JP3993816B2 - DNA encoding granulocyte colony stimulating factor receptor - Google Patents

Description

【図面の簡単な説明】
【図１】 図１はネズミＧ−ＣＳＦレセプターのヌクレオチド配列およびそれより類推されるアミノ酸配列（N末端側）の模式図である。
【図２】 図２はネズミＧ−ＣＳＦレセプターのヌクレオチド配列およびそれより類推されるアミノ酸配列（中間部）の模式図である。
【図３】 図３はネズミＧ−ＣＳＦレセプターのヌクレオチド配列およびそれより類推されるアミノ酸配列（Ｃ末端側）の模式図である。
【図４】 図４はネズミＧ−ＣＳＦレセプターcＤＮＡ(pＩ６２、pＪ１７およびpＦ１)の模式図、制限地図およびヒドロパシープロットを示すグラフ
【図５】 図５は組換えネズミＧ−ＣＳＦを発現するＣＯＳ細胞およびＮＦＳ−６０細胞とネズミ¹²⁵Ｉ−Ｇ−ＣＳＦとの結合を示すグラフであって、Ａはネズミ¹²⁵Ｉ−Ｇ−ＣＳＦとＣＯＳ細胞との飽和結合、ＢはＣＯＳ細胞へのネズミＧ−ＣＳＦ結合データのスキャッチャードプロットを示すグラフ、Ｃはネズミ¹²⁵Ｉ−Ｇ−ＣＳＦのＮＦＳ−６０細胞への飽和結合を示すグラフ、ＤはＮＦＳ−６０細胞へのネズミＧ−ＣＳＦ結合データのスキャッチャードプロットを示すグラフ、
【図６】 図６はＣＯＳ細胞で発現された組換えネズミＧ−ＣＳＦレセプターとネズミＧ−ＣＳＦとの結合特異性を表すグラフ、
【図７】 図７はＣＯＳ細胞およびＮＦ−６０細胞で発現されたネズミＧ−ＣＳＦレセプターのクロスリンキング反応の結果を表すグラフ、
【図８】 図８はネズミＧ−ＣＳＦレセプターmＲＮＡのノーザンハイブリザイゼーション分析の結果を表す写真の模写図、
【図９】 図９はネズミＧ−ＣＳＦレセプターのアミノ酸配列と、プロラクチンおよび成長ホルモンレセプターのアミノ酸配列とを並列に示した配列図である。
【図１０】 図１０はネズミＧ−ＣＳＦレセプターのアミノ酸配列と、ニワトリコンタクチンのアミノ酸配列とを並列に示した配列図である。
【図１１】 図１１はネズミＧ−ＣＳＦレセプターのアミノ酸配列と、ネズミＩＬ−４レセプターのアミノ酸配列とを並列に示した配列図である。
【図１２】 図１２はネズミＧ−ＣＳＦレセプターの模式図である。
【図１３】 図１３はヒトＧ−ＣＳＦレセプターをコードするcＤＮＡ（プラスミドpＨＱ３およびpＨＧ１２）のヌクレオチド配列および推定のアミノ酸配列（N−末端側）を表す図である。
【図１４】 図１４はヒトＧ−ＣＳＦレセプターをコードするcＤＮＡ（プラスミドpＨＱ３およびpＨＧ１２）のヌクレオチド配列および推定のアミノ酸配列（C末端側）を表す図である。
【図１５】 図１５はヒトＧ−ＣＳＦレセプターをコードするcＤＮＡ（プラスミドpＨＱ２およびプラスミドpＨＧ１１）のヌクレオチド配列および推定のアミノ酸配列の一部を表す図である。Ａは図１３−１４に記載のpＨＱ２の配列の内、pＨＱ３の配列と異なる配列部分であって、pＨＱ３のヌクレオチド番号２,０３４から下流に相当する配列のヌクレオチド配列および推定のアミノ酸配列、ＢはpＨＧ１１の配列の内、図１３−１４に記載のpＨＱ２に挿入されている挿入体のヌクレオチド配列および推定のアミノ酸配列を表す図である。
【図１６】 図１６は図１３−１５に記載のpＨＱ３およびpＨＧ１２、pＨＱ２、並びにpＨＧ１１の制限酵素地図の模式図である。
【図１７】 図１７はＣＯＳ細胞により発現された組換えヒトＧ−ＣＳＦレセプターのネズミＧ−ＣＳＦとの結合特性を示すグラフであり、Ａは¹²⁵Ｉ−Ｇ−ＣＳＦのＣＯＳ細胞への飽和結合を示すグラフ、ＢはＧ−ＣＳＦ結合データのスキャッチャード解析の結果を示すグラフである。
【図１８】 図１８はヒトＧ−ＣＳＦレセプターmＲＮＡのノーザンハイブリダイゼーション分析の結果を示す写真の模写図である。
【図１９】 図１９はヒトＧ−ＣＳＦレセプターmＲＮＡのＰＣＲにおける結果を示す写真の模写図である。
【図２０】 図２０はヒトＧ−ＣＳＦレセプターのサザーンハイブリダイゼーション分析の結果を示す写真の模写図、
【図２１】 図２１は発現ベクターpＥＦ−ＢＯＳの模式図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a DNA encoding a granulocyte colony stimulating factor receptor, and more specifically, a DNA encoding a receptor peptide capable of specifically binding to a granulocyte colony stimulating factor (hereinafter referred to as G-CSF), the DNA An expression vector containing the vector, a transformant containing the vector, and a method for producing the receptor by culturing the transformant. Furthermore, the present invention relates to the recombinant G-CSF receptor thus produced.
[0002]
[Prior art]
Blood cell growth and differentiation is regulated by a hormone-like growth and differentiation factor called colony stimulating factor (CSF) [Metcalf, D., Nature,33927-30 (1989)]. CSF is granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and interleukins depending on the situation or stage of action. 3 (IL-3). Among them, G-CSF plays an important role in the proliferation and differentiation of neutrophil granulocytes, and is deeply involved in the control of blood neutrophil concentration and the activation of mature neutrophils. [Nagata, S., Handbook of Experimental Pharmacology, “Peptide Growth Factors and Their Receptors (Peptide Growth Factors and Their] Receptors), edited by Spawn, MB, and Roberts, AB, Springer-Verlag, Heidelberg, Vol. 95 / I, pages 699-722 (1990). ); Nicola, NA, Annu. Rev. Biochem.,5845-77 (1989)]. That is, G-CSF acts on a neutrophil progenitor cell via a receptor (G-CSF receptor) and stimulates its proliferation or differentiation to mainly give neutrophil granulocytes [ Nicola, N.A. and Metcalf, D., Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci. USA),813765-3769 (1984)].
[0003]
G-CSF has a variety of other actions. For example, G-CSF is a regulator of neutrophils by in vivo animal experiments using G-CSF obtained by recombinant DNA technology. (Tuchiya et al., EMBO J.6611-616 (1987); Nicola et al., Annu. Rev. Biochem.5845-77 (1989)).
There are also clinical reports showing that G-CSF administration to cancer patients is advantageous for chemotherapy and bone marrow transplantation (Moustin et al., Trends Pharmacol. Sci.10154-159 (1989)). On the other hand, it is also known that the growth of cancer cells such as myeloid leukemia cells may be stimulated by G-CSF.
[0004]
Thus, despite the fact that G-CSF is a clinically important physiologically active substance, unclear points remain in the mechanism of action, and in order to achieve more effective treatment and diagnosis, the mechanism of action The elucidation of is waiting. For this purpose, it is necessary to characterize the human G-CSF receptor present on the cell surface biochemically and to study the interaction between the receptor and G-CSF.
[0005]
By the way, the cells on which G-CSF acts are limited to neutrophil precursors and mature neutrophils, and various myeloid leukemia cells (Nikola and Metcalf, Proc. Natl. Acad. Sci. USA).813765-3769 (1984), Begley et al., Leukemia.11-8 (1987), Park et al., Blood7456-65 (1989)). Human G-CSF is a polypeptide consisting of 174 amino acids and murine G-CSF is a polypeptide consisting of 178 amino acids. The homology between human G-CSF and murine G-CSF is as high as 72.6% at the amino acid sequence level, and the species specificity is It is also clear that it is rarely recognized (Nikola et al., Nature314625-628 (1985)). On the other hand, G-CSF receptors are non-blood cells such as human endothelial cells (Bussolino et al., Nature337471-473 (1989)) and placenta (Uzumaki et al., Proc. Natl. Acad. Sci. USA).869323-9326 (1989)) was recently reported and the action of G-CSF is more interesting.
[0006]
As described above, elucidation of the interaction between G-CSF and the G-CSF receptor is valuable from an academic point of view, but G-CSF in the prevention and treatment of hematopoietic diseases or other diseases is also clinically relevant. It is important to establish a method for administering CSF and thus achieve a more accurate and effective treatment. On the other hand, as a use of the receptor itself, a solubilized G-CSF receptor is used for certain G-CSF-dependent human leukemia cells (Santori et al., J. Immunol).1393348-3354 (1987)) may also be clinically useful. Furthermore, cancer cells such as myeloid leukemia cells may be proliferated by G-CSF, and the expression of G-CSF receptor in the cells can be examined, and clinical application of G-CSF can be performed more effectively. . As described above, various usefulness has been pointed out for the G-CSF receptor in both research and practical application, and a stable supply of the G-CSF receptor gene and its protein is desired.
