INTRODUCTION

Hepatocyte growth factor/scatter factor (HGF/SF)¹ is a multifunctional protein that acts as a mitogen, motogen, and/or morphogen depending on the cellular target and context (1, 2, 3). It stimulates the proliferation not only of hepatocytes but a wide variety of epithelial cells as well as melanocytes and endothelial and hematopoietic cells (4, 5, 6, 7). It promotes the dispersion of certain epithelial and endothelial cells when they are seeded onto plastic surfaces, hence the term ``scatter factor'' (8, 9). Cells can be induced to invade collagen gels in response to HGF/SF (10), where in some instances they give rise to tubular structures in a manner analogous to branching morphogenesis in vivo. (11, 12). Paradoxically, some sarcoma and carcinoma cells undergo cytotoxic changes following administration of HGF/SF (13, 14). All of these responses appear to be mediated by the c-Met receptor tyrosine kinase (15, 16, 17, 18, 19, 20).

HGF/SF resembles plasminogen in that the two molecules share 38% amino acid sequence identity and several structural motifs (21, 22). Each is synthesized as an inactive monomer, which is proteolytically processed at a conserved site to generate a functional, disulfide-linked heterodimer (23, 24, 25, 26). The heavy chain of the dimer (~60 kDa in HGF/SF) is derived from the amino terminus of the precursor and contains multiple kringle domains (four in HGF/SF and five in plasminogen). These ~80-amino acid domains have a characteristic folding pattern defined by three internal disulfide bonds and additional conserved residues (27). For plasminogen and tissue plasminogen activator, specific kringles have been shown to participate in protein-protein interactions (28). The light chain of HGF/SF (32-34 kDa), like that of plasminogen, has the structure of a serine protease. However, two of the three amino acid residues of the catalytic triad have been replaced with nonconservative substitutions, and HGF/SF is apparently devoid of proteolytic activity (21).

Previous studies identified an alternatively spliced, HGF/SF transcript in which a kringle 2 exon was joined to an exon with an in-frame termination signal (29, 30). Purified HGF/NK2, as the corresponding protein was designated, lacked intrinsic mitogenic activity but specifically blocked the action of HGF/SF in [³H]thymidine incorporation assays (30). Cross-linking studies indicated that HGF/NK2 and HGF/SF competed with each other for binding to Met (30). Thus, HGF/NK2 is a competitive antagonist of HGF/SF mitogenic action. In the course of our analysis of HGF/SF gene expression, we also observed a 2-kb transcript. We report here that this transcript encodes a second naturally occurring, truncated form of HGF/SF. This molecule extends only a few amino acids beyond the first kringle (K1) domain and exhibits biological properties distinct from those of HGF/SF or HGF/NK2. Although it retains the avid heparin binding affinity of HGF/SF and interacts with Met, this novel polypeptide, designated HGF/NK1, exhibits partial agonist/antagonist properties. Thus, the HGF/SF gene encodes three different molecules, each unique in its structural and biological properties.

EXPERIMENTAL PROCEDURES

An M426 cDNA library (31) was screened with probes corresponding to either the heavy or light chain region of HGF/SF, as described previously (30). Restriction enzyme analysis of selected cDNA clones was performed with PstI, XbaI, and EcoRV (New England Biolabs, Inc.) according to the manufacturer's instructions.

A series of polymerase chain reaction (PCR) experiments were conducted using pairs of oligonucleotide primers corresponding to different regions of the HGF/NK1 sequence and either human genomic DNA or pH46 as template. The sense primers were: P1, 5-TGGGTGACCAAACTCCTGCCA-3 (in signal peptide domain); P3, 5-GAGGTACGCTACGAAGTCTGTGAC-3 (in carboxyl-terminal portion of K1); P5, 5-GCAATGGAGACCACCAAACTGTCT-3 (in midregion of pH46 3-untranslated sequence (3-UT)). The antisense primers were: P2, 5-ATTTGTAGTTGCATTTGCACGAAC-3 (~175 base pairs downstream from K1 in 3-UT); P4, 5-AGACAGTTTGGTGGTCTCCATTGC-3 (in midregion of 3-UT, corresponding to sense primer 5); P6, 5-CAATGAAGAGGTTATAGGGAACAGAT-3 (at 3 end of pH46). For PCR (32), 0.3 µg of human genomic DNA or 5 ng of plasmid DNA was subjected to 35 cycles of amplification with the following cycling conditions: 1 min at 94 °C, 2 min at 58 °C, and 3 min at 72 °C. PCR products (5%) were electrophoresed in 1% agarose gels and detected by ethidium bromide staining.

