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J. Biol. Chem., Vol. 278, Issue 44, 43855-43869, October 31, 2003

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Cloning, Expression, Characterization, and Role in Autocrine Cell Growth of Cell Surface Retention Sequence Binding Protein-1^*

Shuan Shian Huang , Fen-Mei Tang¶, Yen-Hua Huang||, I-Hua Liu, Shih-Chi Hsu¶, Shui-Tein Chen**, and Jung San Huang

From the Departments of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, ^¶Institute of Biomedical Sciences and ^**Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, and ^||Department of Biochemistry, Taipei Medical University, Taipei 110, Taiwan

Received for publication, June 17, 2003 , and in revised form, July 30, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell surface retention sequence binding protein-1 (CRSBP-1) is a cell surface binding protein for the cell surface retention sequence (CRS) motif of the v-sis gene product (platelet-derived growth factor-BB). It has been shown to be responsible for cell surface retention of the v-sis gene product in v-sis-transformed cells (fibroblasts) and has been hypothesized to play a role in autocrine growth and transformation of these cells. Here we demonstrate that the CRSBP-1 cDNA cloned from bovine liver libraries encodes a 322-residue type I membrane protein containing a 23-residue signal peptide, a 215-residue cell surface domain, a 21-residue transmembrane domain, and a 63-residue cytoplasmic domain. CRSBP-1 expressed in transfected cells is an 120-kDa disulfide-linked homodimeric glycoprotein and exhibits dual ligand (CRS-containing growth regulators (v-sis gene product and insulin-like growth factor binding protein-3, IGFBP-3) and hyaluronic acid) binding activity. CRSBP-1 overexpression (by stable transfection of cells with CRSBP-1 cDNA) enhances autocrine loop signaling, cell growth, and tumorigenicity (in mice) of v-sis-transformed cells. CRSBP-1 expression also enhances autocrine cell growth mediated by IGFBP-3 in human lung carcinoma cells (H1299 cells), which express very little, if any, endogenous CRSBP-1 and exhibits a mitogenic response to exogenous IGFBP-3, stably transfected with IGFBP-3 cDNA. However, CRSBP-1 overexpression does not affect growth of normal and transformed cells that do not produce these CRS-containing growth regulators. These results suggest that CRSBP-1 plays a role in autocrine regulation of cell growth mediated by growth regulators containing CRS.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell surface retention during secretion is observed with many secreted proteins including growth factors, cytokines, and other biologically important molecules (1–9). Cell surface retention may allow the orchestrated release of growth factors/cytokines at the cell surface, permitting stimulation of spatial orientation, cell growth and differentiation, and organogenesis during embryonic development (10). It may also function as a reservoir of growth factors/cytokines for tissue remodeling in response to injury (11). It has been implicated in the autocrine regulation of cell growth of simian sarcoma virus (SSV)¹-transformed cells (1, 12), in facilitating homodimer formation of insulin-like growth factor binding protein-3 (IGFBP-3) (13), and in promoting tumor angiogenesis and metastasis (9, 14). It is hypothesized to play a role in forming the Wnt-1 signaling complex at the cell surface (15).

The secreted proteins that undergo cell surface retention during secretion as demonstrated by pulse and chase experiments have been shown to possess cell surface retention sequence (CRS) motifs that are responsible for this phenomenon (1, 4, 5, 12, 15, 16). The CRS motifs have clustered basic amino acid residues that are well conserved during evolution from fly to mammal (17), implying their biological importance. Interestingly, for some genes, the CRS motifs are exclusively located in certain exons. The alternative splicing of the CRS-encoding exons during transcription results in the production of various polypeptide isoforms that exhibit distinct but overlapping functions in vitro and in vivo (18–20). For example, vascular endothelial cell growth factor (VEGF) has six alternatively spliced isoforms in humans: VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₃, VEGF₁₈₉, and VEGF₂₀₆. VEGF₁₈₉ contains the CRS motif residues encoded by exons 6 and 7, whereas VEGF₁₆₅ and VEGF₁₂₁ lack exon 6a and exons 6/7, respectively (6, 20–22). Because of the lack of the CRS exons (exons 6 and 7), VEGF₁₂₁ is secreted freely from cells. About 50% of VEGF₁₆₅ (which lacks the major CRS motif) is retained on the cell surface. All of larger VEGF isoforms possess the CRS and are fully retained at the cell surface (6, 20–22). Transgenic mice lacking VEGF isoforms with a CRS exhibit impaired postnatal myocardial angiogenesis and ischemic cardiomyopathy that lead to cardiac failure and death (10, 23). This transgenic animal experiment clearly indicates the biological importance and physiological relevance of cell surface retention of VEGF.

The v-sis gene product, the oncogene product of SSV (24), was reported to exhibit cell surface retention (1). It is hypothesized to play a role in autocrine cell growth and transformation of SSV-transformed cells (12, 25). The cellular homolog of the v-sis gene product, namely the c-sis gene product (PDGF-BB) (26, 27), was also shown to undergo cell surface retention (4, 5). The major CRS of the c-sis gene product is located at residues 214–227 (4, 5). Deletion of the major CRS of the c-sis gene product leads to enhanced secretion of the c-sis gene product but does not significantly affect the transforming activity of the c-sis gene product as demonstrated by transfection and in vitro foci assays (5). This observation suggests that the major CRS is not required for autocrine transformation by the c-sis gene under these experimental conditions. However, it is still consistent with the hypothesis that cell surface retention of the v-sis gene product plays a role in autocrine growth regulation and the overall transformation process in SSV-transformed cells for several reasons. Deletion of the major CRS only partially abolishes cell surface retention of the c-sis gene product (PDGF-BB) (5). This appears to be analogous to VEGF₁₆₅ that lacks the major CRS motif and exhibits partial cell surface retention (6, 20–22). VEGF and PDGF-BB have structural homology. PDGF-BB is a basic protein containing clustered lysine and arginine residues in addition to the major CRS (28). These clustered basic amino acid residues may contribute to partial cell surface retention properties of the c-sis gene product with deletion of the major CRS (5). Also, the overexpression (by transfection) system used by LaRochelle et al. (5), is likely to minimize the role of cell surface retention of the c-sis gene product in autocrine growth regulation and transformation of c-sis-transformed cells. The large amount of the c-sis gene product produced decreases the need for cell surface retention which facilitates the interaction of the c-sis gene product and the PDGF -type receptor (5). v-sis-transformed cells (fibroblasts) are a well accepted system for studying actions of the v-sis gene product and should also be appropriate for defining the function of cell surface retention of the v-sis gene product. For these reasons, the role of cell surface retention of the v-sis gene product in autocrine cell growth and transformation of SSV-transformed cells remains incompletely understood and warrants further investigation.

The molecular mechanism(s) by which secreted proteins with CRS are transported to the cell surface and retained there is not understood. The extracellular matrix (ECM) or heparan sulfate proteoglycans are commonly thought to mediate cell surface retention of secreted proteins having CRS because they are capable of binding secreted proteins with CRS (18, 29–31) and because their interactions with secreted proteins with CRS can be blocked by high salt and a polysulfonate compound (suramin) known for releasing the cell surface-retained secreted proteins from cells (1, 4, 5, 12, 25, 32). However, several lines of evidence suggest that a high affinity membrane-binding protein(s) rather than the ECM or proteoglycans mediate cell surface retention of secreted proteins with CRS. First, cellular proteoglycans are known to represent low affinity (K_d = 0.6–4 µM) but high density binding sites for CRS-containing peptides (30, 31). Second, the Wnt-1 gene product, which possesses CRS, exhibits cell surface retention in both wild-type Chinese hamster ovary cells and mutant Chinese hamster ovary cells lacking expression of proteoglycans (15). Third, cell surface retention of secreted proteins having CRS occurs immediately following synthesis, whereas the association of secreted proteins having CRS with cellular proteoglycans occurs in hours and days following secretion. The cell surface-retained secreted proteins either are released as degraded forms or diffuse slowly and then become associated with the ECM (1, 21, 25, 29, 32). Fourth, a 60–70-kDa membrane glycoprotein, namely cell surface retention sequence binding protein-1 (CRSBP-1), which exhibited high affinity (K_d = 0.5–0.7 nM) binding of the CRS motifs of both the c-sis gene product (PDGF-BB) and VEGF, has been identified in and purified from cultured cells and tissue (25, 32).

