©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification, Purification, and Characterization of Cell-surface Retention Sequence-binding Proteins from Human SK-Hep Cells and Bovine Liver Plasma Membranes (*)

(Received for publication, August 26, 1994; and in revised form, November 1, 1994)

Christian Boensch (1)(§) Ming-Der Kuo (1)(§) Daniel T. Connolly (2) Shuan Shian Huang (1) Jung San Huang (1)(¶)

From the  (1)Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104 and (2)Monsanto Company, St. Louis, Missouri 63167

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cell-surface retention is a newly identified mechanism associated with the secretion of certain polypeptide growth factors and cytokines. This novel form of secretion appears to be mediated by cell-surface retention sequences (CRS) in the polypeptide molecules. To test the hypothesis that high-affinity CRS-binding proteins (CRS-BPs) are responsible for the cell-surface retention, we identified and characterized the high-affinity binding sites on various cell types for I-labeled CRS peptide (sis) and CRS peptide (VEGF), each of which contained the putative CRS motifs of platelet-derived growth factor B (c-sis) and vascular endothelial cell growth factor, respectively. Scatchard plot analysis revealed a single class of high-affinity binding sites with K = 0.5-0.7 nM and 22,000-55,000 sites/cell. High-affinity binding activity could be demonstrated between pH 4.5 and 8.0, but was much greater below 6.0 (maximum pH 5.0-5.5). The ligand binding activity was inhibited by heparin, polylysine, and protamine, but not by cytochrome c. CRS-BPs responsible for the high-affinity binding were identified as 60-72-kDa proteins by ligand affinity labeling.

CRS-BPs were purified from human SK-Hep cells and bovine liver plasma membranes by Triton X-100 extraction followed by affinity column chromatography on wheat germ lectin-Sepharose 4B and CRS peptide (sis)-Affi-Gel 10. Purified CRS-BPs exhibited ligand binding properties (pH profile and inhibitor sensitivity) similar to those of the high-affinity binding sites for CRS peptides on cultured cells. The major CRS-BPs (p60, p66, and p72) purified from bovine liver plasma membranes were found to have identical N-terminal amino acid sequence and were assumed to represent different forms of the same gene product, which we have designated CRS-BP1.


INTRODUCTION

Cell-surface retention during secretion is a newly identified and novel aspect of the secretion of certain polypeptide growth factors and cytokines(1, 2, 3, 4, 5, 6, 7) . These growth factors and cytokines are secreted, but remain associated with the cell surface rather than released into the medium(1, 2, 3, 4, 5, 6, 7) . Such cell surface-bound proteins can be released by incubation with suramin, heparin, or high salt(1, 3, 5) . The functional significance of this novel aspect of secretion is not known. However, in v-sis-transformed cells, cell-surface retention of the v-sis gene product facilitates its binding to the platelet-derived growth factor (PDGF) (^1)receptor(1) . This efficient interaction between the cell surface-bound v-sis gene product and the PDGF receptor on the cell surface may play a role in the autocrine growth of v-sis-transformed cells(1) .

The v-sis gene product (PDGF-B) was first reported to undergo cell-surface retention during secretion(1) , and this result was confirmed by others. PDGF-B (c-sis)(2, 3) , PDGF-A(L) (alternatively spliced longer version of PDGF-A) (2, 3) , vascular endothelial cell growth factor (VEGF)-189 (or VEGF-206), Int-2(5) , Wnt-1(6) , and Cyr 61 (7) proteins have all been shown to exhibit this novel secretion. Among these secretory growth factors and cytokines, the v-sis gene product, PDGF-B (c-sis), PDGF-A(L), and VEGF-189 (or VEGF-206) are structurally homologous growth factors and members of the PDGF superfamily(8, 9, 10, 11, 12, 13) . Int-2 protein is a member of the fibroblast growth factor family(14) . Wnt-1 protein is a proto-oncogene product(15) , and Cyr 61 protein is a growth factor-inducible early gene product(16) . No amino acid sequence homology is found among PDGF family, Int-2, Wnt-1, and Cyr 61 proteins, implying that these secretory proteins may carry analogous sequence motifs mediating their cell-surface retention. Studies of the PDGF-B (c-sis) deletion mutation have revealed that PDGF-B (c-sis) possesses a cell-surface retention sequence (CRS) at residues 219-229 or 212-226(2, 3) . This 11- or 15-amino acid residue CRS contains a 7- or 8-basic amino acid residue sequence that is homologous to sequences present in PDGF-A(L) and VEGF-189 (or VEGF-206). Both PDGF-A(L) and VEGF-189 (or VEGF-206) have been shown to remain associated with the cell surface following secretion(2, 3, 4) . Their alternatively spliced shorter versions (PDGF-A(S), VEGF-121, and VEGF-165), which lack putative CRS, have been shown to be secreted directly into the medium(2, 4, 17) . Deletion of the putative CRS in PDGF-A(L) also results in the promotion of secretion into the medium(17) . These observations argue that the putative CRS of PDGF-A(L) and VEGF-189 (or VEGF-206) are required for their cell-surface retention.

The molecular mechanism(s) by which the secretory proteins with CRS are transported to the cell surface and retained there is not understood. Based on the studies of v-sis product synthesis and secretion by pulse-chase experiments, we hypothesized that the v-sis gene product (a secretory protein with CRS) became associated with the cell-surface components (CRS-binding proteins) in the trans-Golgi complex and exocytosis vesicles, after which the associated complexes appeared at and remained at the cell surface (1) . Recently, Raines and Ross (18) identified binding sites on human smooth muscle cells and umbilical vein endothelial cells for an I-labeled tyrosinated pentadecapeptide and eicosapeptide that contained the CRS motifs of PDGF-B (c-sis) and PDGF-A(L), respectively. The large number (10^8 to 10^9/cell) and low affinity (K = 2-4 µM) of the binding sites identified were consistent with the suggestion that heparan sulfate proteoglycan (HS-PG) was responsible for the binding in the assays. Khachigian et al.(19) also recently identified binding sites on different cell types for an Ilabeled tyrosinated nonadecapeptide containing the CRS motif of PDGF-A(L). 3T3 fibroblasts were found to express a large number of low-affinity binding sites (1.6 times 10^7/cell) with an apparent K of 0.6 µM(19) . The large population of sites bound with low affinity was consistent with HS-PG-binding sites in these assays.

