C-Terminal Truncated Forms of Met, the Hepatocyte Growth Factor Receptor

The MET proto-oncogene encodes a transmembrane tyrosine kinase of 190 kDa (p190MET), which has recently been identified as the receptor for hepatocyte growth factor/scatter factor. pl90MET is a heterodimer composed of two disulfide-linked chains of 50 kDa (p5Oa) and 145 kDa (p14513). We have produced four different monoclonal antibodies that are specific for the extracellular domain of the Met receptor. These antibodies immunoprecipitate with p190MET two additional Met proteins of 140 and 130 kDa. The first protein (pl40MET) is membrane bound and is composed of an a chain (p5Oa) and an 85-kDa C-terminal truncated P chain (p8503). The second protein (p1309ET) is released in the culture supernatant and consists of an a chain (p5Oa) and a 75-kDa C-terminal truncated , chain (p750'). Both truncated forms lack the tyrosine kinase domain. pl40fET and pl30MET are consistently detected in vivo, together with p190MET, in different cell lines or their culture supernatants. pl40MET is preferentially localized at the cell surface, where it is present in roughly half the amount of pl90MET. The two C-terminal truncated forms of the Met receptor are also found in stable transfectants expressing the full-length MET cDNA, thus showing that they originate from posttranslational proteolysis. This process is regulated by protein kinase C activation. Together, these data suggest that the production of the C-terminal truncated Met forms may have a physiological role in modulating the Met receptor function.

domain. pl40fET and pl30MET are consistently detected in vivo, together with p190MET, in different cell lines or their culture supernatants. pl40MET is preferentially localized at the cell surface, where it is present in roughly half the amount of pl90MET. The two C-terminal truncated forms of the Met receptor are also found in stable transfectants expressing the full-length MET cDNA, thus showing that they originate from posttranslational proteolysis. This process is regulated by protein kinase C activation. Together, these data suggest that the production of the C-terminal truncated Met forms may have a physiological role in modulating the Met receptor function.
Truncated forms of growth factor receptors in several cells, tissue culture supernatants, and biological fluids have been described (2,7,11,20,22,24,25,29,33,41,49). C-terminal truncated forms of tyrosine kinase receptors, exposed at the cell surface together with the native receptor, interfere with ligand-induced stimulation of tyrosine kinase activity by forming inactive heterodimers (1,23,45). Soluble forms of C-terminal truncated receptors may also compete with the intact receptor for ligand binding (29). In this study, we describe two distinct C-terminal truncated forms of the protein encoded by the MET proto-oncogene, which has recently been identified as the receptor for hepatocyte growth factor/scatter factor (3,31,32). The MET protooncogene (4,5,35) encodes a transmembrane glycoprotein (pl90ME) with unique features, composed of two disulfidelinked chains of 50 kDa (p50) and 145 kDa (p145'). Both chains are exposed at the cell surface (14,16). The a chain spans the plasma membrane with a hydrophobic amino acid stretch and possesses an intracellular tyrosine kinase domain (5,18,42). Terminal glycosylation and proteolytic cleavage of a single-chain 170-kDa precursor generate the mature form of the Met protein (14,42). The structural characterization described above was derived entirely from studies carried out using antibodies raised against a C-terminal peptide of p190MET. The present work has been carried out using newly developed monoclonal antibodies (MAbs) specific for the Met extracellular domain. Besides the previously described p190WET, these antibodies recognize in all cell lines examined two other Met heterodimers: a 140-kDa complex localized at the cell surface, and a 130-kDa complex released in the culture medium. Both complexes consist of an a chain indistinguishable from p5Oa and a C-terminal precipitation. Lactoperoxidase-H202-catalyzed cell surface radioiodination was performed as previously described (14). ,ug/ml [Boehringer] [14]). Ultracentrifuged tissue culture supernatants or cell extracts were precleaned on protein A-Sepharose and immunoprecipitated with the different antibodies. Immunoprecipitation of molecules exposed at the cell surface was done by incubating intact GTL-16 cells with MAbs before lysis with detergent. Immunocomplexes were collected on protein A-Sepharose, previously reacted with affinity-purified goat anti-mouse immunoglobulin antibodies, washed, and eluted in Laemmli buffer (26)  RPMI medium and further incubated with dimethyl sulfoxide containing or not containing (control) 160 nM TPA (Sigma) for 2 h. To analyze the effect of TPA on Met proteins exposed at the cell surface, GTL-16 cells were treated with TPA as described above and then labelled with 1251 by lactoperoxidase. In both cases, cell proteins solubilized with detergent or supernatants were precipitated as described above. Proteins were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), fixed, fluorographed when required, dried, and exposed to Amersham Hyperfilm for autoradiography. Protein sizes were estimated by using as markers myosin (200 kDa), phosphorylase b (92.5 kDa), bovine serum albumin (69 kDa), egg albumen (43 kDa), and carbonic anhydrase (30 kDa) that had been prelabelled by 14C methylation (Amersham).
