©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Novel Vascular Endothelial Growth Factor (VEGF) Receptors on Tumor Cells That Bind VEGF via Its Exon 7-encoded Domain (*)

(Received for publication, October 3, 1995; and in revised form, December 20, 1995)

Shay Soker (1) Herman Fidder (1) Gera Neufeld (2) Michael Klagsbrun (1)(§)

From the  (1)Departments of Surgery and Pathology, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and the (2)Department of Biology, Technion, Israel Institute of Technology, Haifa 32000, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Vascular endothelial growth factor (VEGF), a potent angiogenic factor, uses two receptor tyrosine kinases, FLK/KDR and FLT, to mediate its activities. We have cross-linked I-VEGF to the cell surface of various tumor cell lines and of human umbilical vein endothelial cells. High molecular mass (220 and 240 kDa) and/or lower molecular mass (165 and 175 kDa) labeled complexes were detected depending on the cell type. The 220- and 240-kDa labeled complexes were shown to contain FLT and FLK/KDR receptors, respectively. On the other hand, the 165- and 175-kDa complexes did not seem to contain FLK/KDR or FLT but instead appeared to contain novel VEGF receptors with relatively low molecular masses of approximately 120 and 130 kDa. These receptors were further characterized in breast cancer MDA MB 231 cells (231), which did not form the high molecular mass complexes and which did not express detectable amounts of flk/kdr or flt mRNA. The 231 cells displayed one VEGF binding site, with a K of 2.8 times 10M and 0.95-1.1 times 10^5 binding sites per cell. By comparison, human umbilical vein endothelial cells had two binding sites, one with a K of 7.5 times 10M, presumably FLK/KDR, and the other with a K of 2 times 10M, a value similar to the VEGF binding sites on 231 cells. These lower affinity/molecular mass receptors on 231 cells cross-linked I-VEGF but not I-VEGF. Accordingly, exon 7 of VEGF, which encodes the 44 amino acids present in VEGF that are absent in VEGF, was fused to glutathione S-transferase (GST). The GST-VEGF-exon 7 fusion protein bound to heparin-Sepharose with a similar affinity as VEGF and inhibited the binding of I-VEGF to 231 cells. Cross-linking of I-GST-VEGF-exon 7 to 231 cells resulted in the formation of 150- and 160-kDa labeled complexes that presumably contained the 120- and 130-kDa lower affinity/molecular mass VEGF receptors. It was concluded that certain tumor-derived cell lines express novel surface-associated receptors that selectively bind VEGF via the exon 7-encoded domain, which is absent in VEGF.


INTRODUCTION

Vascular endothelial growth factor (VEGF) (^1)was initially purified from the conditioned media of folliculostellate cells (1) and a variety of tumor cell lines as a potent angiogenic factor and mitogen for endothelial cells (EC) in vitro(2, 3) . An inducer of blood vessel permeability was concurrently purified from the conditioned medium of U937 cells that was found to be the same as VEGF and was named vascular permeability factor(4, 5) .

Several studies point to VEGF as being an important regulator of angiogenesis. For example, VEGF expression is up-regulated in tissues undergoing vascularization during embryogenesis and during the female reproductive cycle(6, 7) . High levels of VEGF are found in various types of tumors in response to tumor-induced hypoxia but not in normal tissue(8, 9, 10, 11, 12) . A recent study has directly linked VEGF to vascularization-dependent tumor growth by showing that treatment of tumors with monoclonal antibodies directed against VEGF resulted in a dramatic reduction in tumor angiogenesis and tumor mass(13) .

Four different VEGF isoforms, consisting of 121, 165, 189, and 206 amino acids, are produced as a result of alternative splicing from a single gene containing eight exons(14, 15, 16) . The active form of VEGF is a homodimer, and all four isoforms display a similar ability to induce EC proliferation(15) . Although all VEGF isoforms are synthesized with a signal peptide that allows secretion, they differ in their localization probably as a result of differential affinities for heparan sulfate proteoglycans that are found on the cell surface and in the extracellular matrix(15, 17, 18) . VEGF and VEGF have a high affinity for heparan sulfate and are mostly cell and extracellular matrix-associated. VEGF, which has a lower affinity for heparan sulfate, is partially released into the culture medium and partially found on the cell surface and in the extracellular matrix. VEGF is the only VEGF isoform that does not bind to heparin and is exclusively secreted into the culture medium(15, 17) .

VEGF binds to specific receptor tyrosine kinases, KDR and FLT, that are expressed by EC and by several types of non-EC such as human melanoma cells, NIH3T3 cells, HeLa cells, and Balb/c 3T3 cells(19, 20, 21, 22, 23) . The flt and kdr cDNAs were cloned from human libraries prepared from placenta and EC, respectively(24, 25) . KDR has a molecular mass of 190 kDa(26) , and FLT has a lower molecular mass, which is 160 kDa(27) . KDR, whose mouse homologue is known as FLK, is the only known VEGF receptor that is found on the cell surface of bovine aortic endothelial cells(28) . On the other hand, some melanoma cell lines have been shown to synthesize FLT(29) . Efficient binding of I-VEGF to its receptors on EC requires the presence of heparin-like molecules on the cell surface(20) . Heparin has differential effects on the binding of I-VEGF to FLK/KDR and FLT. Soluble heparin seems to enhance the binding of I-VEGF to EC synthesizing FLK/KDR (20, 30) but has the opposite effect on the binding of I-VEGF to melanoma cells synthesizing FLT(28, 29) . I-VEGF binds to alpha2 macroglobulin and as a consequence is not able to bind its receptors on HUVEC. Heparin interferes with this binding and is able to restore the receptor binding capability of I-VEGF(30, 31) .

