CCK-A receptor is more CCK than gastrin
It has a 1000-fold higher affinity for, is present in peripheral tissues such as the pancreas, gastrointestinal tract, gallbladder, and vagus nerve, and also in the central nervous system such as the nucleus of the solitary tract, the nucleus of the posterior area, and the interlimb nucleus. Also exists. The CCK-B receptor has a high affinity for both CCK and gastrin and is widely distributed in brain cells and stomach, especially gastric parietal cells, intestinal chromaffin-like cells and smooth muscle. CCK-B receptors present on brain cell membranes and CCK-B / present on partially isolated gastric parietal cells
The gastrin receptor has a slightly different affinity for CCK-8 or gastrin.

Recently, the complementary DNA of rat CCK-A receptor
Was cloned from the pancreas (Wank, SA et al., Proc.
Natl. Acad. Sci. USA. 89, 3125-3129 (1992)). Kopi
N et al. also isolated a receptor for gastrin, an analogue of CCK, from dog gastric parietal cells. Most recently, we cloned the Mastomys gastrin receptor from a carcinoid derived from intestinal chromaffin-like cells.

CCK derived from brain cells or cells of the digestive tract
As for the receptor, gastrin and CCK-as described above.
Elucidating the mechanism of the difference in affinity of 8 and the role of CCK in the central nervous system and C in mucosal cells of the gastrointestinal tract
The biological function of CK needs to be investigated. For this purpose, first of all, it is important to analyze the structure of the CCK receptor. Such a receptor is directly useful as a reagent for measuring CCK or for screening an agonist or an antagonist of the CCK receptor. Therefore, mass production of the receptor by these structural analyzes and genetic engineering means is desired.

[0007]

OBJECT OF THE INVENTION It is an object of the present invention to provide a novel cholecystokinin receptor derived from human brain cells. Another object of the present invention is to provide a DNA sequence encoding the cholecystokinin receptor, an expression vector having the DNA sequence, and a transformant having the expression vector.

[0008]

The present inventors have conducted various studies to obtain cholecystokinin receptor from human-derived cells.
As a result, cD prepared from human brain cell-derived mRNA
A DNA sequence encoding a cholecystokinin-B receptor was found in NA, and the present invention was completed.

The polypeptide of the present invention comprises the amino acid sequence from Met at position 1 to Gly at position 447 of SEQ ID NO: 2 in the sequence listing. However, this does not exclude a polypeptide that has been cleaved / decomposed / modified / modified in and out of the cell, including removal of the N-terminal Met.

The DNA sequence of the present invention encodes the above-mentioned polypeptide.

According to a preferred embodiment, the DNA of the present invention
The sequence is from A to 153 at position 193 of SEQ ID NO: 2 in the sequence listing.
It has a base sequence consisting of up to C at position 3.

The expression vector of the present invention has the above DNA sequence.

The transformant of the present invention can be obtained by introducing the above expression vector into a host cell. According to a preferred embodiment, the transformant of the present invention contains the above expression vector in CH
Obtained by introduction into O-K1 cells.

(1) Determination of DNA Sequence Encoding Cholecystokinin (CCK-B) Receptor Derived from Human Brain Cells An example of the method for determining the DNA sequence encoding the CCK-B receptor of the present invention will be described in the order of steps. To do.

First, from total RNA derived from human brain cells,
Poly A prepared using an oligo dT cellulose column
⁺ RNA purified, cDNA synthesis kit and λgt1
0 cloning system (Amersham, Arlington Height
s, IL) to create a cDNA library.
Next, for example, a cDNA for a gastrin receptor of a CCK analog (for example, derived from Mastomys of the same mammal) (see Examples below) is used as a probe for screening by hybridization to obtain a positive λ phage clone. CDNA contained in this positive clone
Each A insert is cut out with an appropriate restriction enzyme and subcloned, and then the nucleotide sequence is determined by a known DNA sequencing method such as the dideoxy method. The nucleotide sequence of human CCK-B receptor thus obtained from one cDNA insert and the corresponding amino acid sequence are shown in FIG. 1 and SEQ ID NO: 2 in the sequence listing.

(2) Construction of expression vector, preparation of transformant and expression of CCK-B receptor cDNA encoding the above human CCK-B receptor
Is introduced into an appropriate vector to give an expression vector. A transformant can be obtained by introducing these expression vectors into an appropriate host cell. For example, Chinese hamster ovary cells (CHO) -K1 (ATCC No. C, which lack the expression vector for any CCK receptor).
A transformant capable of expressing and producing the CCK-B receptor can be obtained by transfecting CL-61, Rockville, MD). CCK-B receptors are produced by culturing the transformed CHO-K1 cells under ordinary conditions.

(3) Binding Assay of Product Produced by Transformant The protein produced by the above transformant is human C
C-terminal part of CK (human CCK-8) and / or C
It is subjected to a binding assay for human gastrin I, a CK analog. In this assay, the protein produced by the transformants was labeled with ¹²⁵ I-CCK-8.
^Since it has a high affinity for any of ¹²⁵ I-gastrin I, it is confirmed to be a CCK-B receptor. For example, first, a mixture of the above transformant and a predetermined amount of ¹²⁵ I-labeled CCK-8 and an unlabeled predetermined amount of CCK-8 (C / C); a predetermined amount of ¹²⁵ I-labeled Gastrin I unlabeled with CCK-8
A mixture of (C / G) with a predetermined amount of gastrin I labeled with ¹²⁵ I and a mixture of unlabeled gastrin I (G / G); and a predetermined amount of gastrin I labeled with ¹²⁵ I and labeled Each of the mixture (G / C) with a predetermined amount of CCK-8 which has not been incubated is incubated with the above transformant, and the amount of CCK-8 or gastrin I bound to the transformant is measured. Unlabeled C
The amount of CK-8 or human gastrin I was changed and plotted on the horizontal axis, and labeled CCK-8 or gastrin I
FIG. 2 shows a graph in which the vertical axis represents the amount of radioactivity of the complex with the receptor. For comparison, a graph of the same assay performed using a transformant (CHO-mGR10; described later) into which an expression vector containing mastomys gastrin cDNA was introduced is shown in FIG.