Immunocomplex kinase assay. Proteins were extracted from GTL-16 cells in DIM buffer-1% Triton X-100 and precipitated as described above. Immunocomplexes collected on protein A-Sepharose-goat anti-mouse immunoglobulin antibodies were phosphorylated in 20 pl of the same buffer in the presence of 2.5 ,uCi of [-y-32P]ATP (specific activity, 7,000 Ci/mmol; Amersham) at 30°C for 5 min. The reaction was stopped by adding 1 ml of ice-cold phosphatebuffered saline (pH 7.2) containing 5 mM EDTA. Samples were centrifuged and eluted with boiling Laemmli buffer (26) with or without 2,B-mercaptoethanol. After SDS-PAGE, gels were dried and exposed to Amersham Hyperfilm for autoradiography with intensifying screens.
Peptide mapping. Met a and m chains, derived from surface-radioiodinated GTL-16 cells, were fractionated by SDS-PAGE, excised from the gel, washed twice with 10% methanol to remove SDS, minced, and dried in a lyophilizer. The gel slices were rehydrated with 50 mM NH4CO3 (pH 7.8) containing 50,ug of trypsin-TPCK [L-(tosylamido 2-phenyl)ethyl chloromethyl ketone; Worthington] per band and incubated for 2 h at 37°C. The tryptic digestion was repeated once, and the gel slices were further eluted with 50 mM NH4CO3 (pH 7.8). Eluates were then lyophilized and further incubated with 100,ug of trypsin per ml for 2 h at 37°C. The dried samples were resuspended in buffer A (100% water containing 0.1% trifluoroacetic acid) and filtered on a 0.2-,um-pore-size Acrodisc filter (Gelman Sciences). The iodinated peptides were analyzed on a reverse-phase C2/C18 Superpack Pep-S column (Pharmacia LKB) and resolved on a gradient of acetonitrile in buffer A (0 to 32% acetonitrile in 70 min) with a flow rate of 1 ml/min. The eluted radioactivity was monitored by a Radiomatic A-100 radioactive flow detector (Packard).
Western immunoblotting. Cells were solubilized in the boiling buffer described by Laemmli (26), with or without the reducing agent 2p-mercaptoethanol. Equal amounts of proteins (300 ,ug) were loaded into all lanes. After SDS-PAGE, proteins were transferred to nitrocellulose filters (Hybond; Amersham) and analyzed as described by Towbin et al. (44). Filters were then probed with the indicated antibodies, and specific binding was detected by the enhanced chemiluminescence system (ECL; Amersham).