The binding of VEGF to VEGF receptors on non-EC does not seem to induce cell proliferation. Rather, VEGF induces motility of monocytes(32) , differentiation of osteoblasts(33) , production of insulin by beta cells(34) , and disorganization of actin stress fibers in Balb/c 3T3 cells(23) . Recent studies with cells expressing endogenous or transfected flt and flk/kdr have suggested that VEGF activities, e.g. mitogenicity, chemotaxis, and morphological changes are mediated by FLK/KDR but not through FLT, even though both receptors undergo phosphorylation upon binding of VEGF(35, 36, 37, 38) . Placenta growth factor, a recently purified VEGF homologue (39) that binds to FLT, has no effect on the proliferation rate of EC(36, 40) . These results suggest that although VEGF can bind to multiple receptors on the cell surface, the subsequent VEGF-mediated responses may be different.

Cross-linking of I-VEGF to EC and non-EC results in the formation of multiple I-VEGF-VEGF receptor labeled complexes, with higher molecular masses of about 220-240 kDa and lower molecular masses of about 165-175 kDa(19, 20, 22) . However, to date it has not been clear which of the known VEGF receptors are contained in the various labeled complexes. In this report, we demonstrate (i) that the high molecular mass complexes found in EC and melanoma cells contain FLK or FLT; (ii) that the lower molecular mass complexes do not appear to contain FLK or FLT but rather VEGF receptors of relatively lower affinity; (iii) that the lower affinity/molecular mass receptors found on tumor cells are isoform-specific in that they bind I-VEGF but not I-VEGF, in agreement with previous results obtained for HUVEC(41) ; and (iv) that the binding of I-VEGF to these receptors is mediated by the VEGF exon 7-encoded domain, which is present in VEGF but not VEGF.


EXPERIMENTAL PROCEDURES

Materials

Human recombinant VEGF and VEGF were produced by Sf-9 insect cells infected with a baculovirus-based vector expressing VEGF and VEGF cDNAs, as described previously(29, 42) . VEGF was purified from the conditioned medium of the infected Sf-9 cells by heparin affinity chromatography, and VEGF was purified by hydrophobic chromatography followed by anion exchange chromatography as described(29, 42) . Placenta growth factor was kindly provided by Dr. Y. Cao (Children's Hospital, Boston, MA). Basic fibroblast growth factor was kindly provided by Scios-Nova (Mountain View, CA). Platelet-derived growth factor, epidermal growth factor, and acidic fibroblast growth factor were purchased from R & D systems (Minneapolis, MN). Anti-FLK and anti-FLT antibodies were purchased from Santa-Cruz Biotechnology, Inc. (Santa Cruz, CA). Cell culture media were purchased from Life Technologies, Inc. I-Sodium was purchased from DuPont NEN. Disuccinimidyl suberate and IODO-GEN were purchased from Pierce Chemical Co. Heparin-Sepharose, glutathione-agarose, Sephadex CL-4B, Protein G-coupled Sephadex CL-4B, NAP-5 columns and pGEX-2TK plasmid were purchased from Pharmacia Biotech Inc. TSK-Heparin columns were purchased from TosoHaas (Tokyo, Japan). Molecular weight markers were purchased from Amersham Corp. Porcine intestinal mucosal-derived heparin was purchased from Sigma.

Cell Culture

HUVEC, obtained from American type culture collection (ATCC, Rockville, MD) and bovine aortic endothelial cells, isolated from bovine aortas, were grown as described previously(20) . MDA-MB-231 cells and MDA-MB-453 cells were obtained from ATCC and grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and a mixture of glutamine, penicillin, and streptomycin. RU-mel cells and EP-mel cells were kindly provided by Dr. Randolf Byer (Boston University Medical School, Boston, MA) and grown in Dulbecco's modified Eagle's medium-containing 2% FCS, 8% calf serum, and a mixture of glutamine, penicillin, and streptomycin. Human metastatic prostate adenocarcinoma, LNCaP cells, were kindly provided by Dr. Michael Freeman (Children's Hospital, Boston, MA) and grown in RPMI 1640 medium containing 5% fetal calf serum and a mixture of glutamine, penicillin, and streptomycin.

Radioiodination

The iodination of VEGF, VEGF, and GST-VEGF-exon 7 were carried out using IODO-GEN as described previously(29) . I-VEGF was purified by heparin affinity chromatography, and gelatin was added to a final concentration of 2 mg/ml. I-VEGF and I-GST-VEGF-exon 7 proteins were adjusted to 1 mg/ml bovine serum albumin and purified by size exclusion chromatography using NAP-5 columns. Aliquots of the iodinated proteins were frozen on dry ice and stored at -80 °C. The specific activity ranged between 30,000 and 100,000 cpm/ng protein.

Binding, Cross-linking, and Immunoprecipitation

Binding and cross-linking experiments using I-VEGF, I-VEGF, and I-GST-VEGF-exon 7 were performed as described previously (19, 20) , except that in the binding experiments cells were grown in 48-well dishes and the volumes of the binding reactions were 0.25 ml/well. For the immunoprecipitation of cross-linked complexes, cells were lysed with lysis buffer (phosphate-buffered saline containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) for 20 min on ice. The cell lysate was collected in a tube and spun at 7000 times g for 5 min to remove the cell debris. The lysate was incubated with Sephadex CL-4B for 1 h at 4 °C and then separated from the Sephadex beads by centrifugation at 1000 times g for 3 min. Anti-FLK and anti-FLT antibodies were added to the lysates, and following 1 h of incubation at 4 °C, 20 µl of protein G (coupled to Sephadex CL-4B) were added for an additional 1 h of incubation at 4 °C. Protein G-Sepharose beads were pelleted at 1000 times g for 3 min, washed 3 times with lysis buffer, and resuspended in SDS-PAGE sample buffer. The samples were boiled for 3 min, and proteins were resolved by 6% SDS-PAGE(43) . Gels were exposed to a PhosphorImager screen and scanned after 24 h of exposure. Subsequently, the polyacrylamide gels were exposed to x-ray film. VEGF binding was quantitated by measuring the cell-associated radioactivity in a counter (Beckman, Gamma 5500). The counts represent the average of three wells. All experiments were repeated at least three times, and similar results were obtained.