To test the hypothesis that specific high-affinity binding proteins mediate the cell-surface retention of secretory proteins with CRS(1) , we attempted to identify, purify, and characterize the putative CRS-binding proteins (CRS-BPs) in cultured cells and tissues. In this study, we demonstrate that cultured cells show 22,000-55,000 high-affinity binding sites/cell for CRS-containing peptide ligands with an apparent K of 0.5-0.7 nM. Heparin and basic polypeptides, polylysine and protamine (but not cytochrome c), are potent inhibitors of the ligand binding to these high-affinity binding sites. The major CRS-BPs, which are responsible for high-affinity CRS peptide binding, were identified as protein species with molecular masses of 60-72 kDa by I-labeled ligand affinity labeling. We also report that the major CRS-BPs (p60, p66, and p72) purified from bovine liver plasma membranes possess identical N-terminal amino acid sequence. CRS-BPs p60, p66, and p72 are assumed to represent different forms of the same gene product, which we have designated CRS-BP1. The antiserum raised against the N-terminal peptide of CRS-BP1 immunoprecipitates a ligand affinity-labeled CRS-BP on cultured HepG2 cells.


EXPERIMENTAL PROCEDURES

Materials

NaI (17 Ci/mg) was purchased from ICN Biochemicals (Irvine, CA). Heparitinase (heparin lyase III) from Flavobacterium heparinum, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDAC), -maleimidobutyric acid N-hydroxysuccinimide ester, phenylmethanesulfonyl fluoride, bovine serum albumin (BSA), chloramine T, high molecular mass protein standards (myosin, 205 kDa; beta-galactosidase, 116 kDa; phosphorylase b, 97 kDa; BSA, 66 kDa; ovalbumin, 45 kDa; and carbonic anhydrase, 29 kDa), heparin, protamine sulfate, poly-L-lysine HBr (M(r) 4000-15,000), myelin basic protein, and cytochrome c were obtained from Sigma. IgGsorb was obtained from the Enzyme Center (Malden, MA). Wheat germ lectin-Sepharose 4B was prepared according to the procedure of Kuo et al.(20) . CRS peptide (VEGF) (KSVRGKGKGQKRKRKKSRYKSWSV), CRS peptide (sis) (YVRVRRPPKGKHRKFKHTH), and other peptides were synthesized using t-butyloxycarbonyl chemistry on an Applied Biosystems Model 431A peptide synthesizer and purified by reverse-phase high-performance liquid chromatography (C(18) column) and Sephadex G-25 column chromatography. The amino acid sequences of the synthetic peptides were verified by amino acid sequence analysis using automated Edman degradation. Bio-Rad protein assay reagent and Affi-Gel 10 were obtained from Bio-Rad. CRS peptide (sis)-Affi-Gel 10 was prepared by mixing 10 mg of CRS peptide (sis) and 25 ml of Affi-Gel 10 in 0.1 M NaHCO(3), 0.3 M NaCl, pH 8.0, at 4 °C for 18 h according to the protocol from the manufacturer of Affi-Gel 10. Human liver carcinoma cell lines (SK-Hep and HepG2 cells) were obtained from American Type Culture Collection (Rockville, MD). A large-scale SK-Hep cell culture was carried out in the Department of Cell Culture and Biochemistry, Monsanto Co. (St. Louis, MO). NIH 3T3 cells were cultured and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum. Suramin was obtained from FBA Pharmaceuticals (West Haven, CT). Polyvinylidene difluoride membranes (ProBlott) and chemicals for peptide synthesis and amino acid sequencing were obtained from Applied Biosystems Inc. (Foster City, CA).

Iodination of CRS Peptides

CRS peptides (4 µg) were iodinated with 2 mCi of NaI in 100 µl of 0.2 M sodium phosphate, pH 7.5, containing 5 µl of chloramine T (5 mg/ml). After 2 min, the reaction was stopped by sequential addition of 10 µl of sodium metabisulfite (10 mg/ml), 10 µl of 100 mMN-acetyltyrosine, and 10 µl of 50 mM KI at intervals of 3 min. I-CRS peptides were separated from free iodide by Sephadex G-10 column chromatography (column size, 0.8 times 20 cm). The solvent for gel filtration chromatography was 50 mM sodium phosphate buffer, pH 7.4, containing 0.1% BSA. The specific radioactivities of I-CRS peptides were estimated to be 1-5 times 10^5 cpm/ng.

Binding Assay of I-CRS Peptides on Cultured Cells

SK-Hep, NIH 3T3, and HepG2 cells were grown (3 days) to confluence (4-10 times 10^4 cells/cm^2) on 24-well cluster dishes in DMEM containing 10% fetal calf serum. After washing with serum-free DMEM (0.5 ml), confluent cells were incubated with various concentrations of I-CRS peptide (sis) or I-CRS peptide (VEGF) in the presence and absence of 1 mM suramin in 0.5 ml of buffer A (20 mM HEPES/NaOH, pH 7.4, 0.128 M NaCl, 5 mM KCl, 5 mM MgCl(2), and 1 mM CaCl(2)) containing 0.1% BSA. After 2.5 h at 0 °C, the cells were washed twice with 1 ml of buffer A and dissolved in 0.4 ml of 0.2 N NaOH. The cell-associated radioactivity was then counted in a -counter. For the experiments on pH effect, cells were incubated with 1 nMI-CRS peptides ± 1 mM suramin in Tris acetate buffer (20 mM Tris, 0.128 M NaCl, 5 mM KCl, 1 mM CaCl(2), and 5 mM MgCl(2); the pH was adjusted at 4.5-8.0 with glacial acetic acid) containing 0.1% BSA. After 2.5 h at 0 °C, the cells were washed twice with Tris acetate buffer (1 ml) and dissolved in 0.2 N NaOH for -counting.

Affinity Labeling of CRS-BPs on Cultured Cells

Cells were grown (3 days) to confluence on 60-mm Petri dishes in DMEM containing 10% fetal calf serum. After washing, cells were incubated with 1 nMI-CRS peptide (VEGF) ± 1 mM suramin in 1.6 ml of buffer A containing 0.1% BSA. After 2.5 h at 0 °C, 0.16 ml of 0.11 M EDAC (in H(2)O) was added to the binding medium. The binding medium was further incubated for 20 min at 0 °C. The I-CRS peptide (VEGF) affinity-labeled cells were washed four times with buffer A and detached from the Petri dishes as described previously (21) . The cells were then lysed with 60 µl of 20 mM HEPES/NaOH, pH 7.4, containing 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, and 5 mM EDTA. After centrifugation, the supernatant of the cell lysates was mixed with 60 µl of wheat germ lectin-Sepharose 4B (50% gel suspension) in buffer B (20 mM HEPES/NaOH, pH 7.4, 10% glycerol, and 0.1% Triton X-100). The gel suspension was mixed overnight and centrifuged. After washing, the gels were incubated with 60 µl of 0.2 MN-acetylglucosamine in buffer B containing 0.15 M NaCl for 2 h at 4 °C. The gel suspension was centrifuged. The supernatant (30 µl) was then mixed with 10 µl of 4 times SDS sample buffer for analysis by 7 or 8% SDS-polyacrylamide electrophoresis.