CRSBP-1 is expressed in all cell types and tissues examined including SSV (or v-sis)-transformed cells (fibroblasts) (12, 25, 32). To test the notion that CRSBP-1 is responsible for cell surface retention of the v-sis gene product in SSV-transformed cells, we characterized the interaction of CRSBP-1 (endogenous) with the v-sis gene product in these transformed cells (12, 25). CRSBP-1 appeared to form complexes with the v-sis gene product and PDGF -type receptor as demonstrated by metabolic labeling and co-immunoprecipitation. In these cells, the v-sis gene product, PDGF -type receptor, and CRSBP-1 all underwent rapid intracellular turnover that was blocked in the presence of suramin known to reverse the transformed phenotype of v-sis-transformed cells (1, 4, 5, 12, 25, 33, 34). This was not observed in normal cells and other transformed cells that expressed CRSBP-1 and PDGF -type receptor but did not produce the v-sis gene product or PDGF-BB. In SSV-transformed cells, cell surface CRSBP-1 exhibited ligand-dependent internalization and recycling with a cycling time of 12 min, whereas it was very stable and did not undergo detectable internalization and recycling in cells that do not express the v-sis or c-sis gene product (12, 25). These results suggest that, in SSV-transformed cells, CRSBP-1 forms ternary and binary complexes with newly synthesized v-sis gene product and PDGF -type receptor at the trans-Golgi network (TGN) and that the stable binary (CRSBP-1-v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF -type receptors during internalization/recycling (12, 25). These results also led us to hypothesize that CRSBP-1 plays a role in autocrine regulation of cell growth mediated by the v-sis gene product and other growth regulators with CRS. To test this hypothesis, we cloned the cDNA of CRSBP-1 from bovine liver libraries, expressed it in SSV-transformed cells and cells stably transfected with IGFBP-3, and characterized the biological properties of these cells. In this communication, we demonstrate that the cDNA of CRSBP-1 cloned from bovine liver libraries encodes a 322-amino acid residue type I membrane protein, which is synthesized as a 45-kDa protein post-translationally modified by glycosylation and other as-yet-unidentified processes to yield a 60-kDa glycoprotein that occurs as a disulfide-linked dimer in transfected cells. We also demonstrate that expressed CRSBP-1 possesses dual distinct ligand (CRS (sis)-peptide/IGFBP-3 (a CRS-containing growth regulator) and hyaluronic acid) binding activity and that CRSBP-1 overexpression enhances autocrine regulation of cell growth mediated by the v-sis gene product and IGFBP-3.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Na¹²⁵I (17 Ci/mg), tran³⁵S-label (>1,000 Ci/mmol), L-[³⁵S]cysteine (>800 Ci/mmol), and [-³²P]ATP (4,500 Ci/mmol) were purchased from ICN Biochemicals (Irvine, CA). Suramin was obtained from FBA Pharmaceuticals (West Haven, CT). Cetylpyridinium chloride (CPC), Triton X-100, hyaluronic acid (HA) (M_r 2,000,000) from human umbilical cord (>98% purity), heparan sulfate, pentosan polysulfate, dermatin sulfate, chondroitin sulfate A–C, bovine serum albumin, tunicamycin, beneyl-2-acetylamido-2-deoxy--D-galactopyranoside, chloramine T, molecular mass protein standards (myosin, 200 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa; -lactoglobulin, 18 kDa) were obtained from Sigma. Antisera to PDGF-BB/v-sis gene product, N-terminal peptide (SLRSEEISILGP) and C-terminal peptide (KSPPKTTVRCLEAEV), of bovine CRSBP-1 and antiserum to human IGFBP-3 were raised in rabbits according to our published procedures (1, 12, 33, 35). Anti-IGFBP-3 IgG and preimmune serum IgG were prepared according to standard procedures. Normal NIH 3T3, SSV-, or v-sis-transformed fibroblasts (SSV-NIH 3T3 and SSV-NRK cells) and H1299 cells (human lung carcinoma cells) were cultured and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum as described previously (36). Endo-H and Endo-F were purchased from Glyko (Novato, CA). CRS (sis)-peptide (YVRVRRPPKGKHRKFKHTH)-Affi-Gel 10 was prepared as described previously (25, 32). Protein A-Sepharose was obtained from Amersham Biosciences. Human PDGF-BB and IGFBP-3 were obtained from Austral Biologicals (San Ramon, CA) and R & D Systems, Inc. (Minneapolis, MN).

Cloning and Sequencing of CRSBP-1 cDNA—The determined N-terminal amino acid sequence of the bovine CRSBP-1 purified from bovine liver (25, 32) was used to search the human EST data base. A full-length human homolog EST was identified in the data base (human EST 112782). Using a PST fragment (500 bp) of this EST from American Type Culture Collection (Manassas, VA) as a probe, the CRSBP-1 cDNA was cloned from the bovine liver Uni-ZapXR cDNA library (Stratagene). The cDNA inserts present in the selected clones were isolated by in vivo excision as recombinant pSK- plasmids using filamentous helper phage. The largest cDNA insert (1.6 kb), which was present in one of the isolated clones, was subjected to sequencing. The entire amino acid sequence of human CRSBP-1 was directly deduced from the human EST. Mouse CRSBP-1 cDNA was constructed from three mouse ESTs, AI226003 [GenBank] , AI391129 [GenBank] , and AI318386 [GenBank] (American Type Culture Collection), that contain overlapping sequences.

Transfection—SSV-NIH 3T3, SSV-NRK, NIH 3T3, and H1299 cells were transfected with bovine CRSBP-1 cDNA in pCEP4 (Invitrogen) and with pCEP4 vector only using the calcium phosphate precipitation method. Hygromycin-resistant cells were selected after a 1-week incubation in high glucose-containing DMEM supplements with 10% fetal calf serum, 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, and 200 µg/ml hygromycin. The selected clones, namely H1299/CRSBP-1, SSV-NIH 3T3/CRSBP-1, SSV-NRK/CRSBP-1, NIH 3T3/CRSBP-1, SSV-NIH 3T3/vector, SSV-NRK/vector, NIH 3T3/vector, and H1299/vector cells (8–10 clones for each cell type), were maintained in the same medium with the reduced concentration of hygromycin (100 µg/ml).

pUC119-IGFBP-3 (courtesy of Dr. William I. Wood, Genentech) was digested with EcoRI. The EcoRI fragment (2591 bp) of IGFBP-3 was ligated to pCXN vector (5.8 kb) through the EcoRI site. The sense and antisense orientations of the ligated plasmid were determined using HindIII digestion and sequence determination and named pCXN-IGFBP-3 and pCXN vector, respectively. H1299/CRSBP-1 and H1299/vector (pCEP4) cells were transfected with pCXN-IGFBP-3 and pCXN vector (antisense) using the calcium phosphate method. H1299/CRSBP-1/vector (pCXN), H1299/CRSBP-1/IGFBP-3, H1299/vector (pCEP4)/vector (pCXN), and H1299/vector (pCEP4)/IGFBP-3 cells were selected in medium containing 600 µg/ml G418. Eight clones were selected from each cell type. These selected vector-expressing clones were named in short as H1299/CRSBP-1, H1299/vector, and H1299/IGFBP-3 cells, respectively. The expression of the transfected cDNAs was determined by Western blot analysis.

¹²⁵I-IGFBP-3 Blot Analysis—H1299/CRSBP-1 cells were lysed with 1% Triton X-100 in 25 mM HEPES, pH 7.4. The Triton X-100 extracts were diluted with buffer to make a final concentration of 0.2% and mixed with 0.5 ml of wheat germ lectin-Sepharose 4B gel (50%, v/v) at 4 °C overnight. After centrifugation, the gel pellets were eluted with 0.5 ml of 0.2 M N-acetylglucosamine. One-fifth of the eluents was kept, and the remaining portion of it was subjected to immunoprecipitation with antiserum to CRSBP-1 and non-immune serum. The lectin gel supernatant, 0.2 M N-acetylglucosamine eluents, and immunoprecipitates were subjected to 7.5% SDS-PAGE under reducing conditions and electrotransfer onto nitrocellulose membranes. After transblot, the membranes were washed three times with H₂O for 20 min, incubated with 3% Nonidet P-40 in 50 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl (TBS) for 1.5 h, 1% bovine serum albumin in TBS for 6 h, and 0.1% Tween 20 in TBS for 30 min. The membranes were then incubated with 1 nM ¹²⁵I-IGFBP-3 in TBS containing 1% bovine serum albumin and 0.1% Tween 20 overnight, washed six times with TBS containing 0.1% Tween 20 for 20 min and four times with TBS, and analyzed by autoradiography.

¹²⁵I-IGFBP-3 Binding Assay—The ¹²⁵I-IGFBP-3 binding assay in cultured cells was carried out according to Leal et al. (13, 37, 38). Briefly, H1299/CRSBP-1 and H1299/vector cells were incubated with 6 nM ¹²⁵I-IGFBP-3 with and without 100-fold excess of unlabeled IGFBP-3 in the presence of various concentrations of HA. After 2.5 h at 0 °C, the specific binding of ¹²⁵I-IGFBP-3 was determined by subtracting the nonspecific binding, which was determined in the presence of 100-fold excess of unlabeled IGFBP-3, from the total binding.

Enzymatic Digestion—Cells were labeled with 200 µCi/ml Trans ³⁵S-label (ICN) in methionine-free DMEM for 2 h. The ³⁵S-labeled cells were lysed in 1% Triton X-100, and the cell lysates were reacted with antiserum to CRSBP-1 at 4 °C overnight (33). After incubation with protein A-Sepharose, the immunocomplexes were pelleted by centrifugation. The pellets were then suspended in a solution containing 55 µl of buffer (20 mM sodium phosphate, pH 7.5) containing 0.02% sodium azide and 2.5 µl of 2% SDS, 1 M -mercaptoethanol. After 5 min at 100 °C, the suspension was cooled to room temperature, and 2.5 µl of 15% Nonidet P-40 and 2 µl of Endo-F (5 milliunits) or Endo-H (5 milliunits) were sequentially added into the solution. The enzymatic digestion was carried out at 37 °C overnight. After centrifugation, the supernatant was subjected to SDS-PAGE and fluorography.

¹²⁵I-Labeling of Cell Surface CRSBP-1—Cell surface CRSBP-1 was labeled with ¹²⁵I using the lactoperoxidase method as described previously (12, 25). The cell surface ¹²⁵I-labeled CRSBP-1 in the cell lysates was immunoprecipitated with antiserum to CRSBP-1 and analyzed by 7.5% SDS-PAGE.