[0007]
[Problems to be solved by the invention]
In recent years, genetic engineering techniques have been used for the production of many physiologically active substances. In substance production by genetic engineering, DNA that encodes a target polypeptide is usually cloned, the DNA is incorporated into an appropriate expression vector, and microorganisms, animal cells, etc. are transformed using the obtained recombinant DNA. Convert to express the target substance.
In order to produce a G-CSF receptor by genetic engineering techniques, it is necessary to first clone DNA encoding the target substance, G-CSF receptor. However, G-CSF receptor has a very small presence on the cell surface ( It was difficult to clone the cDNA.
[0008]
[Means for Solving the Problems]
The present inventors have high homology at the amino acid level between human G-CSF and murine G-CSF at 72.6%, and almost no species specificity is observed. Focusing on the fact that cross-reactivity with G-CSF receptor is predicted, first, in order to obtain G-CSF receptor applicable to research and diagnostic analysis, murine (mouse) myeloid leukemia NFS From -60 cells, the receptor was solubilized and purified as a protein with a molecular weight of 100,000 to 130,000. As a purification method, for example, a cell membrane suspension is extracted with CHAPS {3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonic acid}, and then the extract is subjected to G-CSF affinity chromatography ( Recombinant human G-CSF can be treated with affinity chromatography coupled to a gel resin and then purified by gel filtration.
[0009]
On the other hand, mRNA obtained from NFS-60 cells was reverse transcribed, and cDNA encoding the murine G-CSF receptor (hereinafter referred to as G-CSF receptor cDNA) was successfully isolated for the first time. Subsequently, the base sequence of this cDNA was determined, and the amino acid sequence was deduced. The base sequence and the deduced amino acid sequence of murine G-CSF receptor cDNA are shown in FIG.
[0010]
When COS cells were transformed with an expression cloning vector containing murine G-CSF receptor cDNA, the cells expressed a receptor having properties similar to the native murine G-CSF receptor present on NFS-60 cells. As a result of analogizing the amino acid sequence of the G-CSF receptor based on the nucleotide sequence of the murine G-CSF receptor cDNA and comparing it with that of other growth factor receptor families, this murine G-CSF receptor has characteristics in common with them. (See Fig. 7). The probe prepared from the DNA encoding the murine G-CSF receptor thus obtained hybridized with the human G-CSF receptor. This indicates that the G-CSF receptor has no species specificity, and that the human G-CSF receptor is very similar to the mouse G-CSF receptor, so murine G-CSF receptor DNA is human G-CSF receptor. -It is extremely useful as an intermediate for producing a CSF receptor.
[0011]
Next, for the purpose of supplying a sufficient amount of the human G-CSF receptor, the present inventors used a probe prepared from DNA encoding the murine G-CSF receptor and used a total amount derived from human placenta and U937 cells (total ) A cDNA library prepared from mRNA was screened and a cDNA encoding the human G-CSF receptor was successfully cloned.
[0012]
Monkey COS cells transformed with a plasmid encoding the human G-CSF receptor of the present invention produce human G-CSF receptor having specific binding characteristics with G-CSF similar to natural human G-CSF receptor. did.
[0013]
That is, the present invention provides a DNA encoding a G-CSF receptor. The present invention also provides an expression vector encoding a G-CSF receptor. Furthermore, the present invention provides a method for producing a G-CSF receptor, comprising transforming cultured cells with the expression vector, culturing the transformant in a medium, and recovering the G-CSF receptor from the culture. is there.
DETAILED DESCRIPTION OF THE INVENTION
[0014]
In the present specification, the phrase G-CSF receptor peptide refers not only to mature G-CSF receptor but also to all peptide molecules that are part of the receptor molecule and have specific binding activity to G-CSF. Shall.
[0015]
According to the present invention, it has become possible to easily obtain a human G-CSF receptor that has been difficult to isolate by genetic recombination, so that the recombinant G-CSF receptor thus obtained is used. The mechanism of action of G-CSF and G-CSF receptor and research for clinical (diagnostic and therapeutic) applications, and practical application can be promoted. In addition, when clinical application of G-CSF to cancer patients such as leukemia, whether or not the cancer cells express G-CSF receptor should be easily examined using G-CSF receptor cDNA as a probe. Can do. Therefore, such cDNA is thought to be useful for effective clinical application of G-CSF. Furthermore, it is possible to develop proteins and compounds that efficiently bind to the G-CSF receptor by producing a large amount of solubilized G-CSF by genetic recombination and analyzing its tertiary structure. .
[0016]
Cloning of DNA encoding the murine G-CSF receptor was performed by the following method. That is, G-CSF receptor was purified from mouse myeloid leukemia cell NFS-60 with high expression rate of G-CSF receptor, and it was confirmed that its molecular weight was 100,000-130,000 Dalton. Furthermore, total RNA was prepared from the cells by guanidine thioisocyanate / CsCl method, and poly (A) RNA was selected. Subsequently, double-stranded cDNA is synthesized using reverse transcriptase, DNA polymerase, etc., and mammalian expression vector CDM8 (seed, Nature329840-842 (1987)) as a vector was constructed (884 pool of 60-80 clones). Plasmid DNA was prepared from each pool and introduced into COS-7 cells, and two pools I62 and J17 showing binding activity were selected using G-CSF labeled with radioactive iodine. From these pools, two independent clones with high binding activity to G-CSF, plasmids pI62 and pJ17, were identified. COS cells transformed with these plasmids expressed a receptor having binding activity with G-CSF.
[0017]
Subsequently, the nucleotide sequences of plasmids pJ17 and pI62 were determined. As a result, both contained the complete coding region of the murine G-CSF receptor, but also contained the poly (A) sequence and the poly (A) addition signal. I found out. Therefore, the cDNA library was screened again by colony hybridization using 2.5 kb HindIII-XbaI of pJ17 as a probe, and one positive clone (pF1) was selected. This contained a 603 bp 3 ′ non-coding region and two overlapping poly (A) addition signals. The nucleotide sequences of these three cloned cDNAs (pI62, pJ17 and pF1) and the amino acid sequences analogized therefrom are shown in FIGS. 1-3 and SEQ ID NOs: 1 and 2 in the sequence listing. Moreover, the schematic diagram, a restriction enzyme cutting point map, and a hydropathy plot are shown in FIG.
[0018]
The murine G-CSF receptor cDNA cloned according to the present invention has the following characteristics.
It has a long open reading frame (2,511 nucleotides) between the start codon ATG at nucleotides 180-182 and the stop codon TAG at nucleotides 2691-2693. On the 5 ′ upstream side, codons that can be three initiation codons are present at positions 73, 105, and 126, but only a short open reading frame is present downstream. It was found that even when plasmid pI62 was digested with HindIII and these ATG codons were deleted from the cDNA, the expression of recombinant G-CSF receptor in COS cells did not increase or decrease.
[0019]
Furthermore, the N-terminal sequence of the long open reading frame contains a sequence consisting of hydrophobic amino acids, which is considered to be a signal sequence. The N-terminal 25 amino acids were inferred as a signal sequence by comparison with the sequence of a typical signal peptide cleavage site.
[0020]
From the above, it was concluded that the mature murine G-CSF receptor consists of 812 amino acids and has a theoretical molecular weight of 90,814. This value is¹²⁵Compared with the value of the binding experiment with I-iodine-radiated G-CSF (see Example 2 (2), FIG. 7) and the molecular weight of purified murine G-CSF receptor (95,000-125,000) 35,000 daltons less. This difference is probably due to the sugar chain binding to some of the 11 putative N-glycosylation sites (Asn-X-Thr / Ser) found in the extracellular region of the G-CSF receptor. (FIGS. 1-3).
Hydropathy plot of the amino acid sequence of the mature G-CSF receptor (FIG. 4, B) (Kite and Doolite, J. Mol. Biol.157105-132 (1982)), the 24 amino acids from Leu-602 to Cys-625 are uncharged amino acids, followed by 3 basic amino acids. These features are consistent with those found in the transmembrane region of many membrane proteins.
[0021]
Thus, the mature G-CSF receptor consists of an extracellular domain of 601 amino acids, a 24 amino acid transmembrane region (single transmembrane region), and a 187 amino acid cytoplasmic domain. The NH2-terminal 373 amino acids of the extracellular domain are rich in cysteine residues (17/373 amino acids), a property common to the ligand binding regions of many receptors (McDonald et al., British Medical Bulltein).45554-569 (1989)). Erythropoietin receptor (Doandre et al. Cell57277-285 (1989)), the G-CSF receptor is also rich in proline (80 residues, 9.9%), and the murine G-CSF receptor contains a much higher content of tryptophan residues ( 26 residues, 3.2%).
[0022]
As is clear from the above, the murine G-CSF receptor determined according to the present invention is characterized by an N-terminal signal sequence, a single transmembrane region, and an N-terminal portion, which are features common to growth and differentiation factor receptors. It consists of an extracellular domain, an intracellular domain in the C-terminal part.