Preparation and Purification of GST-VEGF-Exon 7 Fusion Protein

Exon 7 of VEGF was amplified by the polymerase chain reaction using the primers CGGGATCCCCTGTGGGCCTTGCTC and GGAATTCTTAACATCTGC-AAGTACGTT. The amplified DNA was digested with BamHI and EcoRI restriction enzymes and cloned in frame into the GST-expressing vector pGEX-2TK (44) to yield the p2TK-exon 7 plasmid. Escherichia coli (DH5alpha) transformed with p2TK-exon 7 were used to produce the GST-VEGF exon 7 (GST-ex 7) fusion protein, and the GST-ex 7 was purified from the bacterial lysate by glutathione-agarose affinity chromatography(44) . Glutathione-agarose-purified GST-ex 7 was applied to a TSK-Heparin column (3.75 ml), which was then washed extensively with 20 mM sodium phosphate, pH 7.2, 0.2 M NaCl. GST-ex 7 was eluted from the column by a linear gradient of 0.2-1.2 M NaCl, pH 7.2. Samples eluted from the column were analyzed by 15% SDS-PAGE (43) and silver staining.


RESULTS

Cross-linking of I-VEGF to the Surface of HUVEC and Tumor-derived Cell Lines

I-VEGF was cross-linked to cells in the presence or the absence of 1 µg/ml heparin (Fig. 1). As reported previously(19, 20) , cross-linking of I-VEGF to HUVEC resulted in the formation of a 240-kDa labeled complex (Fig. 1, lane 1). In the presence of heparin, two additional labeled complexes of approximately 165 and 175 kDa were detected (Fig. 1, lane 2). Cross-linking of I-VEGF to the breast cancer cell line MDA-MB-231 (231 cells) (Fig. 1, lanes 3 and 4) and to the prostate tumor cell line LNCaP (Fig. 1, lanes 5 and 6) produced similar complexes of approximately 165-175 kDa but not the 240-kDa labeled complex. In contrast, the breast cancer cell line MDA-MB-453 (Fig. 1, lanes 7 and 8) did not cross-link I-VEGF at all. Cross-linking of I-VEGF to the surface of two melanoma cell lines, EP-mel and RU-mel, resulted in the formation of several labeled complexes ranging from 150 to 240 kDa (Fig. 2, lanes 7 and 10). The addition of heparin enhanced I-VEGF binding and cross-linking to 231 cells (Fig. 1, lane 4) and LNCaP (Fig. 1, lane 6) cells but did not cause the appearance of any labeled complex in MDA-MB-453. The binding of I-VEGF to the cells was specific because the addition of a 100-fold excess of nonlabeled VEGF completely inhibited the formation of the labeled complexes (not shown).


Figure 1: Cross-linking of -VEGF to the surface of HUVEC and tumor-derived cell lines. I-VEGF (5 ng/ml) was bound and cross-linked to subconfluent cultures of HUVEC (lanes 1 and 2), 231 cells (lanes 3 and 4), LNCaP cells (lanes 5 and 6), and MDA-MB-453 cells (lanes 7 and 8) in 6-cm dishes. The binding was carried out in the presence (lanes 2, 4, 6, and 8) or the absence (lanes 1, 3, 5, and 7) of 1 µg/ml heparin. The cells were lysed, and proteins were resolved by 6% SDS-PAGE as described under ``Experimental Procedures.''




Figure 2: Immunoprecipitation of I-VEGF cross-linked complexes containing VEGF receptors. I-VEGF (5 ng/ml) was bound and cross-linked to subconfluent cultures of HUVEC (lanes 1-3), 231 cells (lanes 4-6), EP-mel cells (lanes 7-9), and RU-mel cells (lanes 10-12) in 10-cm dishes. The binding was carried out in the presence of 1 µg/ml heparin, except for RU-mel. The cells were lysed, and immunoprecipitation was performed with anti-FLK (lanes 2, 5, 8, and 11) and anti-FLT (lanes 3, 6, 9, and 12) antibodies as described under ``Experimental Procedures.'' Samples of cell lysate were kept aside before the antibodies were added to serve as controls (lanes 1, 4, 7, and 10). The immunocomplexes were resolved by 6% SDS-PAGE as described in the legend to Fig. 1.