Chromatography on CRS (sis)-Peptide-Affi-Gel 10 —H1299 cells stably transfected with the bovine CRSBP-1 cDNA and vector only (namely H1299/CRSBP-1 and H1299/vector cells, respectively) were solubilized with 1 ml of 1% Triton X-100 in buffer (20 mM HEPES/NaOH, pH 7.4, 10% glycerol, 0.1 M NaCl). After centrifugation, the Triton X-100 extracts were applied onto a column of CRS (sis)-peptide-Affi-Gel 10 (volume, 10 ml) (25, 32). The column was washed with buffer containing 0.1% Triton X-100 and eluted with 0.5 M NaCl in buffer containing 0.1% Triton X-100. CRSBP-1 was recovered in the 0.5 M NaCl eluents as determined by Western blot analysis using antiserum to CRSBP-1.

Determination of the Cell Surface-retained v-sis Gene Product—The level of the cell surface-retained v-sis gene product was determined as described previously (1). Briefly, cells were pulse-labeled with [³⁵S]cysteine (200 µCi/ml) in cysteine-free DMEM for 30 min and chased in DMEM containing 10 mM unlabeled cysteine for 2 h. ³⁵S-Labeled cells were washed with bicarbonate-free DMEM and incubated with 1 mM suramin at room temperature for 10 min. The suramin washes were then collected and immunoprecipitated with anti-PDGF-BB/v-sis gene product antiserum. The immunoprecipitates were analyzed by 10% SDS-PAGE under non-reducing conditions and fluorography. The relative amount of the v-sis gene product was determined by quantifying the intensity of the bands on the fluorogram.

Metabolic Labeling—Cells were grown to near-confluence and pulse-labeled with [³⁵S]methionine or [³⁵S]cysteine and chased for various times in medium containing 40 mM methionine or cysteine as described previously (1, 12, 33). For determination of the biosynthesis of the v-sis gene product or IGFBP-3, cells were pulse-labeled with [³⁵S]cysteine (for the v-sis gene product) or [³⁵S]methionine (for IGFBP-3) for 30 min only. Cell lysates were immunoprecipitated with antiserum to CRSBP-1, PDGF-BB/v-sis gene product, or IGFBP-3 and analyzed by 7.5 or 10% SDS-PAGE and fluorography. The protein bands on the fluorogram were quantified by densitometry.

Immunoblotting of PLC-, RasGAP, and Shc—After treatment with and without PDGF-BB (100 ng/ml) for 30 min on ice followed by 7 min at 37 °C as described previously (39), cells were lysed. The cell lysates were immunoprecipitated and immunoblotted using antibodies to PLC-, RasGAP, and Shc and antiphosphotyrosine according to the procedures of Valgeirsdóttir et al. (39).

Cell Growth—Cells were plated at a density of 1 x 10⁴ cells/dish in DMEM containing 10 or 1% fetal calf serum with and without anti-IGFBP-3 IgG or preimmune IgG (50 µg/ml). The cell number was counted every day using a hematocytometer. Cells grown in 10 and 1% fetal calf serum showed similar growth profiles except cells grew faster in 10% fetal calf serum than in 1% fetal calf serum.

Tumorigenicity—The tumorigenicity assay was performed using athymic nude mice as described previously (36). The tumor volume size was measured daily for 17 or 19 days.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
CRSBP-1 cDNA Encodes a Type I Membrane Glycoprotein— CRSBP-1 was purified to near-homogeneity from bovine liver plasma membranes as described previously (25, 32). The N-terminal amino acid sequence of the purified CRSBP-1 was determined as NH₂-SLRSEEISILGPXRIMGTLV (25, 32). By using this sequence to search the human EST data base, we identified a full-length human homolog. By using a PST frag-ment (500 bp) as a probe, the full-length bovine CRSBP-1 cDNA was cloned from bovine liver libraries. We also obtained the nucleotide sequence of murine CRSBP-1 by aligning murine CRSBP-1 EST fragments according to the sequence of the bovine CRSBP-1 cDNA. The bovine CRSBP-1 cDNA encodes a 322-residue type I membrane glycoprotein containing a 23-residue signal peptide, a 215-residue cell surface domain, a 21-residue transmembrane domain, and a 63-residue cytoplasmic domain (Fig. 1A). The deduced N-terminal amino acid sequence of the bovine CRSBP-1 cDNA was identical with the determined N-terminal amino acid sequence of CRSBP-1 purified from bovine liver plasma membranes (25, 32). The cell surface ligand-binding domain appeared to include two N-glycosylation sites (NFT and NSS) and 7 half-cystine residues. The cytoplasmic domain had the two potential serine phosphorylation sites but lacked any known internalization motifs. The two potential N-glycosylation sites and 7 half-cystine residues in the cell surface domain and the two potential serine-specific phosphorylation sites in the cytoplasmic domain were conserved in CRSBP-1 from bovine, human, and mouse (Fig. 1B). The deduced amino acid sequence of bovine CRSBP-1 cDNA exhibited 61 and 56% identity with those of human and murine CRSBP-1 cDNAs, respectively (Fig. 1B). These two sequences have been recently identified independently and named LYVE-1 (lymphatic vessel endothelial HA receptor-1) (40, 41). As shown in Fig. 1C, the deduced amino acid sequences of hCRSBP-1 and mCRSBP-1 were identical or almost identical with hLYVE-1 and mLYVE-1. The 9th and 10th residues of mCRSBP-1 are leucine, whereas they are phenylalanine in mLYVE-1. In contrast to the reported cell type-specific expression of LYVE-1 (40, 41), CRSBP-1 was found to be expressed in all cell types examined as determined by ¹²⁵I-cell surface labeling followed by immunoprecipitation (12). It was found in all human tissues examined and was particularly abundant in liver, heart, kidney, skeletal muscle, and placenta as determined by Northern blot analysis (data not shown). LYVE-1 has been shown recently (42) to be expressed in tissues other than the lymphatic vessels.

View larger version (90K): [in this window] [in a new window]   FIG. 1. Nucleotide and deduced amino acid sequences of bovine CRSBP-1 (A) and comparison with human or murine CRSBP-1 (B) and human or murine LYVE-1 (C). A and B, the nucleotide and deduced amino acid sequences of bovine CRSBP-1 (bCRSBP-1) cDNA cloned from bovine liver libraries (A) and the deduced amino acid sequences of human and murine CRSBP-1 (hCRSBP-1 and mCRSBP-1) (B) were determined as described under "Experimental Procedures." The N-terminal amino acid residues of these CRSBP-1 were determined by direct amino acid sequencing of the purified bovine and human CRSBP-1 and marked with the arrowhead. The predicted C-terminal transmembrane domain is underlined and two potential N-linked glycosylation sites are boxed. The half-cystine residue marked by boldface C is believed to be involved in intermolecular disulfide bond formation. Two potential serine phosphorylation sites are circled. Identical amino acid residues in these CRSBP-1 are indicated by the asterisk. C, the deduced amino acid sequences of human and murine LYVE-1 (hLYVE-1 and mLYVE-1) were reported previously (40, 41). The dot indicates the amino acid residues identical with those of bCRSBP-1, and the bar indicates the absence of the corresponding amino acid residues in mCRSBP-1 or mLYVE-1. The deduced amino acid sequence of hCRSBP-1 or mCRSBP-1 is identical with or almost identical with that of hLYVE-1 or mLYVE-1. The 9th and 10th residues in mCRSBP-1 are leucine, and they are phenylalanine in mLYVE-1. Residues 40–138 in hLYVE-1 correspond to the consensus Link module (HA binding domain) (40).

Expressed CRSBP-1 Undergoes Post-translational Modifications—To verify that the cloned bovine CRSBP-1 cDNA encodes functional CRSBP-1 protein, H1299 cells (which expressed very little, if any, endogenous CRSBP-1) were stably transfected with bovine CRSBP-1 cDNA or vector only. The cell lysates of these stably transfected cells, H1299/CRSBP-1 and H1299/vector cells, were analyzed for CRSBP-1 expression on 7.5% SDS-PAGE under reducing conditions followed by Western blot analysis using antiserum to the synthetic N-terminal peptide of bovine CRSBP-1. As shown in Fig. 2A, a significant amount of the 60-kDa CRSBP-1 was detected in the lysates of cells expressing CRSBP-1 (H1299/CRSBP-1 cells) (1st lane) but not in those of cells stably transfected with vector (2nd lane). Because the CRSBP-1 cDNA encodes a polypeptide with an estimated molecular weight of 40,000, which is smaller than the molecular weight (60,000) determined on SDS-PAGE under reducing conditions, CRSBP-1 is presumably post-translationally modified. To characterize the presumed post-translational modifications, we performed pulse-chase experiments using H1299/CRSBP-1 cells. The cells were pulse-labeled with [³⁵S]methionine for 30 min and chased using unlabeled methionine for various periods (Fig. 2B). After the pulse, ³⁵S-labeled CRSBP-1 appeared as an 45-kDa precursor (lane 1). During the chase periods, the precursor was completely converted to the 60-kDa mature form of the protein with a half-time of 45 min (lanes 2–5). After a 2.5-h chase, all newly synthesized CRSBP-1 appeared on the cell surface (lane 5) based on its sensitivity to trypsin digestion (data not shown). The sensitivity of the 45-kDa precursor to Endo-H digestion indicates the presence of high mannose-type oligosaccharides in this precursor molecule, a major product of the 30-min pulse. Endo-H digestion of the 45-kDa precursor yielded a product with molecular mass of 41 kDa (Fig. 2C, lane 1 versus lane 2). The 60-kDa mature form of CRSBP-1 was resistant to Endo-H digestion (data not shown) but sensitive to Endo-F digestion. Endo-F digestion of the mature form and precursor of CRSBP-1 generated products of 53 and 41 kDa, respectively (Fig. 2C, lanes 4 versus 3). The 7-kDa decrease of the molecular mass of the mature form after Endo-F digestion may result from the loss of two N-linked complex-type carbohydrate moieties. The inability of Endo-F to convert the 60-kDa mature form to the 41-kDa precursor Endo-H-digested product suggests that other potential post-translational modifications such as O-glycosylation might have occurred. To test this possibility, H1299/CRSBP-1 cells were treated with N-glycosylation and O-glycosylation inhibitors (tunicamycin (TM) and benzyl-2-acetylamido-2-deoxy--D-galactopyranoside (OI)) and pulse-labeled with [³⁵S]methionine for 2 h. The ³⁵S-labeled CRSBP-1 was immunoprecipitated with antiserum to CRSBP-1 and analyzed by SDS-PAGE under reducing conditions and fluorography. As shown in Fig. 2D, tunicamycin treatment yielded a product with molecular mass of 41 kDa (lane 1). This is identical to that of the precursor Endo-H-digested product. Treatment with OI only slightly altered the molecular mass of the mature form of CRSBP-1 (lane 3). Treatment with both TM and OI yielded a product identical to that obtained from the treatment with TM alone (lane 4). CRSBP-1 was found to be a phosphorylated protein as demonstrated by immunoprecipitation following ³²P-metabolic labeling of H1299/CRSBP-1 cells (data not shown), but the phosphorylation did not appear to affect its mobility on SDS-PAGE. These results suggest that CRSBP-1 undergoes other as-yet-undefined post-translational modifications in addition to N-linked glycosylation, O-linked glycosylation, and phosphorylation and that the tunicamycin-sensitive N-linked glycosylation may be required for the other post-translational modifications.

