RU2124024C1 - Protein, method of protein preparing (variants), pharmaceutical composition - Google Patents

Abstract

FIELD: chemistry of proteins, biochemistry. SUBSTANCE: invention relates to a protein - antagonist of hepatocyte growth factor (HGF) and can be used in biotechnology and medicine. Protein consists of two α-- and β--chains connected by disulfide bond and can bind with HGF but has no domain of cytoplasmic tyrosine kinase. α--Chain is α--chain of receptors of HGF encoded by protooncogen MET (molecular mass is 50 kDa). α--Chain is a form of β--chain of HGF receptors (molecular mass is 75 or 85 kDa) and it is truncated from C-end. Protein is produced by culturing cells expressing MET gene. For isolation from cultural fluid protein is bound with antibodies showing specificity to extracellular domain of β--chain of receptor. Invention ensures to obtain the truncated forms of HGF receptor and use their for pharmaceutical preparations preparing. EFFECT: improved method of preparing and isolation of protein. 5 cl, 13 dwg, 2 ex

Description

 This invention relates to the study of shortened forms of hepatocyte growth factor receptor (HGF) and is a method for producing their preparations and pharmaceutical kits containing them.

The proto-oncogen MET (Cooper et al, Nature 311, 29-33, 1984; Dean et al, Nature 318, 385-388, 1985, Park et al, PNAS USA 84, 6379-6383, 1987) encodes a transmembrane glycoprotein (p190 ^MET or Met receptor) with unique properties, which is a heterodimer consisting of two chains connected by a disulfide bond, the molecular weight of which is 50 kilodaltons (p50 ^α ) and 145 kilodaltons (p145 ^β ) (Giordano et al, Mol. Cell, Biol. 8 3510-3517, 1988, Giordano et al, Nature 339, 155-156, 1989). Both chains face the surface of the cell. The β chain consists of a region of hydrophobic amino acids overlapping the plasma membrane and an intracellular tyrosine kinase domain (Dean et al. 1985, Gonzatti-Haces et al. PNAS USA 85, 21-25, 1988; Tempest et al, FEBS Lett. 209, 357 -361, 1986). The receptor is synthesized as a glycosylated precursor having a molecular weight of 170 kilodaltons, which is enzymatically cleaved during maturation to yield a mature heterodimer (Giordano et al, Oncogene, 4, 1383-1388, 1989; Tempest et al., Br. J. Cancer 58, 3-7, 1988).

 Recently, it has been proposed to use hepatocyte growth factor (HGF) as a ligand for the Met receptor (Bottaro et. Al., Science 251, 803-804, 1991; Naldini et al, Oncogene 6, 501-504, 1991; Naldini et al, 1991 , EMBO J., 10,2867-2878). It is known that HGF, also called hepatopoietin-A / Miyazawa et al., Biochem. Biphys. Res. Commun. 163, 967-973 (1989); Nakamura et al., Nature 342, 440-443, 1989; Zarnegar et al., Cancer Res. 49, 3314, 1989), is the most powerful mitogen for primary cultures of rat and human hepatocytes, as well as a hepatotrophic factor involved in in vivo liver regeneration processes in both humans and rodents (see review of Michalopoulas, FASEB J. 4, 176-187, 1990). In rats and rabbits, HGF has also been found in the pancreas, salivary glands, duodenum, thyroid, and parts of the central nervous system (Tashiro et al., Proc. Natl. Acad-Sci. USA 87, 3200-3204, 1990; Zarneger et al., Proc. Natl. Acad-Sci. USA 87, 1252-1256, 1990).

 The MET oncogen was originally identified by transfection using DNA from human cell lines treated with a chemical carcinogen (Cooper et al., Nature 311, 29-33, 1984). In this cell line, the MET gene was activated during chromosomal rearrangement (Park et. Al., Cell 45, 895 (1986). It was also found that the amplification and activation of the MET gene occurs in cells of the human gastric carcinoma line (Giordano et. Al., Mol. Cell Biol. 8.3510, 1988) and in mouse fibroblasts undergoing spontaneous transformation (cooper et. Al., EMBO J., 5, 2623, 1986).

Currently, we have developed monoclonal antibodies to the extracellular domain and used several human cell lines to screen them. In addition to the well-known p190 ^MET , these antibodies constantly recognized two other Met-heterodimers: a complex of 140 kilodaltons located on the surface of the cells, and a complex of 130 kilodaltons released into the culture medium. Both complexes consist of an α chain indistinguishable from p50 ^α and a shortened β chain of p85 ^β and p75 ^β from the C-terminus, respectively), devoid of the cytoplasmic kinase domain. These truncated Met forms are generated in vivo by post-translational proteolytic processing. Activation of protein kinase C leads to the release of p130 ^MET into the culture medium.

Shortened forms of growth factor receptors have been found in some cells, tissue culture supernatants, and body fluids (Beguin et al., Proc. Natl. Acad. Sci. USA, 85, 637-640; Distefano et al. 1988, Proc. Natl. Acad . Sci. USA 85, 270-274; Johnson et al., 1990 Mol. Cell. Biol., 10, 4728-4736; Mosley et al., 1989, Cell. 59, 335-348; Zabrescky et al., 1991 J. Biol. Chem. 266, 1716-1720). It has recently been reported that soluble C-terminated receptor forms are able to compete for binding to ligands from intact receptors and, by forming inactive heterodimers, can inhibit ligand-induced stimulation of tyrosine kinase activity (Basu et al., 1989, Mol. Cell. Biol., 9 , 671-677; Kashles et al., 1991, Mol. Cell. Biol., 11, 1454-1463; Veno et al., 1991, Science, 252, 844-848). Thus, this invention provides a protein consisting of two disulfide-linked chains of 50 kDa and 75 or 85 kDa, and the protein has the following properties:
- its α-chain is the α-chain of the hepatocyte growth factor receptor (HGF) encoded by the Met proto-oncogen;
- its β-chain is a shortened from the C-terminus form of the β-chain of the indicated HGF receptor;
- the specified protein binds to antibodies specific for the extracellular domain of the β-chain of the specified HGF receptor;
- the specified protein binds to HGF; and
the indicated protein does not have a cytoplasmic tyrosine kinase domain.

According to the invention, two C-terminated Met proteins are a transmembrane form (p140 ^MET ) having a molecular weight of 140 kilodaltons and a soluble protein with a molecular weight of 130 kilodalton (p130 ^MET ) released into the culture medium. These shortened forms can be found in the human gastric carcinoma GTL-16 cell line, in which amplification of the MET gene occurs, leading to super synthesis of the products of this gene, as well as in cell lines of other carcinomas with normal levels of expression of the MET gene.