Immunoprecipitation of Cross-linked I-VEGF-VEGF Receptor Complexes with Anti-FLK and Anti-FLT Antibodies

To test whether the observed I-VEGF-VEGF receptor cross-linked complexes contained FLK/KDR or FLT, specific anti-FLK and anti-FLT antibodies were used to immunoprecipitate these complexes. The 240-kDa complexes of HUVEC (Fig. 2, lane 2), EP-mel cells (Fig. 2, lane 8) and bovine aortic endothelial cells (not shown) were immunoprecipitated by anti-FLK antibodies, whereas the 220-kDa complex of RU-mel was immunoprecipitated by anti-FLT antibodies (Fig. 2, lane 12). These results indicate that HUVEC and EP-mel cells synthesize predominantly FLK/KDR, whereas RU-mel cells synthesize FLT. These anti-VEGF receptor antibodies failed to immunoprecipitate the 165- and 175-kDa complexes from any of the cell lines (Fig. 2), suggesting that these complexes contain VEGF-receptors that are different than FLK/KDR or FLT. For further analysis of the 165- and 175-kDa complexes, we chose 231 cells because they did not produce the higher molecular mass complexes containing FLK/KDR or FLT, thus facilitating analysis of the lower molecular mass complexes. In addition, the expression of flk/kdr or flt mRNA could not be detected by a Northern blot analysis of 231 cell-derived RNA (not shown). Based on the molecular weight of a VEGF dimer (45 kDa), the receptors forming the 165- and 175-kDa labeled complexes were estimated to have molecular masses of approximately 120 and 130 kDa, respectively.

Analysis of VEGF Binding Sites on 231 Cells

To determine the affinity of VEGF for the 120- and 130-kDa receptors, 231 cells and HUVEC were incubated with increasing concentrations of I-VEGF (Fig. 3A). Binding of I-VEGF to 231 cells was carried out in the presence or the absence of heparin. The specific binding of I-VEGF to 231 cells increased in a dose-dependent manner and reached a plateau at approximately 1.9 times 10M (8.5 ng/ml). Heparin (1 µg/ml) induced an 80% increase in the binding of I-VEGF to 231 cells, in agreement with the heparin-induced augmentation of binding shown in Fig. 1(lanes 3 and 4). The binding results were used to generate Scatchard plots (Fig. 3, B-D), which were further analyzed by the LIGAND program(45) . The program predicted the presence of a single class of binding sites on 231 cells with a K(d) of 2.8 times 10M and 0.95-1.1 times 10^5 binding sites per cell (Fig. 3C). Heparin had no significant effect on the affinity of VEGF for its binding sites on 231 cells (K(d) = 2.7 times 10) but induced a 2-fold increase in their number (1.9-2.0 times 10^5 binding sites/cell) (Fig. 3D). Thus, heparin enhances the binding of I-VEGF by increasing the number of available binding sites on 231 cells rather than by changing the affinity of VEGF for its binding sites. In comparison, HUVEC displayed two classes of binding sites for VEGF, the higher affinity binding sites had a K(d) of 7.5 times 10 and approximately 2 times 10^3 binding sites/cell, and the lower affinity binding sites had a K(d) of 2.0 times 10M and 2.5 times 10^4 binding sites/cell (Fig. 3B). The similar K(d) values of VEGF for its binding sites on 231 cells and for the lower affinity sites on HUVEC suggest that these sites may represent the lower molecular mass VEGF receptors that form the 165- and 175-kDa complexes on HUVEC and 231 cells as shown in Fig. 1.


Figure 3: Analysis of VEGF binding sites on 231 cells and HUVEC. A, increasing amounts of I-VEGF (1.5-80 fmol) were added to subconfluent cultures of 231 cells (circles) and HUVEC (triangles) in 48-well dishes. The binding to 231 cells was carried out in the presence (closed circles) or the absence (open circles) of 1 µg/ml heparin. Nonspecific binding was determined by competition with a 200-fold excess of unlabeled VEGF. After binding, the cells were washed and lysed, and the cell-associated radioactivity was determined using a counter. The results shown in A were analyzed by the method of Scatchard, and best fit plots for HUVEC (B), 231 cells (C), and 231 cells in the presence of heparin (D) were obtained using the LIGAND program.



VEGF but Not VEGF Binds to Receptors on 231 Cells

To test the specificity of the interaction between VEGF and the lower affinity/molecular mass receptors, I-VEGF was bound to 231 cells and HUVEC in the presence of a 100-fold excess of nonlabeled growth factors (Fig. 4). Excess VEGF inhibited I-VEGF binding by approximately 90% in both cell types (Fig. 4). On the other hand, neither epidermal growth factor, basic fibroblast growth factor, acidic fibroblast growth factor (none of these shown), placenta growth factor, nor platelet-derived growth factor, of which the latter two have 53 and 18% amino acid sequence homologies to VEGF, respectively(4, 39) , inhibited I-VEGF binding to either cell type (Fig. 4).


Figure 4: The binding of I-VEGF to 231 cells and HUVEC in the presence of excess growth factors. I-VEGF (4 ng/ml) was bound to subconfluent cultures of 231 cells (hatched bars) and HUVEC (solid bars) in 48-well dishes in the presence of 400 ng/ml of unlabeled VEGF, VEGF, PIGF, or platelet-derived growth factor. After binding, cells were washed and lysed, and the cell-associated radioactivity was determined using a counter. The counts obtained are expressed as the percentage of the counts obtained compared with a phosphate-buffered saline (PBS) control.



VEGF is an isoform that is similar to VEGF in its mitogenicity for EC, but unlike VEGF it is not heparin-binding(15) . A 100-fold excess of VEGF inhibited the binding of I-VEGF to HUVEC by 75% but did not inhibit binding of I-VEGF to 231 cells (Fig. 4). In addition, unlike I-VEGF (Fig. 5, lanes 1 and 2), I-VEGF (Fig. 5, lanes 3 and 4) did not form any cross-linked complexes with 231 cells. As a control to ensure that I-VEGF was bioactive, I-VEGF bound to HUVEC but to form only the 240-kDa complex, presumably containing FLK/KDR, (Fig. 5, lanes 5 and 6), confirming previous results(41) . Thus, it appears that VEGF binds only to high affinity receptors such as FLK/KDR to form the 240-kDa complex, whereas VEGF can bind to FLK/KDR and FLT and in addition to lower affinity/molecular mass receptors to form the 165- and 175-kDa complexes.