View larger version (33K): [in this window] [in a new window]   FIG. 2. Western blot analysis (A), kinetic processing (B), enzymatic sensitivity (C), and processing inhibitor sensitivity (D) of expressed CRSBP-1. A, Triton X-100 extracts of H1299/CRSBP-1 and H1299/vector cells were analyzed by Western blot using antiserum to CRSBP-1 following 7.5% SDS-PAGE under reducing conditions. The arrow indicates the location of CRSBP-1. B, H1299/CRSBP-1 cells were pulse-labeled with [35S]methionine for 30 min and chased in the presence of unlabeled methionine for different periods. The 35S-labeled cell lysates were immunoprecipitated with antiserum to CRSBP-1. The immunoprecipitates were then analyzed by 7.5% SDS-PAGE under reducing conditions and fluorography. The intensity of the CRSBP-1 bands on the fluorogram was quantitated by densitometry. The arrowhead and arrow indicate the locations of the precursor and mature form of CRSBP-1, respectively. C, H1299/CRSBP-1 cells were pulse-labeled with [35S]methionine for 30 min and 2 h (lanes 1 and 2 and 3 and 4, respectively). The 35S-labeled CRSBP-1 was immunoprecipitated and then digested with Endo-H (lane 2) and Endo-F (lane 3). The digests were analyzed by 7.5% SDS-PAGE under reducing conditions and fluorography. The closed arrow and arrowhead indicate the locations of the 60-kDa mature form and 45-kDa precursor of CRSBP-1, respectively. The open arrow and arrowhead indicate the locations of the digested products (53 and 41 kDa) of the mature form and precursor of CRSBP-1, respectively. D, H1299/CRSBP-1 cells treated without (control) (lane 2) and with tunicamycin (TM) (lane 1), benzyl-2-acetylamide-2-deoxy--D-galactopyranoside (OI) (lane 3), and both tunicamycin and benzyl-2-acetylamide-2-deoxy--D-galactopyranoside (lane 4) were pulse-labeled with [35S]methionine for 2 h. The 35S-labeled cell lysates were immunoprecipitated with antiserum to CRSBP-1. The immunoprecipitates were then analyzed by 7.5% SDS-PAGE under reducing conditions and fluorography. The arrow, arrowhead, and bar indicate the locations of the mature form (60 kDa), precursor (45 kDa) and a tunicamycin product (41 kDa) of CRSBP-1, respectively.

Expressed CRSBP-1 Is a Disulfide-linked Homodimer— CRSBP-1 contains 7 half-cystine residues in its cell surface domain. This suggests that at least 1 of these 7 half-cystine residues in the cell surface domain of CRSBP-1 may be involved in the formation of intermolecular disulfide bonds. To test this possibility, lysates of H1299/CRSBP-1 cells were analyzed by 7.5% SDS-PAGE under reducing (Fig. 3, lanes 3 and 4) and non-reducing (Fig. 3, lanes 1 and 2) conditions followed by Western blot using antiserum to CRSBP-1. As shown in Fig. 3, CRSBP-1 mainly migrates as 60- and 120-kDa proteins on SDS-PAGE under reducing and non-reducing conditions, respectively. The intensities of the CRSBP-1 antigen bands before and after reduction are comparable (lanes 2 versus 4), suggesting that CRSBP-1 is a disulfide-linked homodimer. The disulfide bond(s) linking the two CRSBP-1 monomers was found to be very sensitive to reducing agents. The cellular level of the disulfide-linked dimer of CRSBP-1 varied depending on culture conditions.

View larger version (37K): [in this window] [in a new window]   FIG. 3. Identification of expressed CRSBP-1 as a disulfide-linked homodimer. An equal protein amount of cell lysates of H1299/CRSBP-1 and H1299/vector cells were analyzed by Western blot following 7.5% SDS-PAGE under reducing and non-reducing conditions (lanes 3 and 4 and lanes 1 and 2, respectively). The closed and opened arrows indicate the locations of the monomer and dimer of CRSBP-1, respectively. The intensity of the CRSBP-1 monomer and dimer was quantified by densitometry. The total intensity of CRSBP-1 before and after reduction with -mercaptoethanol was estimated to be equal.

Expressed CRSBP-1 Exhibits CRS (sis)-Peptide and IGFBP-3 Binding Activity—Because the defining biochemical characteristic of CRSBP-1 is its ability to bind CRS-containing peptides (12, 25, 32), we studied the CRS (sis-specific)-containing peptide binding activity of the lysates of H1299/CRSBP-1 and H1299/vector cells. Triton X-100 extracts of the lysates of H1299/CRSBP-1 and H1299/vector cells were subjected to affinity column chromatography on CRS (sis)-peptide-Affi-Gel 10 which had been used to purify CRSBP-1 from bovine liver plasma membranes and cultured cells (25, 32). The chromatographic fractions were analyzed by Western blot using antiserum to the synthetic N-terminal peptide of bovine CRSBP-1. As shown in Fig. 4A, CRSBP-1 in the lysates of H1299/CRSBP-1 cells was eluted with buffer containing 0.5 M NaCl (lanes 1–5) as reported previously (32) for purification of bovine liver CRSBP-1. In contrast, very little CRSBP-1 was eluted from the lysates of H1299/vector cells (lanes 6–9). CRSBP-1 was not detected in the column flow-through fractions (concentrated) of the lysates from either H1299/CRSBP-1 or H1299/vector cells (data not shown). This result indicates that the expressed CRSBP-1 is capable of binding CRS (sis)-peptide under the experimental conditions.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Affinity column chromatography on CRS (sis)-peptide-Affi-Gel 10 (A), ligand (125I-IGFBP-3) blot analysis (B), and IGFBP-3 co-immunoprecipitation (C) of expressed CRSBP-1. A, Triton X-100 extracts of H1299/CRSBP-1 and H1299/vector cells were applied onto a column of CRS (sis)-peptide-Affi-Gel 10. After extensive washing with buffer, CRSBP-1 was eluted with buffer containing 0.5 M NaCl. The eluents (1 ml/fraction) were analyzed by Western blot using antiserum to CRSBP-1 following 7.5% SDS-PAGE under reducing conditions. The flow-through fractions (1 ml/fraction) did not contain CRSBP-1, and the data only show the 0.5 M NaCl-elution fractions. The arrow and arrowhead indicate the locations of CRSBP-1 monomer and dimer, respectively. B, Triton X-100 extracts of H1299/CRSBP-1 cells were subjected to wheat germ lectin-Sepharose 4B gel absorption. The 0.2 M N-acetylglucosamine eluents of the wheat germ lectin-Sepharose 4B gel were immunoprecipitated with antiserum to CRSBP-1 and non-immune serum. The lectin gel supernatant, lectin gel eluents, and immunoprecipitates were subjected to 7.5% SDS-PAGE under reducing conditions and electrotransfer onto membrane filters followed by renaturation and blotting with 125I-IGFBP-3. The arrow indicates the location of CRSBP-1. C, H1299/CRSBP-1/IGFBP-3 cells were metabolically labeled with [35S]methionine. The 35S-labeled cell lysates were immunoprecipitated with antiserum to CRSBP-1 or IGFBP-3 or non-immune serum. The immunoprecipitates were analyzed by 7.5% SDS-PAGE and fluorography. The arrow indicates the locations of 39- and 42-kDa IGFBP-3, and the open arrow indicates the locations of CRSBP-1 precursor and mature form.