The gastrin gastrin receptor and the CCK-B receptor are so similar that they are used as a comparative sample. When the same test was conducted by using CHO-K1 cells that do not contain the expression vector as a control, ¹²⁵ I- binding of gastrin I or ¹²⁵ I-CCK-8, each polypeptide of excess unlabeled The level of binding of non-specific ¹²⁵ I-labeled polypeptide in the presence of From the above results, it was revealed that the CCK-B receptor of the present invention derived from human brain cells was produced from the transformant.

(4) Measurement of intracellular free calcium concentration of transformant In the presence of gastrin I or CCK-8, intracellular free calcium concentration ([Ca ²⁺ ] i) of the above transformant was measured. This confirms that the protein produced by the transformant is a functional CCK-B receptor. For example, first, the above transformant is incubated with Fura2 / acetoxymethyl ester in an appropriate buffer under an oxygen atmosphere. Next, this transformant was stimulated with gastrin I or CCK-8,
The change in intracellular free calcium is then measured with a fluorescence spectrophotometer. The result is shown in FIG. From FIG. 4 it can be seen that both human gastrin I and CCK-8 induce a significant increase in [Ca ²⁺ ] i. This induction is an antagonist
L365.260 (Merck Sharp and Dohme, West Point, PA)
Blocked by. This indicates that the transformants encode a functional CCK-B receptor.

(5) Presence of CCK Receptor mRNA in Each Human Tissue mRNA is isolated from various human tissues and used as a probe for the cDNA encoding the human CCK-B receptor of the present invention.
The A fragment was radioactively labeled and subjected to Northern blot hybridization. The results are shown in FIG. 5 (lane 1: human cerebrum, lane 2: human gastric body full-thickness layer, lane 3: human gastric body mucosa, lane 4: human gastric antrum mucosa, lane 5: human duodenal mucosa). From FIG. 5, the human CCK of the present invention
A large amount of -B receptor mRNA is present in human brain tissue, stomach tissue, especially mucosal mucosal tissue of the body of the stomach,
It can be seen that the mucosal tissues of the antrum of the stomach and duodenum are almost absent.

[0021]

The present invention will be further described by the following examples.

Example 1 First, the sequence of the cDNA of the DNA encoding the mastomys gastrin receptor used as a probe in order to isolate the DNA encoding the CCK-B receptor derived from human brain cells of the present invention. The method of determining is shown below. Furthermore, a method for producing a transformant that produces a mastomys gastrin receptor used in the binding assay described below is also shown.

(I) Determination of DNA Sequence Encoding Gastrin Receptor Derived from Mastomys ECL A cDNA synthesis kit from poly A ⁺ RNA prepared from an ECL carcinoid cell of Mastomys natalensis using an oligo dT cellulose column. And λgt10 cloning system (Amersham, Arling
ton Heights, IL) is used to create a cDNA library.

Next, the cDNA sequence of the known canine parietal cell gastrin receptor (Kopin, AS et al., Supra) and the rat pancreas-derived CCK receptor (CCK-A), which is considered to be a gastrin receptor analog, were identified. cDNA
Sequence (Wank et al., Proc. Natl. Acad. Sci. USA 89, 3125-3
129 (1992)) was used as a primer to amplify a partial sequence of the gastrin receptor cDNA derived from the Mastomys ECL carcinoid cell by PCR. The sequences of the primer and the antisense primer are as shown in SEQ ID NOS: 3 and 4 of the sequence listing, respectively. The amplified DNA fragment (0.2 kb) was labeled with ³² P by the random primer method and used as a probe, and plaques were screened as follows.

First, a plaque-reproduced nitrocellulose filter was placed in 50% formamide, 1% SD.
S, 1M NaCl, 10% dextran sulfate, 200
It was put in a solution containing μg / ml yeast tRNA and 100 μg / ml denatured salmon sperm DNA, and hybridization was carried out at 42 ° C. for 18 hours using the above probe. Then, the above filter was washed with 0.1% SDS in 0.1%.
1 × SSC (1 × SSC contains 0.15 M NaCl and 15 mM sodium citrate (pH 7.0))
The solution was washed 4 times at 55 ° C. for 30 minutes. Using a Kodak X-ray film and a DuPont enhancing screen (manufactured by DuPont), autoradiography was carried out by leaving it at -80 ° C for 16 hours. As a result, several hybridized clones were found.
One of these cDNA inserts (mGR10)
The restriction enzyme fragment of was subcloned into a plasmid vector. The DNA of this cDNA insert was sequenced by the dideoxy chain termination method (dideoxy method) to Sequenase version 2 (United States Bioche
mical Corp., Cleveland, OH) was used to determine the nucleotide sequence.

Thus, the DN encoding the 2300 bp gastrin receptor obtained from mGR10
The nucleotide sequence of the A fragment and the amino acid sequence corresponding thereto are shown in SEQ ID NO: 1 in the sequence listing. mGR10 has a 1350 bp open reading frame encoding a 450 amino acid protein. This protein is found in the canine parietal cell gastrin receptor and in the murine pancreatic CCK-
It is about the same size as the A receptor. Furthermore, there are 85.7% and 49.0% homology to these receptors, respectively. Hydropathic analysis revealed seven hydrophobic segments, which have the transmembrane domain properties of the G protein coupled receptor superfamily. In addition, this receptor contains three potential asparagine glycosylation sites at amino acids 7, 30, and 36. There are two cysteine residues in the sequence, amino acids 127 and 205, which are likely to be involved in an internal chain disulfide bond similar to the canine parietal cell gastrin receptor. The and carboxy termini are rich in threonine and serine residues, which may be potential phosphorylation sites. As described above, the cloned cDNA is a known G-protein linked receptor (O'Dowd, BF et al., Annu. Rev. Neu.
rosci. 12, 67-83 (1989)) and various structural characteristics in common.