Figure 5: Cross-linking of I-VEGF and I-VEGF to the surface of 231 cells and HUVEC. I-VEGF (5 ng/ml) (lanes 1, 2, 7, and 8) or I-VEGF (10 ng/ml) (lanes 3-6) were bound and cross-linked to subconfluent cultures of 231 cells (lanes 1-4) and HUVEC (lanes 5-8) in 6-cm dishes. The binding was carried out in the presence (lanes 2, 4, 6, and 8) or the absence (lanes 1, 3, 5, and 7) of 1 µg/ml heparin. Cells were lysed, and proteins were resolved by a 6% SDS-PAGE as described in the legend to Fig. 1.



VEGF Interacts with Its Receptors on 231 Cells through the Exon 7-encoded Domain

VEGF differs structurally from VEGF by an insert of a 44-amino acid domain encoded by exon 7(14) . Thus, the ability of VEGF but not VEGF to bind to 231 cells may be due to the presence of the exon 7-encoded domain in VEGF. To test this hypothesis, the VEGF exon 7 was fused to the gene of GST to yield a chimeric protein, GST-VEGF exon 7 (GST-ex 7). GST-ex 7 bound to a TSK-heparin column, and the majority of the protein was eluted with 0.7 M NaCl (Fig. 6A), the same concentration necessary to elute VEGF (not shown). This fusion protein has a molecular mass of 32 kDa, which is the predicted size of GST-ex 7 (Fig. 6B). Thus, it seems that exon 7 encodes a domain that is responsible for the heparin binding capacity of VEGF. Incubation with 2 and 5 µg/ml of purified GST-ex 7 inhibited the binding of I-VEGF to 231 cells by 52 and 95%, respectively, whereas the GST protein had no effect (Fig. 7). The GST-ex 7 fusion protein was radioiodinated and cross-linked to 231 cells in the presence and the absence of 1 µg/ml heparin (Fig. 8). Labeled complexes of approximately 150-160 kDa were formed (Fig. 8, lane 1), and increased binding and cross-linking occurred in the presence of heparin (Fig. 8, lane 2), which is consistent with the ability of heparin to enhance the binding of I-VEGF to its receptors on 231 cells as shown in Fig. 1and Fig. 3. The molecular masses of the I-GST-ex 7 cross-linked complexes, 150 and 160 kDa, are consistent with the binding of 32-kDa GST-ex 7 to 120- and 130-kDa receptors on 231 cells. The ability of the GST-ex 7 fusion protein to bind directly to the 120- and 130-kDa receptors on 231 cells and to inhibit the binding of I-VEGF to these receptors suggests that the exon 7-encoded domain mediates the binding of VEGF to the lower affinity/molecular mass receptors on HUVEC and 231 cells.


Figure 6: Heparin affinity chromatography of the GST-ex 7 fusion protein. GST-ex 7 fusion protein was prepared and purified by glutathione-agarose affinity chromatography as described under ``Experimental Procedures,'' and 300 µg were applied to a TSK-heparin column. Heparin-bound proteins were eluted with a linear gradient of 0.2-1.2 M NaCl (A). An aliquot from the peak fraction (number 58) was resolved by 15% SDS-PAGE, which was silver-stained and photographed (B).




Figure 7: Competition of I-VEGF binding to 231 cells by the GST-ex 7 fusion protein. I-VEGF (4 ng/ml) was bound to subconfluent cultures of 231 cells in 48-well dishes in the presence of 2 and 5 µg/ml GST-ex 7 fusion protein (fraction 58) (solid bars) or 5 µg/ml GST protein (hatched bar). The cells were washed and lysed, and the cell-associated radioactivity was determined in a counter. The counts obtained are expressed as the percentage of the counts obtained in a control experiment that used phosphate-buffered saline (PBS, open bar).




Figure 8: Cross-linking of I-GST-ex 7 fusion protein to the surface of 231 cells. I-GST-ex 7 (10 ng/ml) was bound and cross-linked to subconfluent cultures of 231 cells in 6-cm dishes. The binding was carried out in the presence (lane 2) or the absence (lane 1) of 1 µg/ml heparin. The cells were lysed, and proteins were resolved by 6% SDS-PAGE as described in the legend to Fig. 1.




DISCUSSION

In recent years, there has been growing evidence that VEGF plays a major role in regulating angiogenesis during normal development and in tumors(6, 11, 13, 38, 46) . VEGF binds to specific high affinity receptors, FLK/KDR and FLT, which mediate VEGF responses(26, 27) , and which were initially shown to be associated with various types of EC (25, 47, 48) . In addition to EC, VEGF also binds to receptors on the surface of cell types such as HeLa, human melanoma, and NIH3T3(20, 22, 23) . Analysis of I-VEGF-VEGF receptor cross-linking patterns have demonstrated that there are multiple VEGF binding sites, for example, on EC and on melanoma cells(19, 20, 22) . However, the actual identities of the multiple VEGF receptors in these cross-linked complexes have not been determined, and thus our goal in this study was to characterize the VEGF receptors in the various complexes. Accordingly, I-VEGF was cross-linked to the cell surface of several cell types. Analysis of the cross-linking products revealed the presence of several I-VEGF-cross-linked complexes of higher (220 and 240 kDa) and lower (165 and 175 kDa) molecular mass. HUVEC formed both the higher and lower molecular mass complexes as shown previously(20, 41) , as did several melanoma cell types(22, 29) . On the other hand, the breast cancer-derived cell line, MDA MB 231 (231 cells), and the prostate carcinoma-derived cell-line, LNCaP, formed only the lower molecular mass complexes. There was no case in which the high but not the low molecular mass complexes were formed.