Furthermore, the following results were obtained by comparing the amino acid sequence of the murine G-CSF receptor with that of other growth factors (FIGS. 9-11). The receptors used for comparison were growth hormone, prolactin, erythropoietin, IL-6, IL-2, IL-4, IL-3, and GM-GSF, all of which belong to the growth factor receptor group. As shown in FIG. 9, cysteine and tryptophan residues commonly found in the growth factor receptor group are also conserved in the G-CSF receptor, and the “WSXWS” motif (Gearing, EMBO J. Biol.83667-3676 (1989), Ito et al., Science.247324-326 (1990)) are also found at amino acid residues 294-298, suggesting that the G-CSF belongs to the same group. According to the comparison of this G-CSF receptor with other blood cell growth factor receptor groups, the homology between G-CSF and IL-6 is 44.6%, but the homology between the receptors is G-CSF receptor and prolactin. Low compared to homology with receptor. In addition, as shown in FIG. 10, the amino acid sequence (376-601) of the extracellular region of G-CSF receptor and chicken contactin (Rancito, J. Cell. Biol.1071561-1573 (1988)), a significant homology is observed with a part of the extracellular region. Contactin is a nerve cell surface glycoprotein (130 kD) and is considered to be related to the transmission of information between cells of the nervous system. The amino acid residues 737-818 of contactin retain homology with fibronectin type III segments related to binding to cells, heparin and DNA, and this region seems to play an important role in cell-cell adhesion. It is. Granulocytes are always formed in the bone marrow, indicating the presence of a direct interaction between neutrophil progenitor cells and myeloid cells (Robert et al., Nature332376-378 (1988)). The similarity between the extracellular region of the murine G-CSF receptor and contactin suggests that this region is involved in the interaction between neutrophil progenitor cells and myeloid cells.
[0023]
The cytoplasmic domain of G-CSF, like other growth factor receptors, is rich in serine (12.8%) and proline (12.3%). And the transmembrane and cytoplasmic region sequences of G-CSF receptor are significantly similar to IL-4 receptor.
[0024]
As shown in FIG. 11, the first 46 amino acids of the transmembrane and cytoplasmic regions of the G-CSF receptor are homologous to the corresponding regions of murine IL-4 (50.0%). Furthermore, amino acid residues 672-808 of the G-CSF receptor have significant similarity (45.4%) with amino acid residues 557-694 of the IL-4 receptor. This suggests that information transmission to cells by G-CSF and information transmission by IL-4 occur by the same mechanism.
[0025]
In the present invention, cDNA was obtained by reverse transcription of mRNA obtained from murine leukemia cell NFS-60 cells. This cDNA was obtained from other cells of the same animal (for example, WEHI-3BD).⁺Cells and mouse bone marrow cells) (see FIG. 8). It is also homologous to the genome of other animal species (eg human).
[0026]
The 3.7-kb mRNA of G-CSF receptor is not only NFS-60 cells but also WEHI-3BD.⁺(Figure). This means that the growth of NFS-60 cells by G-CSF and WEHI-3BD⁺This suggests that cell differentiation is related to the same G-CSF receptor. NFS-60 and WEHI-3BD⁺It seems that G-CSF action on cells has different results of proliferation and differentiation due to different signaling mechanisms downstream from the receptor. For example, the c-myb and evi-1 sites that appear to be involved in bone marrow cell differentiation are rearranged in NFS-60 cells, but WEHI-3BD⁺Differences have been reported that are not rearranged within the cell (Morishita et al., Cell54831-840 (1989)).
[0027]
DNA encoding the murine G-CSF receptor of the present invention can be obtained according to the nucleotide sequence shown in FIGS. This was deposited with the Ministry of International Trade and Industry, Institute of Industrial Science, Microbial Industrial Technology Research Institute under the accession number, Mikuken Bacteria No. 11353 (FERM P-11353) (Deposit Date: March 9, 1990) Escherichia coli. Transferred to an international deposit under the Budapest Treaty. It can be isolated from pI62 in a conventional manner (Accession number in international deposits: MICRO KUJI No. 3312 (FERM BP-3312; transfer date: March 16, 1991), or chemically synthesized. It is possible and can also be obtained by synthesizing a probe of usually about 30 nucleotides based on the sequence and using it as a hybridization probe for a genomic library or cDNA library. The library can be prepared from any species that expresses the G-CSF receptor, such as humans, mice, rats, and other animals, as described above, and methods for synthesizing and hybridizing DNA used as probes are known to those skilled in the art. Preparation of genomic and cDNA libraries is also known to those skilled in the art. It is known.
[0028]
The cDNA encoding the human G-CSF receptor of the present invention was cloned by the following method. That is, total RNA was prepared from human placenta and U937 cells (human histiocytic lymphoma, ATCC CRL 1593) with high expression rate of G-CSF receptor, and poly (A) RNA was selected. Subsequently, double-stranded cDNA was synthesized using reverse transcriptase, DNA polymerase, etc., and a cDNA library was constructed using mammalian expression vector pEF-BOS (see FIG. 21). On the other hand, a hybridization probe was prepared from DNA encoding the above-mentioned murine G-CSF, and the cDNA library was screened by colony hybridization or plaque hybridization using the probe, and a positive clone was selected.
[0029]
Five positive clones were isolated from a cDNA library prepared from U937 cells (pHQ1-pHQ5). On the other hand, more than 100 positive clones were obtained from a cDNA library prepared from human placenta, 6 positive clones were isolated and EcoRI-digested, and the resulting EcoRI fragment was subcloned with pBluescript SK (+). . The isolated cDNA was then analyzed by restriction enzyme mapping and DNA sequencing. As a result, it was found that clones were classified into the following three classes. Most of the cDNAs obtained from placenta and U937 cells belonged to class 1.
[0030]
Class 1: Plasmids pHQ3 (U937 cells) and pH12 (placenta)
This class of clones has a large open reading frame encoding a protein of 836 amino acids. The nucleotide sequence and the deduced amino acid sequence are shown in FIGS. According to the hydropathic analysis of the deduced amino acid sequence, the G-CSF receptor encoded by this class of cDNA is transmembrane consisting of a signal sequence of 23 amino acid residues from the N-terminus, an extracellular domain of 604 residues, and 26 residues. It was shown to contain a region (transmembrane domain) and a cytoplasmic domain of 183 residues. The restriction enzyme cleavage points of plasmid pHQ3 (identical to pH12) are described in FIG.
[0031]
The estimated molecular weight of the human G-CSF receptor (813 amino acids) encoded in this plasmid differs from the molecular weight of the conventional human G-CSF receptor by 30,000-60,000 daltons. This is thought to be due to the glycosylation of nine potential N-glycosylation sites present in the extracellular region.
[0032]
The homology between the human G-CSF receptor cloned according to the present invention and the murine G-CSF receptor is 72% at the nucleotide sequence level and 62.5% at the amino acid sequence level. The homology in the amino acid sequence is the entire region of the molecule. Uniform throughout. There are 17 cysteine residues in the extracellular domain of this human G-CSF receptor, 14 of which are conserved in humans and mice. Furthermore, a conserved “WSXWS” motif common to cytokine receptors also exists in the extracellular domain, indicating that the human G-CSF receptor belongs to the cytokine receptor group.
[0033]
Class 2: Plasmid pHQ2 (U937 cells)
The nucleotide sequence of plasmid pHQ2 is identical to that of pHQ3 except that 88 nucleotides of nucleotide number 2,034-2,121 are deleted from the sequence of pHQ3. This 88 nucleotide region includes a transmembrane region. The nucleotide sequence downstream from the deletion start site of the plasmid is shown in FIG. 15A. The nucleotide sequence and the deduced amino acid sequence of the class 2 clone are shown in SEQ ID NOs: 5 and 6, respectively.
[0034]
As is apparent from the figure, in pHQ2, the sequence of the translational reading frame encoding 150 amino acids downstream from the deletion site is different from that of pHQ3, and it encodes a secreted and solubilized G-CSF receptor. Conceivable. This receptor consists of 748 amino acids, and the calculated molecular weight is 82,707.
[0035]
Class 3: Plasmids pHG11 and pHG5 (placenta)
These plasmids contain an 81 bp insertion at nucleotide position 2,210 in the pHQ3 sequence. The insertion position is the cytoplasmic domain of the G-CSF receptor, and the translational open reading frame is not changed. The nucleotide sequence of the insert and the deduced amino acid sequence are shown in FIG. 15B. In addition, the base sequences and deduced amino acid sequences of class 3 clones are shown in SEQ ID NOs: 7 and 8, respectively. The receptor encoded by plasmid pHQ2 has 27 more amino acids than the class 1 G-CSF receptor and a molecular weight of 2,957. FIG. 16 shows a restriction map of cDNA having this insertion part.
[0036]
When the binding activity of the above three human G-CSF receptor cDNAs to G-CSF was examined, monkey COS cells transformed with class 1 plasmid pHQ3 were found to be murine.¹²⁵It had high affinity with IG-CSF (dissociation constant in binding was 550 pM and the number of receptors was 3.4 × 10^FourCells / cell). The affinity for this binding was almost identical to the affinity for binding of murine G-CSF to the murine G-CSF receptor expressed by COS cells. The binding affinity of human G-CSF to the receptor naturally present on the surface of U937 cells has a dissociation constant of 424 pM [Park, L.S., Waldron, P.E., Friend (Friend, D. Sassenfeld, H.M., Price, V., Anderson, D., Cosman, D., Andrews, R.G., Bernstein (Bernstein, I.D.) and Ardal (D.L.), Blood,7456-65 (1989)], the polypeptide encoded by pHQ3 is a receptor with sufficiently high affinity for G-CSF.
[0037]
On the other hand, since the polypeptide encoded by class 2 pHQ2 is partially deleted, COS cells and mice transformed with pHQ2 cDNA¹²⁵Binding to IG-CSF was very low. The dissociation constant is 440 pM and the binding site is 6 × 10³/ Cells. This also suggests that the receptor encoded by pHQ2 lacking the transmembrane region is a solubilized form secreted from cells.