Heparitinase Treatment of Cultured Cells

Cells were grown to confluence on 24-well cluster dishes in DMEM containing 10% fetal calf serum. After washing with serum-free DMEM, confluent cells were treated with 10 units/ml heparitinase in 0.16 ml of buffer A for 2 h at 37 °C. The cells were then washed and subjected to binding assay of I-CRS peptides. In control experiments under the same conditions, the activity of heparitinase was ascertained by its ability to remove the proteoglycan moieties of the transforming growth factor-beta type III receptor as described previously(21) .

I-CRS Peptide (VEGF) Cross-linking Assay of Chromatographic Fractions

The reaction mixture (50-µl volume) contained 5 µl of chromatographic fractions, 10-30 nMI-CRS peptide (VEGF), and suramin (0 and 1 mM) in buffer B or Mes buffer (20 M Mes/NaOH, pH 4.5-6.0, 10% glycerol, and 0.1% Triton X-100) containing 50 mM NaCl. The reaction mixture was incubated at 4 °C for 2.5 h. The reaction mixture was further incubated with 10 mM EDAC (5 µl of 0.11 M EDAC in H(2)O) for 20 min. The reaction mixture was then mixed with 4 times SDS sample buffer for analysis by 8% SDS-polyacrylamide gel electrophoresis followed by autoradiography. The relative intensity of the I-CRS peptide (VEGF)bulletCRS-BP complex bands on the autoradiograph was quantitated by a PhosphorImager.

In some experiments, after binding and cross-linking, the reaction mixture was incubated with 3 µl of 1 M Tris-HCl, pH 7.4, 1 M glycine, which was used to inactivate excess EDAC. After 5 h at 4 °C, 100 µl of wheat germ lectin-Sepharose 4B (50% suspension in 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.2% Triton X-100) was added to the reaction mixture. The gel suspension was incubated at 4 °C for 5 h. Following centrifugation, the gels were washed five times with 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.2% Triton X-100 and incubated with 100 µl of 0.4 MN-acetylglucosamine in 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.2% Triton X-100 (2 h at 4 °C). The N-acetylglucosamine eluents were analyzed by 7% SDS-polyacrylamide gel electrophoresis.

Purification of CRS-BPs from SK-Hep Cells

Frozen SK-Hep cells (180-ml volume) were thawed and mixed with 400 ml of Tris buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, and 130 mM NaCl) containing 1 mM phenylmethanesulfonyl fluoride and 1 mM suramin at 4 °C for 50 min. After centrifugation, the cell pellets were washed twice with Tris buffer and solubilized with 200 ml of 2% Triton X-100 in 20 mM HEPES/NaOH, pH 7.4, 10% glycerol. After centrifugation, the Triton X-100 extracts were mixed with wheat germ lectin-Sepharose 4B (30-ml gel volume) at 4 °C for 6 h. The gel suspension was then packed onto a column. After washing sequentially with 0.15 M NaCl in buffer B and with buffer B, the column was eluted with 0.2 MN-acetylglucosamine in buffer B.

The CRS-BP-containing fractions from wheat germ lectin-Sepharose 4B column chromatography were pooled and applied onto a column (15-ml volume) of CRS peptide (sis)-Affi-Gel 10. After washing with buffer B, the column was eluted with 0.5 M NaCl in buffer B. CRS-BPs were recovered in the 0.5 M NaCl eluents.

Purification of CRS-BPs from Bovine Liver Plasma Membranes

Bovine liver plasma membranes (5 g protein) were prepared as described previously (20) and mixed with 200 ml of Tris buffer containing 1 mM phenylmethanesulfonyl fluoride and 1 mM suramin. After centrifugation, the membrane pellets were washed twice with Tris buffer. The washed pellets were then extracted with 200 ml of 2% Triton X-100 in 20 mM HEPES/NaOH, pH 7.4, 10% glycerol at 4 °C for 30 min. After centrifugation, the Triton X-100 extracts were subjected to wheat germ lectin-Sepharose 4B column chromatography and CRS peptide (sis)-Affi-Gel 10 affinity column chromatography as described above.

N-terminal Amino Acid Sequence Analysis of CRS-BPs Purified from Bovine Liver Plasma Membranes

The major CRS-BPs (60, 66, and 72 kDa) obtained by CRS peptide (sis)-Affi-Gel 10 column chromatography were separated by 7% SDS-polyacrylamide gel electrophoresis and subsequently transblotted onto polyvinylidene difluoride membrane filters according to the procedure of Matsudaira (22) . After staining with Coomassie Brilliant Blue, each of the major CRS-BP bands (60, 66, and 72 kDa) was cut and subjected to amino acid sequence analysis by automated Edman degradation on an Applied Biosystems Model 477A gas/liquid-phase protein sequencer with an on-line Applied Biosystems Model 120A phenylthiohydantoin-derivative analyzer.

Preparation of Antiserum to CRS-BP1

A C-terminal cysteinated peptide whose amino acid sequence was derived from the N-terminal amino acid sequence of CRS-BP1 was synthesized and conjugated with bovine thyroglobulin using -maleimidobutyric acid N-hydroxysuccinimide ester according to the procedure described previously(23) . CRS-BP1 N-terminal peptide-thyroglobulin conjugate with adjuvants was injected subcutaneously into rabbits for generation of antiserum as described by Huang and Huang(24) . The specificity of the antiserum to CRS-BP1 was verified by Western blot analysis and enzyme-linked immunosorbent assay.

Immunoprecipitation of Ligand Affinity-labeled CRS-BPs on HepG2 Cells

HepG2 cells were grown to confluence on 60-mm Petri dishes and affinity-labeled with 10 nMI-CRS peptide (VEGF) in the presence and absence of 1 mM suramin using 10 mM EDAC as described above. After washing with buffer A, the cells were lysed with 0.5 ml of 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 5 mM EDTA, 5 mM NaF, and 1% Triton X-100. After centrifugation, 100 µl of the cell lysates was incubated with 10 µl of antiserum to CRS-BP1 at 4 °C overnight. About 60 µl of IgGsorb (50% suspension) was then added to the reaction mixture. After 2 h at 4 °C, the reaction mixture was centrifuged, and the pellets were washed five times with 20 mM Tris-HCl, 0.15 M NaCl containing 0.2% Triton X-100. The washed pellets were mixed with SDS sample buffer for analysis by 7% SDS-polyacrylamide gel electrophoresis.

The remaining 400 µl of the cell lysates was mixed with 100 µl of wheat germ lectin-Sepharose 4B (50% gel suspension). After mixing at 4 °C for 16 h, the gels were washed five times with 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl containing 0.2% Triton X-100 and incubated with 100 µl of 0.4 MN-acetylglucosamine in 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.2% Triton X-100 at 4 °C for 2 h. The N-acetylglucosamine eluents were then analyzed by 7% SDS-polyacrylamide gel electrophoresis.