Immunoprecipitation studies with anti-FLK/KDR and anti-FLT antibodies were used to identify the VEGF receptors on the various cell types. The 240-kDa complex formed by I-VEGF binding to HUVEC was shown to contain FLK/KDR, confirming previous results(41) , whereas the 220-kDa complex formed with RU-mel cells was shown to contain FLT. Although EC have been reported to express flt mRNA(47, 48) , we have not been able to identify FLT as part of the I-VEGF-cross-linked complexes formed with HUVEC or bovine aortic endothelial cells. On the other hand, immunoprecipitation with anti-FLK/KDR and anti-FLT antibodies showed no cross-reactivity with proteins in the 165- and 175-kDa complexes in 231 cells, consistent with similar results for HUVEC(41) , suggesting that these complexes contain VEGF receptors that are different than FLK/KDR and FLT. Based on the molecular mass of a VEGF dimer, we estimated the size of these lower molecular mass VEGF receptors to be 120 and 130 kDa.

We chose 231 cells to characterize the lower molecular mass VEGF receptors because they do not produce FLK/KDR- or FLT-containing complexes and do not express flk/kdr or flt mRNA. Scatchard analysis of the binding of I-VEGF to 231 cells revealed the presence of one class of binding sites that bind VEGF with a K(d) of 2.8 times 10M and 0.95-1.1 times 10^5 binding sites/cell. Heparin induced a 2-fold increase in the binding of I-VEGF to 231 cells by increasing the number of binding sites/cell without significantly changing the affinity for VEGF. On the other hand, HUVEC displayed two binding sites for VEGF, with K(d) values of 7.5 times 10 and 2.0 times 10M, respectively. These results indicate that the VEGF binding sites on 231 cells have an affinity for VEGF similar to the lower affinity binding sites on HUVEC. Because both cell types produce labeled complexes of 165 and 175 kDa upon cross-linking with I-VEGF, it seems that the lower molecular mass VEGF receptors detected on the cell surface of 231 cells may be the same as the lower affinity VEGF receptors found on HUVEC. Accordingly, we have designated these binding sites as lower affinity/molecular mass VEGF receptors.

Although excess nonlabeled VEGF inhibited I-VEGF binding to 231 cells, a 100-fold excess of VEGF did not. In addition, no radiolabeled complexes, neither of higher nor lower molecular mass, could be detected upon cross-linking of I-VEGF to 231 cells. As a control, I-VEGF was shown to be active in that it formed a 240-kDa complex, presumably containing FLK/KDR, upon cross-linking to HUVEC. On the other hand, I-VEGF did not form 165- and 175-kDa complexes with HUVEC, consistent with the 231 cell results. The ability of I-VEGF, but not I-VEGF, to form 165- and 175-kDa complexes with HUVEC has been reported recently(41) . Taken together, these results demonstrate a VEGF isoform specificity of binding in which I-VEGF is capable of binding to FLK/KDR but not to the lower affinity/molecular mass receptors, whereas I-VEGF binds to both. Thus, VEGF activities may be specific for FLK/KDR and/or FLT, whereas VEGF has a wider range of activities by using more receptor types.

Because the only structural difference between VEGF and VEGF resides in the 44-amino acid insert encoded by exon 7(14) , we assumed that this domain might mediate the binding of VEGF to 231 cells. To test this hypothesis, a chimeric protein made of GST and the exon 7-encoded domain of VEGF was prepared. This GST-ex 7 fusion protein bound to heparin-Sepharose with a similar affinity as VEGF(17) , indicating that the heparin-binding domain of VEGF is localized to the exon 7-encoded domain. In addition, GST-ex 7 competed with the binding of I-VEGF to 231 cells and could bind and be cross-linked directly to the lower affinity/molecular mass receptors. Taken together, these results indicate that the exon 7-encoded domain is responsible for both VEGF heparin-binding and the binding to the lower affinity/molecular mass receptors.

An important question to consider is whether the 120- and 130-kDa lower affinity receptors expressed by 231 cells are novel or whether they may be truncated forms of FLK/KDR or FLT lacking about 40-50 kDa in molecular mass. The latter possibility is unlikely because (i) VEGF does not bind to these receptors on 231 cells or EC but is fully capable of binding to FLK/KDR on EC (Fig. 5) (41) and to FLT on melanoma cells(29) , suggesting that the lower affinity/molecular mass receptors do not contain the extracellular domain of FLK or FLT. However, it is possible that FLT or FLK could be truncated in such a way that would allow VEGF but not VEGF binding, but this possibility would suggest different binding sites for the two VEGF isoforms, which has yet to be demonstrated; (ii) the 120- and 130-kDa lower affinity receptors are not recognized by anti-FLK or anti-FLT antibodies. These are polyclonal antibodies directed against the C-terminal 20 and 17 amino acids of FLK and FLT, respectively. Thus, as a minimum the lower affinity receptors do not appear to contain the C-terminal of FLK or FLT, although it is possible that they do contain some other cytoplasmic sequences; and (iii) the 231 cells do not express detectable levels of flk/kdr or flt mRNA. Taken together, the circumstantial evidence suggests that the lower affinity/molecular mass VEGF receptors are probably not related to FLK or FLT. However, this question will not be resolved definitively until these receptors are purified or cloned and sequence information is available.