Transfection of MET cDNA in NIH 3T3 cells. The expression vector used for these studies was based on plasmid pMT2, containing the major late adenovirus promoter. The expression plasmid was constructed with a 4.3-kb cDNA encompassing the entire MET coding sequence (16). As this plasmid does not contain any selectable marker, cells were cotransfected with pSV2neo carrying the neomycin resistance gene. The plasmid was cotransfected into NIH 3T3 cells by the lipofection procedure. Two days after transfection, the neomycin analog G418 was added to the culture medium to select for resistant clones. Stable transfectants expressing Met receptors were identified by assaying for the ability to synthesize Met receptors by Western blot analysis.

RESULTS
A truncated pl4OMET coexists with pl9MET at the cell surface. Proteins exposed at the surface of GTL-16 cells were labelled with 125I by lactoperoxidase. Cells were extracted with 1% Triton X-100, and solubilized molecules were precipitated with a MAb against the C-terminal peptide of the Met protein or with MAbs recognizing different epitopes of the extracellular domain. Immunoprecipitates were analyzed by SDS-PAGE under nonreducing or reducing conditions. The antibody against the C-terminal peptide precipitated the p190MET heterodimer (Fig. 1A, lane 1), which was resolved in p50a and p1450 chains under reducing conditions (Fig. 1B, lane 1). MAbs raised against the extracellular domain precipitated together with p190MET another molecule migrating with an apparent size of 140 kDa (p140AET; Fig. 1A, lanes 2 to 4). These immunoprecipitates under reducing conditions were resolved in three chains of, respectively, 145, 85, and 50 kDa (Fig. 1B, lanes 2 to 4), suggesting that p140MET is a heterodimer of p85 and p50. The experiment whose results are shown in Fig. 1C formally proves this interpretation of the heterodimeric structure of pl40MET. The 140-kDa band excised from panel A, rerun under reducing conditions, originated p85 and p50 (Fig. 1C, lane 1). The latter comigrated with p5O0 dissociated from the pl90MET a4p complex (Fig. 1C, lane 2). The ratio of p190QET to pl40MET varied between 1:1 and 2:1. Identical results were obtained when surface-iodinated proteins from other cell lines were examined (data not shown).
High-pressure liquid chromatography (HPLC) analysis of tryptic peptides derived from surface-iodinated p1450 and p85P chains yielded indistinguishable profiles (Fig. 2). In conclusion, p140MET is a heterodimer of p50a and an 85-kDa a chain (p85P) identical to the extracellular domain of p1450.
The truncated pl40MET is not phosphorylated on tyrosine. C-terminal peptide can be phosphorylated in vitro on the P subunit (14). Incubation of immunoprecipitates obtained with the four MAbs directed against the Met extracellular domain in the presence of [y-32P]ATP resulted in labelling of pl90MET but not of p140MET (Fig. 3A). Under reducing conditions, labelled p1450 and the coprecipitated Prl70 were the only molecular species observed (Fig. 3B). Similarly, pl4OMET was never detected by antiphosphotyrosine antibodies in Western blots of GTL-16 proteins, while pl90MET was consistently visualized (data not shown; 14). These data suggest that the truncated form of the Met protein lacks the tyrosine residues that can be phosphorylated in vitro or in vivo. Control antibodies directed against the C-terminal peptide also failed to detect the truncated Met form, while in in vitro kinase assays they did precipitate a low amount of a phosphorylated 45-kDa molecular species ( Fig. 3A and B, lanes 6). We have previously shown that this small-molecular-size band is generated by limited proteolysis of p1451 during immunoprecipitation in the presence of detergent (30).