(II) Construction of Expression Vector, Preparation of Transformant The mGR10 (about 2.3 kbp) fragment obtained in the above section I was further digested with EcoRI (mGR10: 1.8 k).
b) to the pHβAPrlneo plasmid (Gunning, P. et al., Pro
c. Natl. Acad. Sci. USA 84, 4831-4835 (1987))
To obtain an expression vector. 100 μg / ml kanamycin and 10% calf serum (Hazleton, Lenex
a, KS) was added to Ham's F-12 medium and subcultured to CHO-K1 cells, and the Lee-Okayama method (Chen, C.
Et al., Mol. Cell. Biol. 7, 2745-2752 (1987)) were used to transfect the expression vector. After transfection, the cells were treated with 700 μg / ml G41
It was kept in a medium containing 8 (Geneticin, GIBCO). 3-
After culturing for 4 weeks, G418-resistant transformant cells (CHO
-MGR10) was selected.

Example 2 (I) Determination of DNA Sequence Coding for CCK Receptor Derived from Human Brain Cells Poly A ⁺ RNA derived from human brain cells was obtained from Clontech labora.
Purchased from tories, Inc., CA. This poly A ⁺ RNA
Is obtained from all normal brain tissues except the pituitary gland, including the human cerebral cortex, diencephalon, midbrain, pons, medulla, and cerebellum. Poly A ⁺ RN prepared from this poly A ⁺ RNA using an oligo dT cellulose column
From A, a cDNA library is prepared using a cDNA synthesis kit and a λgt10 cloning system (Amersham, Arlington Heights, IL).

Next, the plasties were screened using ³² P-labeled cDNA fragment of gastrin receptor of Mastomys as a probe. The probe was a 0.2 kb DNA fragment described in Section I of Example 1 and an EcoRI-digested DNA fragment of mGR10 (1.8 kb: mGR10).
Were prepared using the Megaprime DNA labeling system (2 × 10 ⁹ cpm per μg of DNA, Amersham). First, a plaque-replicated nitrocellulose filter was treated with 30% (w / v) formamide, 5 × SSC (1 × SSC was 0.15M NaCl).
And 15 mM sodium citrate (pH 7.0)), 1 × Denhardt's solution (0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 5 mM NaH ₂ PO ₄ , 0.1%. Place in a solution containing SDS and 100 μg / ml denatured salmon sperm DNA, and use one of the above probes at 42 ° C. for 1 hour.
Hybridization was performed for 8 hours. Next, the above filter was placed in 2 × SSC (pH 7.
Solution 0) was washed 4 times for 15 minutes each at room temperature, and then 2 times for 30 minutes each at 50 ° C. with 1 × SSC (pH 7.0) solution containing 0.1% SDS. Using a Kodak X-ray film and a DuPont enhancing screen (manufactured by DuPont), autoradiography was carried out by leaving it at -80 ° C for 16 hours. As a result, 5 clones hybridized to both probes were found. One of these cDNA inserts (h-
The restriction enzyme fragment of CCKB3) was subcloned into a plasmid vector. The nucleotide sequence of this cDNA insert was sequenced by the dideoxy chain termination method (dideoxy method) to Sequenase version 2 (United
States Biochemical Corp., Cleveland, OH). Thus, the nucleotide sequence of the 2200 bp human CCK receptor-encoding DNA fragment obtained from h-CCKB3 and the corresponding amino acid sequence are shown in FIG. 1 and SEQ ID NO: 2 in the sequence listing. h-CC
KB3 has a 1341 bp open reading frame encoding a 447 amino acid protein. This protein is derived from canine parietal cell gastrin receptor (c-CCK-B), Mastomys ECL carcinoid cell-derived gastrin receptor (m-CCK-B),
And rat pancreatic CCK-A receptor (r-CCK-
It is almost the same size as A). In addition, they have 88.8%, 90.3%, and 44.7% homology to these receptors, respectively. A comparison diagram of the amino acid sequences of these receptors and h-CCK-B of the present invention is shown in FIG. Hydropathic analysis revealed seven hydrophobic segments (underlined I-VI in Figure 1).
I), they have the transmembrane domain properties of the G protein coupled receptor superfamily. In addition, this receptor contains three potential asparagine glycosylation sites at amino acids 7, 30, and 36. Two cysteine residues (amino acid 127
And positions 205), which may be involved in an internal chain disulfide bond similar to the canine parietal cell gastrin receptor. The third cytoplasmic loop and carboxy terminus are rich in threonine and serine residues, which may be potential phosphorylation sites. As described above, the cloned cDNA was used as a known G-protein linked receptor (O'Dowd, BF et al., Annu. Rev. Neuro.
sci. 12, 67-83 (1989)) with various structural features in common.