CRSBP-1 is the cell surface major membrane protein identified in cultured cells and tissues with binding activity for the CSR motifs of the v-sis gene product (PDGF-BB) and VEGF (12, 32). It may also bind other CRS-containing proteins such as IGFBP-3. IGFBP-3 and other high affinity IGFBPs possess a putative CRS near their C termini and have been shown to be retained on the cell surface following synthesis and secretion (43–46). We therefore determined the IGFBP-3 binding activity of CRSBP-1 expressed in H1299/CRSBP-1 cells by ligand blot analysis. Membrane glycoproteins of H1299/CRSBP-1 cells were solubilized in 1% Triton X-100-containing buffer and concentrated by wheat germ lectin-Sepharose 4B gel absorption followed by elution with 0.2 M N-acetylglucosamine. The lectin gel eluents were then immunoprecipitated with antiserum to CRSBP-1 and non-immune serum. The IGFBP-3 binding activity of the immunoprecipitates was determined using ligand blot analysis after SDS-PAGE under reducing conditions and renaturation. As shown in Fig. 4B, a major ¹²⁵I-IGFBP-3-binding protein was detected in the glycoprotein fractions eluted from wheat germ lectin-Sepharose 4B gel (lane 4), whereas the wheat germ lectin gel supernatant (unabsorbed fractions) showed very little ¹²⁵I-IGFBP-3-binding protein (lane 3). This ¹²⁵I-IGFBP-3-binding protein could be immunoprecipitated with antiserum to CRSBP-1 (lane 1) but not with non-immune serum (lane 2). This result indicates that CRSBP-1 is capable of binding IGFBP-3 in vitro. This result also suggests that the CRSBP-1 monomer binds IGFBP-3.

To define the biological relevance of the IGFBP-3 binding activity of CRSBP-1, H1299/CRSBP-1 cells were stably transfected with IGFBP-3 cDNA and designated as H1299/CRSBP-1/IGFBP-3 cells. These cells constitutively expressed both CRSBP-1 and IGFBP-3. The interaction of CRSBP-1 and IGFBP-3 in these cells was investigated by immunoprecipitation either with antiserum to CRSBP-1 or with antiserum to IGFBP-3 following metabolic labeling of cells with [³⁵S]methionine. As shown in Fig. 4C, ³⁵S-labeled IGFBP-3, which was glycosylated to yield 39- and 42-kDa products (lane 1), was capable of forming complexes with ³⁵S-labeled CRSBP-1 as demonstrated by co-immunoprecipitation using antiserum to CRSBP-1 (lane 3). This result indicates that IGFBP-3 interacts with CRSBP-1 in H1299/CRSBP-1/IGFBP-3 cells. Together with the results shown in Fig. 4, A and B, this also suggests that the expressed CRSBP-1 is functional with respect to the ligand (CRS-containing peptides or proteins) binding activity.

Expressed CRSBP-1 Possesses Dual Ligand Binding Activity—The amino acid sequence of bovine CRSBP-1 exhibited 61 and 56% identity with those of human and murine LYVE-1 cDNAs (40, 41), respectively, suggesting that bovine CRSBP-1 is the bovine homolog of LYVE-1. LYVE-1 has been shown to bind fluorescein-conjugated HA (41). To characterize the HA binding activity of the expressed bovine CRSBP-1, we performed CPC precipitation and then SDS-PAGE under reducing conditions followed by Western blot analysis after incubation of the lysates of H1299/CRSBP-1 cells with HA (M_r 2,000,000) and other glycosaminoglycans. CPC precipitation has been used to determine the HA binding activity of HA-binding proteins such as CD44 and RHAMM (47–49). The binding proteins in the CPC precipitates represent the HA complexes. As shown in Fig. 5A, CRSBP-1 formed complexes with HA in a concentration-dependent manner (lanes 2–6). In the control, the reaction mixture lacking HA did not show CRSBP-1-HA complexes in the CPC precipitates (lane 1). The pretreatment of the cell lysates with -mercaptoethanol appeared to block the formation of the CRSBP-1-HA complex presumably via cleavage of the intrachain disulfide bonds of CRSBP-1 (lane 7). CRSBP-1 also significantly formed complexes with heparan sulfate and pentosan polysulfate but not with other glycosaminoglycans (Fig. 5B). To characterize the HA binding affinity, the lysates of H1299/CRSBP-1 cells were incubated with various concentrations of HA. The CRSBP-1-HA complex was then determined by CPC precipitation followed by SDS-PAGE and Western blot analysis. As shown in Fig. 5C, increasing concentrations of HA correspondingly increased formation of the CRSBP-1-HA complex with a half-maximum concentration of 20 µg/ml (10 nM). These results suggest that CRSBP-1 is capable of binding HA with high affinity.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Specific binding of HA to CRSBP-1. A, various amounts of the lysates of H1299/CRSBP-1 cells were incubated with HA (0 or 50 µg/ml) after treatment with and without -mercaptoethanol (MSH). After incubation at room temperature for 1 h, the reaction mixture was subjected to CPC precipitation. After washing, the pellets were analyzed by 7.5% SDS-PAGE followed by Western blot analysis using antiserum to CRSBP-1. The arrow indicates the location of CRSBP-1. B, the lysates of H1299/CRSBP-1 cells were incubated with 50 µg/ml of HA, heparan sulfate (HS), pentosan polysulfate (PS), dermatin sulfate (DS), chondroitin sulfate A (CSA), chondroitin sulfate B (CSB), or chondroitin sulfate C (CSC). The reaction mixtures were subjected to CPC precipitation and then analyzed by 7.5% SDS-PAGE followed by Western blot analysis using antiserum to CRSBP-1. The absence of cell lysates, CPC precipitation, or glycosaminoglycan (GAG) (lanes 1, 2, and 10) was used as a negative control to determine nonspecific precipitation. The arrow indicates the location of CRSBP-1. C, the lysates of H1299/CRSBP-1 cells were incubated with various concentrations of HA (0, 5, 10, 20, 30, and 50 µg/ml). After incubation, the reaction mixtures were subjected to CPC precipitation and analyzed by 7.5% SDS-PAGE followed by Western blot and densitometry. The arrow indicates the location of CRSBP-1.

The apparent dual ligand binding activity for HA and CRS-containing proteins (the v-sis gene product and IGFBP-3) of CRSBP-1 raised the following question: does the binding of one ligand by CRSBP-1 affect the binding activity of the other ligand? To address this question, we examined the effect of CRS (sis)-peptide on the HA binding activity of CRSBP-1 or vice versa using H1299/CRSBP-1 and H1299/vector cells. CRS (sis)-peptide, whose amino acid sequence corresponds to the major CRS of PDGF-BB, has been shown to inhibit the binding of the v-sis gene product and IGFBP-3 to CRSBP-1 (12, 25, 32). As shown in Fig. 6, at 60 and 120 µM, CRS (sis)-peptide did not inhibit HA binding to CRSBP-1 in H1299/CRSBP-1 cells (Fig. 6A, lanes 3 and 4), whereas HA at 60 µg/ml slightly enhanced the binding of ¹²⁵I-IGFBP-3 to these cells (Fig. 6B). This HA (60 µg/ml)-enhanced binding (15%) of ¹²⁵I-IGFBP-3 was statically significant when compared with the control without HA treatment (Student's t test, p < 0.05). It was mediated by CRSBP-1 expressed in H1299/CRSBP-1 cells as shown by the finding that HA did not enhance, but rather slightly inhibited, ¹²⁵I-IGFBP-3 binding to H1299/vector cells (Fig. 6B). These cells expressed very little, if any, endogenous CRSBP-1. HA may inhibit ¹²⁵I-IGFBP-3 binding to the IGFBP-3 receptor in H1299/vector cells. Glycosaminoglycans such as heparin have been shown to inhibit ¹²⁵I-IGFBP-3 binding to the IGFBP-3 receptor in cells (13). These results suggest that CRSBP-1 possesses two distinct ligand-binding sites.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Effect of CRS (sis)-peptide (A) or HA (B) on HA or 125I-IGFBP-3 binding to CRSBP-1 expressed in H1299 cells. A, cell lysates of H1299/CRSBP-1 cells were incubated with HA in the presence of different concentrations of CRS (sis)-peptide (0, 60, and 120 µM). After incubation, the reaction mixtures were subjected to CPC precipitation and analyzed by SDS-PAGE followed by Western blot analysis using antiserum to CRSBP-1. The absence of HA in the reaction mixture was used as a negative control. The arrow indicates the location of CRSBP-1. B, H1299/CRSBP-1 and H1299/vector cells were incubated with 6 nM 125I-IGFBP-3 with and without unlabeled IGFBP-3 in the presence of various concentrations of HA (0, 20, 40, and 60 µg/ml). After 2.5 h at 0 °C, the specific binding of 125I-IGFBP-3 to the cells was determined by subtracting the nonspecific binding, which was determined in the presence of 100-fold excess of unlabeled IGFBP-3, from the total binding. The bars represent the mean ± S.D. of quadruplicate determination. The increased 125I-IGFBP-3 binding (at 60 µg/ml of HA) is statistically significant (p < 0.05) when compared with the control without HA.