pl40MET is not the result of experimentally induced proteolysis. To ascertain whether the truncated form of the Met protein was the result of proteolytic degradation introduced during the experimental procedures or was present in vivo, living cells were solubilized with boiling Laemmli buffer to block protease activity. Western blots of different human cell lines were probed with MAbs specific for the Met extracellular domain. Under nonreducing conditions, both pl9QMET and pl4OMET forms were detected in four epithelial cell lines expressing the MET gene (Fig. 4). When the same blots were probed with antibodies against the Met C-terminal peptide, p190MET was the only species observed (data not shown). Notably, the 45-kDa band observed in kinase MAbs anti-extracellular domain from either surface-radioiodinated GTL-16 cells or spent tissue culture medium harvested from radioiodinated GTL-16 cells. Proteins were separated by SDS-PAGE, and the excised bands were subjected to exhaustive tryptic digestion. About 20,000 cpm of each digest was analyzed on a reversephase C2/C18 Superpack Pep-S column (Pharmacia LKB) developed with a 0 to 32% acetonitrile gradient in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. Radioactive peptides were detected by an on-line gamma counter. ft, flowthrough. assays after detergent treatment was never detected under these conditions, further supporting the postlytic origin of this fragment (30). These data show that in living cells the truncated form of the Met protein is present in physiological conditions.
Uneven cellular distribution of pl90kET and p1401ET . To quantitate the relative amounts of pl90MET and p140MET in  Living cells were incubated for 2 h on ice with MAbs before being lysed with detergent (lanes 1 and 3). Alternatively, cells were lysed first and then incubated with MAbs (lanes 2 and 4). In both cases, immunocomplexes were recovered with protein A-Sepharose; normalized amounts of radiolabelled proteins were analyzed by SDS-PAGE under reducing conditions. MAbs directed against the Met extracellular domain (DO-24; lanes 1 and 2) or against the C-terminal peptide (DR-6; lanes 3 and 4) were used. Gels were dried and exposed for autoradiography for 4 days. exposed at the cell surface was carried out (see Materials and Methods), and the amount of protein recovered was compared with that immunoprecipitated from whole cell extract. At the cell surface, the ratio of p1451 to p850 was approximately 2:1 (Fig. 5, lane 1), a ratio similar to that observed after surface iodination and immunoprecipitation (Fig. 1). Conversely, in the whole cell extract, the ratio of p1450 to p851 was approximately 10:1 (Fig. 5, lane 2). Anti-C terminus MAbs precipitated only p1451 after cell lysis (Fig.  5, lanes 3 and 4). It thus can be concluded that the truncated Met form preferentially localizes at the cell surface. Since the same lysis procedure was used to precipitate Met proteins from the surface or from the whole cell extracts, this experiment further indicates that the potential proteolytic activity induced by detergent does not play a significant role in the generation of p851.
A truncated soluble pl30MET is released in the culture medium. GTL-16 cells were metabolically labelled with [35S]methionine. The conditioned medium, clarified by ultracentrifugation, was precipitated with MAbs anti-extracellular domain and analyzed by SDS-PAGE. Under nonreducing conditions, a molecule with an apparent molecular size of 130 kDa (p13ET) was observed (Fig. 6A, lane 1). Upon reduction, this molecule was resolved in two subunits of 50 kDa (pSOa) and 75 kDa (p750) (Fig. 6C, lane 1). No proteins were precipitated from the same medium by MAbs against the Met C-terminal peptide ( Fig. 6A and C, lanes 2). Similar results were obtained with a supematant harvested from the lung carcinoma cell line A549 (Fig. 6B, lane 1).
HPLC analysis was performed on tryptic peptides of the soluble p751 chain. As starting material, we used immunoprecipitates of p130JET released in the culture medium from surface-radioiodinated cells. p750 was eluted from SDS- polyacrylamide gels run under reducing conditions. Figure 3 shows that the 1251I-labelled tryptic peptides were indistinguishable from those obtained from p1451 and p851.
The soluble p130MET is devoid of kinase activity, as assessed by the immunocomplex kinase assay in vitro (data not shown).
These data show that the Met form released in the culture medium is an cup complex devoid of the cytoplasmic and transmembrane domains of p1451 and that its presence is not restricted to GTL-16 cells.