We have not yet determined the biological role of the lower affinity/molecular mass VEGF receptors. To date, we have not been able to demonstrate VEGF-induced proliferation or protein phosphorylation of 231 cells. It may be that these receptors mediate other effects of VEGF such as migration (49) or morphological changes(23) . Alternatively, they may potentiate the responses of FLK/KDR or FLT to VEGF. In addition, it has been demonstrated that although VEGF and VEGF are both released from cells, some VEGF is also associated with the cell surface, possibly due to its interaction with heparan sulfate proteoglycans(17, 18) . Thus, the binding of VEGF to the lower affinity/molecular mass VEGF receptors via its exon 7-encoded domain suggests that these proteins might contain heparan sulfate because exon 7 contains within it the heparin-binding domain of VEGF However, the lower affinity/molecular mass VEGF receptors on 231 cells are probably not heparan sulfate-containing proteoglycans because heparin augments rather than inhibits their I-VEGF binding and the relative sharpness of the 165- and 175-kDa complexes is not characteristic of proteoglycans.

In summary, we have characterized a new class of lower affinity/molecular mass VEGF isoform-specific receptors found on EC and tumor cell surfaces, that bind VEGF but not VEGF. Their structure and function is, however, unclear at present. Purification of these receptors is now underway in order to better determine their role in modulating VEGF activity.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA37392 and GM47397 (to M. K.) and by funds from the ``De Drie Lichten'' and ``Dr. Saal van Zwanenberg Stichting'' Foundations (to H. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-7503; Fax: 617-355-7291; klagsbrun{at}a1.tch.harvard.edu.

(^1)
The abbreviations used are: VEGF, vascular endothelial growth factor; EC, endothelial cells; GST, glutathione S-transferase; HUVEC, human umbilical vein-derived endothelial cells; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Smitha Gollamudi-Payne for technical assistance, Dr. Seiji Takashima for assistance with heparin affinity chromatography, and Dr. Helen Gitay-Goren for preparation of VEGF. We thank Drs. Gerhard Raab and Michael Gagnon for reading this manuscript critically.