CRSBP-1 Overexpression Influences Growth and Tumorigenicity of v-sis-transformed Cells—Based on the findings that CRSBP-1 forms binary and ternary complexes with the v-sis gene product and the activated, phosphorylated PDGF -type receptor in SSV- or v-sis-transformed cells, and that all three of them undergo rapid intracellular turnover in these cells, which can be blocked by suramin (1, 12, 33, 34), we hypothesized that CRSBP-1 plays a role in autocrine regulation of growth and transformation of v-sis-transformed cells (12). v-sis-transformed cells produce a large amount of the v-sis gene product which may minimize the role of endogenous CRSBP-1 (expressed at low levels) in the regulation of autocrine growth of these cells, and to test this hypothesis, we used an overexpression approach to investigate the role of CRSBP-1 in the growth and tumorigenicity of v-sis-transformed cells (SSV-NRK and SSV-NIH 3T3 cells) (36) and normal NIH 3T3 cells which do not express the v-sis gene product. SSV-NRK, SSV-NIH 3T3, and NIH 3T3 cells were stably transfected with bovine CRSBP-1 cDNA and vector only. The expression of CRSBP-1 in these cells was determined by Western blot analysis and cell surface ¹²⁵I labeling. As shown in Fig. 7, the cells stably transfected with CRSBP-1 cDNA (SSV-NRK/CRSBP-1, SSV-NIH 3T3/CRSBP-1, and NIH 3T3/CRSBP-1 cells) exhibited 3–5-fold (for SSV-NRK/CRSBP-1 and SSV-NIH 3T3/CRSBP-1 cells) or 1.5–2-fold (for NIH 3T3 cells/CRSBP-1 cells) higher amounts of CRSBP-1 than their counterparts stably transfected with vector only, based on both Western blot (Fig. 7A) and cell surface ¹²⁵I-labeling assays (Fig. 7B). SSV-NRK/CRSBP-1 and SSV-NIH 3T3/CRSBP-1 cells showed more rapid growth and greater tumorigenicity than SSV-NRK/vector and SSV-NIH 3T3/vector cells (Fig. 8, A and B, respectively). In contrast, NIH 3T3 cells and H1299 cells (human lung carcinoma cells) stably transfected with CRSBP-1 cDNA and vector only did not show any significant difference in cell growth (Fig. 8C). These cells do not express the v-sis or c-sis gene product. Taken together, these results suggest that CRSBP-1 overexpression enhances growth and tumorigenicity of v-sis-transformed cells and that it does not affect growth of normal and transformed cells that do not express the v-sis or c-sis gene product.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Comparison of CRSBP-1 expression in v-sis-transformed and normal NIH 3T3 cells stably transfected with CRSBP-1 cDNA and their counterparts stably transfected with vector only. An equal protein amount of cell lysates of v-sis-transformed cells and normal NIH 3T3 cells stably transfected with CRSBP-1 cDNA (SSV-NRK/CRSBP-1, SSV-NIH 3T3/CRSBP-1, and NIH 3T3/CRSBP-1 cells) and their counterparts stably transfected with vector only (SSV-NRK/vector, SSV-NIH 3T3/vector, and NIH 3T3/vector cells) was subjected to SDS-PAGE under reducing conditions and Western blot analysis directly (A) or to immunoprecipitation with antiserum to CRSBP-1 followed by SDS-PAGE under reducing conditions and autoradiography after cell surface 125I labeling (B). The arrow indicates the location of CRSBP-1. The proteolytic products of cell surface 125I-labeled CRSBP-1 as indicated by bars were found in these v-sis-transformed cells. The intensity of the 125I-labeled CRSBP-1 band on the autoradiogram was determined by a PhosphorImager.

View larger version (17K): [in this window] [in a new window]   FIG. 8. Cell growth (A and C) and tumorigenicity (B)ofv-sis-transformed cells, normal NIH 3T3 and H1299 cells stably transfected with CRSBP-1 cDNA, and their counterparts stably transfected with vector only. The cell growth in cultures of SSV-NRK/CRSBP-1, SSV-NRK/vector, SSV-NIH 3T3/CRSBP-1, SSV-NIH 3T3/vector, NIH 3T3/CRSBP-1, NIH 3T3/vector, H1299/CRSBP-1, and H1299/vector cells was determined by counting cell numbers daily after plating at the density of 1 x 104 cells/dish. The tumorigenicity of these v-sis-transformed cells overexpressing CRSBP-1 and expressing vector only was determined by inoculating 5 x 106 cells in each nude mouse. The tumor size was measured in volume daily after inoculation. The bars represent the mean ± S.D. of quadruplicate determination. The cell numbers (on the 4th or 5th day) of v-sis-transformed cells stably transfected with CRSBP-1 were significantly higher than those of their counterparts stably transfected with vector only (Student's t test, p < 0.001).

CRSBP-1 Overexpression Increases the Level of Cell Surface-retained v-sis Gene Product in v-sis-transformed Cells—Based on the hypothesis that cell surface retention of the v-sis gene product plays a positive role in autocrine regulation of growth and transformation of v-sis-transformed cells (12), CRSBP-1 overexpression was anticipated to enhance growth and tumorigenicity of v-sis-transformed cells via increasing cell surface retention of the v-sis gene product in these cells. To test this, we determined the relative levels of cell surface-retained (or associated) v-sis gene product using published procedures (1). In this experiment, after [³⁵S]cysteine pulse labeling and chase in the presence of unlabeled cysteine (to allow the transport of the newly synthesized v-sis gene product to the cell surface), the cell surface-retained ³⁵S-labeled v-sis gene product in these v-sis-transformed cells was washed with 1 mM suramin which is known to remove the v-sis gene product from the cell surface (1, 4, 5). The suramin washes were immunoprecipitated with antiserum to the v-sis gene product, and the immunoprecipitates were then analyzed by 10% SDS-PAGE under non-reducing conditions and fluorography. As shown in Fig. 9 and Table I, 3-fold more cell surface ³⁵S-labeled v-sis gene product was detected in SSV-NRK/CRSBP-1 and SSV-NIH 3T3/CRSBP-1 cells than in their counterparts stably transfected with vector only (Fig. 9, lanes 2 versus 1 and lanes 4 versus 3, respectively). CRSBP-1 overexpression did not affect the biosynthesis of the v-sis gene product in these v-sis-transformed cells as determined by [³⁵S]cysteine metabolic labeling (30-min pulse labeling) followed by immunoprecipitation and SDS-PAGE/fluorography (data not shown). It appeared to increase the level of the cell surface-retained v-sis gene product by increasing the transport of the v-sis gene product to the cell surface (Table I). Together with the finding shown in Fig. 8, these results indicate that the apparent cell surface retention of the v-sis gene product correlates positively with growth and tumorigenicity in v-sis-transformed cells.

View larger version (19K): [in this window] [in a new window]   FIG. 9. Cell surface retention of the v-sis gene product in v-sis-transformed cells stably transfected with CRSBP-1 cDNA and vector only. v-sis-transformed cells stably transfected with CRSBP-1 cDNA (SSV-NRK/CRSBP-1 and SSV-NIH 3T3/CRSBP-1 cells) and vector only (SSV-NRK/vector and SSV-NIH 3T3/vector cells) were pulse-labeled with [35S]cysteine for 30 min and chased in the presence of unlabeled cysteine for 2 h. After the 2-h chase, a fraction of newly synthesized v-sis gene product appeared on the cell surface. The cell surface-retained v-sis gene product was released by washing with 1 mM suramin. The suramin washes were immunoprecipitated with antiserum to PDGF-BB/v-sis gene product and then analyzed by 10% SDS-PAGE under non-reducing conditions and fluorography. The arrow and bar indicate the locations of the v-sis gene product and its degradation products, respectively. The v-sis gene product without degradation is equivalent to p44sis identified previously (1). The intensity of the 35S-labeled v-sis gene product band was quantified with densitometry.

CRSBP-1 Overexpression Regulates Autocrine Loop Signaling in v-sis-transformed Cells—The correlation of the apparent cell surface retention of the v-sis gene product with the growth and tumorigenicity in v-sis-transformed cells raises the possibility that the cell surface-retained v-sis gene product activates exclusively unoccupied cell surface PDGF -type receptors, resulting in autocrine regulation of growth and transformation of these cells. The cell surface-retained v-sis gene product is known to be mainly associated with CRSBP-1 (1, 12). To test this possibility, we determined the effect of exogenous PDGF-BB on the activation of signaling molecules known to be mediated via PDGF -type receptors in SSV-NIH 3T3 and normal NIH 3T3 cells (as control) by immunoprecipitation and immunoblotting using specific antibodies to PLC-, RasGAP, Shc and anti-phosphotyrosine. PLC-, RasGAP, and Shc were chosen for study because they represent the suramin-insensitive (PLC- and RasGAP) and suramin-sensitive (Shc) molecules involved in the PDGF -type receptor-mediated autocrine loop signaling (39). If v-sis-transformed cells possess unoccupied cell surface PDGF -type receptors that are accessible to the cell surface-retained v-sis gene product, exogenous PDGF-BB should be able to activate cell surface PDGF -type receptors and these signaling molecules. As shown in Fig. 10A, exogenous PDGF-BB failed to stimulate the activation of the PDGF -type receptor, PLC-, RasGAP, and Shc in SSV-NIH 3T3 cells (Fig. 10A-a, lanes 2 versus 1, lanes 6 versus 5 and lanes 10 versus 9, respectively). In fact, PDGF -type receptor and these signaling molecules were already activated due to autocrine expression of the v-sis gene product (Fig. 10A-a, 1st versus 3rd lanes and 5th versus 7th lanes). In contrast, it stimulated the activation of these molecules in normal NIH 3T3 cells (Fig. 10A-a, 4th versus 3rd lanes, 8th versus 7th lanes, and 12th versus 11th lanes, respectively). The protein levels of these signaling molecules in these cells did not change after treatment with PDGF-BB (Fig. 10A-b). These results suggest that, unlike normal NIH 3T3 cells, their v-sis-transformed counterparts do not express a significant level of activable cell surface PDGF -type receptor-mediated signaling. This is supported by previous experiments on ¹²⁵I-PDGF-BB cell surface binding and cross-linking that demonstrated fewer PDGF -type receptors on the cell surface of SSV-NIH 3T3 cells (36) (data not shown).