The truncated p140AIET and pj30MET originate by posttranslational processing. p140MET either could arise from translation of an alternatively spliced mRNA encoding only the extracellular and the transmembrane domains of the Met protein or could result from posttranslational processing. Indeed, four different MET transcripts are detected in GTL-16 cells (16,34), but none of them lacks the region coding for the cytoplasmic domain (see Discussion). To investigate the origin of the C-terminal truncated Met proteins, NIH 3T3 cells were cotransfected with the full-length human MET cDNA and pSV2neo. Stable transformants were selected with G418 and analyzed for Met expression. Northern (RNA) blot analysis of positive cells detected a single mRNA of the expected size (approximately 4.3 kb; data not shown). Cells were solubilized in boiling Laemmli buffer, and proteins were examined under reducing conditions by Western blot. The antibodies against the Met C-terminal peptide detected the 170-kDa Met precursor and the p1451 chain (Fig. 7, lane 1). In addition, the antibodies directed against the Met extracellular domain detected a molecule with antigenic properties and molecular mass consistent with those of the truncated p8513 (Fig. 7, lane 2). Moreover, in the supernatants of the transfected cells, these antibodies detected a molecule comparable to the soluble FIG. 7. Expression by mouse fibroblasts transfected with the human MET cDNA of intact p190MET as well as truncated forms. Solubilized proteins from NIH 3T3 cells transfected with the fulllength MET cDNA derived from the major 9-kb mRNA (lanes 1 and 2) or from their conditioned medium (lane 3) were analyzed by Western blot under reducing conditions. Proteins solubilized from NIH 3T3 cells transfected with the vector clone (lane 4) were also processed as described for lanes 1 through 3. Lanes: 1, MAbs against the C-terminal peptide of the Met protein; 2 to 4, MAb (DO-24) directed against the Met extracellular domain. Specific binding was detected by the enhanced chemiluminescence system (ECL; Amersham). Bands A to D have sizes consistent with the 170-kDa Met precursor (A), with the intact p14513 (B), with the truncated p8513 (C), and with the soluble p7513 (D). p750 (Fig. 7, lane 3). In transfected NIH 3T3 cells, the ratio of the truncated Met ,1 chains to the intact one was higher than in human cells. These data show that the Met truncated forms originate from posttranslational processing of proteins encoded by the full-length transcript.
pl30fET is generated by a proteolytic process. To prove that the soluble p130MET derives from the membrane-bound Met proteins, the following experiment was carried out. GTL-16 cells adhering to tissue culture plates were surface labelled with 125I by the lactoperoxidase method and cultured for 4 h. Culture medium was harvested and immunoprecipitated with MAbs anti-extracellular domain. The immunoprecipitate, analyzed by SDS-PAGE under reducing conditions, yielded p7513 and p50, the expected subunits of soluble p130'ET (Fig. 8, lane 2). The corresponding cell extracts treated with the same MAbs yielded p1451, p851, and p5Oa (Fig. 8, lane 3). MAbs directed against the Met C-terminal peptide were used as controls. These antibodies did not precipitate any protein from the culture medium (Fig.  8, lane 1), while they precipitated only the intact p1451 and p5O0 chains from cell extracts (Fig. 8, lane 4). These results demonstrate that the soluble Met protein is released in the culture medium by proteolytic cleavage of the membranebound Met proteins.
pl30MET release is up-regulated by TPA treatment. To investigate whether the release of p130MET can be modulated in living cells, protein kinase C was activated by TPA treatment. GTL-16 cells, metabolically labelled with [35S]methionine, were stimulated by 160 nM TPA for 2 h. Cell extracts and supernatants were then harvested, ultracentrifuged, and precipitated with MAbs against Met extra- . Immunocomplexes, collected on protein A-Sepharose, were analyzed by SDS-PAGE under reducing conditions. Gels were fluorographed, dried, and exposed for autoradiography for 6 h. (C) GTL-16 cells were treated with TPA (lane T) or dimethyl sulfoxide (lane C) as described above, labelled with 125I by lactoperoxidase, washed, and extracted with nonionic detergent. Clarified cell extracts were processed for immunoprecipitation with MAb DO-24, and immunocomplexes were analyzed as described above. cellular domain. Immunoprecipitates were analyzed by SDS-PAGE under reducing conditions. Following TPA treatment, the amount of p75Y detected in the tissue culture supernatant was significantly increased compared with control levels (Fig. 9A). The amount of total cellular p1451 and p851 was apparently unaffected (Fig. 9B). To analyze the effect of TPA on Met proteins exposed at the cell surface, GTL-16 cells were treated with TPA and then labelled with 125I by lactoperoxidase before immunoprecipitation. When only the surfaced-labelled Met proteins were considered, the amount of p850 was significantly decreased after TPA treatment. p1450 was less affected than p850 (Fig. 9C). It is thus concluded that protein kinase C activation up-regulates the release of the extracellular p130MET by stimulating proteolytic processing of the membrane-bound pl40MET and pl90MET forms.