(II) Construction of expression vector, preparation of transformant and expression of human CCK-B receptor h-CCKB3 fragment (about 2.2 kbp) obtained in the above section I.
PHβA Prlneo plasmid (Gunning, P. et al., Proc.
Atl. Acad. Sci. USA 84, 4831-4835 (1987)) Sal
It was introduced into the I site to obtain an expression vector. 100 μg / m
l kanamycin and 10% calf serum (Hazleton,
Lenexa, KS) was added to Ham's F-12 medium, which was stored in CHO-K1 cells (in a 6 cm dish).
The expression vector was transfected using the ayama method (Chen, C. et al., supra). Separately, CHO-K1 cells containing no plasmid were prepared and used as a control. After transfection, the cells should be 700 μg / ml
The cells were cultured in the above medium containing G418 (Genenticin, GIBCO). After 3 to 4 weeks, G418-resistant transformants were selected.

(III) Binding Assay of Human CCK-B Receptor Produced by Transformant to CCK and Gastrin ¹²⁵ I-sulfated CCK-8 (hereinafter simply referred to as ¹²⁵ I-CCK-8) was prepared by using Amersham, Buckinghamshire, Purchased from UK). The transformant obtained in the above section II (CHO-h
-CCKB) and the transformant (CHO-mGR10) obtained in Example 1 as a comparison, as follows.
Binding assay was performed. First, the above cells in a dish were prepared by adding 0.1% bovine serum albumin (BSA) to Hanks' balanced salt solution (binding medium).
Washed with 1 ml. Next, 70 pM of ¹²⁵ I-CCK
-8 and various concentrations of unlabeled CCK-8 (Peninsul
a, Belmont, CA) was added to each well (C / C mixed wells: black squares in Figures 2 and 3). Separately, solutions containing the analog Gastrin I instead of CCK-8 were also prepared and added to each well (C
/ G mixed well: white circles in FIGS. 2 and 3.). 2 each
Incubation was carried out at 4 ° C for 60 minutes. After incubation, the cells were incubated with 1 ml of ice-cold binding medium,
After washing 3 times, it was dissolved in 0.5 ml of NaOH.

The radioactivity bound to the cells was measured with a gamma counter (Aloka). All measurements were performed in triplicate. The total amount of specific binding of labeled CCK-8 is calculated as follows: ¹²⁵ I-CCK-8 unlabeled CC.
The total amount of binding in the absence of K-8 was set to 100%, and the above CC
The amount of ¹²⁵ I-CCK-8 bound when K-8 or gastrin I was contained at various concentrations was expressed as a percentage. The results are shown in FIG. 2 (transformant CHO-h-CCKB) and FIG. 3 (transformant CHO-mGR10). In addition,
¹²⁵ I-CCK-8 did not bind to control CHO-K1 cells.

[0033] was performed using ¹²⁵ I- gastrin I further similar experiment instead of ^{125 I-CCK-8 (G} / C
Wells (marked with X in FIGS. 2 and 3) and G / G wells (see FIG. 2)
And the black circles in FIG. 3)). The results are also shown in FIGS. 2 and 3. From this figure, it was revealed that both CHO-h-CCKB4 and CHO-mGR10 bind to both CCK-8 and gastrin with high affinity. However, it can be seen that the human CCK-B receptor showed a slightly higher affinity for CCK-8 than that for gastrin I, although the mastomys gastrin receptor showed almost the same affinity for CCK and gastrin. The binding specificity for such receptors was shown to be slightly different.

(IV) Measurement of Free Calcium Concentration in Transformant The transformant containing h-CCKB3 described above was functional C
To confirm that it expresses the CK-B receptor,
The effect of CCK-8 and gastrin I on intracellular free calcium concentration in transformants was investigated.

First, the transformant was treated with Hepes buffered Krebs-Ringer medium (125 mM NaCl, 5 mM K).
Cl, 1.2 mM MgSO ₄ , 1.2 mM KH ₂ PO ₄ ,
Incubated with Fura2 / acetoxymethyl ester (1 μM) in ₂ mM CaCl ₂ , 6 mM glucose, 25 mM Hepes, pH 7.4) for 30 minutes at 37 ° C. under oxygen atmosphere. After incubation,
Gastrin I (10 ^-9 M) or CCK-8 (10 ^-9
M) stimulate the cells and then by a fluorescence spectrophotometer
The intracellular free calcium concentration was measured from the 340 nm / 380 nm ratio.

Both gastrin I and CCK-8 (10 ^-9 M) induced a significant increase in [Ca ²⁺ ]. This trigger is CCK
-B and a gastrin receptor antagonist
L365,260 (10 ^-5 M) (Merck Sharp and Dohme, West Poin
Control cells were unresponsive (blocked by t, PA) (19) (data not shown). Therefore, like the natural cholecystokinin-B receptor derived from human brain, CCKB3 was found to encode a functional CCK receptor that is coupled to an intracellular signal transduction system leading to an increase in [Ca ²⁺ ].

(V) Presence of CCK receptor mRNA in various human tissues Total RNA was isolated from human brain tissue and stomach tissue by the guanidinium isocyanate / cesium chloride method. All RN
A was separated on a 1% agarose gel and nitrocellulose membrane (Schleicher & Schuell
Inc., Keene, NH).

Blots with 50% formamide, 4 × SS
C, 5 × Denhardt's solution, 0.5% SDS, 10% dextran sulfate, 250 μg / ml denatured salmon sperm DNA and h-CCK prepared in (II) above as a probe
Hybridization was carried out at 42 ° C. for 16 hours in a solution containing the 1.8 kb BamHI cDNA fragment of B3 (labeled with megaprime). Blot at room temperature for 20
Wash twice with a solution containing 2 × SSC and 0.1% SDS for 2 minutes each, and further wash twice at 55 ° C. (30 minutes each) with 0.1.
It was washed with a solution containing × SSC and 0.1% SDS.

Kodak X-O Matte AR film (Koda
(K, Rochester, NY) and DuPont's enhancing screen and left overnight at -70 ° C for autoradiography. The result is shown in FIG. From this figure, in addition to human brain cells, a small amount of mRNA expression was confirmed in the gastric mucosal cells, particularly in the mucosa of the gastric corpus, in the gastric antrum, and the expression of mRNA was not detectable in the duodenal mucosa. It can be seen that the CCK-B receptor mRNA of E. coli exists in these specific tissues.