View larger version (38K): [in this window] [in a new window]   FIG. 10. Effects of exogenous PDGF-BB (A) and CRSBP-1 overexpression (B) on tyrosine phosphorylation of PLC-, RasGAP, and Shc in SSV-NIH 3T3 cells and normal NIH 3T3 cells. A, after treatment of cells with and without 100 ng/ml PDGF-B for 30 min on ice followed by 7 min at 37 °C as described previously (39), an equal protein amount of cell lysates of SSV-NIH 3T3 and normal NIH 3T3 cells were immunoprecipitated with specific antibodies to PLC-, RasGAP, and Shc. The immunoprecipitates were subjected to 7.5% SDS-PAGE and immunoblot using anti-phosphotyrosine (a) or specific antibodies to PLC-, RasGAP, and Shc (b). The relative intensity of protein bands is quantified by densitometry of the x-ray film. The arrow indicates the location of the PDGF -type receptor (PDGFR), PLC-, RasGAP, or Shc. B, an equal protein amount of cell lysates of SSV-NIH 3T3/CRSBP-1 and SSV-NIH 3T3/vector cells was subjected to immunoprecipitation (using specific antibody to PLC-, RasGAP, or Shc) followed by SDS-PAGE and immunoblot using antibody to phosphotyrosine (a) or antibodies to PLC-, RasGAP and Shc (b). The relative intensity of protein bands is quantified by densitometry of the x-ray film. The arrow indicates the location of the PDGF -type receptor (PDGFR), PLC-, RasGAP, or Shc.

As shown in Fig. 10A, SSV-NIH 3T3 cells expressed significant levels of the activated PDGF -type receptor, PLC-, RasGAP, and Shc. In these cells, the activation of these molecules might be mediated in part via the interaction of the cell surface-retained v-sis gene product and newly synthesized PDGF -type receptor (which is on the way to the cell surface) in the endosomes and prelysosomal compartments during internalization and recycling of the CRSBP-1 and v-sis gene product complex (12). Therefore, we examined the effect of CRSBP-1 overexpression on the activation of these signaling molecules in SSV-NIH 3T3 cells. As shown in Fig. 10B-a, the levels of tyrosine-phosphorylated PLC-, RasGAP, and Shc were higher in SSV-NIH 3T3 cells overexpressing CRSBP-1 than in their counterparts stably transfected with vector only (2nd versus 1st lanes, 4th versus 3rd lanes, and 6th versus 5th lanes, respectively). CRSBP-1 overexpression also slightly increased tyrosine phosphorylation of the PDGF -type receptor in SSV-NIH 3T3 cells (Fig. 10B, 2nd versus 1st lane). In the control experiments, CRSBP-1 overexpression did not affect the protein levels of those signaling molecules as demonstrated by immunoblot analysis using specific antibodies to the signaling molecules (Fig. 10B-b). These results suggest that CRSBP-1 overexpression enhances the generation of activated PLC-, RasGAP, and Shc in SSV-NIH 3T3. In the same way, CRSBP-1 overexpression increases the level of cell surface retention of the v-sis gene product and enhances cell growth and tumorigenicity in these v-sis-transformed cells.

CRSBP-1 Expression Regulates Autocrine Cell Growth Mediated by IGFBP-3 in H1299 Cells—According to the classical autocrine (extracellular loop) model (50–55), the growth factor should stimulate cell growth exclusively through interaction with its specific cell surface receptor. In fact, in autocrine cell growth and transformation mediated by the v-sis gene product in SSV-transformed cells (56), the newly synthesized v-sis gene product (PDGF-BB) mainly interacts with the PDGF -type receptor in the intracellular compartments (1, 12, 33, 34, 39, 57). These interactions result in initiating suramin-sensitive and suramin-insensitive signaling that lead to cell growth and transformation (39). CRSBP-1 appeared to facilitate such interactions and augments the autocrine regulation of cell growth and tumorigenicity. To see if CRSBP-1 is also capable of regulating autocrine growth mediated by other growth regulators with CRS, we investigated the effect of CRSBP-1 expression (by stable transfection of cells with CRSBP-1 cDNA) on H1299/IGFBP-3 cells which were stably transfected with the IGFBP-3 cDNA. This cell system was chosen because of the following reasons. 1) H1299 cells expressed very little, if any, endogenous CRSBP-1 and did not produce detectable endogenous IGFBP-3. 2) H1299 cells responded to growth stimulation by exogenous IGFBP-3 (58). IGFBP-3 is a bifunctional growth regulator depending on cell type (13, 37, 38, 43–46): a growth inhibitor for epithelial cells, endothelial cells, and other cell types, and a growth stimulator for certain fibroblasts and carcinoma cells. The growth regulatory (IGF-independent and TGF- antagonist-sensitive) activity of IGFBP-3 is mediated by the IGFBP-3 receptor which is identical to the type V TGF- receptor (13, 37, 38, 58). Both IGFBP-3 and TGF- are weak mitogens or growth factors toward H1299 cells (58). 3) IGFBP-3 has been shown to interact with CRSBP-1 in these cells as demonstrated by ligand blot analysis and co-immunoprecipitation (Fig. 4, B and C). As shown in Fig. 11, A and B, cells expressing IGFBP-3 (H1299/IGFBP-3 and H1299/CRSBP-1/IGFBP-3 cells) grew more rapidly than their counterparts stably transfected with vector only (H1299/vector and H1299/CRSBP-1 cells). Because exogenous IGFBP-3 had a stimulatory effect on the growth of H1299 cells (58), this result suggests that IGFBP-3 is an effective autocrine growth factor in the H1299 cell system. CRSBP-1 expression appeared to augment autocrine cell growth mediated by IGFBP-3 (in H1299/CRSBP-1/IGFBP-3 cells) when compared with H1299/IGFBP-3 cells (Table II). As shown in Table II, anti-IGFBP-3 IgG inhibited growth of H1299 cells expressing IGFBP-3 (H1299/IGFBP-3 and H1299/CRSBP-1/IGFBP-3 cells) but did not affect growth of H1299 cells stably transfected with vector only (H1299/vector and H1299/CRSBP-1 cells). CRSBP-1 expression did not affect the biosynthesis of IGFBP-3 in H1299/IGFBP-3 cells as determined by [³⁵S]methionine metabolic labeling (30-min pulse) followed by immunoprecipitation and SDS-PAGE/fluorography (data not shown). These results indicate that CRSBP-1 expression is indeed capable of enhancing cell growth in another autocrine cell growth system.

View larger version (15K): [in this window] [in a new window]   FIG. 11. Increase of cell growth by IGFBP-3 expression in H1299/vector cells and H1299/CRSBP-1 cells. Cells (H1299/IGFBP-3 cells, H1299/vector cells; H1299/CRSBP-1/IGFBP-3 cells, H1299/CRSBP-1 cells) were plated in DMEM containing 1% fetal calf serum at a cell density of 1 x 104 cells/dish. The cell number was counted daily. The bars represent the mean ± S.D. of quadruplicate determination. The cell numbers (on the 4th or 5th day) of H1299/CRSBP-1 and H1299 cells stably transfected with IGFBP-3 cDNA were significantly higher than those of their counterparts stably transfected with vector only (Student's t test, p < 0.001).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Several lines of evidence presented herein indicate that the CRSBP-1 protein encoded by the CRSBP-1 cDNA cloned from bovine liver libraries is identical with that purified from bovine liver (12, 25, 32). First, the deduced N-terminal amino acid sequence of bovine CRSBP-1 cDNA is identical with the determined N-terminal amino acid sequence of CRSBP-1 purified from bovine liver. Second, expressed CRSBP-1 reacts with specific antisera to CRSBP-1 purified from bovine liver as determined by Western blot analysis and by metabolic labeling followed by immunoprecipitation. Third, both expressed CRSBP-1 and CRSBP-1 purified from bovine liver exist as disulfide-linked homodimers with a molecular mass of the monomer of 60–70 kDa. Fourth, the C-terminal cytoplasmic domain in the CRSBP-1 deduced amino acid sequence does not contain any known internalization motifs. This is consistent with the observation that cell surface CRSBP-1 was relatively stable in NIH 3T3 cells and other cell types that do not express the v-sis gene or c-sis product (12, 25). Fifth, expressed CRSBP-1 exhibits ligand binding activity toward CRS (sis)-peptide and IGFBP-3 as demonstrated by CRS (sis)-peptide affinity column chromatography and ligand blot/co-immunoprecipitation, respectively. CRSBP-1 purified from bovine liver binds CRS (sis)-peptide and CRS (VEGF)-peptide as demonstrated by CRS (sis)-peptide affinity column chromatography and CRS (sis/VEGF)-peptide cross-linking (25, 32).