The truncated Met proteins have the same heterodimeric structure as the intact p190 ^MET , consisting of two chains connected by a disulfide bond, the α chains of the shortened forms are completely identical to the α chain of p190 ^MET ; whereas their β-chains have a lower molecular weight. The molecular weight of the β chain of the p140 ^MET protein is approximately 85 kilodaltons (p85 ^β ), and the β chain of the p130 ^MET protein is approximately 75 kilodaltons (p75 ^β ).
Both p85 ^β and p75 ^β have a cut off C-terminus. They are not recognized by antibodies acting on the C-terminal nonadecapeptide presented on the basis of the MET gene sequence. They lack the cytoplasmic tyrosine kinase domain containing Tyr ¹²³⁵ , which is the main site of in vitro phosphorylation, as evidenced by their inability to phosphorylate along horizon, both in vivo and in vitro. The N-terminal domain of p85 ^β and p75 ^β is identical to the corresponding domain of the p145 ^β protein, since they are all recognized by monoclonal antibodies that define four different epitopes and have the same extracellular tryptic peptides.

It is believed that the p85 ^β chain is a transmembrane glycoprotein that has lost most of its cytoplasmic domain. Based on the predictions of the amino acid sequence, it can be concluded that the cleavage of the last 435 C-terminal amino acids should lead to a decrease in molecular weight of almost 50 kD. This value is consistent with the observed differences in the electrophoretic mobility of the intact and truncated β-chains. P75 ^β lacks the cytoplasmic domain and transmembrane segment, since it is released into the culture supernatant and is not associated with the cell membrane. In addition, it is 10 kD lower compared to the p85 ^β transmembrane chain. This reduction in size is consistent with the loss of a segment including the transmembrane domain.

The proteins of this invention can be isolated from cultures of cell lines in which the MET gene is expressed. Typical representatives of such cell lines are carcinoma cell lines. Therefore, this invention provides a method for producing protein, comprising
1) the cultivation of cells expressing the Met gene in the medium;
2) the interaction of the resulting conditioned medium with antibodies and
3) the release and isolation of the aforementioned protein from the resulting immunocomplex, characterized in that the antibody is specific for the extracellular β-chain of the HGF receptor, and said protein that is released and secreted is a protein of the invention in which the β-chain has a mole. weight 75 kD.

 As cells expressing the MET gene, cells of any suitable cell line can be used. For this purpose, cells of such cell lines as gastric carcinoma, lung adenocarcinoma, dental carcinoma and colon carcinoma are suitable. Preferably the use of cells superproducing the MET gene. The culture medium can be any medium containing digestible sources of carbon and nitrogen and, preferably, mineral salts. Suitable for this is RPMI 1640 medium supplemented with fetal calf serum.

 Antibodies specific for the extracellular domain of the tyrosine kinase β chain encoded by the MET gene can be prepared in the usual manner. These antibodies are typically monoclonal (Kohler and Milstein, Natere 256, 495-497, 1975). Hybridoma cells producing monoclonal antibodies can be obtained by fusion of spleen cells isolated from an immunized animal with a tumor cell. The immunized mammal may be a rat or mouse. The mammal may be immunized with whole cells or extracts of cells that express the MET gene. It is preferable to select cells possessing superexpression of the MET gene. Antibodies produced by hybridomas are selected by evaluating their ability to immunoprecipitate the Met protein extracted from the cell line producing the Met protein. Hybridomas can be grown in culture or introduced intraperitoneally to form ascites fluid or into the bloodstream of an allogeneic or immunocompromising host (a host with impaired immunological reactivity).

An immunoprecipitate is formed between the antibody and p130 ^MET , which is present in the conditioned medium. The immunoprecipitate is collected, for example, on a column, such as protein-A-Sepharose (trademark), to which specific anti-Met antibodies have been linked, and cross-linked. P130 ^{MET is} released from the immunocomplex by treatment with low pH or high salt.

The invention also provides a method for producing protein, comprising
1) extraction of cells expressing the Met gene using a detergent;
2) the interaction of the extract with antibodies;
3) the release and isolation of the specified protein from the resulting immunocomplex; characterized in that the antibody is specific for the extracellular domain of the β-chain of the HGF receptor, and said protein, which is released and secreted, is a protein of the invention in which the β-chain has a mole. weight 85 kD.

Cells that express the MET gene and the culture medium in which they are grown are the same as described above. Cells are extracted with a detergent, usually a non-ionic detergent is used, such as TritonX100 or CHAPS / 3 - [(3-Cholamidopropyl) dimethylammonio-1-propanosulfonate] /. CHAPS is manufactured by Fluka and is used as a 1% solution in HEPS buffer as described in Naldini et al., Molecular and Cellular Biology, vol. 11, 4 (April 1991), pages 1793-1803). Typically, the cells are extracted into a buffer to which protease inhibitors, such as pepstatin, leupeptin, aprotinin, trypsin inhibitor, can be added. The resulting extract binds to antibodies specific for the β-chain of tyrosine kinase encoded by the MET gene, and p140 ^{MET is} released from the resulting immunoprecipitate as described above.

 The protein of the invention can be isolated and purified. Almost complete purification of this protein can be achieved both from impurities of the whole tyrosine kinase encoded by the MET gene, and from other components of cells expressing the MET gene. This protein can be used as an antagonist of HGF. The protein retains the ability to bind to HGF, but, being deprived of the tyrosine kinase domain, it is not able to transmit an intracellular mitogenic signal. Therefore, this protein can be used to treat tumors, including tumors of the gastrointestinal tract, liver, thyroid gland and brain. Indeed, a large percentage of cells from these tumors show HGF receptor expression, and HGF is required for the growth of these tumors. This protein can also be used as an agent that suppresses liver regeneration, for the treatment of liver hyperplasia or adenomatosis.

 The protein of the invention may be administered to the patient by any convenient parenteral route. The choice of method of administration of the drug - subcutaneously, intramuscularly or intravenously, the dose and frequency of administration depends on many factors. It takes into account the purpose of taking the drug, the age and weight of the patient, as well as his state of health. However, usually protein is prescribed in an amount of from 10 to 1000 μg per dose, preferably from 50 to 500 μg per dose for each course of treatment.

 Protein may be added to the pharmaceutical composition. The pharmaceutical composition also contains a pharmaceutically acceptable carrier or diluent. Depending on the course of treatment, any suitable carrier or diluent may be used.

 The following examples illustrate the invention. They are presented in the figures.