[0038]
In order to examine the binding characteristics of class 3 cDNA, the expression plasmid pQW11 was constructed by inserting the 5 ′ sequence of pHQ3 cDNA and the 3 ′ sequence of pH11 into the mammalian expression vector pEF-BOS. COS cells were transformed with this plasmid, and the resulting transformants and mice¹²⁵-As a result of binding analysis with G-CSF, it was found that the 27 amino acid insert in the cytoplasmic domain had little influence on the binding activity with G-CSF.
[0039]
The nucleotide sequences of DNAs encoding the human G-CSF receptor of the present invention are described in FIGS. 13 and 14 and SEQ ID NOs: 3, 5, and 7. This DNA was deposited with the Institute of Microbial Industrial Technology, Ministry of International Trade and Industry under the deposit number No. 11666, No. 11567, and No. 11568 (Deposit date: June 28, 1990). After that, under the Budapest Treaty, Escherichia coli transferred to the international deposit on March 16, 1991. pHQ2, Escherichia coli. pHQ3 and Escherichia coli. It can be isolated from pHG11 as usual. The accession numbers and transfer dates for these international deposits are as follows. Escherichia coli. pHQ2; accession number: fine work Kenjo No. 3313 (FERM BP-3313); Escherichia coli. pHQ3: Fine Work Research Institute No. 3314 (FERM BP-3314); Escherichia coli. pH11: Fine Work Research Institute, No. 3315 (FERM BP-3315).
[0040]
We also analyzed various human tissue cells for the presence of G-CSF receptor RNA by Northern hybridization using probes prepared from these human G-CSF receptor cDNAs. U937 cells, placenta and KG-1 cells gave a single band of 3.7 kb, and among them, it was confirmed that the placenta expressed a large amount of mRNA of G-CSF receptor.
[0041]
Furthermore, the type of receptor expressed by the cells was examined by PCR method. Class 1G-CSF receptor was U937 and placental cells, class 2 solubilized receptor was U937 cells, and class 3 receptor having an insert. Was confirmed to be expressed by placental cells.
[0042]
Further, as a result of examining the number of genes encoding the G-CSF receptor by Southern hybridization, it was found that a single gene exists per human haploid genome.
[0043]
According to the present invention, the nucleotide sequence of DNA encoding the human G-CSF receptor has been clarified. An expression vector that functions in an appropriate host system is constructed using this DNA, and a host cell is transformed with the expression vector. The recombinant human G-CSF receptor can be produced by culturing the obtained transformant. The recombinant human G-CSF receptor thus obtained is useful for various purposes such as diagnosis of leukemia and elucidation of the mechanism of action of human G-CSF.
[0044]
The nucleotide sequence of the cDNA encoding the G-CSF receptor of the present invention is described in FIGS. 1-3 and SEQ ID NO: 1 for mice, and FIGS. 13-15 and SEQ ID NOs: 3, 5, and 7 for humans. Those skilled in the art will understand that derivatives of this activity can be easily derived from these DNAs by nucleotide insertions, substitutions or deletions. Therefore, the DNA thus derived is also included in the scope of the present invention.
[0045]
The polypeptide that binds to G-CSF used to achieve the object of the present invention is not necessarily required to be a mature G-CSF receptor. Rather, it may be preferable that the fragment has a binding activity to G-CSF. Similarly, a DNA fragment encoding a part of the mature G-CSF receptor having binding activity with the entire sequence of DNA encoding the mature G-CSF receptor and DNA encoding the G-CSF is also useful.
[0046]
Therefore, the present invention provides a receptor peptide capable of binding to G-CSF, and a DNA encoding the peptide.
[0047]
Thus, the DNA encoding the G-CSF receptor peptide of the present invention includes DNA encoding a mature G-CSF receptor and a peptide fragment having the binding activity of the mature G-CSF receptor to G-CSF. .
[0048]
Using the DNA of the present invention, DNA encoding G-CSF receptor of other animal species is obtained, an expression vector for G-CSF receptor is constructed, and introduced into an appropriate cultured cell to express G-CSF receptor. be able to.
[0049]
Since an expression vector containing DNA encoding the G-CSF receptor of the present invention can be constructed by various methods, those skilled in the art may select as appropriate. A vector suitable for expression of the G-CSF receptor gene preferably has a promoter for initiation of transcription immediately upstream of the insertion site of the G-CSF receptor DNA sequence. Appropriate promoters are also known in the art and can be selected according to functional properties in the host cell. For example, the mouse metallothionein promoter and SV-40 small T promoter can be used for murine and monkey cells, respectively. Bacterial promoters can also be used to express G-CSF in bacteria. It is desirable that there is a poly-A signal downstream of the insertion site of the G-CSF receptor sequence. Desirably, a selectable marker such as a drug resistance marker is present in the vector. A particularly desirable marker is a neomycin resistance gene.
[0050]
An expression vector can be constructed by inserting DNA encoding the G-CSF receptor into an appropriate vector. A suitable vector is selected from those known in the art in consideration of the promoter, polyA signal, selection marker and other conditions. Examples of DNA vectors that can be used for the purpose of inserting the cDNA of the present invention and introducing it into cultured cells to express the cDNA include pSV2 and bovine papilloma virus DNA.
[0051]
The cultured cells that can be used for expression of the G-CSF receptor of the present invention may be any cells that can replicate and can express the DNA described in FIG. 1-3 or FIG. 13-15. For example, prokaryotic microorganisms such as E. coli, eukaryotic microorganisms such as S. cerevisiae, and mammalian cells are used. Tissue culture cells include avian or mammalian cells such as murine, rat and monkey cells. Selection and use methods of an appropriate host cell-vector system are known to those skilled in the art, and a system suitable for expression of the cDNA encoding the G-CSF receptor of the present invention can be arbitrarily selected from them. it can.
[0052]
The present invention will be described in more detail with reference to examples below, but these examples do not limit the present invention.
【Example】
Example 1  Cloning of murine G-CSF receptor DNA
1) Cells
Murine myeloid leukemia cells NFS-60 (Weinstein et al., Proc. Natl. Acad. Sci. USA)835010-5014 (1986); donated by J. Ihle of St. Jude Childress Research Hospital) 10% fetal calf serum (FCS) and 10-20 units / ml recombinant mouse IL-3 Cultured in RPMI 1640 medium supplemented with
COS-7 cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% FCS as usual.
[0053]
2) Growth and differentiation factors such as recombinant G-CSF
Bovine papillomavirus expression vector (Fukunaga et al., Proc. Natl. Acad. Sci. USA) carrying human G-CSF cDNA (Tsuchiya et al., 1987, supra).81, 5086-5090 (1984)), human recombinant G-CSF was purified from the culture solution of mouse C127I cells transformed.
Mouse G-CSF was purified as a homogeneous protein using a similar expression system. Human recombinant G-CSF and M-CSF produced by Chinese hamster ovary cells were obtained from Chugai Pharmaceutical, Japan.
Human recombinant G-CSF produced by E. coli was purchased from Amersham.
Mouse recombinant IL-3 and GM-CSF were obtained from Miyajima and Allai (DNAX Institute).
Mouse recombinant IL-6 and mouse recombinant LIF were obtained from Mr. Hirano (Osaka University) and Mr. Nicola (Walter Eliza Hall Institute), respectively.
Rat prolactin was purchased from Chemicon International, Inc.
Murine recombinant G-CSF labeled with radioactive iodine is produced by the modified IODO-GEN method (Fraker and Spec, Biochem. Biophys. Res. Commun.80849-857 (1978)) [specific activity: 6-8 × 10^Fourcpm / ng protein (1,200-1,600 cpm / fmole)].
[0054]
3) CDM8 cDNA library
Total RNA was prepared from logarithmic growth phase NFS-60 cells by guanidine isothiocyanate / CsC1 method, and poly (A) RNA was selected by oligo (dT) -cellulose column chromatography. Using the reverse transcriptase (purchased from Seikagaku) and Amersham kit, the literature description (Nagata et al., Nature319415-418 (1986)), and double-stranded cDNA was synthesized.
[0055]
A BstXI adapter was added to the resulting blunt-end cDNA and electrophoresed on a 1% agarose gel. A cDNA of 1.8 kb or more was recovered from the gel, ligated to BstXI-digested mammalian expression vector CDM8 (Seed, 1987, supra), and E. coli MC1061 / p3 cells were transformed with the obtained DNA ( Electroporation, Dowell et al., Nucleic Acids Res.166127-6145 (1988)).
[0056]
4) Preparation of DNA
6x10^FourIndividual bacterial colonies were plated in 24-well microtiter plates at a density of 60-80 colonies per well. Glycerin cultures were prepared for each pool of colonies. A part of each glycerin culture was inoculated into LB broth, and plasmid DNA was prepared from each pool by boiling method (boiling method) (Maniatis et al., Molecular Cloning: A Laboratory Manual 1982), and subjected to phenol extraction and ethanol precipitation. .
[0057]
5) Transfection of COS-7 cells
A monolayer culture of COS-7 cells was obtained in a 6-well microtiter plate and the cells were treated with the modified DEAE dextran method (Sonpyirak and Danna, Proc. Natl. Acad. Sci. USA).787575-7578 (1981)), and was transfected with the plasmid DNA prepared in 4) above.
That is, about 50% of the fully grown cells were washed three times with serum-free DMEM and contained 50 mM Tris-HCl (pH 7.3), 0.3 mg / ml DEAE-dextran and 1 μg of plasmid DNA for 8 hours at 37 ° C. Incubated in 0.6 ml DMEM. After 20% glycerin-containing Tris-HCl buffered saline for 2 minutes at room temperature, the cells were washed twice with DMEM and incubated in DMEM containing 10% FCS.