RESULTS

High-affinity Binding Sites for CRS Peptides on Cultured Cells

For characterization of the binding sites for CRS peptides on cultured cells, two synthetic peptides, namely CRS peptide (sis) and CRS peptide (VEGF), which contained the CRS of PDGF-B (c-sis) and VEGF-189 (residues 213-230 and 116-139, respectively), were used as the ligands. CRS peptide (sis) was a tyrosinated nonadecapeptide, whereas CRS peptide (VEGF) was a tetracosapeptide. Both peptides contained tyrosine residues that served as the sites for I labeling. In the binding assays, the cells were incubated with various concentrations of I-CRS peptides. Binding was determined after 2.5 h at 0 °C when the binding reached equilibrium. Specific binding was estimated by subtracting nonspecific binding from total binding. Nonspecific binding was determined in the presence of 1 mM suramin. Suramin has been shown to effectively displace cell surface-bound growth factors(1, 3) . As shown in Fig. 1A, I-CRS peptide (sis) bound to SK-Hep cells in a dose-dependent manner with a saturating concentration of 4 ng/ml. Scatchard plot analysis of the binding data revealed a single class of high-affinity binding sites with an apparent K(d) of 0.5 nM and 22,000 sites/cell (Fig. 1B). Similar values of K(d) and binding sites/cell were obtained when I-CRS peptide (VEGF) was used as the ligand (Table 1). High-affinity binding sites for I-CRS peptides were also observed on other cell types, such as NIH 3T3 cells and HepG2 cells (Table 1).


Figure 1: Binding of I-CRS peptide (sis) to SK-Hep cells. A shows the concentration dependence of the binding of I-CRS peptide (sis) to SK-Hep cells at pH 7.4. The nonspecific binding was determined in the presence of 1 mM suramin. Each data point represents the mean ± S.D. from three assays. B shows the Scatchard plot analysis of the specific binding data from A.





Since CRS peptides are basic in nature and heparin has been shown to displace the cell surface-bound growth factors(4, 5) , the effects of heparin and basic polypeptides on the binding of I-CRS peptide (VEGF) to SK-Hep cells were examined. As shown in Fig. 2(A and B), heparin, polylysine, and protamine (but not cytochrome c) inhibited the I-CRS peptide (VEGF) binding to SK-Hep cells. The half-maximal inhibition concentrations (IC) of heparin, polylysine, and protamine were estimated to be 0.4, 8, and 1 µg/ml, respectively. Similar IC values for heparin and basic polypeptides were also obtained when I-CRS peptide (sis) was used as the ligand (data not shown).


Figure 2: Effects of heparin (A) and basic polypeptides (B) on the binding of I-CRS peptide (VEGF) to SK-Hep cells. The binding of I-CRS peptide (VEGF) to SK-Hep cells was carried out at pH 7.4 using 1 nMI-CRS peptide (VEGF) and various concentrations of heparin or basic polypeptides. The specific binding of I-CRS peptide (VEGF) in the absence of heparin or basic polypeptides is taken as 100% bound.



To investigate whether HS-PG was responsible for the high-affinity binding sites, the effect of pretreatment with heparitinase on the ligand binding activity of the high-affinity binding sites was examined. Without pretreatment of SK-Hep cells with heparitinase, the specific binding at 1 nMI-CRS peptide (sis) was estimated to be 1250 ± 100 cpm/well. After pretreatment with heparitinase, the specific binding of I-CRS peptide (sis) (1 nM) was determined to be 1219 ± 152 cpm/well. This result suggests that the heparitinase pretreatment does not affect the high-affinity binding of I-CRS peptide (sis) to SK-Hep cells and that the high-affinity binding of I-CRS peptide (sis) is not mediated through HS-PG.

Broad pH Activity of CRS Peptide High-affinity Binding Sites on SK-Hep Cells

As described previously(1) , secretory proteins with CRS are believed to interact with CRS-BPs in the trans-Golgi complex and exocytosis vesicles. Since the lumens in the trans-Golgi complex and exocytosis vesicles and culture media (or extracellular compartment) have different pH values ranging from <6.0 to 7.4(25) , CRS-BPs would be expected to show ligand binding activity within a broad pH range. To test this prediction, the effect of various pH values on the binding of I-CRS peptides (sis) to SK-Hep cells was examined. Although, as shown in Fig. 3, I-CRS peptide (sis) bound to SK-Hep cells at various pH values from 4.5 to 8.0, the maximal activity was at pH 5.0-5.5, when binding was consistently increased 3-6-fold (compared with that observed at pH 7.4). A similar pH profile was observed when I-CRS peptide (VEGF) was used as the ligand (data not shown).


Figure 3: Effect of pH on the binding of I-CRS peptide (sis) to SK-Hep cells. The binding assay was performed in Tris acetate buffer, pH 4.5-8.0, with 1 nMI-CRS peptide (sis) ± 1 mM suramin. The specific binding of I-CRS peptide (sis) at the pH indicated was determined after incubation at 0 °C for 2.5 h.



Identification of CRS-BPs on SK-Hep Cells by Ligand Affinity Labeling

To identify CRS-BPs, which serve as high-affinity binding sites on SK-Hep cells, SK-Hep cells were incubated with 1 nMI-CRS peptide (VEGF). After 2.5 h at 0 °C, the CRS-BPs were affinity-labeled with I-CRS peptide (VEGF) in the presence of 10 mM EDAC and then analyzed by 8% SDS-polyacrylamide gel electrophoresis followed by autoradiography. After affinity labeling, several I-CRS peptide (VEGF)bulletCRS-BP complexes with molecular masses of 63-75 kDa were identified as a broad band on the autoradiograph of the SDS gel (Fig. 4, lane1). The complex formation of I-CRS peptide (VEGF) and CRS-BPs was abrogated in the presence of 1 mM suramin (lane2). This result suggests that SK-Hep cells express CRS-BPs that possess molecular masses ranging from 60 to 72 kDa (the molecular mass of I-CRS peptide (VEGF) is 3 kDa).


Figure 4: Ligand affinity labeling of CRS-BPs on SK-Hep cells. CRS-BPs on SK-Hep cells were affinity-labeled by I-CRS peptide (VEGF) in the presence (+) and absence (-) of 1 mM suramin using 10 mM EDAC. Affinity-labeled CRS-BPs were analyzed by 8% SDS-polyacrylamide gel electrophoresis followed by autoradiography. The bracket indicates the location of the major I-CRS peptide (VEGF)bulletCRS-BP affinity-labeled complexes (63-75 kDa).