DISCUSSION
Cell surface receptors endowed with tyrosine kinase activity share structural features. Their mature forms consist of at least three distinct domains with specific functions: an extracellular N-terminal ligand-binding domain, a transmembrane anchoring domain, and a cytoplasmic C-terminal domain. The latter contains the kinase region, regulatory sequences, and sites responsible for the interaction with cellular substrates (20,46,48). The MET oncogene (4, 5, 35) encodes a tyrosine kinase receptor whose ligand has recently been reported to be hepatocyte growth factor/scatter factor (3,31,32). The Met receptor is a transmembrane heterodimeric disulfide-linked complex of 190 kDa (p190MET).
This complex consists of an extracellular a chain of 50 kDa and a transmembrane 1 chain of 145 kDa (14,16). Terminal glycosylation and proteolytic cleavage of a single-chain precursor of 170 kDa generate the mature form of p190AIET (15,43). On the basis of the cDNA sequence (EMBL data bank accession number X54559) derived from the major transcript (36), the 1 chain would consist of 1,084 or 1,085 amino acids, depending on the position of the cleavage site. The predicted ,B-chain extracellular domain would include amino acids 306 to 932, the single transmembrane hydrophobic segment would include amino acids 933 to 955, and the intracellular domnain would include amino acids 956 to 1390.
In this study, by using a panel .of MAbs reacting with the extracellular domain of the Met receptor, two C-terminal truncated Met proteins were identified: a 140-kDa transmembrane form (pl40'ET) and a 130-kDa soluble protein, which is released in the culture medium (pl30MET). These truncated forms are detectable in the GTL-16 human gastric carcinoma cell line, in which the MET gene is amplified and overexpressed (14), as well as in other carcinoma cell lines with normal levels of MET expression.
The truncated Met proteins have the heterodimeric structure of the intact p190MET, consisting of two disulfidebonded chains (Fig. 10). The a chains of the truncated forms are indistinguishable from the a chain of p190MET, while their 1 chains have a smaller molecular size. The 1 chain of p140MET is about 85 kDa (p850), while the 1 chain of p130ME is about 75 kDa (p75).