FIG. 1 shows that the truncated form of the Met protein is identified by monoclonal antibodies to the extracellular domain. Pictures A and B: GTL-16 cells surface-labeled with ¹²⁵ I in the presence of lactoperoxidase. Cellular proteins were solubilized by Triton X-100 and immunoprecipitated with various monoclonal antibodies (Mabs) directed either to the C-terminal peptide (DR-6, lane 1) or to the extracellular domain (DO-24, DN-30, DN-31, lanes 2, 3, 4) Met protein. Immunoprecipitating proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) under conditions that prevent the restoration of disulfide bonds (image A), or after treatment with 2β-mercaptoethanol, which leads to the restoration of these bonds (image B). In image C: p140 ^MET (lane 1) and p190 ^MET complexes (lane 2) obtained after immunoprecipitation with DO-24 monoclonal antibodies were excised from polyacrylamide gel (SDS-PAGE), eluted in Laemmli buffer, recovered with 2β-mercaptoethanol and analyzed in SDS-PAGE under conditions of reduction of disulfide bonds. The gels were dried and exposed to obtain autoradiograms for 3 days.

In FIG. Figure 2 shows two-dimensional tryptic peptide maps of extracellular domains of β-chains isolated from intact and truncated Met receptors. After immunoprecipitation with monoclonal antibodies (Mabs) of the anti-extracellular domain p145 ^β (image A), p85 ^β (image B) and p75 ^β (image C), isolated either from surface radioiodinated GTL-16 cells or from the supernatant of radioiodinated GTL-16 cells were divided into SDS-PAGE. Proteins were electrophoretically transferred to the Immobilon P membrane and subjected to exhaustive tryptic hydrolysis. About 3000 cpm of each hydrolyzate was placed in a hole in the right corner corresponding to the anode and subjected to electrophoresis at pH 1.9 and upward chromatography in the pyridine-acetic acid-butanol-water system. The main tryptic peptide (marked by an arrow in the figure) obtained from the α-chain of the Met protein is shown for comparison (in picture D). Maps were exposed for 21 weeks at -70 ^° C with / reinforcing / intensifying screens. The position of the source in the figure is indicated by a circle.

не фосфорилируется in vitro. Белки из GTL-16 клеток, солюбилизированные в неионном детергенте, иммунопреципитировались моноклональными антителами к экстраклеточному домену Met-белка (DL-21, DO-24, DN-30, DN-31, дорожки 1, 2, 3, 4) или моноклональными антителами к неродственному белку (дорожка 5). Промытые иммунокомплексы фосфорилировались в присутствии [γ-³²p] ATP и меченые белки разделялись в SDS-ПААГ в условиях, препятствующих восстановлению дисульфидных связей (снимок A), или в условиях восстановления этих связей (в присутствии 2β-меркаптоэтанола, снимок B). Гели высушивались и экспонировались в течение 2 дней для получения авторадиограммы. Видно, что метятся только интактный p90^MET или интактная цепь p145^β и Pr 170 Met-предшественник.In FIG. 3 shows that

not phosphorylated in vitro. Proteins from GTL-16 cells solubilized in a non-ionic detergent were immunoprecipitated with monoclonal antibodies to the extracellular domain of the Met protein (DL-21, DO-24, DN-30, DN-31, lanes 1, 2, 3, 4) or monoclonal antibodies unrelated protein (lane 5). The washed immunocomplexes were phosphorylated in the presence of [γ- ³² p] ATP and the labeled proteins were separated in SDS-PAGE under conditions that prevent the restoration of disulfide bonds (image A), or under conditions of restoration of these bonds (in the presence of 2β-mercaptoethanol, image B). The gels were dried and exposed for 2 days to obtain an autoradiogram. It is seen that only the intact p90 ^MET or the intact p145 ^β chain and Pr 170 Met-precursor are labeled.

In FIG. Figure 4 shows that murine fibroblasts transfected with human MET cDNA express intact p190 ^MET as well as shortened forms. Proteins from NIH 3T3 cells (lanes 1, 2) or from their conditioned medium / lanes 3, 4 / obtained after transcription of these cells with full-length MET cDNA synthesized on a 9 kb dominant mRNA matrix (thousand nucleotide pairs) were solubilized by boiling in Laemmli buffer and analyzed by Western blot hybridization under reducing conditions. Lanes: 1 and 4, obtained using monoclonal antibodies to the C-terminal peptide of the Met protein; 2 and 3 - with monoclonal antibodies (DO-24) to the Met-extracellular domain. Specific binding was detected using an ECLTM enhancing chemiluminescent system, Amersham). The bands have dimensions corresponding to 170 kD of Met-precursor (A), intact p145 ^β (B), shortened p85 ^β (C) and soluble p75 ^β (D).

In FIG. 5 shows the uneven cell localization of p190 ^MET and p140 ^MET . GTL-16 cells were labeled with [ ³ H] glucosamine for 18 hours. Living cells were incubated for 2 hours on ice with monoclonal antibodies, and then lysed with a detergent (lanes 1, 3, and 5). Or alternatively, the cells were first lysed and then incubated with monoclonal antibodies (lanes 2, 4 and 6). In both cases, immunocomplexes were isolated on an A-Sepharose protein column, and radiolabeled proteins were analyzed in SDS-PAGE under reducing conditions. The following antibodies were used: monoclonal antibodies directed to the extracellular domain (DO-24, lanes 1 and 2) or to the C-terminal peptide of the Met protein (DR-6, lanes 5 and 6), and monoclonal antibodies to the extracellular domain of the receptor for EGF (lanes 3 and 4). The gels were dried and exposed for 4 days to obtain a radio autograph.

In FIG. Figure 6 shows the expression of p140 ^MET in various human cell lines under physiological conditions (Western blot). Cellular proteins were solubilized in boiling Laemmli buffer, separated in SDS-PAGE under non-reducing conditions, transferred to a nitrocellulose filter and sequentially treated with DL-21 monoclonal antibodies and [ ¹²⁵ 1] - sheep α-mouse Ig. 300 μg of cellular protein was applied to each lane; 100 μg applied to the GTL-16 lane. The following cell lines were used: lane 1-A549, lung carcinoma; lane 2: GTL-16, gastric carcinoma, lane 3: SK-BR3, breast carcinoma, lane 4: HT-29, colon carcinoma, lane 5: KB, dental carcinoma.

In FIG. 7 shows that the soluble truncated form of the Met protein is identified by monoclonal antibodies to the extracellular domain. GTL-16 (images A, C) or A549 cells (image B) were labeled with [ ³⁵ S] methionine overnight (100 μCi / ml), washed and extracted with detergent. The clarified cell extracts (lane 1) and the conditioned medium (lanes 2 and 3) were precipitated with monoclonal antibodies to the extracellular domain (DO-24, lanes 1 and 3) or to the C-terminal peptide (DR-6, lane 2) of the Met protein. Immunoprecipitating proteins were separated in SDS-PAGE under non-reducing conditions (images A, B) or after reductive treatment with 2β-mercaptoethanol (image C). The gels were x-rayed, dried and exposed for 3 days to obtain a radio autograph.