Purification of CRS-BPs from SK-Hep Cells and Bovine Liver Plasma Membranes

The above observations provided the background for developing a I-CRS peptide (VEGF) cross-linking assay to detect CRS-BPs in the chromatographic fractions. In this cross-linking assay, I-CRS peptide (VEGF) was incubated with an aliquot of the chromatographic fractions in the presence and absence of 1 mM suramin. After 2.5 h at 0 °C, the reaction mixture was further incubated for 20 min with 10 mM EDAC and then analyzed by 8% SDS-polyacrylamide gel electrophoresis followed by autoradiography.

In preliminary experiments, we found that CRS-BPs in plasma membranes could be extracted with Triton X-100, but not by high salt (2 M NaCl) or high pH (0.5 M Na(2)CO(3), pH 11.5). This result suggests that CRS-BPs are membrane proteins. For isolation of CRS-BPs, Triton X-100 was therefore used to solubilize CRS-BPs in plasma membranes and was included in the solvent buffers for the subsequent chromatographies.

Since many cell-surface receptor proteins are glycoproteins, which can be absorbed by wheat germ lectin affinity gel(20, 26) , the Triton X-100 extracts of suramin-prewashed SK-Hep cells or bovine liver plasma membranes were mixed with a 30-ml gel volume of wheat germ lectin-Sepharose 4B gel. After mixing overnight, the gel suspension was packed onto a column. After washing extensively with buffer, the column was eluted with 0.2 MN-acetylglucosamine. As shown in Fig. 5A (SK-Hep cells) and Fig. 6A (bovine liver plasma membranes), a single protein peak was detected by the absorbance at 595 nm (Bio-Rad reagent assay) in the eluents of 0.2 M N-acetylglucosamine. This protein peak contained CRS-BPs, which were identified by the I-CRS peptide (VEGF) cross-linking assay. As shown in Fig. 5B (SK-Hep cells) and Fig. 6B (bovine liver plasma membranes), the major CRS-BPs were identified as the I-CRS peptide (VEGF) cross-linked complexes with molecular masses of 58-63 and 69-75 kDa. These CRS-BP-containing fractions were then pooled and subjected to ligand affinity column chromatography on CRS peptide (sis)-Affi-Gel 10 (Fig. 5C and Fig. 6C). After washing with buffer, the affinity column was eluted with 0.5 M NaCl (Fig. 5C for SK-Hep cells and Fig. 6C for bovine liver plasma membranes). The major CRS-BPs, identified as the I-CRS peptide (VEGF) cross-linked complexes with molecular masses of 58-63 and 69-75 kDa, were found in the 0.5 M NaCl eluents (Fig. 5D (lanes 1-10) and 6D (lanes3-12)), but not in the flow-through fractions (Fig. 6D, lanes1 and 2). The I-CRS peptide (VEGF)bulletCRS-BP complexes with molecular masses of 69-75 kDa appeared to comigrate with I-CRS peptide (VEGF)bulletBSA complexes (Fig. 5D (lanes11 and 12) and 6D (lanes13 and 14)). The I-CRS peptides (VEGF)bulletBSA complexes were generated during storage of I-CRS peptide (VEGF) and cross-linking with EDAC (BSA was used as the carrier protein). The preparations of CRS-BPs purified by ligand affinity column chromatography were used for the following activity and biochemical characterization of CRS-BPs.


Figure 5: Wheat germ lectin-Sepharose 4B column chromatography (A and B) and CRS peptide (sis)-Affi-Gel 10 column chromatography (C and D) of CRS-BPs from SK-Hep cells. A shows the wheat germ lectin-Sepharose 4B column chromatography of Triton X-100 extracts of SK-Hep cells. After exhaustively washing with the equilibration buffer, CRS-BPs were eluted with 0.2 MN-acetylglucosamine. The protein concentrations of chromatographic fractions were spectrophotometrically monitored at 595 nm using Bio-Rad protein assay reagent. The arrow indicates the start of elution with 0.2 MN-acetylglucosamine. The flow rate was 20 ml/h, and the fractional volume was 1 ml. The fractions indicated by the hatchedbox were pooled and subjected to CRS peptide (sis)-Affi-Gel 10 column chromatography. B shows the detection of CRS-BPs in the column fractions shown in A by cross-linking with I-CRS peptide (VEGF) and EDAC. Cross-linking with I-CRS peptide (VEGF) was performed in the presence (+) and absence(-) of 1 mM suramin. The brackets indicate the locations of I-CRS peptidebulletCRS-BP complexes (58-63 and 69-75 kDa). C shows the CRS peptide (sis)-Affi-Gel 10 column chromatography of CRS-BPs obtained by wheat germ lectin-Sepharose 4B column chromatography. The fractional volume prior to elution with 0.5 M NaCl was 0.8 ml, and the fractional volume in the 0.5 M NaCl elution was 1 ml. The flow rate was 20 ml/h. D shows the detection of CRS-BPs by cross-linking with I-CRS peptide (VEGF) and EDAC. Cross-linking in the absence of fractions (buffer alone) was used as control (lanes11 and 12). The brackets indicate the locations of I-CRS peptide (VEGF)bulletCRS-BPs complexes (58-63 and 69-75 kDa).




Figure 6: Wheat germ lectin-Sepharose 4B column chromatography (A and B) and CRS peptide (sis)-Affi-Gel 10 column chromatography (C and D) of CRS-BPs from bovine liver plasma membranes. A shows wheat germ lectin-Sepharose 4B column chromatography. The chromatographic conditions are the same as described in the legend of Fig. 5A. B shows the identification of CRS-BPs by cross-linking with I-CRS peptide (VEGF) and EDAC. C shows the CRS peptide (sis)-Affi-Gel 10 column chromatography of CRS-BPs obtained by wheat germ lectin-Sepharose 4B column chromatography. The conditions for elution of CRS-BPs are essentially identical to those described in the legend of Fig. 5C. D shows the identification of CRS-BPs in the fractions from CRS peptide (sis)-Affi-Gel 10 chromatography by cross-linking with I-CRS peptide (VEGF) and EDAC.



Ligand Binding Activity of Purified CRS-BPs

To define the ligand specificity of CRS-BPs purified by ligand affinity column chromatography, the effect of various concentrations of unlabeled CRS peptide (sis) or CRS peptide (VEGF) on the binding of I-CRS peptide (VEGF) to purified CRS-BPs was examined. As shown in Fig. 7(A and B), increasing concentrations of unlabeled CRS peptide (VEGF) and CRS peptide (sis) inhibited the complex formation of I-CRS peptide (VEGF) and CRS-BPs. At a 243-fold molar excess, unlabeled CRS peptide (VEGF) or CRS peptide (sis) inhibited >95% of the complex formation. Other unrelated peptides (AERFSELEHLDRLSGRS, NMRPVYPTKAFPNHYS, and CTGCSEYQQRKEPVSDILK) did not show any inhibitory effect on complex formation even at a 500-fold molar excess (data not shown). These results suggest that purified CRS-BPs have ligand specificity for CRS peptides.