[0040]

According to the present invention, as described above, a novel human CCK receptor and D encoding the receptor are obtained.
An NA sequence, an expression vector having the DNA sequence, and a transformant containing the expression vector are provided. Thus, D encoding human CCK-B receptor
This is the first time NA has been isolated. C obtained
The CK-B receptor is useful as a reagent for measuring CCK or for screening CCK agonists and antagonists.

[0041]

[Sequence list]

[0042]

[SEQ ID NO: 1] Sequence length: 2321 Sequence type: Nucleic acid chain number: Double-strand topology: Linear Sequence type: cDNA Origin organism name: Mastomys matalensis Symbol representing sequence characteristics: CDS existence Location: 49..1398 Method of characterizing: S sequence GGAGCTCGCT TAACGCTTAG CGGACGTGAG TGGGACTTCA CTGGAGCC ATG GAG CTG 57 Met Glu Leu 1 CTC AAG CTG AAC AGC AGC GTG CAG GGA CCA GGA CCC GGG TCG GGG TCT 105 Leu Sers Leu Lys Val Gln Gly Pro Gly Pro Gly Ser Gly Ser 5 10 15 TCT TTG TGC CAC CCG GGT GTC TCC CTC CTC AAC AGC AGT AGT GCG GGC 153 Ser Leu Cys His Pro Gly Val Ser Leu Leu Asn Ser Ser Ser Ala Gly 20 25 30 35 AAC CTC AGC TGC GAG CCC CCT CGT ATC CGC GGA ACC GGG ACC AGA GAA 201 Asn Leu Ser Cys Glu Pro Pro Arg Ile Arg Gly Thr Gly Thr Arg Glu 40 45 50 CTG GAG TTG GCC ATT AGA ATC ACC CTT TAT GCG GTG ATC TTT CTG ATG 249 Leu Glu Leu Ala Ile Arg Ile Thr Leu Tyr Ala Val Ile Phe Leu Met 55 60 65 AGC ATT GGC GGA AAC ATG CTC ATC ATT GTG GTC CTG GGA CTG AGC C GA 297 Ser Ile Gly Gly Asn Met Leu Ile Ile Val Val Leu Gly Leu Ser Arg 70 75 80 CGC CTA AGA ACA GTC ACC AAC GCC TTC CTG CTC TCC CTG GCA GTC AGT 345 Arg Leu Arg Thr Val Thr Asn Ala Phe Leu Leu Ser Leu Ala Val Ser 85 90 95 GAC CTC CTG CTG GCT GTC GCT TGC ATG CCC TTC ACA CTC CTG CCC AAC 393 Asp Leu Leu Leu Leu Ala Val Ala Cys Met Pro Phe Thr Leu Leu Pro Asn 100 105 110 115 CTC ATG GGC ACA TTC ATC TTC GGC ACA GTC ATC TGC AAG GCC GTT TCC 441 Leu Met Gly Thr Phe Ile Phe Gly Thr Val Ile Cys Lys Ala Val Ser 120 125 130 TAC CTC ATG GGG GTA TCA GTG AGT GTA TCC ACT CTA AAT CTC GTG GCC 489 Tyr Leu Met Gly Val Ser Val Ser Val Ser Thr Leu Asn Leu Val Ala 135 140 145 ATC GCC CTG GAG CGA TAC AGT GCC ATC TGC CGA CCA CTG CAA GCA CGA 537 Ile Ala Leu Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu Gln Ala Arg 150 155 160 GTG TGG CAG ACA CGC TCC CAC GCA GCT CGG GTG ATC CTA GCC ACG TGG 585 Val Trp Gln Thr Arg Ser His Ala Ala Arg Val Ile Leu Ala Thr Trp 165 170 175 CTG CTG TCT GGA CTG CTT ATG GTA CCC TAT CCT GTG TAC ACC GTG GTA 633 Leu Leu Ser Gly Leu Leu Met Val Pro Tyr Pro Val Tyr Thr Val Val 180 185 190 195 CAG CCA GTG GGA CCT CGA GTG CTG CAG TGC ATG CAT CGC TGG CCC AGT 681 Gln Pro Val Gly Pro Arg Val Leu Gln Cys Met His Arg Trp Pro Ser 200 205 210 GCA CGC GTC CGA CAA ACC TGG TCC GTG CTA CTG CTA ATG CTT TTG TTC 729 Ala Arg Val Arg Gln Thr Trp Ser Val Leu Leu Leu Met Leu Leu Phe 215 220 225 TTC ATC CCG GGT GTG GTT ATG GCG GTG GCC TAT GGA CTC ATC TCC CGC 777 Phe Ile Pro Gly Val Val Met Ala Val Ala Tyr Gly Leu Ile Ser Arg 