Here we also show that CRSBP-1 overexpression enhances growth and tumorigenicity of SSV-NRK and SSV-NIH 3T3 cells but does not affect growth of human lung carcinoma cells (H1299) and normal NIH 3T3 cells. Recently, we have also found that CRSBP-1 overexpression does not influence cell growth and tumorigenicity of myc-transformed NIH 3T3 cells (33).² The specific effect of CRSBP-1 overexpression on v-sis-transformed cells support a role of CRSBP-1 in v-sis-induced growth and transformation. CRSBP-1 overexpression appears to enhance growth/tumorigenicity of SSV-NRK/SSV-NIH 3T3 cells via increasing the level of cell surface-retained v-sis gene product in these cells. The apparent cell surface retention of the v-sis gene product may serve as an indicator of formation of binary CRSBP-1-v-sis gene products complexes that are capable of being transported (from the TGN) to and retained at the cell surface and of interacting with and forming ternary complexes with the PDGF -type receptor in TGN and endosomal/prelysosomal compartments (during internalization and recycling at the cell surface) (12, 25). There are two lines of support for this. First, CRSBP-1 forms ternary complexes with the v-sis gene product and the activated PDGF -type receptor in TGN and/or endosomal/prelysosomal compartments in SSV-NIH 3T3 cells (12). Second, in SSV-NIH 3T3 cells, the cell surface v-sis gene product does not associate with the PDGF -type receptor but does form CRSBP-1 complexes, undergoing internalization and recycling with a cycling time of 12 min (12).

Valgeirsdottir et al. (39) classified autocrine loop signaling in c-sis-transformed cells into suramin-sensitive and suramininsensitive pathways. The former includes Shc, Src, and Raf1, and the latter involves phosphatidylinositol 3-kinase, PLC-, and RasGAP. Here we demonstrate that CRSBP-1 overexpression enhances activation of both the suramin-sensitive (e.g. Shc) and suramin-insensitive (e.g. PLC- and RasGAP) pathways in SSV-NIH 3T3 cells as it does for increasing the level of cell surface-retained v-sis gene product and for enhancing growth/tumorigenicity in these two cell types. The activation of these signaling pathways is mainly mediated by the intracellularly activated PDGF -type receptor. There are several lines of evidence for this. First, exogenous PDGF-BB does not significantly affect signaling in SSV-NIH 3T3 cells. Second, protamine, an inhibitor which blocks the binding of v-sis or c-sis gene product to cell surface PDGF -type receptors (1, 25, 33), does not influence the activation of PLC-, RasGAP and Shc in SSV-NIH 3T3 cells.² Third, suramin, which enters cells and accumulates in intracellular acidic compartments, e.g. TGN (59–61), blocks the interaction of the v-sis gene product and PDGF -type receptor in the intracellular compartments (1, 33, 34, 56, 62).

IGFBP-3 is a bifunctional growth regulator; it inhibits growth of epithelial cells, endothelial cells, and other cell types in IGF-dependent and -independent manner and stimulates growth of fibroblasts (e.g. NIH 3T3 cells) and certain carcinoma cells (e.g. H1299 human lung carcinoma cells) (13, 37, 38, 44, 58). It inhibits cell growth by either scavenging IGFs from the IGF-I receptor or directly interacting with its specific receptor in responsive cells (44–46). The IGF-independent growth inhibitory and growth stimulatory activities are mediated by the type V TGF-/IGFBP-3 receptor and are sensitive to a TGF- peptide antagonist (13, 37, 38). Both IGFBP-3 and TGF-₁ are weak mitogens or growth factors for H1299 cells (58). Here we demonstrate that CRSBP-1 expression enhances autocrine regulation of cell growth mediated by IGFBP-3 in H1299/CRSBP-1/IGFBP-3 cells. Although the signaling pathways induced by autocrine stimulation of IGFBP-3 are currently unknown, based on these findings in SSV-transformed cells, we propose a modified model (12) for autocrine loop signaling involved in growth regulation mediated by growth regulators (dimeric proteins) with CRS and CRSBP-1. In this model, CRSBP-1 forms complexes with growth regulator with CRS and presents it to the unoccupied growth regulator receptor during routing of these three proteins from the endoplasmic reticulum to the TGN. At TGN, the transient ternary complex of CRSBP-1-growth regulator-receptor (dimerized or oligomerized) is formed and mediates suramin-sensitive signaling (e.g. Shc) (39, 63). During transport from the endoplasmic reticulum to medial Golgi, CRSBP-1 may also be able to form transient ternary complexes with growth regulator and its receptor, which mediates suramin-insensitive signaling (e.g. PLC- and RasGAP) (39). The binary complex of CRSBP-1 and growth regulator formed at TGN and/or Golgi complexes is transported to the cell surface and retained there (cell surface retention) where it undergoes internalization and recycling. During internalization and recycling, CRSBP-1 presents the growth regulator to its receptor at the cell surface and in the endosomal/prelysosomal compartments, resulting in generation of suramin-sensitive signaling. Suramin has been shown to enter cells and accumulate in intracellular acidic compartments such as TGN and endosomal/prelysosomal compartments (59–61). Both the suramin-sensitive and suramin-insensitive signaling cascades appear to be enhanced by CRSBP-1 overexpression.

The cell biological properties of CRSBP-1 have been studied using cultured cells (12, 25, 32). Its physiological role remains to be defined. The prominent ligands of CRSBP-1 including PDGF-BB and the VEGF family are known to be involved in the development and growth of the vascular endothelial system (64, 65). This suggests that CRSBP-1 may play a role in the autocrine regulation of cell growth of endothelial cells during formation of vessels. The finding of structural identity of CRSBP-1 and LYVE-1 may unravel its involvement in lymphangiogenesis (64, 65). LYVE-1 is localized in the luminal and abluminal faces of lymphatic vessels and has been used as a marker for lymphatic vessels. Two of the VEGF family members, VEGF-C and VEGF-D, have been shown to be localized specifically in lymphatic vessels and to regulate growth of the lymphatic endothelial cells via interaction with their receptor VEGFR-3 which is also specifically localized in lymphatic vessels (65). Because VEGF-C and VEGF-D possess potential CRS motifs near the C termini of the molecules (KGKKFHH and KHKLFH, respectively) (66–68), it is likely that CRSBP-1 interacts with VEGF-C and VEGF-D in lymphatic endothelial cells or that both VEGF-C and VEGF-D serve as ligands for CRSBP-1 in these cells. Metabolic pulse and chase experiments have revealed that they have a potential to be transported to the cell surface and retained there (69). CRSBP-1 may regulate growth of lymphatic endothelial cells stimulated by VEGF-C and VEGF-D through an autocrine mechanism.

Prevo et al. (41) hypothesized that LYVE-1 was involved in the transport of HA from tissue to lymph because HA was shown to bind to 293 T fibroblasts transiently transfected with the LYVE-1 cDNA and was taken up by these cells. The physiological importance of the HA internalization mediated by LYVE-1 remains to be defined for two reasons. First, the internalization efficacy of HA in these transfected cells appears to be relatively low. The internalization of fluorescein isothiocyanate-HA only reached a maximum after 4 h of incubation. Only about 50% of cell surface-bound fluorescein isothiocyanate-HA was internalized even after a 5-h incubation. In SSV-transformed cells, cell surface CRSBP-1 undergoes ligand-dependent internalization and recycling with a cycling time of 12 min when it is occupied by the ligand (the v-sis gene product) (12, 25). Second, the overexpression system was used in their experiments. In fact, CRSBP-1 is present at relatively low levels in cells when compared with those in the overexpression system (12, 25). The exact biological function of the HA binding activity of CRSBP-1 remains to be defined. It is suspected that HA may modulate the VEGF binding activity of CRSBP-1 in these lymphatic endothelial cells as it enhances IGFBP-3 binding to CRSBP-1 in H1299/CRSBP-1 cells. However, HA does not exhibit significant effect on autocrine cell growth mediated by v-sis (in SSV-NIH 3T3 cells) or IGFBP-3 (in H1299/CRSBP-1/IGFBP-3 cells).² It is also possible that CRSBP-1 may mediate the transport of the IGFBP-3-IGF-1 complex from tissues to lymph (70–72), which may be enhanced by HA. The IGFBP-3-IGF-1 complex is unable to interact with either the IGFBP-3 receptor or the IGF-1 receptor in endothelial cells but is capable of binding to CRSBP-1 (13, 25, 44–46).

	FOOTNOTES

 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY372937 [GenBank] .

^* This work was supported by The National Institutes of Health Grant CA38808 and grants from Academia Sinica and National Science Council, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8148; Fax: 314-577-8156; E-mail: huangss{at}slu.edu. To whom correspondence may 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: 314-577-8156; E-mail: huangjs{at}slu.edu.

¹ The abbreviations used are: SSV, simian sarcoma virus; CRSBP-1, cell surface retention sequence binding protein-1; CRS, cell surface retention sequence; IGFBP-3, insulin-like growth factor binding protein-3; PDGF, platelet-derived growth factor; DMEM, Dulbecco's modified Eagle's medium; HA, hyaluronic acid; CPC, cetylpyridinium chloride; PLC, phospholipase C; VEGF, vascular endothelial cell growth factor; ECM, extracellular matrix; TGN, trans-Golgi network; Endo, endo--N-acetylglucosaminidase; IGF, insulin-like growth factor; TGF, transforming growth factor; TM, tunicamycin; OI, benzyl-2-acetylamide-2-deoxy--D-galactopyranoside.

² S. S. Huang, F.-M. Tang, Y.-H. Huang, I.-H. Liu, S.-C. Hsu, S.-T. Chen, and J. S. Huang, unpublished results.

	ACKNOWLEDGMENTS

 
We thank Drs. William S. Sly and Frank E. Johnson for critical review of the manuscript and John McAlpin for typing the manuscript. We also thank Jeff Grubb and Dr. William I. Wood for providing pCXN and pUC119-IGFBP-3 plasmids, respectively.

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