Figure 7: Inhibition of the I-CRS peptide (VEGF) binding to CRS-BPs by unlabeled CRS peptides. The binding of I-CRS peptide (VEGF) to CRS-BPs purified from SK-Hep cells (A) and `bovine liver plasma membranes (B) was determined by the I-CRS peptide (VEGF) cross-linking assay. In the assay, the concentrations of I-CRS peptide (VEGF) and unlabeled CRS peptides were 10 nM and -fold molar excess as indicated, respectively. The I-CRS peptide (VEGF) cross-linked complexes were identified by 8% SDS-polyacrylamide gel electrophoresis followed by autoradiography. The brackets indicate the locations of I-CRS peptide (VEGF)bulletCRS-BP complexes (58-63 and 69-75 kDa).



As shown in Fig. 3, intact SK-Hep cells showed a high-affinity binding activity for I-CRS peptides within a broad pH range. If purified CRS-BPs are responsible for the binding activity of SK-Hep cells for I-CRS peptides, purified CRS-BPs should show a similar pH activity. To test this prediction, purified CRS-BPs were incubated with I-CRS peptide (VEGF) at various pH values (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0). After 2.5 h at 0 °C, the I-CRS peptide (VEGF)bulletCRS-BP complexes were cross-linked by 10 mM EDAC and analyzed by 8% SDS-polyacrylamide gel electrophoresis. As shown in Fig. 8, the complex formation of I-CRS peptide (VEGF) and CRS-BPs occurred at various pH values from 5.0 to 8.0, with a maximum at pH 5.0-5.5. This pH profile is similar to that of the I-CRS peptide (VEGF) binding activity observed in intact SK-Hep cells (Fig. 3). CRS-BPs that were identified as the I-CRS peptide (VEGF) cross-linked complexes with molecular masses of 69-75 kDa showed a maximal ligand binding activity at pH 5.0-5.5 and appeared to be the major CRS-BPs at this pH. It is of importance to note that the cross-linking efficiency of EDAC does not vary greatly within the pH range from 5.0 to 8.0 under the cross-linking conditions(27) .


Figure 8: Effect of pH on the I-CRS peptide (VEGF) binding to CRS-BPs purified from bovine liver plasma membranes. The binding of I-CRS peptide (VEGF) (10 nM) to CRS-BPs was carried out at various pH values (in buffer B and Mes buffer for pH 6.5-8.0 and pH 5.0-6.0, respectively) in the presence (+) and absence (-) of 1 mM suramin. The I-CRS peptide (VEGF)bulletCRS-BP complexes were cross-linked using EDAC and analyzed by 8% SDS-polyacrylamide gel electrophoresis followed by autoradiography. The brackets indicate the locations of I-CRS peptide (VEGF)bulletCRS-BP cross-linked complexes (58-63 and 69-75 kDa).



The ligand binding activities of the high-affinity binding sites (or CRS-BPs) on SK-Hep cells were found to be inhibited by heparin and basic polypeptides. The effects of heparin and basic polypeptides on the complex formation at pH 5.0 of I-CRS peptide (VEGF) and purified CRS-BPs were therefore investigated. At pH 5.0, the I-CRS peptide (VEGF)bulletCRS-BP complexes with molecular masses of 69-75 kDa were identified as the major complexes. As shown in Fig. 9, like in intact cells, the ligand binding activities of purified CRS-BPs were inhibited by heparin, polylysine, protamine, and myelin basic protein, but not by cytochrome c. The inhibition of I-CRS peptide (VEGF)bulletCRS-BP complex formation by heparin and basic polypeptides suggests that electrostatic forces are involved in the interactions of I-CRS peptides and CRS-BPs. To further define the electrostatic interactions between CRS peptides and CRS-BPs, we investigated the complex formation at pH 5.0 of I-CRS peptide (VEGF) and CRS-BPs in the presence of different concentrations of NaCl. As shown in Fig. 10, at 300 mM NaCl, very little complex formation of I-CRS peptide (VEGF) and CRS-BPs was detected on the autoradiograph. Interestingly, the presence of 50 mM NaCl appeared to enhance complex formation. About 1.5-2.0-fold enhancement was consistently observed when compared with that observed in the absence of NaCl. These results support that electrostatic interactions are involved in the complex formation of I-CRS peptides and CRS-BPs. Because cytochrome c, which is a basic protein with a pI of 11.7 (28) , failed to inhibit the complex formation of I-CRS peptides and CRS-BPs, these results also suggest that in addition to being basic in nature, certain structural features (e.g. clustering of basic amino acid residues in a certain configuration) are required for the basic polypeptides to inhibit complex formation.


Figure 9: Effects of heparin and basic polypeptides on the binding at pH 5.0 of I-CRS peptide (VEGF) to CRS-BPs purified from bovine liver plasma membranes. The binding of I-CRS peptide (VEGF) (10 nM) to CRS-BPs purified from bovine liver plasma membranes was performed at pH 5.0 in the absence and presence of CRS peptides, heparin, basic polypeptides, or suramin as indicated. The I-CRS peptide (VEGF)bulletCRS-BP complexes were cross-linked and then analyzed by 8% SDS-polyacrylamide gel electrophoresis followed by autoradiography. The bracket indicates the location of the I-CRS peptide (VEGF)bulletCRS-BP complexes (69-75 kDa). The relative intensity of CRS-BPs (69-75 kDa) was quantitated by a PhosphorImager.




Figure 10: Effect of NaCl concentration on the binding at pH 5.0 of I-CRS peptide (VEGF) to CRS-BPs purified from bovine liver plasma membranes. The binding of I-CRS peptide (VEGF) to CRS-BPs purified from bovine liver plasma membranes, which had been ultrafiltrated to remove endogenous NaCl, was carried out at pH 5.0 in the presence of various concentrations of NaCl with (+) or without(-) 1 mM suramin. The I-CRS peptide (VEGF)bulletCRS-BP complexes were cross-linked by EDAC and then analyzed by 8% SDS-polyacrylamide gel electrophoresis followed by autoradiography. The bracket indicates the location of I-CRS peptide (VEGF)bulletCRS-BP complexes (69-75 kDa). The relative intensity of CRS-BP complexes (69-75 kDa) was quantitated by a PhosphorImager.