[0058]
6) Screening of COS-7 cells (transformants) expressing G-CSF receptor
72 hours after transfection, COS-7 cells were washed with DMEM (pH 7.3) (binding medium) containing 10% FCS and 20 mM HEPES in 0.6 ml of binding medium.¹²⁵I-G-CSF (1.7 × 10^Fivecpm (200 pM)) at 37 ° C. for 2 hours. Unbound radioactive G-CSF was removed and the cells were treated with 0.7 mM CaCl.₂And 0.5 mM MgCl₂Washed 3 times with phosphate buffered saline (PBS) supplemented with 1 and once with PBS. Cells were then harvested by trypsinization and the radioactivity bound to the cells was counted with an AUTO-GAMMA 5000 MINAXI gamma counter (Packard).¹²⁵Background binding between I-labeled G-CSF and COS cells transfected with CDM8 vector was 308 ± 38 (SD) cpm. On the other hand, among COS cells transformed with a plasmid DNA pool containing cDNA, COS cells into which plasmid DNAs of two pools (I62 and J17) were introduced were¹²⁵Significant binding with IG-CSF was observed (500 cpm and 912 cpm, respectively).
[0059]
144 independent clones from each positive pool (I62 and J17) were cultured in 24-well microtiter plates (6 plates) and subjected to sib selection using 12 × 12 clones (Maniatis et al., Supra, 1982). After plasmid mini-preparation and COS-7 cell transfection,¹²⁵A binding reaction with IG-CSF was performed and a single clone was selected from each positive pool.
[0060]
That is, pool I62 and J17 bacterial clones were divided into 12 subgroups of 12 clones each. Some subgroups are directed to COS cells¹²⁵The binding reaction of IG-CSF was 3710 and 4010 cpm. A single clone of each positive subgroup was then analyzed to identify two independent clones pI62 and pJ17. When COS-7 cells were transfected with plasmid DNAs of pI62 and pJ17¹²⁵The IG-CSF binding values were 30300 cpm and 31600 cpm, respectively. Subsequently, the nucleotide sequences of plasmids pJ17 and pI62 were determined. Both of these contained the complete coding region of the G-CSF receptor, but also contained the poly (A) sequence and the poly (A) addition signal. I found that there was no. Therefore, the cDNA library was screened again by colony hybridization using 2.5 kb HindIII-XbaI of pJ17 as a probe, and one positive clone (pF1) was selected. This contained a 603 bp 3 'non-coding region, in which there were two overlapping poly (A) addition signals. The base sequences of these three cloned cDNAs (pI62, pJ17 and pF1) and the amino acid sequences deduced therefrom are shown in FIG. 1-3. Moreover, the schematic diagram, a restriction enzyme cutting point map, and a hydropathy plot are shown in FIG.
[0061]
Example 2  Characterization of cloned murine G-CSF receptor DNA
1) Binding activity of cloned G-CSF receptor
¹²⁵The binding of IG-CSF to COS cells and NFS-60 cells was examined.
COS cells cultured on 15 cm plates were transfected with 20 μg pI62 or pJ17. Twelve hours after glycerin shock, the cells were divided into 6-well microtiter plates and cultured in DMEM containing 10% FCS for 60 hours. Cells are washed with binding medium and various amounts of¹²⁵Incubated with I-G-CSF (range 10 pM-1.2 nM) at 4 ° C. for 4 hours.¹²⁵In order to measure non-specific binding between IG-CSF and cells, a binding reaction is performed in the presence of a large excess of unlabeled G-CSF (800 nM), and the non-specifically bound radioactivity is subtracted from the total binding activity. Specific binding activity was determined.
[0062]
On the other hand, 5.2 × 10⁶Individual NFS-60 cells at various concentrations¹²⁵By incubating at 4 ° C. for 4 hours in 0.3 ml of RPMI-1640 medium consisting of 10% FCS containing I-G-CSF and 20 mM HEPES (pH 7.3), the cells were allowed to bind to G-CSF. It was. The results are shown in FIG. A is¹²⁵Saturation binding between IG-CSF and COS cells is shown. As above, 1 × 10 5 with plasmid pJ17⁶After transfection of COS cells, various amounts of¹²⁵Incubation was performed in the presence or absence of excess amounts of unlabeled G-CSF in the presence of IG-CSF. White circles indicate total binding, white triangles indicate non-specific binding, black circles indicate specific binding, and specific binding is a value obtained as a difference between total binding and non-specific binding. B is a graph showing a Scatchard plot of G-CSF binding data to COS cells. C is¹²⁵FIG. 2 is a graph showing saturation binding of I-G-CSF to NFS-60 cells, in which white circles indicate total binding, white triangles indicate non-specific binding, black circles indicate specific binding, and specific binding indicates total binding and non-specific binding. It is a value obtained as a difference. D is a graph showing a Scatchard plot of G-CSF binding data to NFS-60 cells. From the graph, the equilibrium dissociation constant of G-CSF receptor expressed in COS cells is 290 pM, and 3.0 × 10 6 per cell.^FourIt can be seen that there are individual receptors. COS cell transfection efficiency of 10-20% (Songpilac and Donna, Proc. Natl. Acad. Sci. USA)78, 7575-7578 (1981)) and transfection-positive COS cells expressed recombinant G-CSF receptor 1.5-3.0 × 10^FiveIt is predicted that it is expressed at the frequency of individual / cell. Since the equilibrium dissociation constant of the intrinsic G-CSF receptor on NFS-60 cells is 180 pM (FIG. 5D), these results indicate that the cDNA encoded by plasmid pJ17 has a high affinity for murine G-CSF. Suggesting that it is sufficient to express
[0063]
Subsequently, the binding specificity of the recombinant G-CSF receptor expressed in COS cells with G-CSF was examined. As described above, COS cells transfected with G-CSF receptor cDNA (pJ17) were treated with 1 μg of unlabeled murine G-CSF, human G-CSF, murine GM-CSF, murine IL-6, murine LIF, rat prolactin. Or 2 ng in the presence or absence of human M-CSF¹²⁵Incubated with IG-CSF. Recombinant human G-CSF produced by murine C127 cells, Chinese hamster ovary cells or E. coli was used as human G-CSF. Bound to COS cells in each experiment¹²⁵The radioactivity of IG-CSF was expressed as a percentage of the value in the absence of competitor. The results are shown in FIG.
[0064]
Human G-CSF is murine G-CSF and murine WEHI-3B D⁺Compete for binding with cells (Nicola et al., 1985, supra). Unlabeled recombinant human G-CSF produced by either mammalian cells or E. coli competed well with labeled murine G-CSF in the binding reaction of COS cells transfected with plasmid pJ17 (FIG. 6). In contrast, depending on unlabeled recombinant murine GM-CSF, murine IL-3, murine IL-6, murine leukemia inhibitory factor (LIF), rat prolactin or human M-CSF¹²⁵The binding of IG-CSF to COS cells was not inhibited at all.
[0065]
2) Cross-linking reaction
G-CSF¹²⁵A cross-linking reaction between IG-CSF and a receptor expressed by COS cells was performed as follows.
8 × 10 transfected with plasmid pI62 as above^FiveCOS cells (3.5 cm plate) of 1.2 nM¹²⁵Incubated with I-G-CSF for 2.5 hours at 4 ° C. in 0.6 ml binding medium in the presence or absence of 1.5 μM unlabeled G-CSF. Cells were scraped from the plate using a cell lifter and washed 3 times with 1 ml of PBS. The cross-linking reaction was performed on ice for 20 minutes in 1 ml PBS containing 150 μM disuccinimidyl suberate (DSS) and 150 μM disuccinimidyl tartrate (DST). The reaction was stopped by adding 50 μl of 1 M Tris-HCl (pH 7.4), centrifuged to collect the cells, protease inhibitor mixture (2 mM EDTA, 2 mM (p-aminophenyl) methanesulfonyl fluoride hydrochloride, 2 mM O-phenanthroline). The cells were lysed with 15 μl of 1% Triton X-100 containing 0.1 mM leupeptin, 1 μg / ml pepstatin A and 100 units / ml aprotinin). The supernatant was obtained by centrifugation, and this clear lysate (10 μl) was analyzed by 4-20% polyacrylamide gel gradient electrophoresis in the presence of SDS (Lemli, Nature).227680-685 (1970)). The film was exposed to X-ray film at -80 ° C for 2 days using a reinforced screen. As a size marker¹⁴A C-label molecular weight standard (rainbow marker, Amersham) was electrophoresed in parallel.
[0066]
The results are shown in FIG. Lane 2 represents the result of the cross-linking reaction in the presence of an excess amount of unlabeled murine G-CSF, and lanes 3 and 4 represent the absence. However, in lane 3, it is a result of reaction which does not contain DSS and DST. Lane 5 shows murine NFS-60 cells (3 x 10⁶Cell / lane) in the same way¹²⁵Lane 6 represents the results of an experiment that was incubated with IG-CSF in the presence of excess unlabeled G-CSF and in the absence and cross-linked by DSS and DST. Lanes 1 and 7 were each used as a size marker¹⁴C label molecular weight standard (rainbow marker, Amersham). The molecular weight of the protein standard is indicated in kD.
