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The Heparin Binding Domain of Vitronectin Is the Region that Is Required to Enhance Insulin-Like Growth Factor-I Signaling

Division of Endocrinology, University of North Carolina, Chapel Hill, North Carolina 27599-7170

Address all correspondence and requests for reprints to: Laura A. Maile, 6111 Thurston Bowles, University of North Carolina, Chapel Hill, North Carolina 27599-7170. E-mail: laura_maile{at}med.unc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
We have shown that vitronectin (Vn) binding to a cysteine loop sequence within the extracellular domain of the ß3-subunit (amino acids 177–184) of Vß3 is required for the positive effects of Vn on IGF-I signaling. When Vn binding to this sequence is blocked, IGF-I signaling in smooth muscle cells is impaired. Because this binding site is distinct from the site on ß3 to which the Arg-Gly-Asp sequence of extracellular matrix ligands bind (amino acids 107–171), we hypothesized that the region of Vn that binds to the cysteine loop on ß3 is distinct from the region that contains the Arg-Gly-Asp sequence. The results presented in this study demonstrate that this heparin binding domain (HBD) is the region of Vn that binds to the cysteine loop region of ß3 and that this region is sufficient to mediate the positive effects of Vn on IGF-I signaling. We provide evidence that binding of the HBD of Vn to Vß3 has direct effects on the activation state of ß3 as measured by ß3 phosphorylation. The increase in ß3 phosphorylation associated with exposure of cells to this HBD is associated with enhanced phosphorylation of the adaptor protein Src homology 2 domain-containing transforming protein C and enhanced activation MAPK, a downstream mediator of IGF-I signaling. We conclude that the interaction of the HBD of Vn binding to the cysteine loop sequence of ß3 is necessary and sufficient for the positive effects of Vn on IGF-I-mediated effects in smooth muscle cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
IGF-I IS A POTENT stimulator of the growth, differentiation, survival, and migration of both normal and transformed cells (1). Activation of the intrinsic tyrosine kinase activity of the IGF-I receptor (IGF-IR) after ligand binding couples IGF-I to downstream signaling pathways including the MAPK and phosphatidyl inositol-3 kinase pathways (1). The response of a cell to IGF-I is determined not only by its ability to bind to and activate its receptor but also by the activation state of integrins (2, 3, 4, 5, 6, 7). The ability of IGF-I to mediate its effects in several different cell types including, smooth muscle cells (SMCs), preadipocytes (8), osteoblasts (9), and various malignant cells (10) has been shown to be regulated by integrin ligand occupancy. We have shown that blocking Vß3 ligand occupancy with either a monoclonal anti-Vß3 antibody or the disintegrin echistatin blocks IGF-I-stimulated SMC migration and proliferation (2, 3). ß1 integrin ligand occupancy has been implicated in the regulation of the response of chondrocytes to IGF-I (11).

The cross-talk between integrins and growth factors is not limited to IGF-I. Direct association between Vß3 and the platelet-derived growth factor, insulin, or vascular endothelial growth factor receptors has been demonstrated (12, 13). The epidermal growth factor receptor can associate directly with 5ß1 and 6ß4 (14). In other cases, the integrin and growth factor receptor signaling pathways converge further downstream, for example, insulin receptor substrate 1 associates with Vß3 in response to insulin receptor activation (15). Recently, it was reported that the ß1 integrin subunit could bind to insulin receptor substrate 1 and to a GRB-2-associated binding protein-1/Src homology 2 containing protein tyrosine phosphatase complex and that these interactions modified IGF-IR signaling (10). Although these interactions have suggested a significant role for the interaction between integrin and growth factor signaling pathways, the consequences of this interaction for downstream signaling have not been reported.