Biochemical Characterization of Major CRS-BPs Purified from Bovine Liver Plasma Membranes

The preparations of CRS-BPs purified from SK-Hep cells were found to contain a significant quantity of contaminant proteins. For this reason, CRS-BPs purified from bovine liver plasma membranes were used for their biochemical characterization. CRS-BPs purified from bovine liver plasma membranes were subjected to analysis by 7% SDS-polyacrylamide gel electrophoresis followed by silver staining. As shown in Fig. 11A, the major CRS-BPs showed molecular masses of 60, 66, and 72 kDa and were designated p60, p66, and p72, respectively. To define the ligand binding activities of these three major CRS-BPs, ligand binding and cross-linking were performed, and the I-CRS peptide (VEGF)bulletCRS-BP cross-linked complexes were separated by wheat germ lectin-Sepharose 4B absorption from I-CRS peptide (VEGF)bulletBSA covalent-linked complexes. After removal of I-CRS peptide (VEGF)bulletBSA complexes by wheat germ lectin-Sepharose 4B absorption, three distinct I-CRS peptide (VEGF) complexes could be clearly identified by 7% SDS-polyacrylamide gel electrophoresis. As shown in Fig. 11B, the major CRS-BPs (p60, p66, and p72) formed I-CRS peptide (VEGF) cross-linked complexes with apparent molecular masses of 63, 69, and 75 kDa, respectively. These results suggest that in the presence of I-CRS peptide (VEGF)bulletBSA complexes, these major I-CRS peptide (VEGF)bulletCRS-BP cross-linked complexes appear as a broad band on SDS-polyacrylamide gel electrophoresis (Fig. 5Fig. 6Fig. 7Fig. 8). This broad band can be resolved into individual I-CRS peptide (VEGF)bulletCRS-BP complexes if the I-CRS peptidebulletBSA complexes are removed prior to 7% SDS-polyacrylamide gel electrophoresis.


Figure 11: SDS-polyacrylamide gel patterns of CRS-BPs purified from bovine liver plasma membranes (A) and their I-CRS peptide (VEGF) cross-linked complexes (B). A shows the SDS gel pattern of purified CRS-BPs (total protein, 0.3 µg) after electrophoresis and silver staining. The arrows indicate the locations of the major CRS-BPs (60, 66, and 72 kDa). B shows the autoradiograph of the I-CRS peptide (VEGF) cross-linked complexes of CRS-BPs purified from bovine liver plasma membranes. After binding and cross-linking at pH 7.4, the I-CRS peptide (VEGF) cross-linked complexes were subjected to wheat germ lectin-Sepharose 4B absorption and elution with 0.2 MN-acetylglucosamine prior to 7% SDS-polyacrylamide gel electrophoresis. The arrows indicate the locations of the major I-CRS peptide (VEGF)bulletCRS-BP complexes (63, 69, and 75 kDa).



To define the structural relationship of p60, p66, and p72, the N-terminal amino acid sequence of each of these CRS-BPs was analyzed using automated Edman degradation after further purification by 7% SDS-polyacrylamide gel electrophoresis followed by polyvinylidene difluoride membrane transblotting. CRS-BPs p60, p66, and p72 were found to share the same N-terminal sequence of H(2)N-Ser-Leu-Arg-Ser-Glu-Glu. These results suggest that p60 and p66 may be the proteolytic products of p72. Alternatively, these CRS-BPs may be the products due to differential glycosylation (or other post-translational modifications) of the same gene product.

Expression of CRS-BP1 on Cultured Cells

Since it is likely that CRS-BPs p60, p66, and p72 are encoded by the same gene and these CRS-BPs are the first CRS-binding proteins identified and isolated, CRS-BPs p60, p66, and p72 are collectively designated CRS-BP1. To investigate the expression of CRS-BP1 on cultured cells, we performed immunoprecipitation of CRS-BP1 expressed on HepG2 cells with antiserum to the N-terminal peptide of CRS-BP1 following affinity labeling with I-CRS peptide (VEGF). HepG2 cells have been found to express more CRS-BP1 than other cell types examined (Table 1). As shown in Fig. 12, HepG2 cells expressed several CRS-BPs that formed I-CRS peptide (VEGF) cross-linked complexes with molecular masses of 150, 75, and 63 kDa (lane3). The I-CRS peptide (VEGF)bulletCRS-BP complexes with a molecular mass of 75 kDa appeared to be immunoprecipitated by anti-CRS-BP1 antiserum (lanes1 and 2). It is of importance to note that only one-fifth of the cells were used for immunoprecipitation. The 150- and 63-kDa complexes may be the dimer and degradation products of the 75-kDa complex, respectively, which may not be recognized by anti-N-terminal peptide antiserum to CRS-BP1. Alternatively, the 150- and 63-kDa complexes may be I-CRS peptide (VEGF) affinity-labeled products of other unrelated CRS-BPs. This result suggests that CRS-BP1 is expressed on cultured cells and is at least partially responsible for the high-affinity binding of CRS peptides on HepG2 cells.


Figure 12: Immunoprecipitation of ligand affinity-labeled CRS-BPs on HepG2 cells by antiserum to CRS-BP1. Confluent HepG2 cells were incubated with 10 nMI-CRS peptide (VEGF) in the presence (+) and absence (-) of 1 mM suramin. The I-CRS peptide (VEGF)bulletCRS-BP complexes were then cross-linked by EDAC, concentrated by wheat germ lectin-Sepharose 4B absorption, and analyzed by 7% SDS-polyacrylamide gel electrophoresis followed by autoradiography (lanes3 and 4). One-fifth of the cells were immunoprecipitated by anti-N-terminal peptide antiserum to CRS-BP1 and then analyzed by 7% SDS-polyacrylamide gel electrophoresis followed by autoradiography (lanes1 and 2). The arrow indicates the location of a I-CRS peptide (VEGF)bulletCRS-BP complex with a molecular mass of 75 kDa.




DISCUSSION

Cell-surface retention is an intriguing aspect of secretion of some proteins that has been defined by pulse-chase experiments(1, 2, 3, 4, 5, 6, 7) . During chase, this class of newly synthesized S-labeled secretory proteins undergo processing of carbohydrate moieties in the endoplasmic reticulum and Golgi complex and appear and remain at the cell surface for many hours(1, 2, 3, 4, 5, 6, 7) . The cell surface-bound secretory proteins can be released upon treatment with suramin or heparin or by protease digestion(1, 3, 5) . Upon longer incubations (days), the cell surface-bound secretory proteins either are released (mainly as degraded forms) or diffuse slowly and become associated with extracellular matrix(1, 29) . Several secretory growth factors and cytokines, which have been shown to undergo cell-surface retention by pulse-chase experiments, possess putative CRS motifs (Table 2). The CRS motifs appear to share some of the following structural features. 1) Four (or 5) or more residues out of a stretch of 7 (or 8) contiguous residues including the N- and C-terminal residues are basic amino acids. 2) No acidic amino acid is included in these 7 or 8 contiguous amino acid residues. 3) No more than 2 contiguous residues are nonbasic amino acid residues. 4) Most of the CRS motifs are located in the C-terminal parts of the molecules. The CRS motifs appeared to be homologous to nucleus localization sequence motifs (31) and may function as nucleus localization sequences when appropriately expressed(32, 33) .