In FIG. Figure 8 shows that soluble p130 ^{MET is} generated by proteolysis of Met proteins exposed on the cell surface. GTL-16 cells were superficially labeled with ¹²⁵ I in the presence of lactoperoxidase. After an incubation of 4 hours, labeled proteins were isolated either from culture supernatants (lanes 1 and 2) or from cell extracts (lanes 3 and 4) and were immunoprecipitated with monoclonal antibodies directed to the extracellular domain (DO-24, lanes 2 and 3) or to the C-terminal peptide (DR-6, lanes 1 and 4) of the Met protein. Immunoprecipitating proteins were separated in SDS - PAGE under reducing conditions. The gels were dried and exposed for 18 hours to obtain an autoradiogram.

In FIG. Figure 9 shows the stimulation of the release of p130 ^MET from the cell surface upon activation of protein kinase C. GTL-16 cells were labeled overnight [ ³⁵ S] with methionine (100 μCi / ml, treated for 2 hours with 160 nM TPA dissolved in dimethyl sulfoxide (lane T), or with dimethyl sulfoxide alone (lane C), washed and extracted with a non-ionic detergent. Conditioned medium (A) and clarified cell extracts B were released by monoclonal antibodies directed to the Met-extracellular domain (DO-24). Immunocomplexes were collected on a protein-A-ste column pharose and analyzed in SDS-PAGE under reducing conditions. Gels were X-rayed, dried and exposed for 6 hours to obtain an autoradiogram. Image C: GTL-16 cells were treated with TPA (lane T) or dimethyl sulfoxide (lane C), as described above, were labeled ¹²⁵ I using lactoperoxidase, washed and extracted with a non-ionic detergent, clarified cell extracts were immunoprecipitated with DO-24 monoclonal antibodies, and immunocomplexes were analyzed as described above.

In FIG. Figure 10 shows the binding of ¹²⁵ I-HGF to soluble p130 ^MET PVC microtiter cells coated with DN-30 monoclonal antibodies directed to the extracellular domain of the Met protein and used as a trap for various concentrations of p130 ^MET or conditioned medium lacking p130 ^Met (-). ¹²⁵ I-HGF was then added as described in Example 2 in the Materials and Methods section. The plates were incubated overnight at 4 ^° C., washed 3 times and the bound radioactivity was eluted with 1% SDS and measured in a γ counter.

In FIG. 11 shows the inhibition of tyrosine phosphorylation of the HGF receptor (p145 ^β subunit) by soluble p130 ^MET . GTL-16 cells were incubated with p130 ^MET partially purified by Lentil Lectin affinity chromatography as described in Example 22 of the Materials and Methods section. The relative amount of phosphotyrosine presented in p145 ^β was evaluated by measuring the optical density of the corresponding break, observed on the radiograms of Western blots displayed by P-Tyr antibodies. The drawing shows the values obtained in one of the typical experiments. A: values obtained with cells incubated with conditioned medium lacking p130 ^MET ; B: cells incubated with p130 ^MET purified on Lectil Lectin.

In FIG. 12 shows the in vitro inhibition of tyrosine kinase activity of Met (HGF receptor) p145 ^β subunit by soluble p130 ^MET . The receptor was precipitated by polyclonal anti-Met-serum and incubated for 5 min at room temperature with 60 μl of various dilutions of the conditioned medium depleted in protein p130 ^MET (A) or partially purified protein p130 ^MET (B). The kinase reaction was carried out on ice for 2 min in the presence of 10 μm [γ- ³² p] ATP and 5 mM MgCl ₂ . After electrophoresis in SDS-PAGE and autoradiography, kinase activity was evaluated by measuring the optical density of the ³² P-labeled band on a laser densitometer. The diagram shows the values obtained in a typical experiment.

 FIG. 13 illustrates a radioimmunometric determination of soluble p130 Met released from GTL-16 cells into tissue culture medium. White circles indicate the results obtained using the supernatant of GNL-16 cells, black circles indicate RPMI medium (negative control).

 Example 1. Materials and methods.

 Cells.

All lines used in this work are human cell lines. GTL-16 cells are a clonal line derived from poorly differentiated gastric carcinoma (Giordano et al., Bol. Cell. Biol. 8, 3510-3517, 1988). A549 lung adenocarcinoma cells, KB dental carcinoma cells, HT-29 colon carcinoma cells, and SK-BP-3 breast carcinoma cells were obtained from the American Type Culture Collection. Cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum (Flow Laboratories, Inc. McLean, Va) in an atmosphere of water vapor saturated with 5% CO ₂ .

 Antibodies

 Monoclonal antibodies to the extracellular domain of the Met protein were obtained after immunization of whole live GTL-16 cells expressing Met protein according to the method of Kohler and Milstein (Nature 256, 495-497, 1975).

 Spleen immune cells were fused to Ag 8.653 myeloma cells and hybrid supernatants were selected by enzyme-linked immunosorbent assay for their distinctive ability to bind to GTL - 16 cells / Prat et al, Cancer Detection, and Prevention, 10, 293-301, 1987). Hybrid supernatants were selected for their ability to selectively bind to cells that overexpress Met protein by enzyme-linked immunosorbent assay (Prat et al, 1985).

For immunoprecipitation experiments, cells were labeled for ³⁵ hours with ³⁵ S-methionine, washed extensively and lysed with the nonionic detergent Triton X100, as previously described (Prat et al, Cancer Research, 45, 5799-5807, 1985). Samples were analyzed in 8% SDS-PAGE and autoradiographed for 1 day at -70 ^° C using an ultra-sensitive Amersham (Hyper-film) X-ray film. Four different types of monoclonal antibodies (DL-21, DN-30, DN-31, DO-24) were selected for these studies. These monoclonal antibodies interact with various β-chain epitopes based on reciprocal (mutual) cross-competition. Since the same results were obtained with different monoclonal antibodies, we will talk about this panel as Mabs anti-extracellular domain. Monoclonal antibodies (DR-6) were also prepared for a peptide corresponding to nineteen C-terminal amino acids (Ser ¹³⁷² to Ser ¹³⁹⁰ ) of the MET gene sequence (EMBL Data-Bank reference N X54559). Monoclonal antibodies to the extracellular domain of the epidermal growth factor receptor are the same as described by Honegger et al, EMBO J. 7 (10), 3053-3060, 1988. Unrelated monoclonal antibodies were used as a negative control.

 Cell surface iodination, metabolic labeling and immunoprecipitation.