230 235 240 GAA CTC TAC CTA GGG CTC CGC TTT GAT GGT GAC AAT GAC AGC GAC ACC 825 Glu Leu Tyr Leu Gly Leu Arg Phe Asp Gly Asp Asn Asp Ser Asp Thr 245 250 255 CAA AGC CGG GTC AGA AAC CAA GGG GGC CTG CCG GGT GGG ACA GCA CCT 873 Gln Ser Arg Val Arg Asn Gln Gly Gly Leu Pro Gly Gly Thr Ala Pro 260 265 270 275 GGG CCT GTC CAC CAG AAC GGG GGT TGC CGG CAT GTG ACC GTG GCT GGA 921 Gly Pro Val His Gln Asn Gly Gly Cys Arg His Val Thr Val Ala Gly 280 285 290 GAA GAC AAT GAT GGC TGC TAC GTG CAA CTT CCG CGT TCC CGA CTG GAG 969 Glu Asp Asn Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser Arg Leu Glu 295 300 305 ATG ACA ACA CTG ACC ACG CCC ACT CCT GGG CCC GGC CTG GCC TCG GCC 1017 Met Thr Thr Leu Thr Thr Pro Thr Pro Gly Pro Gly Leu Ala Ser Ala 310 315 320 AAC CAG GCC AAG CTG CTG GCT AAG AAG CGG GTG GTG CGA ATG CTG CTG 1065 Asn Gln Ala Lys Leu Leu Ala Lys Lys Arg Val Val Arg Met Leu Leu 325 330 335 GTG ATT GTT TTG CTT TTT TTC CTG TGT TGG CTG CCA ATA TAC AGC GCC 1113 Val Ile Val Leu Leu Phe Phe Leu Cys Trp Leu Pro Ile Tyr Ser Ala 340 345 345 350 355 AAC ACA TGG TGC GCG TTC GAT GGT CCA GGA GCG CAC CGA GCA CTC TCA 1161 Asn Thr Trp Cys Ala Phe Asp Gly Pro Gly Ala His Arg Ala Leu Ser 360 365 370 GGA GCC CCT ATC TCT TTC ATT CAC TTG CTG AGT TAT GCC TCC GCT TGT 1209 Gly Ala Pro Ile Ser Phe Ile His Leu Leu Ser Tyr Ala Ser Ala Cys 375 380 385 GTC AAC CCC CTG GTC TAC TGT TTC ATG CAC CGC CGC TTC CGC CAG GCC 1257 Val Asn Pro Leu Val Tyr Cys Phe Met His Arg Arg Phe Arg Gln Ala 390 395 400 TGC CTG GAC ACA TGT G CC CGC TGT TGC CCA CGC CCT CCA CGA GCT CGC 1305 Cys Leu Asp Thr Cys Ala Arg Cys Cys Pro Arg Pro Pro Arg Ala Arg 405 410 415 CCA CGG CCT CTT CCG GAT GAG GAT CCT CCT ACC CCC TCC ATC GCT TCT 1353 Pro Arg Pro Leu Pro Asp Glu Asp Pro Pro Thr Pro Ser Ile Ala Ser 420 425 430 435 CTA TCC AGG CTA AGC TAT ACC ACC ATC AGC ACA CTA GGG CCT GGC 1398 Leu Ser Arg Leu Ser Tyr Thr Thr Ile Ser Thr Leu Gly Pro Gly 440 445 450 TGAGGGGTTG GAGAGTAGAG AATGTGACAA GGTACATAAT TACTATGAAG TGACCCATCC 1458 AAACACATAA GAAACAAAAT CCAGGATTAA TCCAGTTGGT ACTCAATACC ATGGACAGAC 1518 CCCTACACAG AGAAAATAGT GTCTTTGCTG CCCCCACCTG AAACAGACAG GAATCTTACA 1578 TAGGAAAGGA AGCTCACTTG TGATATGGAC TGAGTCCCTT CCTAGATGTC TGGCACTGAC 1638 CCCATTTCAT AGACAGACAC AAGGTCTATA ACAGTAAACT TTCCCTATTC AAGGGGAATT 1698 CTGGCAAGGG CTGATTGGCT CCTCACATGA ATACATTAAT GGCACTACTC TAAAGTGCCC 1758 GTAGCCTAGT GCAGTAGTGA CTTAGGACAC TGCAGCTGTC CCCAGTTTGA CCTCATCATT 1818 GCCTTCCTCA TCCAGCACTG AAATCATCCA CAGACCTAAT CACATGCCTT TCAGAGCTGC 1878 AGATCTTTCA GCCCTGAAAA GTCCCCTCAT CCCTTTCCAT TGGGGACTGT AGAAAGTCCT 1938 CTTCCCTCCG GCCTCTCCTC CCTCACATCA TAAAAGAATA TGTGACTTAG TGCTGTCCTG 1998 GACTACCTGA GCTTTGGCAG GCAGGTGAAC AGGTGTGGTT TATGGGAAGC TTCTTCATGT 2058 ATTTGCTCCT ATGACTACTC AATCCCATGT CCAACTTTGT GCAAAAAGGC CTTGGTATGA 2118 AGACAGAGGT GAGCATATCC TCTGTTGACC TAACCATACT CCAGTGATGT AGTAGCTGGG 2178 GACTCATTAT GGCAGGTGGA ATACCCATGA CACTTCATGA CCCCTGAGTA CATCCTCTCA 2238 TTTGGTGTCA TTGTTAAAGT ACTATACTAA TGGCTGCCTG TTAATTTTCT GATGAAAAGA 2298 TAAAAGACAA AAGACTAAAA AAA 2321