The persistent cell-surface retention of secretory proteins with CRS implies high-affinity binding sites for cell-surface retention of these proteins. Attempts to identify these high-affinity binding sites resulted in little success due to the presence of an enormous number (10^7 to 10^8 sites/cell) of low-affinity (K(d) 0.6-4 µM) binding sites(18, 19) . In the presence of a relatively high number of low-affinity binding sites(18, 19) , the higher affinity binding sites cannot be detected by binding analysis using a 100-fold molar excess of unlabeled ligand to estimate the nonspecific binding. The 100-fold molar excess of unlabeled ligand only increases the ligand binding to the low-affinity sites at the low concentrations of radioactive ligand so that the apparent nonspecific bindings are higher than the total bindings of the ligand.

To identify the high-affinity binding sites for CRS peptides, we determined the suramin-displaceable specific binding of I-CRS peptides on cells. Suramin inhibits the high-affinity binding of I-CRS peptides. Scatchard plot analysis of the suramin-displaceable specific binding revealed a single class of high-affinity binding sites for I-CRS peptides on various cell types. The ligand binding activity of these high-affinity binding sites is not influenced by pretreatment of cells with heparitinase, suggesting that HS-PG is not responsible for the high-affinity binding of I-CRS peptides. This suggestion has been supported by the observation of cross-linked complex formation of I-CRS peptide (VEGF) and high-affinity binding sites (CRS-BPs). HS-PG has not been reported to be able to form cross-linked complexes with proteins under cross-linking conditions(34) .

We hypothesized that the cell-surface retention of secretory proteins with CRS results from the consistent interaction with high-affinity binding sites (or binding proteins) during intracellular routing of both secretory proteins and high-affinity binding sites (or binding proteins) from the trans-Golgi complex to the plasma membrane(1) . If the cell-surface high-affinity binding sites are the same components involved in the interaction with secretory proteins in the intracellular organelles (the trans-Golgi complex and exocytosis vesicles), the high-affinity binding sites should show a broad pH activity. As expected, the cell-surface high-affinity binding sites were found to exhibit a broad pH activity, with a maximum at pH 5.0-5.5. This result suggests that the high-affinity binding sites (or binding proteins) have the potential to interact with secretory proteins with CRS in the intracellular organelles, that have lower luminal pH.

For biochemical characterization of CRS-BPs, we have developed a purification procedure involving Triton X-100 extraction of liver plasma membranes or cultured cells and sequential affinity column chromatography on wheat germ lectin-Sepharose 4B and CRS peptide (sis)-Affi-Gel 10. This procedure enabled us to obtain 20 µg of CRS-BPs from 5 g protein of liver plasma membranes. Since the cross-linking assay for CRS-BPs is semiquantitative, we are unable to estimate the exact yield and -fold purification of CRS-BPs purified by this procedure. However, the recovery of CRS-BPs from each of the column chromatographies on wheat germ lectin-Sepharose 4B and CRS peptide (sis)-Affi-Gel 10 was estimated to be >80% by applying a known quantity of purified CRS-BPs onto these affinity columns.

Major CRS-BPs purified from bovine liver plasma membranes and SK-Hep cells are glycoproteins (retained by wheat germ lectin-Sepharose 4B gel) with molecular masses of 60, 66, and 72 kDa, which are designated p60, p66, and p72, respectively. These CRS-BPs form I-CRS peptide (VEGF) cross-linked complexes with molecular masses of 63, 69, and 75 kDa, respectively. Based on the molecular mass (3 kDa) of I-CRS peptide (VEGF), it is estimated that CRS-BPs form 1:1 stoichiometric complexes with I-CRS peptide (VEGF). N-terminal amino acid sequence analysis revealed that CRS-BPs p60, p66, and p70 purified from bovine liver plasma membranes possess the same N-terminal amino acid sequence, suggesting that p60 and p66 may be the degradation products of p72. Alternatively, these CRS-BPs may be differential post-translational modification products of the same gene product.

Several lines of evidence support the suggestion that the major CRS-BPs purified are involved in the high-affinity binding of I-CRS peptide (VEGF) to the cell surface of cultured cells. These include the following. 1) The molecular masses (63-75 kDa) of I-CRS peptide (VEGF) cross-linked complexes of purified major CRS-BPs are very similar to those of I-CRS peptide (VEGF) affinity-labeled binding sites on intact SK-Hep cells; 2) purified major CRS-BPs show a pH activity (with a maximum at pH 5.0-5.5) similar to that of cell-surface high-affinity binding sites; 3) the ligand binding activities of purified CRS-BPs and high-affinity binding sites are inhibited by polylysine, protamine, and heparin, but not cytochrome c; 4) antiserum to the N-terminal peptide of CRS-BP1 immunoprecipitates a I-CRS peptide (VEGF)bulletCRS-BP cross-linked complex on intact HepG2 cells; and 5) CRS-BP1 is identified as an integral membrane protein based on the observations that CRS-BPs in plasma membranes cannot be extracted by high salt or high pH (11.5) and that CRS-BP1 is recovered in the detergent phase during phase partition in Triton X-114. (^2)

This study is the first to identify and characterize high-affinity binding sites for CRS peptides on cultured cells and to isolate and characterize CRS-BPs from SK-Hep cells and bovine liver plasma membranes. The finding of the acidic pH activity of the high-affinity binding sites or CRS-BPs supports the hypothesis that the high-affinity binding sites (or CRS-BPs) interact with secretory proteins with CRS in the intracellular organelles during intracellular routing of both proteins from the trans-Golgi complex to the plasma membrane or extracellular compartment. Experiments are currently in progress to investigate the interaction of CRS-BP1 with the sis gene product in the intracellular organelles.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA-38808 and by a grant from Monsanto Co. 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.

§
Contributed equally to this work.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8135; Fax: 314-772-1307.

(^1)
The abbreviations used are: PDGF, platelet-derived growth factor; c-sis, cellular homolog of the viral oncogene v-sis; VEGF, vascular endothelial cell growth factor; CRS, cell-surface retention sequence(s); CRS-BP, CRS-binding protein; HS-PG, heparan sulfate proteoglycan; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; Mes, 2-(N-morpholino)ethanesulfonic acid.

(^2)
C. Boensch, M.-D. Kuo, D. T. Connolly, S. S. Huang, and J. S. Huang, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. William S. Sly for critical review of this manuscript, Ute Schaeper for early involvement in this research project, and Maggie Klevorn for preparing the manuscript.


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