[0067]
A cross-linking reaction between G-CSF receptor on NFS-60 cells and labeled murine G-CSF (molecular weight 25,000) yields an apparent molecular weight of 125,000-155,000 (lane 6), which is NFS This suggests that the molecular weight of the murine G-CSF receptor on -60 is 100,000-130,000. Similarly, mice¹²⁵A main band was obtained with a molecular weight of 120,000-150,000 (lane 4) by cross-linking reaction between IG-CSF and a receptor expressed on COS cells, which is slightly different from the value detected in NFS-60 cells. ing. These bands were not observed for cross-linking reactions in the presence of unlabeled G-CSF (lanes 2 and 5) or in the absence of binding reagent (lane 3). Slight differences in the molecular weights of COS and NFS-60 cells can be explained by the different glycosylation of the receptors in these cell lines.
[0068]
3) Hybridization
Colony hybridization and Northern hybridization were performed according to the method described in the literature (Maniatis, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory (1982)). As a probe, a 2.5 kb HindIII-XbaI fragment of clone pJ17 was subjected to random primer labeling (Fineberg and Bogelstein, Anal. Biochem.1326-13 (1983))³²Those labeled with P were used. The result of Northern hybridization is shown in FIG.
[0069]
Total RNA or poly (A) RNA prepared from various murine tissues was used. They are L929 (lane 1), NFS-60 (lanes 2 and 3), FDC-P1 (lane 4), WHEI-3BD.⁺(Lane 5) or mouse tissue, brain (lane 6), lung (lane 7), spleen (lane 8), bone marrow (lane 9), liver (lane 10), and kidney (lane 11). 30 μg of total RNA (lanes 1 and 3-11) or 2 μg of poly (A) RNA (lane 2) were subjected to electrophoresis on 1.3% agarose gel containing 6.6% formaldehyde, and Northern hybridization was performed according to the method described in the above literature. Analyzed with
[0070]
Example 3  The base sequence of the cloned murine G-CSF receptor DNA and the amino acid sequence of the polypeptide encoded by the DNA
1) Nucleotide sequencing
DNA sequencing was performed using T7-DNA polymerase (Pharmacia) and α-³⁵This was performed by a dideoxynucleotide chain determination method using S dATPαS (Amersham). The results are shown in FIGS. 1-3 and FIG.
FIG. 1-3 shows the nucleotide sequence of murine G-CSF receptor cDNA (pI62, pJ17 and pF1) and the amino acid sequence deduced therefrom. The numbers above and below each line represent the nucleotide position and amino acid position, respectively, starting with the mature G-CSF receptor Cys-1. In the amino acid sequence, the signal sequence and the transmembrane region are underlined. Two overlapping poly (A) addition signals (AATAAA) are also underlined. Potential N-glycosylation sites (Asn-X-Ser / Thr) (11 in the extracellular region and 2 in the cytoplasmic region) are boxed.
[0071]
FIG. 4 is a murine G-CSF receptor cDNA, and A is a schematic diagram and restriction map of three independent cDNAs (pI62, pJ17, pF1). The part enclosed by a rectangle is an open reading frame. The stippled and filled portions represent the signal sequence and the transmembrane region, respectively. Restriction enzyme cleavage sites are also shown. B is a hydropathy plot of the amino acid sequence of the murine G-CSF receptor, which was obtained by the Kite and Dolite method (1982) using a 10 residue window. The numbers at the bottom of the figure represent the positions of the amino acid residues of the precursor protein.
[0072]
2) Comparison of amino acid sequence of G-CSF receptor with other growth factor receptor amino acid sequences (FIGS. 9-10).
FIG. 9 shows the G-CSF receptor, prolactin and growth hormone receptor in parallel. A comparison of the murine G-CSF receptor 96-317 amino acid sequence with rat prolactin and human growth hormone receptors shows the maximum homology obtained by introducing several gaps (-). Residues that are considered identical in the two sequences are surrounded by a solid line, and residues that are considered favored substitutions are surrounded by a dashed line. Preferred amino acid substitutions are defined as substitutions between pairs of amino acids belonging to any one of the following groups: S, T, P, A and G; N, D, E and Q; H, R and K; M , I, L and V; F, Y and W. Conserved among 9 members of the growth factor receptors (family) (G-CSF, prolactin, growth hormone, eleslopoietin, GM-CSF, IL-2β, IL-3, IL-4 and IL-6) Amino acids are listed with parentheses below each line and without parentheses. Residues without parentheses were conserved in more than 8 members, whereas residues in parentheses were conserved in 5-7 members of the family.
[0073]
FIG. 10, like FIG. 9, shows the 376-601 amino acid sequence of murine G-CSF receptor and the amino acid sequence of chicken contactin in parallel.
FIG. 11 shows the murine G-CSF receptor amino acid sequence 602-808 in parallel with the murine IL-4 receptor as described above.
FIG. 12 is a schematic diagram of the murine G-CSF receptor, and the boxed area represents the mature G-CSF receptor. “TM” represents a transmembrane region, region “A” represents a domain (222 amino acids) similar to other growth factor receptors (prolactin and growth hormone receptors), and a “WSXWS” motif can be seen. The region "B" (226 amino acids) of the murine G-CSF receptor is similar to chicken contactin. Region "C" (211 amino acids) contains the transmembrane domain (underlined) and the cytoplasmic domain of the G-CSF receptor and is similar to the two regions of the murine IL-4 receptor.
[0074]
Example 4  Cloning of human G-CSF receptor cDNA
(1) Isolation of human G-CSF receptor cDNA clone
Poly (A) RNA was prepared from U937 cells, and reverse transcriptase (purchased from Seikagaku Corporation) and Amersham cDNA synthesis kit were used to describe the literature (Nagata et al., Nature,319415-418 (1986)), a double-stranded cDNA was synthesized. A BstX1 adapter was added to the resulting blunt-end cDNA, and electropermanent was performed on a 1% agarose gel. A cDNA of 2.5 kb or more was recovered from the gel and bound to the mammalian expression vector pEF-BOS (FIG. 21).E .coli DH1 cells were electroporated (Dowell et al., Nucleic Acids Res.,166127-6145 (1988)).
The total number of libraries obtained was then 3.4 x 10^FourIndividual clones were screened by colony hybridization.
[0075]
Hybridization probes were prepared by random oligonucleotide primer labeling of 2.5 kb HindIII-XbaI fragment of murine G-CSF receptor cDNA (Sambrook, J., Fritsch, EF and Maniatis, T). .. Molecular Cloning (1989): A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York: Cold Spring Harbor・ In the laboratory (Cold Spring Harbor Laboratory)]³²Those labeled with P were used. The probe used here is murine G-CSF receptor cDNA (pI62). The nucleotide sequence and the deduced amino acid sequence are shown in FIGS. 1-3. In FIG. 1-3, the signal sequence and the transmembrane region are underlined. Potential N-glyphsyl sites are also boxed.
[0076]
Hybridization was achieved by lowering the hybridization temperature to 28 ° C. and filtering the filter at 37 ° C. with 150 mM NaCl / 15 mM sodium citrate, pH 7.0 / 0.1% NaDodSo.₄Other than washing in the literature [Fukunaga, R., Matsuyama, M., Okamura, H., Nagata, K., Nagata, S. and Sokawa] , Y.), Nucleic Acids Research,144421-4436 (1986)]. That is, a colony replica filter was prepared, and each nitrocellulose filter and a probe heated at 95 ° C. for 5 minutes and then rapidly cooled were hybridized at 28 ° C. for 12 hours. Subsequently, after the filter was washed by the method described above, the target clone was searched for by autoradiography.
[0077]
On the other hand, a human placenta cDNA library constructed using λgt11 was purchased from Clonetech and screened by plaque hybridization using murine G-CSF receptor cDNA.
That is, about 1.5 × 10⁶The phage DNA of individual clones was obtained by the method described by Benton and Divis (Benton, WD & Davis, RW, Science,196, 180-182 (1977)), followed by screening by plaque hybridization. The probe DNA and hybridization conditions used are the same as the probes and conditions used for screening the U983 cDNA library described above.
[0078]
Five positive clones were isolated from a cDNA library prepared from U937 cells (pHQ1-pHQ5).
On the other hand, human placental cDNA library (1.5 × 10 5⁶Clones showing 100 or more positive signals in plaque hybridization of mouse G-CSF receptor cDNA and EcoRI fragments of 6 positive clones (λHG4, 5, 11, 12, 14 and 18) Was subcloned into the pBluescript SK (+) vector and designated pHG4, 5, 11, 12, 14 or 18.
[0079]
For DNA sequencing, a series of plasmids deleted approximately 300 bp in length were prepared using exonuclease III and mung bean nuclease (Sunbrook et al., Supra). Sequencing was then performed using T7 DNA polymerase, deaza dGTP and α-³⁵This was performed by the dideoxy chain termination method using SdATPα-S.
As a result of sequencing, it was found that cDNAs isolated from U937 and placental cDNA libraries were classified into the following three classes.
[0080]
Class 1: Plasmids pHQ3 (U937) and pH12 (placenta)
Most cDNAs isolated from U937 and placental cDNA libraries belong to this class. The nucleotide sequences of these plasmids and the deduced amino acid sequences are shown in FIGS. 13-14. A map showing the restriction enzyme cleavage site is shown in FIG.
[0081]
These class 1 plasmids have a large open reading frame encoding a protein of 836 amino acids. As a result of hydropathic analysis of the deduced amino acid sequence, as is apparent from the figure, the N-terminal 23 amino acid residues correspond to the signal sequence, a 604 residue extracellular domain, a 26 residue transmembrane region, and 183 Followed by the cytoplasmic domain of the residue. There are nine potential N-glycosylation sites in this extracellular region.