Poly(A)⁺ RNA was isolated from M426 and SK-LMS-1 cells as described (33). Samples (5 µg each) were electrophoresed in a 1% denaturing formaldehyde agarose gel and transferred to nitrocellulose filters. Filters were prehybridized for 2 h at 42 °C in Hybrisol (Oncor; 40% formamide, 10% dextran sulfate, 1% SDS, 6 × SSC, and blocking agents) and then hybridized for 15 h in the same solution with [³²P]dCTP-labeled randomly primed probes corresponding either to the heavy chain of HGF/SF or to a segment of the 3-UT of pH46. The heavy chain probe was generated by PCR using HGF/SF cDNA as template with sense primer 5-GGACAAAGGAAAAGAAGAAATACAATT-3 and antisense primer 5-ATTGTCAGCGCATGTTTTAATTGCACA-3 corresponding to sequences 91-117 and 847-873, respectively numbering according to Ref. 22. The 3-UT probe was prepared using pH46 as template with sense primer 5-GGTAAATAAACCTGAATGCCA-3 and antisense primer 5-TTTCTGTGGAAGCAGGTGCTG-3 corresponding to sequences 679-699 and 1057-1077, respectively (numbering based on HGF/NK1 cDNA sequence submitted to GenBank). Filters were washed twice (30 min each) in 2 × SSC, 0.1% SDS at room temperature and twice (30 min each) in 0.1 × SSC, 0.1% SDS at 50 °C and exposed to Kodak X-Omat AR film for 24 h.

The HGF/NK1 coding sequence tagged with BamHI restriction sites was subcloned into the BamHI site of the baculovirus vector pVL941 (Pharmingen) (34). Recombinant baculovirus was produced by cotransfecting Sf9 (Spodoptera frugiperda) insect cells with HGF/NK1-pVL941 and AcNPV (Autographa californica) baculovirus DNA by the calcium phosphate method as suggested by the manufacturer (BaculoGold transfection kit, Pharmingen). Similarly, viral plaque purification, amplification, stock production, and infections were performed according to protocols provided by the manufacturer.

For production of HGF/NK1 protein, 2 × 10⁸ Sf9 cells were seeded in a 175-cm² T flask containing Sf 900 medium (Life Technologies, Inc.) plus 10% fetal bovine serum (Biofluids). After a 1-h incubation to facilitate cell attachment, the medium was replaced with fresh medium containing recombinant virus at a multiplicity of infection of 10:1. 1 h later, the culture was aspirated, and fresh medium was added. After 3 days, conditioned medium was harvested and either frozen at 20 °C or directly loaded onto a heparin-Poros HPLC column (2.7 ml of bed volume; Perseptive Biosystems) at a flow rate of 5 ml/min. After washing the column with 20 mM phosphate buffer, pH 7.4/0.3 M NaCl, protein was eluted with a linear gradient of 0.3-1.5 M NaCl. Fractions containing HGF/NK1 were identified by immunoblotting.

The full-length coding region of HGF/SF was subcloned into the baculovirus vector pVL941 essentially as described for HGF/NK1. Recombinant HGF/SF protein was generated and purified as outlined for HGF/NK1, using either a heparin-TSK (ToyoHaas) or heparin-Poros column. Typically, HGF/SF preparations were ~90% pure and contained varying proportions of both monomeric and heterodimeric forms.