REFERENCES

  1. Ferrara, N., and Henzel, W. J. (1989) Biochem. Biophys. Res. Commun. 161, 851-858 [Medline] [Order article via Infotrieve]
  2. Myoken, Y., Kayada, Y., Okamoto, T., Kan, M., Sato, G. H., and Sato, J. D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5819-5823 [Abstract]
  3. Plouet, J., Schilling, J., and Gospodarowicz, D. (1989) EMBO J. 8, 3801-3806 [Abstract]
  4. Keck, R. J., Hauser, S. D., Krivi, G., Sanzo, K., Warren, T., Feder, J., and Connolly, D. T. (1989) Science 246, 1309-1312 [Medline] [Order article via Infotrieve]
  5. Connolly, D. T., Olander, J. V., Heuvelman, D., Nelson, R., Monsell, R., Siegel, N., Haymore, B. L., Leimgruber, R., and Feder, J. (1989) J. Biol. Chem. 264, 20017-20024 [Abstract/Free Full Text]
  6. Shweiki, D., Itin, A., Neufeld, G., Gitay-Goren, H., and Keshet, E. (1993) J. Clin. Invest. 91, 2235-2243 [Medline] [Order article via Infotrieve]
  7. Breier, G., Albrecht, U., Sterrer, S., and Risau, W. (1992) Development 114, 521-532 [Abstract]
  8. Plate, K. H., Beier, G., Millauer, B., Ullrich, A., and Risau, W. (1993) Cancer Res. 53, 5822-5827 [Abstract]
  9. Shweiki, D., Itin, A., Soffer, D., and Keshet, E. (1992) Nature 359, 843-845 [CrossRef][Medline] [Order article via Infotrieve]
  10. Dvorak, H. E,, Sioussat, T. M., Brown, L. F., Berse, B., Nagy, J. A., Sotrel, A., Manseau, E. J., Van de Water, L., and Senger, D. R. (1991) J. Exp. Med. 174, 1275-1278 [Abstract]
  11. Klagsbrun, M., and Soker, S. (1995) Curr. Biol. 3, 699-702
  12. Kondo, S., Asano, M., Matsuo, K., Ohmori, I., and Suzuki, H. (1994) Biochim. Biophys. Acta 1221, 211-214 [Medline] [Order article via Infotrieve]
  13. Kim, K. J., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, H. S., and Ferrara, N. (1993) Nature 362, 841-844 [CrossRef][Medline] [Order article via Infotrieve]
  14. Tischer, E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz, D., Fiddes, J. C., and Abraham, J. A. (1991) J. Biol. Chem. 266, 11947-11954 [Abstract/Free Full Text]
  15. Houck, K. A., Ferrara, N., Winer, J., Cachianes, G., Li, B., and Leung, D. W. (1991) Mol. Endocrinol. 5, 1806-1814 [Abstract]
  16. Ferrara, N., Houck, K., Jakeman, L., and Leung, D. W. (1992) Endocr. Rev. 13, 18-32 [Medline] [Order article via Infotrieve]
  17. Houck, K. A., Leung, D. W., Rowland, A. M., Winer, J., and Ferrara, N. (1992) Cancer Res. 52, 4821-4823 [Abstract]
  18. Park, J. E., Keller, G. A., and Ferrara, N. (1993) Mol. Biol. Cell 4, 1317-1326 [Abstract]
  19. Vaisman, N., Gospodarowicz, D., and Neufeld, G. (1990) J. Biol. Chem. 265, 19461-19466 [Abstract/Free Full Text]
  20. Gitay-Goren, H., Soker, S., Vlodavsky, I., and Neufeld, G. (1991) J. Biol. Chem. 267, 6093-6098 [Abstract/Free Full Text]
  21. Jakeman, L. B., Winer, J., Bennett, G. L., Altar, C. A., and Ferrara, N. (1992) J. Clin. Invest. 89, 244-253 [Medline] [Order article via Infotrieve]
  22. Gitay-Goren, H., Halaban, R., and Neufeld, G. (1993) Biochem. Biophys. Res. Commun. 190, 702-709 [CrossRef][Medline] [Order article via Infotrieve]
  23. Enomoto, T., Okamoto, T., and Sato, J. D. (1994) Biochem. Biophys. Res. Commun. 202, 1716-1723 [CrossRef][Medline] [Order article via Infotrieve]
  24. Shibuya, M., Yamaguchi, S., Yamane, A., Ikeda, T., Tojo, A., Matsushime, H., and Sato, M. (1990) Oncogene 5, 519-524 [Medline] [Order article via Infotrieve]
  25. Terman, B. I., Carrion, M. E., Kovacs, E., Rasmussen, B. A., Eddy, R. L., and Shows, T. B. (1991) Oncogene 6, 1677-1683 [Medline] [Order article via Infotrieve]
  26. Terman, B. I., Dougher-Vermazen, M., Carrion, M. E., Dimitrov, D., Armellino, D. C., Gospodarowicz, D., and Bohlen, P. (1992) Biochem. Biophys. Res. Commun. 187, 1579-1586 [Medline] [Order article via Infotrieve]
  27. De Vries, C., Escobedo, J. A., Ueno, H., Houck, K., Ferrara, N., and Williams, L. T. (1992) Science 255, 989-991 [Medline] [Order article via Infotrieve]
  28. Terman, B., Khandke, L., Dougher-Vennazan, M., Maglione, D., Lassam, N. J., Gospodarowicz, D., Persico, M. G., Bohlen, P., and Eisinger, M. (1994) Growth Factors 11, 187-195 [Medline] [Order article via Infotrieve]
  29. Cohen, T., Gitay-Goren, H., Sharon, R., Shibuya, M., Halaban, R., Levi, B. Z., and Neufeld G. (1995) J. Biol. Chem. 270, 11322-11326 [Abstract/Free Full Text]
  30. Soker, S., Goldstaub, D., Svahn, C. M., Vlodavsky, I., Levi, B. Z., and Neufeld, G. (1994) Biochem. Biophys. Res. Commun. 203, 1339-1347 [CrossRef][Medline] [Order article via Infotrieve]
  31. Soker, S., Svahn, C. M., and Neufeld, G. (1993) J. Biol. Chem. 268, 7685-7691 [Abstract/Free Full Text]
  32. Clauss, M., Gerlach, M., Gerlach, H., Brett, J., Wang, F., Familletti, P. C., Pan, Y. C., Olander, J. V., Connolly, D. T., and Stern, D. (1990) J. Exp. Med. 172, 1535-1545 [Abstract]
  33. Midy, V., and Plouet, J. (1994) Biochem. Biophys. Res. Commun. 199, 380-386 [CrossRef][Medline] [Order article via Infotrieve]
  34. Oberg, C., Waltenberger, J., Claesson-Welsh, L., and Welsh, M. (1994) Growth Factors 10, 115-126 [Medline] [Order article via Infotrieve]
  35. Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M., and Heldin, C. H. (1994) J. Biol. Chem. 269, 26988-26995 [Abstract/Free Full Text]
  36. Park, J. E., Chen, H. H., Winer, J., Houck, K. A., and Ferrara, N. (1994) J. Biol. Chem. 269, 25646-25654 [Abstract/Free Full Text]
  37. Seetharam, L., Gotoh, N., Maru, Y., Neufeld, G., Yamaguchi, S., and Shibuya, M. (1995) Oncogene 10, 135-147 [Medline] [Order article via Infotrieve]
  38. Millauer, B., Wizigmann-Voos, S., Schnurch, H., Martinez, R., Moller, N. P. H., Risau, W., and Ullrich, A. (1993) Cell 72, 835-846 [Medline] [Order article via Infotrieve]
  39. Maglione, D., Guerriero, V., Viglietto, G., Delli-Bovi, P., and Persico, M. G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9267-9271 [Abstract]
  40. Kendall, R. L., Wang, G., DiSalvo, J., and Thomas, K. A. (1994) Biochem. Biophys. Res. Commun. 201, 326-330 [CrossRef][Medline] [Order article via Infotrieve]
  41. Gitay-Goren, H., Cohen, T., Tessler, S., Soker, S., Gengrinovitch, S., Rockwell, P., Klagsbrun, M., Levi, B.-Z., and Neufeld, G. (1996) J. Biol. Chem. 271, in press
  42. Cohen, T., Gitay-Goren, H., Neufeld, G., and Levi, B. Z. (1992) Growth Factors 7, 131-138 [Medline] [Order article via Infotrieve]
  43. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  44. Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.) 67, 31-40
  45. Munson, P. J., and Rodbard, D. (1980) Anal. Biochem. 107, 220-239 [Medline] [Order article via Infotrieve]
  46. Millauer, B., Shawver, L. K., Plate, K. H., Risau, W., and Ullrich, A. (1994) Nature 367, 576-579 [CrossRef][Medline] [Order article via Infotrieve]
  47. Barleon, B., Hauser, S., Scholimann, C., Weindel, K., Marme, D., Yayon, A., and Weich, H. A. (1994) J. Cell. Biochem. 54, 56-66 [Medline] [Order article via Infotrieve]
  48. Peters, K. G., DeVries, C., and Williams, L. T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 915-919
  49. Yoshida, A., Anand-Apte, B., and Zetter, B. (1996) Growth Factors , 12, in press

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.