[0043]

[SEQ ID NO: 2] Sequence length: 2136 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence type: cDNA origin Organism name: human Sequence features Characteristic symbol: CDS Location: 193..1533 How the features were determined: S   Array CGGGAACCCA GGCGGGGCGA GCCGCGGGAG AGTGGAGGGC AGGCGCCTGG GCTGGGGGCG 60 GGGACCAGGC GGGGCAGGGG GCAGGGAGAG GAGGGCGGCG GGAGCTGAGC CGGAATCGCA 120 GCGTGAGCAG GTGGAGCCGC GGTGGGAGCC CGCCGGGTCG AGCTGAGTAA GGCGGCGGGC 180 TCGGCGGGGG CC ATG GAG CTG CTA AAG CTG AAC CGG AGC GTG CAG GGA ACC 231               Met Glu Leu Leu Lys Leu Asn Arg Ser Val Gln Gly Thr                 1 5 10 GGA CCC GGG CCG GGG GCT TCC CTG TGC CGC CCG GGG GCG CCT CTC CTC 279 Gly Pro Gly Pro Gly Ala Ser Leu Cys Arg Pro Gly Ala Pro Leu Leu      15 20 25 AAC AGC AGC AGT GTG GGC AAC CTC AGC TGC GAG CCC CCT CGC ATT CGC 327 Asn Ser Ser Ser Val Gly Asn Leu Ser Cys Glu Pro Pro Arg Ile Arg  30 35 40 45 GGA GCC GGG ACA CGA GAA TTG GAG CTG GCC ATT AGA ATC ACT CTT TAC 375 Gly Ala Gly Thr Arg Glu Leu Glu Leu Ala Ile Arg Ile Thr Leu Tyr                  50 55 60 GCA GTG ATC TTC CTG ATG AGC GTT GGA GGA AAT ATG CTC ATC ATC GTG 423 Ala Val Ile Phe Leu Met Ser Val Gly Gly Asn Met Leu Ile Ile Val              65 70 75 GTC CTG GGA CTG AGC CGC CGC CTG AGG ACT GTC ACC AAT GCC TTC CTC 471 Val Leu Gly Leu Ser Arg Arg Leu Arg Thr Val Thr Asn Ala Phe Leu          80 85 90 CTC TCA CTG GCA GTC AGC GAC CTC CTG CTG GCT GTG GCT TGC ATG CCC 519 Leu Ser Leu Ala Val Ser Asp Leu Leu Leu Ala Val Ala Cys Met Pro      95 100 105 TTC ACC CTC CTG CCC AAT CTC ATG GGC ACA TTC ATC TTT GGC ACC GTC 567 Phe Thr Leu Leu Pro Asn Leu Met Gly Thr Phe Ile Phe Gly Thr Val 110 115 120 125 ATC TGC AAG GCG GTT TCC TAC CTC ATG GGG GTG TCT GTG AGT GTG TCC 615 Ile Cys Lys Ala Val Ser Tyr Leu Met Gly Val Ser Val Ser Val Ser                 130 135 140 ACG CTA AGC CTC GTG GCC ATC GCA CTG GAG CGG TAC AGC GCC ATC TGC 663 Thr Leu Ser Leu Val Ala Ile Ala Leu Glu Arg Tyr Ser Ala Ile Cys             145 150 155 CGA CCA CTG CAG GCA CGA GTG TGG CAG ACG CGC TCC CAC GCG GCT CGC 711 Arg Pro Leu Gln Ala Arg Val Trp Gln Thr Arg Ser His Ala Ala Arg         160 165 170 GTG ATT GTA GCC ACG TGG CTG CTG TCC GGA CTA CTC ATG GTG CCC TAC 759 Val Ile Val Ala Thr Trp Leu Leu Ser Gly Leu Leu Met Val Pro Tyr     175 180 185 CCC GTG TAC ACT GTC GTG CAA CCA GTG GGG CCT CGT GTG CTG CAG TGC 807 Pro Val Tyr Thr Val Val Gln Pro Val Gly Pro Arg Val Leu Gln Cys 190 195 200 205 GTG CAT CGC TGG CCC AGT GCG CGG GTC CGC CAG ACC TGG TCC GTA CTG 855 Val His Arg Trp Pro Ser Ala Arg Val Arg Gln Thr Trp Ser Val Leu                 210 215 220 CTG CTT CTG CTC TTG TTC TTC ATC CCG GGT GTG GTT ATG GCC GTG GCC 903 Leu Leu Leu Leu Leu Phe Phe Ile Pro Gly Val Val Met Ala Val Ala             225 230 235 TAC GGG CTT ATC TCT CGC GAG CTC TAC TTA GGG CTT CGC TTT GAC GGC 951 Tyr Gly Leu Ile Ser Arg Glu Leu Tyr Leu Gly Leu Arg Phe Asp Gly         240 245 250 GAC AGT GAC AGC GAC AGC CAA AGC AGG GTC CGA AAC CAA GGC GGG CTG 999 Asp Ser Asp Ser Asp Ser Gln Ser Arg Val Arg Asn Gln Gly Gly Leu     255 260 265 CCA GGG GCT GTT CAC CAG AAC GGG CGT TGC CGG CCT GAG ACT GGC GCG 1047 Pro Gly Ala Val His Gln Asn Gly Arg Cys Arg Pro Glu Thr Gly Ala 270 275 280 285 GTT GGC GAA GAC AGC GAT GGC TGC TAC GTG CAA CTT CCA CGT TCC CGG 1095 Val Gly Glu Asp Ser Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser Arg                 290 295 300 CCT GCC CTG GAG CTG ACG GCG CTG ACG GCT CCT GGG CCG GGA TCC GGC 1143 Pro Ala Leu Glu Leu Thr Ala Leu Thr Ala Pro Gly Pro Gly Ser Gly             305 310 315 TCC CGG CCC ACC CAG GCC AAG CTG CTG GCT AAG AAG CGC GTG GTG CGA 1191 Ser Arg Pro Thr Gln Ala Lys Leu Leu Ala Lys Lys Arg Val Val Arg         320 325 330 ATG TTG CTG GTG ATC GTT GTG CTT TTT TTT CTG TGT TGG TTG CCA GTT 1239 Met Leu Leu Val Ile Val Val Leu Phe Phe Leu Cys Trp Leu Pro Val     335 340 345 TAT AGT GCC AAC ACG TGG CGC GCC TTT GAT GGC CCG GGT GCA CAC CGA 1287 Tyr Ser Ala Asn Thr Trp Arg Ala Phe Asp Gly Pro Gly Ala His Arg 350 355 360 365 GCA CTC TCG GGT GCT CCT ATC TCC TTC ATT CAC TTG CTG AGC TAC GCC 1335 Ala Leu Ser Gly Ala Pro Ile Ser Phe Ile His Leu Leu Ser Tyr Ala                 370 375 380 TCG GCC TGT GTC AAC CCC CTG GTC TAC TGC TTC ATG CAC CGT CGC TTT 1383 Ser Ala Cys Val Asn Pro Leu Val Tyr Cys Phe Met His Arg Arg Phe             385 390 395 CGC CAG GCC TGC CTG GAA ACT TGC GCT CGC TGC TGC CCC CGG CCT CCA 1431 Arg Gln Ala Cys Leu Glu Thr Cys Ala Arg Cys Cys Pro Arg Pro Pro         400 405 410 CGA GCT CGC CCC AGG GCT CTT CCC GAT GAG GAC CCT CCC ACT CCC TCC 1479 Arg Ala Arg Pro Arg Ala Leu Pro Asp Glu Asp Pro Pro Thr Pro Ser     415 420 425 ATT GCT TCG CTG TCC AGG CTT AGC TAC ACC ACC ATC AGC ACA CTG GGC 1527 Ile Ala Ser Leu Ser Arg Leu Ser Tyr Thr Thr Ile Ser Thr Leu Gly 430 435 440 445 CCT GGC TGAGGAGTAG AGGGGCCGTG GGGGTTGAGG CAGGGCAAAT GACATGCACT 1583 Pro Gly     447 GACCCTTCCA GACATAGAAA ACACAAACCA CAACTGACAC AGGAAACCAA CACCCAAAGC 1643 ATGGACTAAC CCCAACGCAC AGGAAAAGGT AGCTTACCTG ACACAAGAGG AATAAGAATG 1703 GAGCAGTACA TGGGAAAGGA GGCATGCCTC TGATATGGGA CTGAGCCTGG CCCATAGAAA 1763 CATGACACTG ACCTTGGAGA GACACAGCGT CCCTAGCAGT GAACTATTTC TACACAGTGG 1823 GAACTCTGAC AAGGGCTGAC CTGCCTCTCA CACACATAGA TTAATGGCAC TGATTGTTTT 1883 AGAGACTATG GAGCCTGGCA CAGGACTGAC TCTGGGATGC TCCTAGTTTG ACCTCACAGT 1943 GCCCCTTCCC AATCAGCACT GAAAATACCA TCAGGCCTAA TCTCATACCT CTGACCAACA 2003 GGCTGTTCTG CACTGAAAAG GTTCTTCATC CCTTTCCAGT TAAGGACCGA GGCCCTGCCC 2063 TCTCCTTCCT TACCCAAACT GTTCAAGAAA TAATAAATTG TTTGGCTTCC TCCTGAAAAA 2123 AAAAAAAAAA AAA 2136