For immunoblotting, proteins were resolved in 12.5% polyacrylamide-SDS gels under reducing or nonreducing conditions and transferred to Immobilon (polyvinylidene difluoride) filters (Millipore). Blocking and detection of proteins with diluted, GammaBind (Pharmacia Biotech Inc.)-purified antiserum to HGF/SF (4) were as described previously (35). Silver staining of protein resolved by SDS-PAGE was performed with Silver Stain Plus (Bio-Rad) following the manufacturer's protocol.

DNA synthesis by B5/589 human mammary epithelial cells was measured by [³H]thymidine incorporation as described (36). Epidermal growth factor (recombinant murine) was from Collaborative Research. The scatter assay was performed with a subclone of Madin-Darby canine kidney cells, kindly provided by Dr. Robert Furlong, according to published methods (37).

Purified HGF/NK1 (2 µg) was radiolabeled with chloramine T as described for HGF/NK2 (17). B5/589 cells were incubated with Hepes binding buffer (35) containing 0.9 nM [¹²⁵I]HGF/NK1 (2.5 × 10⁵ cpm; specific activity ~10 µCi/µg) for 45 min at room temperature, washed with cold Hepes-buffered saline, pH 7.4, and treated as described for cross-linking with [¹²⁵I]HGF/NK2 (17) except that 100 µM of water-soluble bis (sulfosuccinimidyl) suberate (BS3; Pierce) was used instead of disuccinimidyl suberate. In some experiments, varying concentrations of unlabeled HGF/NK1 or HGF/SF were included in the binding buffer with [¹²⁵I]HGF/NK1.

After lysing cells with Hepes solubilizer buffer (50 mM Hepes, pH 7.4, 1% Triton X-100 (v/v), 100 mM NaF, 2.5 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin), proteins were immunoprecipitated with a rabbit polyclonal antiserum (50 µg/ml) to the carboxyl-terminal 28 amino acid residues of human Met protein in the absence or the presence of competing peptide (1 µg/ml). Immunoprecipitated proteins were pelleted with immobilized protein-G (GammaBind, Pharmacia) and eluted with Laemmli buffer. Following separation in 6% SDS-PAGE, gels were dried and exposed to Kodak X-Omat AR film or exposed to a storage screen and analyzed using a PhosphorImager (Molecular Dynamics). Alternatively, cells were solubilized directly in SDS and boiled for 3 min in the presence of 100 mM -mercaptoethanol, and lysates were subjected to electrophoresis for autoradiography as above.

Confluent B5/589 cells were serum-starved for 24 h, exposed to HGF/SF or HGF/NK1 for 10 min at 37 °C, lysed in Hepes solubilizer buffer, and immunoprecipitated with anti-phosphotyrosine monoclonal antibody 4G10 bound to agarose beads (50 µl of beads/lysate; Upstate Biotechnology, Inc.). Immunoprecipitated proteins were resolved by 7.5% SDS-PAGE and immunoblotted with the Met peptide antiserum (7.5 µg/ml) as described previously (17).

RESULTS

In the course of studies that identified HGF/NK2 as the product of a 1.3-kb alternative HGF/SF transcript, we detected several cDNA clones in an M426 library that hybridized to a HGF/SF heavy but not light chain oligonucleotide probe. Restriction enzyme analysis indicated multiple distinct patterns among these clones. One was typified by a 1.2-kb insert that encoded HGF/NK2, as previously reported (30). A different pattern was exhibited by three other clones that contained a 2-kb insert and lacked EcoRV and XbaI sites present in HGF/SF (Fig. 1A). Sequence analysis of one of these clones, designated pH46, revealed a distinct, 210-amino acid truncated version of HGF/SF consisting of the signal peptide and the amino-terminal and K1 domains. The coding sequence of HGF/NK1 terminated immediately downstream of the K1 domain with two additional amino acids and a translational stop codon not found in the corresponding region of HGF/SF. The open reading frame was flanked by 54 base pairs of 5-untranslated sequence previously observed in HGF/SF transcripts as well as a unique ~1.2-kb 3-UT containing a polyadenylation signal (AATAAA).