Both p85P and p750 are truncated at their C termini. In fact, they are not recognized by antibodies directed against the C-terminal nonadecapeptide (Ser-1372 to Ser-1390) predicted from the MET sequence. Moreover, they lack the cytoplasmic tyrosine kinase domain containing Tyr-1235, the major phosphorylation site (10). This is shown by the fact that they are not phosphorylated on tyrosine, neither in vivo nor in vitro. p85f and p75P share the N-terminal domain with p145P, since they are all recognized by MAbs defining four different epitopes, and they yield identical extracellular tryptic peptides. The p85 chain is likely a transmembrane glycoprotein that has lost most of its cytoplasmic domain. Given the presence of a consensus sequence for proteolysis (-K-974 KRKQ-) within the predicted amino acid sequence, a cleavage of the last 435 C-terminal amino acids would reduce the molecular mass by about 50 kDa. This value is consistent with the observed difference in electrophoretic migration of the intact versus the truncated chains. p75P lacks the cytoplasmic domain and the transmembrane segment, since it is released in the culture supernatant and does not associate with the cell membrane. Moreover, it shows a 10-kDa reduction compared with the membrane-spanning p85 chain. This reduction in size is compatible with the loss of a segment including the transmembrane domain. p140MET and p130MET could originate either by translation of alternatively spliced mRNAs or by posttranslational proteolytic cleavage of the intact p190'' or its precursor. The second hypothesis is supported by the following data. Four MET transcripts of 9, 7, 5.2, and 3.4 kb have indeed been detected in different cell lines (16,34); however, none of them lacks the region encoding the cytoplasmic domain, as shown by studies performed with probes for defined regions of the gene (17). Moreover, mouse cells transfected with the full-length human cDNA express pl9%MET as well as the truncated forms p140MET and p130MET* Three lines of evidence show that the C-terminal truncated forms of the Met protein arise from a proteolytic process occurring in vivo and are not the result of experimental manipulations: (i) p140MET is detected in samples prepared by extracting living cells with boiling Laemmli buffer to block protease activity, (ii) uneven amounts of p140MET and pl90MET are precipitated from the cell surface and from the whole cell extract solubilized with identical procedures, and (iii) the amount of detectable p140MET is modulated in vivo by activation of protein kinase C.
The predicted reciprocal intracellular polypeptide generated by proteolytic cleavage of a precursor Met protein was never detected in Western blot experiments performed with antibodies against either the Met C-terminal peptide or phosphotyrosine, which suggests that this fragment, if generated, is rapidly degraded in vivo. A previously reported 45-kDa C-terminal fragment generated in vitro by detergentinduced proteolysis (30) cannot be correlated with the presence of the truncated Met forms in vivo. Proteolysis giving rise to p140MET could occur both within the endoplasmic lumen and at the cytoplasmic face of the cell membrane, as suggested by its preferential localization at the cell surface. This localization could also be due to the fact that, in analogy with what has been reported for the epidermal growth factor receptor (19), p140MET, lacking tyrosine kinase activity, cannot be targeted to lysosomes but is recycled to the cell surface.
To our knowledge, this is the first example of a transmembrane truncated receptor generated by proteolytic processing in the absence of the ligand. The truncated forms of the receptor encoded by trkB (24,28), the murine interleukin-4 (IL-4) receptor (29), and the human epidermal growth factor receptor (47) are generated by translation of alternatively spliced mRNAs.
The conserved expression of the truncated forms of the Met receptor among different cell lines suggests that they may play a physiological role. The soluble p130MET might represent a breakdown product of the receptor. We have previously shown that activation of protein kinase C negatively regulates the Met receptor tyrosine kinase activity by increasing serine phosphorylation of the P chain (12). The present finding that TPA also increases the levels of soluble p130MET suggests that activation of protein kinase C induces concomitant receptor down-modulation by proteolytic activity, as described for the colony-stimulating factor 1 receptor (8). Whether a similar receptor down-modulation occurs also in response to the hepatocyte growth factor/scatter factor ligand is presently under investigation. In this respect, it has been reported that epidermal growth factor induces the N-terminal truncation of its receptor by a proteolytic process (6). It is tempting to speculate that the truncated forms may also interfere with the Met receptor signal transduction pathway by competing with the intact receptor for binding to the ligand. Such a negative regulatory role has already been shown for the soluble form of the IL-4 receptor (29). Moreover, the transmembrane truncated receptors, devoid of tyrosine kinase activity, may form inactive heterodimers with the intact receptors. We have already shown that trans phosphorylation is required for activation of the Met kinase (30). It has been reported that truncated epidermal growth factor and platelet-derived growth factor receptors can inhibit wild-type receptor function (1,23,45). However, an excess or at least equimolar amount of truncated receptor is required for the effect of the dominant negative mutant (45 there may be more truncated protein transiently expressed than is apparent in the steady-state conditions analyzed in this culture system.