[0082]
In addition, there are 17 cysteine residues in the extracellular domain of the peptide encoded by this cDNA, 14 of which are conserved in humans and mice. The domain also has a “WSXWS” motif conserved in cytokine receptors, indicating that the human G-CSF receptor belongs to cytokine receptors.
[0083]
The homology between human G-CSF receptor (813 amino acids) and murine G-CSF receptor encoded by this class 1 plasmid was 72% at the nucleotide sequence level and 62.5% at the amino acid sequence level. Also, the homology in the amino acid sequence was uniform throughout the entire region of the molecule.
[0084]
Class 2: Plasmid pHQ2 (U937 cells)
The nucleotide sequence of plasmid pHQ2 is identical to that of pHQ3 except that 88 nucleotides of nucleotide number 2,034-2,121 are deleted from the sequence of pHQ3. The end of the deleted sequence is indicated by a black arrowhead in the sequence described in FIGS. 13-14.
[0085]
As is apparent from the figure, the 88 nucleotide deletion region includes a transmembrane region. The nucleotide sequence downstream from the deletion is shown in FIG. 15A.
In pHQ2, the translational reading frame encoding 150 amino acids downstream from the deletion site has been altered by this deletion (see FIG. 16), and is thought to encode a secreted and solubilized G-CSF receptor. It is done. This solubilized receptor consists of 748 amino acids, and the calculated molecular weight is 82,707.
[0086]
Class 3: Plasmids pHG11 and pHG5 (placenta)
The nucleotide sequences of these plasmids are those in which an 81 bp DNA fragment is inserted at nucleotide number 2,210 of pHQ3. The insertion position is in the cytoplasmic domain of the G-CSF receptor, and is the site indicated by the thick arrow in FIGS. 13-14. The translation open reading frame has not changed. The nucleotide sequence of the insert and the deduced base sequence are shown in FIG. 15B. Therefore, the receptor encoded by plasmid pHQ2 is 27 amino acids larger and 2,957 larger in molecular weight than the first class G-CSF receptor.
[0087]
The nucleotide sequences and deduced amino acid sequences of these three classes of plasmids are set forth in FIGS. 13-15 and SEQ ID NOs: 3-8. 13-14, various symbols in the sequence of pHQ2 in A have the following meanings. The amino acid sequence number is such that the amino acid Glu is number 1. The signal sequence and transmembrane region are indicated by bold underlining, and the N-glycosylation site (Asn-X-Thr / Ser) is indicated by a square. The “WSXWS” motif portion conserved in the cytokine receptor family is indicated by a double underline. The black arrowhead represents the end of the sequence deleted in pHQ2, and the thick arrow represents the insertion site of the insert in pH11. A thin arrow indicates an oligonucleotide primer part used in the PCR method described later. In FIG. 15A, the sequence downstream from Δ indicates the nucleotide sequence corresponding to the sequence downstream of nucleotide 2,034 from which pHQ3 has been deleted, and the deduced amino acid sequence in pHQ2. FIG. 15B shows the nucleotide sequence and deduced amino acid sequence of the insert at pH 657 at amino acid position 657, contained in pH11. The inserted sequence is enclosed in parentheses.
[0088]
A restriction map of the above three classes of plasmids is shown in FIG. In the figure, the part enclosed by a square represents an open reading frame, the shaded part represents a signal sequence, and the filled part represents a transmembrane region. The hatched portion in pHQ2 is a portion changed from the open reading frame of other cDNA, and represents a sequence encoding a different amino acid. The hatched portion in pHG11 represents an insert sequence portion encoding 27 amino acids.
[0089]
(2) Detection of G-CSF receptor mRNA in human cells
Human G-CSF receptor mRNA was detected by polymerase chain reaction (PCR) using primers prepared from the above cDNA.
The synthesis and PCR of single-stranded cDNA is substantially performed by Kawasaki [Kawasaki, ES, PCR Protocols, “A guide to methods and applications (A guide to methods and applications]). and application), Innis, M.A., gel fund (Gelfand, D.H.), Shinsky, J.J. and white (White, T.J.), Academic Press, San Diego, CA (Academic Press, San Diego, CA), pages 21-27 (1990)], and the results are shown in FIG.
[0090]
That is, random RNA (lanes 2 and 5) or poly (A) RNA (lanes 21 and 4) obtained from human placenta or U937 cells, or 2 μg of human placenta (lanes 3 and 6) in a random amount of 6 μl in a reaction mixture of 50 μl. In the presence of 0.5 μg of the body (hexamer) and 80 units of AMV reverse transcriptase, it was subjected to cDNA synthesis as previously described (Kawasaki et al., Supra) (Kawasaki et al., Supra).
5 μl of the reaction mixture is taken, diluted with 100 μl of PCR buffer containing 50 pmol each of the following forward and reverse primers and placed in a DNA thermal cycler (Perkin-Elmer-Setas) preheated to 80 ° C. .
[0091]
The reaction was then started by adding 2.5 units of Tag polymerase. The conditions for PCR are as follows. A cycle of 1.5 minutes at 95 ° C., 1.5 minutes at 70 ° C., and 1.5 minutes at 72 ° C. is performed 30 times.
[0092]
Finally, the product (10% of the reaction mixture) was subjected to 1.5% agarose gel electrophoresis in TBE buffer and observed with ethidium bromide fluorescence. As size markers, BamHI and MvaI digested pBR322 were electrophoresed simultaneously to indicate the size of the DNA fragment in base pairs. Amplified DNA fragments (A1, A2, B1 and B2) corresponding to the isolated DNA are indicated by arrows.
[0093]
In FIG. 19, the samples in lanes 1 to 3 are amplified using a pair of a forward primer of nucleotide numbers 1,790-1,810 and a reverse primer of 2,179-2,156. Lanes 4 to 6 are samples obtained by amplifying the second oligonucleotides (nucleotide numbers 2,086 to 2,105 and 2,322 to 2,303) as specific primers.
As is apparent from the figure, both U937 and placental cells express class IG-CSF receptors, U937 cells express soluble G-CSF receptors, and placental cells have G-CSF with an insert in the cytoplasmic region. It was confirmed to express the receptor.
[0094]
Example 5  COS cell transformation and cloned binding activity of cloned human G-CSF receptor cDNA
mouse or rat¹²⁵The binding between SF and COS cells transformed with the cDNA obtained in Example 4 was examined.
Labeling of murine recombinant G-CSF, transformation of COS cells with an expression vector containing human G-CSF receptor cDNA, and¹²⁵The binding assay of IG-CSF to COS cells was performed according to the method disclosed in the aforementioned murine examples.
[0095]
Plasmid pHG11 was used to construct a full length cDNA with an insert in the cytoplasmic region. Specifically, the plasmid pHG11 was completely digested with restriction enzymes (NheI, purchased from Takara Shuzo) and partially digested with BstXI (1,425) (purchased from Takara Shuzo). The reaction conditions for the restriction enzyme were the same as those attached to the enzyme from Takara Shuzo.
Next, the 1.38 kb BstXI-NheI fragment and the 6.9 kb BstXI-NheI fragment of pHQ3 were ligated using T4-DNA ligase (purchased from Takara Shuzo) to construct pQW11. Then, COS cells are transformed with expression plasmids (pHQ2, pHQ3 and pHQW11) containing these cDNAs,¹²⁵Binding to IG-CSF was examined.
[0096]
That is, COS cells cultured on a 15 cm plate were transfected with 20 μg of pHQ2, pHQ3, or pHQ11. Twelve hours after glycerin shock, the cells were divided into 6-well microtiter plates and cultured in DMEM containing 10% FCS for 60 hours. Cells were washed with binding medium (DMEM containing 10% FCSF and 20 mM HEPES (pH 7.3)) and various amounts of¹²⁵Incubated with I-G-CSF (range 10 pM to 1.2 nM) at 4 ° C. for 4 hours.¹²⁵In order to measure non-specific binding between IG-CSF and cells, the binding reaction was performed in the presence of a large excess of unlabeled G-CSF (800 nM), and the radioactivity bound nonspecifically from the total binding activity. The specific binding activity was determined by subtracting. FIG.¹²⁵Saturation binding activity of I-G-CSF to COS cells, FIG. 17B shows the results of its Scatchard analysis. At this time, murine G-CSF receptor cDNA was similarly introduced into COS cells, and the binding activity of G-CSF was examined.
[0097]
In FIG. 17, black triangles indicate COS cells transformed with murine G-CSF receptor cDNA. Among the COS cells transformed with the human G-CSF receptor cDNA, the transformant by pHQ3 is indicated by a white circle, the transformant by pHQ2 is indicated by a black circle, and the transformant by pQW11 is indicated by a white triangle.
FIG. 17 shows that monkey COS cells transformed with pHQ3 or pHQ11 are murine.¹²⁵It has high affinity with IG-CSF, dissociation constant in binding is 550 pM, and the number of receptors is 3.4 × 10^FourIt turns out that it is an individual / cell. Also, COS cells and mice transformed with pHQ2 cDNA¹²⁵Binding to IG-CSF was at a very low level because the plasmid pHQ2 encodes a partially deleted peptide (dissociation constant 440 pM, binding site 6 × 10³/cell). This strongly suggests that the receptor encoded by pHQ2 lacking the transmembrane region may be secreted from the cell and accumulated in the medium.