The presence of novel sequence downstream from the K1 domain suggested that HGF/NK1 mRNA resulted from alternative processing of the nascent HGF/SF transcript. To determine the mechanism responsible for the generation of HGF/NK1, we examined the genomic structure near exon K1b (38, 39). PCR analysis of total cellular and pH46 DNA was performed with various pairs of oligonucleotide primers corresponding to different regions of the HGF/NK1 cDNA sequence. As shown in Fig. 1 (A and B), amplification of sequences extending from the 3 end of K1 to the middle or downstream end of the 3-UT (Fig. 1B, lanes 3-6) and within the 3-UT (Fig. 1B, lanes 7 and 8) yielded fragments of the same apparent size when either genomic or HGF/NK1 cDNA was used as template. To rule out the possibility of plasmid contamination of the genomic DNA preparation, PCR was carried out with primers (P1 and P2) to exons spanning >10 kb of genomic sequence (Fig. 1A and Refs. 38 and 39). Although a fragment of the expected length was obtained with pH46 DNA as template, no product was observed with the genomic DNA preparation (Fig. 1B, lanes 1 and 2). These data indicated that the unique sequence at the 3 end of the HGF/NK1 cDNA was contiguous with exon K1b in the human genome. This finding was reinforced by the fact that the first 10 nucleotides of the 3-UT were identical to the published intronic sequence (38) located immediately downstream from exon K1b (Fig. 1C). As illustrated in Fig. 1C, the 3-UT of pH46 corresponds to the 5 end of the ~6.6-kb intron separating exons K1b and K2a in the HGF/SF gene (38).

Although retention of intronic sequence in pH46 raised the possibility that the HGF/NK1 clone might have resulted from incomplete or aberrant RNA processing, this cDNA possessed a polyadenylation signal approximately 30 base pairs upstream from a poly(A) tail. Moreover, using a probe derived from the unique 3-UT, Northern blot analysis of poly(A)⁺ RNA from M426 fibroblasts and SK-LMS-1 cells revealed a 2-kb transcript, corresponding to a faint band seen with an HGF/SF heavy chain probe (Fig. 2). Taken altogether, these results established that HGF/NK1 was a naturally occurring variant encoded by an alternative HGF/SF transcript.

In preliminary experiments, the HGF/NK1 coding sequence was placed into an MMTneo vector (40) and introduced into NIH/3T3 cells using standard calcium phosphate transfection methodology. Immunoblot analysis of conditioned medium from transfected cells revealed the presence of a ~20-kDa HGF/SF cross-reactive protein that was absent from the medium of control cells (data not shown). This protein bound avidly to heparin-Sepharose and eluted with 0.9-1.0 M NaCl, comparable with conditions employed for HGF/SF (4). A highly purified preparation was obtained by subsequent sizing chromatography and ion exchange or reverse-phase HPLC (data not shown). However, the amounts recovered (typically a few µg/liter conditioned medium) were not sufficient to perform extensive biological analysis.

As an alternative, we expressed the protein in Sf9 insect cells with a baculovirus vector. This approach had proven successful in generating HGF/SF with biological activity comparable with that of the naturally occurring factor (18). The chromatographic, electrophoretic, and immunologic properties of baculovirus-expressed HGF/NK1 matched those of the recombinant NIH/3T3-derived material. Benefitting from the higher level of expression in the baculovirus system, we were able to obtain a highly purified preparation of recombinant HGF/NK1 with a one-step purification process based on heparin affinity chromatography (Fig. 3). The yield was approximately 40 µg of HGF/NK1 from 1 liter of Sf9-conditioned medium. Like HGF/SF and HGF/NK2 (4, 30), HGF/NK1 migrates as a more compact molecule in SDS-PAGE when its disulfide bonds are intact rather than reduced (Fig. 3).