Radioiodination of the cell surface catalyzed by lactoperoxidase and H ₂ O ₂ was carried out as previously described (Giordano et al, 1988). For pulse chase experiments, cells were pretreated with methionine-free medium for 30 min, 400 μCi / ml [ ³⁵ S] -methionine were pulse-labeled for 15 min, washed twice with complete medium and then grown for various periods of time. Cells were labeled with 100 μCi / ml of either [ ³ H] -glucosamine or [ ³⁵ S] -methionine for 18 hours. All radioactive isotopes were obtained from Amersham (Amersham Corp., Arlington Heights, Ill). After labeling, the cells were extracted with ice-cold buffer containing 10 mM Pipers pH 7.6, 100 mM NaCl, 5 mM MgCl ₂ 300 mM sucrose, 5 mM EDTA (DIM buffer), 1% Triton X100 and protease inhibitors (pepstatin, 50 μg / ml; leupeptin, 500 μg / ml; aprotinin, 1 μg / ml; 2 mM PMSF, Sigma; soybean trypsin inhibitor, 500 μg / ml, Boehringer). Ultracentrifuged tissue culture supernatants or cell extracts were pre-purified on a protein-A-Sepharose column and immunoprecipitated with various antibodies. Immunoprecipitation of molecules exposed on the cell surface was carried out by incubation of intact GTL - 16 cells with monoclonal antibodies before detergent lysis. Immunocomplexes were collected on a protein-A-Sepharose column pretreated with affinity purified goat anti-mouse Ig antibodies (GαMIg), washed and eluted in Laemmli buffer (Nature 230, 680-685, 1970) with or without 2β-mercaptoethanol. The treatment with tetradecanoyl phorbol acetate (TPA) was carried out on GTL - 16 cells labeled with [ ³⁵ S] methionine for 18 hours. Cells were washed with fresh RPMI medium and then incubated with dimethyl sulfoxide containing or not containing (control) 160 nM TPA (Sigma) for 2 hours. To analyze the effect of TPA on Met proteins exposed on the cell surface, GTL - 16 cells were treated with TPA as described above, and then ¹²⁵ I was labeled with lactoperoxidase. In both cases, cell proteins solubilized with a detergent or supernanants were precipitated as described above. Proteins were electrophoresed in SDS-PAGE, fixed, fluorographed when necessary, dried and exposed to obtain an autoradiogram on Amersham Hyperfilm film.

 Analysis of the kinase activity of immunocomplexes.

Proteins were extracted from GTL - 16 cells in DIM buffer, 1% Triton X-100 and precipitated as described above. Immunocomplexes collected for A-Sepharose protein - G α MIg, were phosphorylated in 20 μl of the same buffer in the presence of 2.5 μCi / [γ- ³² p] ATP (specific activity 7000 cells / Mm; Amersham) at 30 ^° C for 5 minutes. The reaction was stopped by adding 1 ml of cold physiological saline prepared on phosphate buffer, pH 7.2, containing 5 mM EDTA. Samples were centrifuged and eluted with Laemmli boiling buffer with or without 2β-mercaptoethanol. After electrophoresis in SDS-PAGE, the gels were dried and exposed to obtain autoradiograms on Amersham Hyperfilm film with amplifying screens.

 Peptide mapping.

 Met-β chains obtained from surface radioiodinated GTL - 16 cells were fractionated by SDS - PAGE electrophoresis and electrophoretically transferred to Immobilon membranes (Millipore, Towbin et al, PNAS USA 76, 4350-4353, 1979).

Membrane fragments containing radioactive β-chains were impregnated with 0.5% polyvinylpyrrolidone in 100 mM acetic acid at 37 ^° C for 30 minutes, washed briefly with water and then with freshly prepared 0.05 M NH ₄ HCO ₃ . The immobilized protein was tryptically treated with 10 μg TRPC-treated trypsin (Worthington) for 16 hours at 37 ^° C and then another 2 hours at 37 ^° C with the addition of 10 μg of fresh enzyme (Hunter et al, PNAS USA 77, 1311-1315, 1980). The hydrolysates were applied onto cellulose thin-layer plates / 13255 Cellulose, Kodak), at an angle corresponding to the anode, and were subjected to electrophoresis at pH 1.9 (2.5% formic acid, 7% glacial acetic acid) at 1200 V. The plates were then subjected to upward chromatography (n-butanol: pyridine: acetic acid: water, 20: 30: 6: 24 (v / v) volume / volume /. Peptides were visualized autoradiographically on Amersham Hyperfilm with amplification screens.

 Western blotting.

Cells were solubilized by boiling in Laemmli buffer with or without a 2β-mercaptoethanol reducing agent. Equal amounts of proteins (300 μg) were applied to each lane. Samples were analyzed by Western blotting as described by Towbin et al, 1979. Blots (probed) were manifested with a hybridoma supernatant containing DL-21 monoclonal antibodies and then ¹²⁵ I-labeled sheep α-MIg (Amersham).

Autoradiograms were exposed with amplifying screens for 2-3 days. As markers, pre-labeled [ ¹⁴ C] methylation (Amersham) myosin (200 kD), phosphorylase (92.5 kD), bovine serum albumin (69 kD), egg albumin (43 kD) and carbonic anhydrase (30 kD) were used.

 Transfection of MET cDNA into NIH 3T3 cells.

 The expression of the vector used for these studies is based on the fact that the plasmid pMT2 contains the main late adenovirus promoter. An expression plasmid was constructed using a 4.3 kb DNA fragment containing the complete coding sequence of the MET gene (Giordano et al, 1989, Nature 339 155-156). Since this plasmid does not contain any selective marker, the cells were cotransfected with plasmid p SV2 neo carrying the neomycin resistance gene. The plasmid was cotransfected into NIH 3T3 cells by lipofection. Two days after transfection, a neomycin G418 analog was added to the culture medium in order to select resistant clones. Stable transfectants expressing Met receptors were identified by the ability to synthesize Met receptors using Western blot analysis.

 Results.

The truncated p140 ^MET coexists with the p190 ^MET on the cell surface.

Proteins exposed on the surface of GTL - 16 cells were labeled with ¹²⁵ I using lactoperoxidase. Cells were extracted with 1% Triton and solubilized molecules were precipitated with monoclonal antibodies to the C-terminal peptide of the Met protein or monoclonal antibodies recognizing various epitopes of the extracellular domain. Immunoprecipitates were analyzed in SDS-PAGE under non-reducing and reducing conditions. Antibodies to the C-terminal peptide precipitated a p190 ^MET heterodimer (Fig. 1A, lane 1), which, upon reduction, decomposed into the p50 ^α and p145 ^β chains (Fig. 1B, lane 1). Monoclonal antibodies to the extracellular domain precipitated together with p190 ^MET other molecules migrating with an apparent molecular weight of 140 kD (p140 ^MET , Fig. 1A, lanes 2-4). These immunoprecipitates under reduction conditions decomposed into three chains of 145, 85 and 50 kD, respectively (Fig. 1B, lanes 2-4), suggesting that p140 ^MET is a heterodimer consisting of p85 and p50. The results of the experiment shown in FIG. 1C confirm this interpretation of the p140 ^MET heterodimeric structure. The 140 kD band cut from the gel shown in image A, upon repeated phoresis under conditions of recovery, generated p85 and p50 (Fig. 1C, lane 1). The latter migrated with p50 ^α dissociated from the p190 ^MET αβ complex (Fig. 1C, lane 2). The ratio of p190 ^MET to p140 ^MET ranged from 1: 1 to 2: 1. Similar results were obtained in the study of surface-iodinated proteins from other cell lines.