[0044]

[SEQ ID NO: 3] Sequence length: 30 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Synthetic DNA Array TTCCTGCTCT CCCTGGCAGT CAGTGACCTC

[0045]

[SEQ ID NO: 4] Sequence length: 21 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Synthetic DNA Array TTGCAGTGGT CGGCAGATGG C

[Brief description of drawings]

FIG. 1 is a sequence diagram showing the DNA sequence of the human CCK-B receptor of the present invention and the corresponding amino acid sequence.

FIG. 2 is a graph showing the results of a binding assay for examining the binding properties of the human CCK-B receptor of the present invention and gastrin I or CCK-8.

FIG. 3 is a graph showing the results of a binding assay for investigating the binding properties of the Mastomys gastrin receptor and gastrin I or CCK-8.

FIG. 4 is a graph showing the effect of gastrin I and CCK-8 on the intracellular free calcium concentration of the transformant producing the human CCK receptor of the present invention.

FIG. 5 shows the isolation of mRNA isolated from each human tissue,
1 is an autoradiography chart showing the results of hybridization with a cDNA fragment of the human CCK-B receptor of the present invention.

FIG. 6: CCK-B derived from human brain cells, Mastomys ECL
It is a comparison diagram of the amino acid sequences of gastrin receptor derived from cells, dog parietal cell gastrin receptor, and rat pancreas CCK-A.

Front page continuation (51) Int.Cl. ⁷ Identification code FI C12R 1:91) C12N 15/00 ZNAA (C12P 21/02 C12R 1:91) (72) Inventor Mitsuhiro Ito Arata-cho, Hyogo-ku, Hyogo-ku 1-10-24- 206 (72) Inventor Taizo Taniguchi 5-10-1-801 (72) Inventor Hirohisa Nakata 1-16-15 Souyama-cho, Kita-ku, Kobe-shi, Hyogo (58) ) Fields surveyed (Int.Cl. ⁷ , DB name) C07K 14/595 C12N 5/00 C12N 15/09 C12P 21/02 C12R 1:91 BIOSIS (DIALOG) CAPLUS (STN) MEDLINE (STN) REGISTRY (STN) EMBASE (STN) SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq

Claims (6)

(57) [Claims] 1. From Met at position 1 of SEQ ID No. 2 in the sequence listing to 4
Human cholesiform containing an amino acid sequence consisting of Gly at position 47
Stokinin B receptor. 2. A DNA encoding the polypeptide according to claim 1 . 3. A to 1 at position 193 of SEQ ID NO: 2 in the sequence listing
The DNA according to claim 2, which has a base sequence consisting of up to C at position 533 . 4. An expression vector having the DNA according to claim 2. 5. A transformant obtained by introducing the expression vector according to claim 4 into a host cell. 6. The transformant according to claim 5, wherein the host cell is a Chinese hamster ovary cell (CHO-K1).