HGF/NK1 exhibited mitogenic activity as determined by [³H]thymidine incorporation in B5/589 human mammary epithelial cells (Fig. 4A). However, it was significantly less potent than baculovirus-expressed HGF/SF tested under the same conditions. Even at a concentration as high as 8 nM, HGF/NK1 stimulated only 20-25% of the maximal DNA synthesis elicited by HGF/SF at 0.5 nM. Moreover, HGF/NK1 behaved as a specific antagonist of HGF/SF in the same assay. A 40-fold molar excess of HGF/NK1 reduced the mitogenic activity of 0.1 nM HGF/SF by ~70%, whereas no inhibition of epidermal growth factor activity was observed under the same conditions (Fig. 4B). At high concentrations (5-10 nM), HGF/NK1 also promoted scattering of Madin-Darby canine kidney cells, with an effect comparable with that of HGF/SF at a 50-fold lower molar concentration (data not shown).

3

To establish that the activities of HGF/NK1 were attributable to a direct interaction with the high affinity HGF/SF receptor, a series of covalent cross-linking experiments were performed. Following incubation of B5/589 cells with ¹²⁵I-labeled HGF/NK1 and cross-linking agent, cell lysates were immunoprecipitated with a Met-specific peptide antiserum in the presence or the absence of competing synthetic peptide. When the immunoprecipitates were resolved by SDS-PAGE, autoradiography revealed a single major Met peptide-specific band corresponding in size to a complex consisting of [¹²⁵I]HGF/NK1 and the Met subunit at a stoichiometry of 1:1 (Fig. 5A). Cross-linking in the presence of either excess unlabeled HGF/NK1 or HGF/SF suggested that the affinity of HGF/NK1 for Met was within an order of magnitude of that of the full-length growth factor (Fig. 5B).

To further study the interaction of HGF/NK1 with its receptor, we examined the ability of this ligand to stimulate tyrosine phosphorylation of Met. Using concentrations corresponding to those employed in the bioassays described above, a significant increase in Met tyrosine phosphorylation was detected in response to HGF/NK1 (Fig. 6). In fact, the intensity of the phosphotyrosine signal induced by HGF/NK1 at 8 nM was comparable with that observed with HGF/SF at 0.5 nM. These results demonstrated that HGF/NK1 and HGF/SF were capable of stimulating a similar level of Met tyrosine phosphorylation, even though HGF/NK1 elicited a weaker mitogenic response.

DISCUSSION

In the present study, we identified a new naturally occurring truncated form of HGF/SF. This molecule, designated HGF/NK1, is encoded by a 2-kb alternative HGF/SF transcript, which results from retention of a portion of the intron separating exons K1b and K2a. The biological properties of HGF/NK1 are different than those of the other previously characterized HGF/SF isoforms. In contrast to HGF/NK2, HGF/NK1 has intrinsic agonist activity in the B5/589 DNA synthesis bioassay. However, its potency was considerably less than HGF/SF. Moreover, a 40-fold molar excess HGF/NK1 inhibited the mitogenic activity of HGF/SF. Chemical cross-linking experiments with B5/589 cells demonstrated that HGF/NK1 bound directly to Met, with an apparent affinity estimated to be within an order of magnitude that of HGF/SF. Thus, HGF/NK1 behaves as a partial agonist/antagonist of HGF/SF mitogenic activity on these human mammary epithelial cells.

The different biological effects of HGF/SF and HGF/NK1 cannot be attributed simply to the level of ligand-induced Met autophosphorylation. Under conditions in which they elicited a similar degree of Met tyrosine phosphorylation, HGF/NK1 was less efficient in stimulating DNA synthesis. However, whereas the total phosphotyrosine content of Met following exposure to each isoform was comparable, the distribution of phosphotyrosine residues in the receptor sequence may vary. The major sites of Met autophosphorylation (Tyr¹²³⁴ and Tyr¹²³⁵), which reside in the catalytic domain and are required for tyrosine kinase activity (41, 42), presumably are phosphorylated in response to both ligands. However, other tyrosine residues, Tyr¹³⁴⁹ and Tyr¹³⁵⁶, near the carboxyl terminus of Met are believed to be critical for docking of effector molecules such as phosphatidylinositol 3-kinase, phospholipase C, pp60c-src, and the GRB-2-Sos complex (43). Site-directed mutagenesis of the above-mentioned as well as other tyrosine residues in Met had either a positive or negative effect on specific cellular responses (44). Thus, variation in phosphorylation of these residues is likely to have a significant impact on signaling pathways and could account for the unique patterns of activity associated with the different HGF/SF isoforms. In addition, differential regulation of Met tyrosine kinase activity by serine phosphorylation (45, 46, 47) or Met-associated tyrosine phosphatase activity (48) also might contribute to the particular effects of the various HGF/SF isoforms.