Bidimensional tryptic maps of surface iodinated p145 ^β and p85 ^β chains show that the extracellular domains of the intact and truncated chains of the Met protein contain identical peptides (Figs. 2A, 2B). Based on the results obtained, it was concluded that p140 ^MET is a heterodimer consisting of p50 ^β and an 85 kD β chain (p85 ^β ) formed from p145 ^β when the C-terminus is cut off.

The truncated p140 ^{MET is} not tyrosine phosphorylated.

Using antibodies specific for the C-terminal peptide p190 ^MET , it was previously shown that this protein can be phosphorylated in vitro by the β subunit (Giordano et al, Mol. Cell. Biol. 8, 3510-3517, 1988). Incubation of immunoprecipitates obtained with four monoclonal antibodies directed to the Met-extracellular domain in the presence of [γ- ³² p] ATP leads to labeling of p190 ^MET , but p140 ^MET is not labeled (Fig. 3A). Under reducing conditions, only p145 ^{β was} labeled (Fig. 3B). In addition, p140 ^{MET was} never manifested by anti-phosphotyrosine antibodies in Western blots of GTL-16 proteins, while p190 ^MET could always be visualized (Giordano et al, 1988). These data suggest that the truncated form of the Met protein lacks tyrosine residues, which can be followed by in vitro or in vivo phosphorylation.

The truncated p140 ^MET and p130 ^MET occur during processing of the p190 ^MET .

p140 ^MET can arise either during translation of alternatively spliced with mRNA encoding only the extracellular and transmembrane domains of the Met protein, or as a result of post-translational processing of an intact molecule. Indeed, four different MET transcripts were found in GTL-16 cells (Giordano et al, Nature 339, 155-156, 1989; Park et al, Cell 45, 895-907, 1986), but they all have a region encoding the cytoplasmic domain.

In order to investigate the origin of the cut-C-terminated Met protein, NIH 3T3 cells cotransfected the full length human MET cDNA and p SV2 neo. Stable transformants were selected with G418 and analyzed for their ability to Met-expression. Northern (RNA) blot analysis of positive cells revealed the only mRNA of the expected size (approximately 4.3 kb). Cells were solubilized in a Laemmli boiling buffer, and proteins were examined under reduced conditions by Western block analysis. Using antibodies to the C-terminal Met peptide, a 170 kD Met precursor and a p145 ^β chain were detected (Fig. 4, lane 1). In addition, when developing antibodies directed towards the extracellular domain of Met, a molecule with antigenic properties and molecular weight corresponding to the truncated form of p85 ^{β was} detected (Fig. 4, lane 2). Moreover, a molecule comparable to soluble p75 ^{β was} detected in the supernatants of transfected cells with these antibodies (Fig. 4, lane 3). In transfected NIH 3T3 cells, the ratio of truncated Met-β chains to intact was higher than in human cells. These data indicate that truncated Met forms occur during translation of a full-sized transcript.

составляет приблизительно 2:1 (фиг. 5, дорожка 1). Напротив, в целом клеточном экстракте p85^β составлял не более чем 1/10 от p145^β (фиг. 5, дорожка 2). Анти-C-концевые моноклональные антитела осаждали p145 только из клеточных экстрактов (фиг. 5, дорожки 5 и 6). Таким образом, можно считать, что укороченная форма Met преимущественно локализована на поверхности клетки.Uneven cell distribution of p190 ^MET and p140 ^MET
To evaluate the relative amounts of p190 ^MET and p140 ^MET in the whole cell and on the cell surface, GTL-16 cells were labeled with [ ³ H] -glucosamine. Since glycosylation affects only the common extracellular domain, two molecular species must be labeled with this isotope with equal efficiency. The selective immunoprecipitation of molecules exposed on the surface of the cells was carried out (see the "Methods" section). The amount of protein precipitated in this way was comparable to the number of proteins immunoprecipitated from the whole cell extract. FIG. 5 shows that on the cell surface the ratio of p145 ^β to

is approximately 2: 1 (FIG. 5, lane 1). In contrast, in the whole cell extract, p85 ^β was no more than 1/10 of p145 ^β (Fig. 5, lane 2). Anti-C-terminal monoclonal antibodies precipitated p145 only from cell extracts (Fig. 5, lanes 5 and 6). Thus, we can assume that the truncated form of Met is predominantly localized on the cell surface.

Since the same lysis method was used to precipitate the Met proteins located on the cell surface or in whole cell extracts, this experiment also indicates that the potential proteolytic activity induced by the detergent does not play a significant role in the generation of p85 protein ^β
p140 ^{MET is} not the result of experimentally induced proteolysis.

Proteolytic cleavage of p190 ^MET could be induced by activation of endogenous proteases during cell extrusion. As an internal control of post-lytic proteolysis, we used antibodies specific to the extracellular domain, capable of immunoprecipitating the epidermal growth factor receptor. This receptor is expressed in GTL-16 cells and is known to be sensitive to proteolysis (Basu et al, Nature 311, 477 - 480, 1984). FIG. Figure 5 shows that under the conditions under which the shortened form of p140 ^MET is observed, only intact 175 kDa EGF receptor molecules are deposited, both from whole cell lysates and from protein lysates located on the cell surface.

In order to completely exclude the possibility of the occurrence of a shortened form of Met protein as a result of proteolytic degradation induced during extraction or immunoprecipitation, the presence of this form was analyzed directly in Western blots of various human cell lines. In these experiments, living cells were solubilized with boiling Laemmli buffer, which led to the blocking of protease activity. Total proteins were separated in SDS-PAGE under conditions not leading to the restoration of disulfide groups, and manifested by monoclonal antibodies directed to the extracellular domain. In four carcinoma cell lines expressing the MET gene, both forms of p190 ^MET and p140 ^{MET were} observed (Fig. 6). These data suggest that the truncated form of the Met protein is present in living cells under physiological conditions.

The truncated soluble protein p130 ^{MET is} released into the culture medium.

GTL-16 cells were metabolically labeled with ³⁵ S-methionine and the conditioned medium clarified by ultracentrifugation was precipitated with monoclonal antibodies to the extracellular domain and analyzed in SDS-PAGE. Under non-reducing conditions, molecules with an apparent mole were observed. weighing 130 kDa (p130 ^MET ) (Fig. 7A, lane 3). Upon reduction, this molecule disintegrated into two subunits of 50 kDa (p50) and 75 kDa (p75), respectively (Fig. 7c, lane 3). Monoclonal antibodies to the C-terminal Met peptide did not precipitate any proteins from the same medium (FIGS. 7A and 7C, lane 2). Similar results were obtained with a supernatant collected from the A549 pulmonary carcinoma cell line / Fig. 7B, lane 3 /.