[0098]
In addition, the transformant with the plasmid pQW11 and the transformant with the pHQ3 are almost the same mice.¹²⁵Showing binding activity for IG-CSF indicates that the 27 amino acid insert in the cytoplasmic domain has little effect on G-CSF binding.
For purification of human G-CSF receptor obtained by expression of the transformant, the above-described method for purifying natural mouse G-CSF receptor can be used.
[0099]
Example 6  Analysis of DNA and mRNA encoding human G-CSF receptor DNA or RNA encoding human G-CSF receptor was analyzed by Northern hybridization and Southern hybridization.
Total RNA was prepared from various cell lines and fresh human placenta using the guanidine isothiocyanate / CsCl method as previously described (Saintbrook, supra).
[0100]
On the other hand, cellular DNA was derived from human T lymphocytes (Fukunaga et al., Nucleic. Acids Res.,144421-4436 (1986)).
Southern and Northern blot hybridization was performed according to the method described in the literature (Maniatis et al., Molecular Cloning, Cold Spring Laboratory (1982)) using the XhoI DNA fragment (3 kb) of plasmid pHQ3 containing human G-CSF receptor cDNA as a probe. I did it.
[0101]
1) Analysis of G-CSF receptor transcript and genomic DNA
MRNA from various cells is converted into human G-CSF receptor cDNA.
The probe was tested by Northern hybridization.
The results are shown in FIG. The cells used are as follows. Human U937 cells (lanes 1 and 2), human KG-1 (lane 3), human HL-60 (lane 4), human FL (lane 6), human CHU-2 (lane 7), and human placenta (lane 5) And 8).
U937: human histiocytic lymphoma, ATCC CRL 1593
KG-1: human acute myelogenous leukemia, ATCC CCL246
HL-60: human promyelocyte leukemia, ATCC CCL240
FL: human amnion, ATCC CCL62
Lanes 2 to 6 and 8 represent total RNA (20 μg), and lanes 1 and 7 represent poly (A) RNA (1 μg).
[0102]
FIG. 18A is a copy of an image obtained by exposing human G-CSF receptor cDNA to a DNA probe and exposing the filter to X-ray film after hybridization for 40 hours (lane 8 is 2 hours).
In RNA obtained from U937, placenta and KG-1, a single band is observed at 3.7 kb. Moreover, the signal detected in placenta-derived RNA is 20 times or more that in U937-derived RNA.
B: Blot again,³²P-labeled human elongation factor (elongation factor) 1α cDNA [Uetsuki, T., Naito, A., Nagata, S. and Kaziro, Y., Journal of Bisio Logical chemistry (J. Biol. Chem.),H.2645791-5798 (1990)], and a copy of an image obtained by exposing a filter to an X-ray film for 1 hour. In this case, almost the same signal was obtained with RNA prepared from any cell. These results indicate that the placenta expresses a very large amount of G-CSF receptor mRNA, suggesting a placental growth promoting action of G-CSF.
[0103]
2) Southern hybridization
The number of genes encoding human G-CSF receptor was examined by Southern hybridization.
10 μg of human genomic DNA was digested with EcoRI, HindIII, BamHI, BglII, XbaI, PstI, SacI and ApaI and subjected to 0.8% agarose gel electrophoresis. Transfer the DNA to a nitrocellulose filter,³²Hybridized with P-labeled human G-CSF receptor cDNA. At this time, the DNA size marker was run simultaneously.
[0104]
The results are shown in FIG. The figure shows EcoRI (lane 1), HindIII (lane 2), BamHI (lane 3), BglII (lane 4), XbaI (lane 5), PstI (lane 6), SacI (lane 7) and ApaI (lane 8). The analysis result regarding each digestion fragment is shown. Only one to two bands are observed in DNA digested with restriction enzymes EcoRI, HindIII, BglII, and XbaI, whereas DNA digested with BamHI, PstI, SacI, and ApaI has 4-5 bands each. It is done. As shown in FIG. 16, since human G-CSF receptor cDNA has 3BamHI, 6PstI, 2SacI, and 3ApaI restriction enzyme cleavage sites, it is estimated from this result that there is one G-CSF receptor gene per human haploid genome. .
[0105]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic diagram of a nucleotide sequence of a murine G-CSF receptor and an amino acid sequence deduced therefrom (N-terminal side).
FIG. 2 is a schematic diagram of the nucleotide sequence of murine G-CSF receptor and the amino acid sequence (intermediate part) inferred therefrom.
FIG. 3 is a schematic diagram of the nucleotide sequence of the murine G-CSF receptor and the amino acid sequence deduced therefrom (C-terminal side).
FIG. 4 is a graph showing a schematic diagram, restriction map and hydropathy plot of murine G-CSF receptor cDNA (pI62, pJ17 and pF1).
FIG. 5 shows COS cells expressing recombinant murine G-CSF, NFS-60 cells and murine.¹²⁵1 is a graph showing binding to I-G-CSF, where A is a mouse.¹²⁵Saturated binding between IG-CSF and COS cells, B is a graph showing a Scatchard plot of murine G-CSF binding data to COS cells, C is a mouse¹²⁵Graph showing saturation binding of IG-CSF to NFS-60 cells, D is a graph showing a Scatchard plot of murine G-CSF binding data to NFS-60 cells,
FIG. 6 is a graph showing the binding specificity of recombinant murine G-CSF receptor expressed in COS cells and murine G-CSF,
FIG. 7 is a graph showing the results of cross-linking reaction of murine G-CSF receptor expressed in COS cells and NF-60 cells;
FIG. 8 is a copy of a photograph showing the results of Northern hybridization analysis of murine G-CSF receptor mRNA.
FIG. 9 is a sequence diagram showing the amino acid sequence of murine G-CSF receptor and the amino acid sequences of prolactin and growth hormone receptor in parallel.
FIG. 10 is a sequence diagram showing the amino acid sequence of a murine G-CSF receptor and the amino acid sequence of chicken contactin in parallel.
FIG. 11 is a sequence diagram showing the amino acid sequence of the murine G-CSF receptor and the amino acid sequence of the murine IL-4 receptor in parallel.
FIG. 12 is a schematic diagram of the murine G-CSF receptor.
FIG. 13 is a diagram showing the nucleotide sequence and the deduced amino acid sequence (N-terminal side) of cDNAs (plasmids pHQ3 and pHG12) encoding human G-CSF receptor.
FIG. 14 is a diagram showing the nucleotide sequence and the deduced amino acid sequence (C-terminal side) of cDNAs (plasmids pHQ3 and pHG12) encoding human G-CSF receptor.
FIG. 15 is a diagram showing a part of the nucleotide sequence and the deduced amino acid sequence of cDNAs encoding human G-CSF receptor (plasmid pHQ2 and plasmid pH11). A is a sequence portion different from the pHQ3 sequence in the pHQ2 sequence shown in FIGS. 13-14, the nucleotide sequence of the sequence corresponding to the downstream from the nucleotide number 2,034 of the pHQ3 and the deduced amino acid sequence, B is It is a figure showing the nucleotide sequence and the deduced amino acid sequence of the insert inserted in pHQ2 described in FIGS. 13-14 in the sequence of pHG11.
FIG. 16 is a schematic diagram of restriction enzyme maps of pHQ3 and pHG12, pHQ2 and pHG11 described in FIGS. 13-15.
FIG. 17 is a graph showing the binding characteristics of recombinant human G-CSF receptor expressed by COS cells with murine G-CSF, and A is¹²⁵The graph which shows the saturation binding to COS cell of IG-CSF, B is a graph which shows the result of the Scatchard analysis of G-CSF binding data.
FIG. 18 is a copy of a photograph showing the results of Northern hybridization analysis of human G-CSF receptor mRNA.
FIG. 19 is a copy of a photograph showing the results of PCR of human G-CSF receptor mRNA.
FIG. 20 is a copy of a photograph showing the result of Southern hybridization analysis of human G-CSF receptor;
FIG. 21 is a schematic diagram of an expression vector pEF-BOS.

Claims (7)

A DNA encoding a protein having any of the following amino acid sequences and having a binding activity to a granulocyte colony-stimulating factor.
(1) an amino acid sequence represented by amino acid numbers 26 to 837 in the amino acid sequence described in SEQ ID NO: 2, wherein one or several amino acids are deleted, substituted or added; and
(2) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by amino acid numbers 26 to 626 in the amino acid sequence described in SEQ ID NO: 2.
However, DNA encoding the amino acid sequence represented by amino acid numbers 26 to 626 or 26 to 837 in the amino acid sequence described in SEQ ID NO: 2 is excluded. A DNA that hybridizes under stringent conditions with DNA comprising any of the following base sequences, and that encodes a protein having a binding activity to granulocyte colony-stimulating factor.
(1) the base sequence represented by nucleotide numbers 255 to 2690 in the base sequence set forth in SEQ ID NO: 1, and
(2) The base sequence represented by nucleotide numbers 255 to 2057 in the base sequence described in SEQ ID NO: 1.
However, DNA encoding the amino acid sequence represented by amino acid numbers 26 to 626 or 26 to 837 in the amino acid sequence described in SEQ ID NO: 2 is excluded.   A protein encoded by the DNA according to claim 1 or 2.   An expression vector containing the DNA according to claim 1 or 2.   A transformant containing the expression vector according to claim 4.   A method for producing a granulocyte colony stimulating factor receptor comprising culturing the transformant according to claim 5 in a medium and recovering a product capable of binding to granulocyte colony stimulating factor from the obtained culture.   A granulocyte colony-stimulating factor receptor produced by the method according to claim 6.

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