Our experiments established that the alternative transcript encoding HGF/NK1 resulted from retention of intronic sequence adjacent to exon 1Kb. Although this mechanism for generating alternative transcripts is unusual, other examples of retained introns have been described (49, 50, 51, 52, 53). Of note, three cases involve transcripts that are expressed at relatively high levels in placenta: a soluble form of the HLA-G antigen (52), a variant of human growth hormone-V encoding a unique 104-amino acid carboxyl terminus (51), and an isoform of human gonadotropin-releasing hormone (50). The relative preponderance of cases in placenta led to the hypothesis that this organ may contain specific factors that facilitate export of mRNA with introns into the cytoplasm (52). This may be relevant to the expression of HGF/NK1. Miyazawa et al. detected a relatively abundant ~2-kb alternative HGF/SF transcript in placenta (29). Although the extent of truncation had not been fully determined, this mRNA hybridized to the coding sequence of HGF/NK2 but not to probes for K3, K4, or the serine-protease domain. Given its size and hybridization pattern, we surmise that it probably corresponded to the HGF/NK1 transcript described here. Recently, gene targeting experiments revealed that loss of the HGF/SF gene resulted in death in utero due to placental insufficiency (54, 55). This finding demonstrated that the HGF/SF gene is crucial for placental development and, in view of the observed expression pattern in placenta, raises the possibility that HGF/NK1 might participate in the development of this organ.

Our results concerning HGF/NK1 activity differ in some respects from the analysis of an artificially engineered version of HGF/NK1 (56). In the latter instance, HGF/NK1 behaved as a pure antagonist of HGF/SF mitogenic activity, showing no agonist activity at concentrations up to 100 nM in an assay using rat hepatocyte primary cultures. It also barely stimulated Met tyrosine phosphorylation when tested at 20 and 100 nM. There are several possible reasons for these differences. The artificially constructed version of HGF/NK1 was expressed in bacteria as a fusion protein containing a 10-amino acid FLAG epitope at its amino terminus to target secretion of the protein into the periplasmic space. The engineered protein also lacked the two amino acid extension to the K1 domain that is present in the naturally occurring isoform. Conceivably, either of these structural differences could affect biological activity by modifying receptor-ligand interactions. Recently, we expressed the naturally occurring HGF/NK1 sequence in bacteria and observed biological activity very similar to that described above for the baculovirus-expressed protein.² Thus, bacterial expression per se does not result in a molecule having activities at variance with the results obtained in the present study. However, our bacterial expression strategy included a series of steps to optimize protein refolding and fidelity of disulfide bond formation that could influence the activity of the final product. Independent of differences in HGF/NK1 preparations, the discrepancies in our data relative to the earlier report might be attributable to the cells used in our respective bioassays. For instance, proteoglycan composition, which varies enormously among cells (57) and affects signaling by fibroblast growth factors (58, 59, 60) and HGF/SF (61, 62)³ might account for the contrasting responses of different cell types to HGF/NK1.

HGF/NK1 should prove to be a useful tool in the structure-function analysis of HGF/SF. Although its affinity for both heparin and Met are similar to that of HGF/SF and it retains biological activity, the smaller size and lack of glycosylation sites render HGF/NK1 particularly suitable for crystallographic and NMR structural analysis. Systematic modification of HGF/NK1 should provide additional insight into the interaction of HGF/SF with its receptors. This, in turn, may lead to the development of more potent, clinically useful agonists or antagonists of HGF/SF signaling.