Tryptic peptide maps have also been created for the p75 ^β chain. The initial material for these experiments was immunoprecipitate p130 ^MET released into the culture medium from surface radioiodinated cells. p75 ^{β was} eluted from SDS-PAGE gels after electrophoresis under reduction conditions. In FIG. 3C shows that labeled tryptic peptides were indistinguishable from their analogues derived from p145 ^β and p85 ^β .
According to estimates of the kinase analysis of in vitro immuno-complexes, p130 ^MET lacks kinase activity. These data show that the Met form released into the culture medium is an αβ complex lacking the cytoplasmic and transmembrane domains of p145 ^β , and the formation of this molecule is not limited to GTL-16 cells.

p130 ^{MET is} formed during the proteolytic process.

To prove that soluble p130 ^{MET is} formed from membrane-bound Met proteins, the following experiment was performed. GTL-16 cells adsorbed on tissue culture plates were superficially labeled with the ¹²⁵ I lactoperoxidase method and cultured for another 4 hours. The culture medium was collected and immunoprecipitated with monoclonal antibodies to the extracellular domain. Immunoprecipitate when analyzed in SDS-PAGE under reduction conditions yielded p75 ^β and p50 ^β , the expected subunits of soluble p130 ^MET (Fig. 8, lane 2). The corresponding cell extracts treated with the same monoclonal antibodies gave p145 ^β , p85 ^β and p50 ^α (FIG. 8, lane 3). Monoclonal antibodies directed to the Met-C terminal peptide were used as a control. These antibodies did not precipitate any proteins from the culture medium (FIG. 8, lane 1), and only intact chains p145 ^β and p50 ^α were precipitated from cell extracts (FIG. 8, lane 4). These results indicate that soluble Met protein is released into the culture medium by proteolytic cleavage of membrane-bound Met proteins.

The release of p130 ^{MET is} stimulated by TPA treatment.

To investigate whether p130 ^MET release can be modulated in living cells, protein kinase C was activated by treatment with TPA. GTL-16 cells metabolically labeled with [ ³⁵ S] methionine were stimulated with 160 nM TPA for 2 hours.

Cell extracts and supernatants were then harvested, ultracentrifuged, and precipitated with monoclonal antibodies to the Met-extracellular domain. Immunoprecipitates were analyzed by SDS-PAGE by recovery by electrophoresis. After TPA treatment, the amount of p75 ^β detected in the tissue culture supernatant increased markedly compared to the control (Fig. 9A). The number of total cellular proteins p145 ^β and p85 ^β did not seem to change (Fig. 9B). In order to analyze the effect of TPA on Met proteins exposed on the cell surface, GTL - 16 cells were treated with TPA and then ¹²⁵ I were labeled with lactoperoxidase and immunoprecipitated. When only surface-labeled Met proteins were taken into account, the amount of p85 ^β decreased significantly after treatment with TPA. p145 ^{β was} less affected by TPA than p85 ^β (Fig. 9C). Thus, it was concluded that activation of protein kinase C accelerates the release of extracellular p130 ^MET by stimulating proteolytic processing of membrane-bound p140 ^MET and p190 ^MET forms.

 Example 2. Materials and methods.

 Reagents

[γ- ³² p] ATP (specific activity: 7000 Ku mmol ^-1 ) and [ ¹²⁵ I] protein A were obtained from Amersham. ¹²⁵ I-HGF was prepared as described by Naldini et al, (1991, EMBO J., 10, 2867-2878 /. Briefly, the method is as follows: purified HGF / 1 μg / was radiolabeled with free Na ¹²⁵ I carrier (2 mCi) and iodogen (Pierce). 200 μl of iodogen from a solution of 100 μg / ml in chloroform were dried in a polypropylene propylene in a stream of nitrogen, then HGF and ¹²⁵ I in 0.525 M phosphate buffer, pH 7.4 were added, the reaction was left for 15 minutes at 4 ^o C, and then the mixture was transferred to another vial and left on ice for 10 minutes, BSA was added to a final concentration of 0.1% in 0.4 M NaCl, 0.1% CHAPS, 20 mM PO ₄ buffer, pH 7, 4 and m The ligand was separated from free Na ¹²⁵ I by affinity chromatography on a 1 ml heparin-sepharose column (Pierce), previously equilibrated with the same buffer. After intensive washing of the column, 1.3 M NaCl was eluted in the same buffer and 0 fractions were collected , 5 ml Fractions containing TCA (TXY) - precipitated radioactivity were combined and concentrated on a Centrisart (Sartorius) diafiltration apparatus with a membrane that did not allow proteins larger than 20 kDa and stored at 4 ^° C. The specific activity of the labeled atom was approximately 8 * 10 ⁷ CPM / μg / 5700 Ci / mmol /, which corresponds to a molar ratio I / HGF of approximately 3/1. Thus, the preparation did not contain a significant amount of unlabeled molecules.

 We used Lentil lectin Sepharose and Sepharose Protein A from Pharmacia, Triton X-100 from Fluka.

 Cells.

We used the GTL-16 cell line described in Example 1. Cells were cultured in (Flow) RPML 1640 medium containing 10% fetal bovine serum / Flow (Laboratories, Netherlands) and stored at 37 ^° C in a humidified atmosphere with 5% CO ₂ .

 Antibodies

 Phosphotyrosine antibodies were induced against p-amino-benzene phosphonate and affinity purified as previously described (Comoglio ei al., 1984, EMBO J., 3, 483-489). Polyclonal anti-Met serum was produced in rabbits immunized with the synthetic VDTRPASFWETS peptide corresponding to the C-terminal amino acid sequence predicted based on the MET gene sequence. Monoclonal antibodies DN-30, DN-31, and DO-24 to the extracellular domain of the Met protein were obtained from mice after immunization with whole living GTL - 16 cells.

Monoclonal antibodies were purified from ascites by precipitation with ammonium sulfate and affinity chromatography on a protein A column - Sepharose 4B (Pharmacia, Uppsala, Sweden). Purified DO-24 monoclonal antibodies were labeled with ¹²⁵ I using chloramine T with a specific activity of 2-4 mKu / mg protein.

Partial cleaning p130 ^MET .

The serum-free conditioned medium was collected from GTL - 16 cofluent cultures were ultracentrifuged at 50,000 g, concentrated ten times on amine filters (100 K) (stock solution). Purification was carried out using affinity chromatography on lentil lectin sepharose. After reaching an affinity interaction lasting 2 hours at 4 ^° C., the column was washed several times with RPMI medium, and glycoproteins were eluted with RPMI containing 0.2 M α-methyl-D-mannoside (Fluka). After overnight dialysis against RPMI, p130 ^MET was removed from a portion of the material purified by affinity chromatography by exhaustive adsorption on DN-31 monoclonal antibodies not solubilized on Sepharose Protein A. This material was used as a negative control. The partially purified p130 ^MET solution was standardized so that the protein concentration was 0.4 OD units (280 nm). The glycoprotein was stored at -20 ^o C.

 Two-determinant immunoassay (DDIA).

96-well EL ISA boards (Titertek Immuno Assay-Plate, Flow Laboratories, Netherlands) coated with DN-30 monoclonal antibodies and saturated with BSA were incubated overnight at 4 ^° C with various concentrations of partially purified p130 ^MET and radiolabeled HGF. The DDIA method, designed to estimate the amount of p130 ^MET , used two types of monoclonal antibodies directed to different epitopes of the extracellular domain of Met proteins. Purified monoclonal antibodies DN-30 were adsorbed on 96-well ELISA plates, the plates were then saturated and various concentrations of gross supernatant collected from GTL-16 cell cultures were added. Binding was detected by radio-labeled DO-24 monoclonal antibodies used as labeled substances. The boards were then washed extensively and the bound radioactivity was eluted with 1% boiling SDS and counted in a gamma counter (Autogamma, Packard).

 Immunoprecipitation and in vitro autophosphorylation assay.

GTL - 16 cells were washed twice with cold PBS and lysed in IM buffer (10 mM Pipers, pH 6.8, 100 mM NaCl; 5 mM MgCl ₂ , 300 mM sucrose, 5 mM EDTA) plus 1% Triton x-100 and a mixture of inhibitors protease. Cell lysates were centrifuged at 10,000 rpm at 4 ^° C for 30 minutes and incubated with anti-MET serum bound to Sepharose-Protein A. The bound proteins were washed several times with DIM buffer and incubated for 5 minutes at room temperature with 60 μl of various dilutions GTL lentil lectin eluate - 16 supernatant lacking p130 ^MET or containing p130 ^MET . The kinase reaction was carried out on ice for 2 minutes. 10 μCi [γ- ³² p] ATP per sample was diluted with unlabeled ATP to a final concentration of 100 μmol. The kinase reaction was stopped by adding 1 ml of physiological saline on a trisbuffer with 10 mM EDTA µmol Na ₃ OV ₄ . After a short centrifugation, the proteins eluted in boiling Laemmli; buffer and were subjected to electrophoresis in 8% SDS-PAGE followed by autoradiography for 12 hours at -80 ^o C with amplifying screens. The relative amount of phosphate incorporated in the p145 ^MET β-subunit was evaluated by measuring the optical density of the corresponding autoradiographic band using a laser densitometer (Pharmacia LKB 2202 Ultroscan).

 Western blotting.

The GTL-16 cell monolayer was washed twice with cold saline on phosphate buffer, and the cells were solubilized by boiling in Laemmli buffer (Laemmli, UK, 1970, Nature 230, 680-685). Samples were equalized at a protein concentration of 300 μg / well, subjected to electrophoresis in 8% SDS-PAGE and transferred to nitrocellulose filters. Blots showed 10 μg ml ^{-1 of} purified P-Tyr antibodies and then ¹²⁵ I with labeled protein A, as described in detail (DI Renzo et al., 1986, Eur. J. Biochem. 158, 383-391). Filters were subjected to autoradiography for 24 hours at -70 ^o C using reinforcing screens. The relative amount of phosphotyrosine included in the p145 ^MET β-subunit was evaluated by measuring the optical density of the corresponding autoradiographic band using a laser densitometer.

Results. HGF binds to p130 ^MET .

FIG. 10 shows that increasing amounts of soluble truncated p130 ^{MET not} solubilized on microtiter wells coated with specific antibodies bind HGF. The binding is saturable and dose-dependent.

Inhibition of p130 ^MET tyrosine phosphorylation of Met / HGF receptor in vivo.

GTL - 16 cells express a large amount of the HGF receptor, the β subunit of which (p145) is constitutively phosphorylated by tyrosine in vivo. This can be confirmed by Western blotting of total cellular proteins solubilized with an ionic detergent, analyzed in SDS-PAGE and probed with P-Tyr antibodies (Fig. 11, A). Treatment of these cells for 10 minutes with p130 ^MET purified on lentyl lactin induced a 30% reduction in tyrosine phosphorylation of the HGF / SF receptor (FIG. 11, B).

Inhibition of p130 ^MET tyrosine kinase activity of Met / HGF receptor in vitro.

The HGF receptor was immunoprecipitated from GTL-16 cells with antiserum directed to a synthetic peptide derived from the C-terminal sequence of the protein. Incubation of immune complexes with p130 ^MET before kinase analysis led to dose-dependent inhibition of autophosphorylation of the intact receptor subunit (p145 ^MET ; Fig. 12, B). With the same concentrations of lentil-lectin eluates lacking p130 ^MET , only a mild inhibitory effect was observed (Fig. 12, A).

Quantification of soluble p130 ^MET by DDIA.

p130 ^MET can be quantified by solid-phase two-determinant immunoassay using a tandem of monoclonal antibodies recognizing different epitopes. FIG. 13 shows a typical binding curve obtained with p130 ^MET released from GTL-16 cells into tissue culture medium. The binding is saturable, dose dependent and specific.

Claims (4)

 1. A protein consisting of two chains connected by a disulfide bond, namely an α-chain with a mol.m. 50 kDa and β-chains with mol.m. 75 or 85 kDa having the following properties: the α chain is the α chain of the hepatocyte growth factor receptor (HGF) encoded by the MET proto-oncogen; the β chain is a C-terminally shortened HGF receptor β chain; said protein binds to antibodies specific for the extracellular domain of the β-chain of the HGF receptor; said protein binds to HGF; said protein does not have a cytoplasmic tyrosine kinase domain.  2. The method of obtaining the protein according to claim 1, providing for the cultivation of cells expressing the MET gene in a nutrient medium, the interaction of the culture fluid with antibodies with the formation of the immunocomplex, the release and extraction of the target product from the immunocomplex, characterized in that they use antibodies specific for the extracellular domain HGF receptor β-chains and is isolated in white with a β-chain having a mol.m. 75 cd.  3. The method of obtaining the protein according to claim 1, providing for the cultivation of cells expressing the MET gene in a nutrient medium, extracting the cells with detergent, the interaction of the extract with antibodies to form an immunocomplex, releasing and isolating the target product from the immunocomplex, characterized in that antibodies specific to the extracellular domain of the β-chain of the HGF receptor, and a protein with a β-chain having a mol.m. 85 kD.  4. A pharmaceutical composition that inhibits hepatocyte growth factor (HGF), containing the active ingredient and a physiologically acceptable carrier or diluent, characterized in that the active ingredient contains the protein of claim 1 in an amount of 10-1000 μg per dose.

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