We have shown previously that vitronectin (Vn) association with Vß3 is necessary for IGF-I-stimulated biological actions (2, 3) and that Vn binding to a cysteine loop sequence within the extracellular domain of ß3 (amino acids 177–184) is required for IGF-I signaling (16). Because this site is distinct from the site on ß3 that binds the Arg-Gly-Asp (RGD) domain (i.e. amino acids 107–171), we hypothesized that the region of Vn that binds to the ß3 cysteine loop is distinct from the domain containing the RGD sequence and that binding of this region to the ß3 cysteine loop is required for the positive effect of Vn on IGF-I signaling. Because the heparin binding domain (HBD) of Vn has been shown to bind to cell surface proteins including integrins, we determined whether the HBD of Vn mediated the interaction of Vn with the cysteine loop region of ß3 and whether interaction between the HBD of Vn and the cysteine loop contributed to the positive effects of Vn on IGF-I signaling.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Binding of the HBD Region of Vn to the Cell Surface Is Blocked in the Presence of an Antibody against the Cysteine Loop Region of ß3
Cell surface binding experiments were performed as described in Materials and Methods, and the results are presented in Table 1. When cells were incubated with an excess of unlabeled Vn HBD peptide (10 µM), the binding of the labeled peptide (4 nM) was specifically reduced by 97 ± 7% (mean ± SEM; n = 3). Similarly, a peptide derived from the HBD of IGF binding protein-5 (IGFBP-5) reduced binding by 73 ± 2.5% (mean ± SEM; n = 3). In the presence of peptide corresponding to the HBD of IGFBP-5 but with one amino acid substitution (K208A) the binding of the Vn HBD was reduced by 83 ± 4% (mean ± SEM; n = 3). A control peptide containing an irrelevant sequence did not compete for binding [i.e. a 3 ± 1.5% reduction (mean ± SEM; n = 3; P = not significant (ns)]. We have shown previously that the K208A substitution eliminates the ability of native IGFBP-5 to bind to heparin (17). Because the mutant peptide (BP-5M) still prevents binding of the Vn HBD to the cell surface, this suggests that the Vn HBD is binding to a cell surface site that is distinct from cell surface proteoglycans.

View this table: [in this window] [in a new window]   Table 1. Vn HBD Peptide Binds to Vß3

To further confirm the ability of the HBD to bind to ß3, we used an affinity purification column that had been prepared by cross-linking Vn HBD peptide to Sepharose. After biotinylation of cell surface proteins with a cell-impermeable form of biotin, cells were lysed and the cell lysate circulated over the column. After washing the column to remove proteins that were bound nonspecifically to the column, we eluted those that were bound with higher affinity. Figure 1 shows a Western immunoblot using avidin horseradish peroxidase (HRP) after SDS-PAGE of the total cell lysate (St), the proteins that flowed through the column (Ft 1 & 2) and the three-column fractions that contained proteins that had adhered to the column (Fx 3–5). The starting material contained a substantial number of cell surface proteins labeled with biotin and analysis of lanes 2 and 3 (Ft 1 and 2) showed that the majority of these proteins flowed through the column. In the lanes containing proteins that were specifically eluted from the affinity column, it can be seen that there were several distinct bands (indicated with arrows). Based upon their electrophoretic mobility, the estimated molecular sizes of the biotinylated proteins detected were as follows: band 1, 220 kDa; band 2, 103 kDa; band 3, 97 kDa; and band 4, 46 kDa. Because we were particularly interested in whether the Vn HBD peptide bound to ß3, we compared the bands of protein bands detected with a ß3 immunoblot of whole-cell lysates. It can be seen that each biotinylated protein corresponds to a band that is detected after Western blotting with a ß3 antibody. ß3 migrates as several different sized bands, when separated by SDS-PAGE, depending upon its conformation (18), and this accounts for the several distinct bands detected. Intact ß3 has an apparent electrophoretic mobility of 97 and 103 kDa, and this corresponds to bands 2 and 3. ß3, which has remained associated with V, has an apparent electrophoretic mobility greater than 200 kDa, and this presumably accounts for band 1. Band 4 corresponds to a fragment of ß3 (data not shown). When the experiment was repeated without biotin labeling of the cell surface, it can be seen in the immunoblot in Fig. 1B that bands corresponding to the apparent molecular sizes of ß3 were detected in the fraction that was eluted from the column. Although we cannot conclude that the Vn HBD only binds to ß3, the data support the conclusion drawn from the binding assays that ß3 is a major binding site for Vn HBD.

View larger version (63K): [in this window] [in a new window]   Fig. 1. The HBD of Vn Binds to the Cysteine Loop Region of ß3 A (Large panel), Quiescent SMCs (after biotin labeling) were lyzed and then circulated over a Vn HBD affinity column. Aliquots of the starting material (St), flow through (Ft) and fractions (containing proteins that eluted specifically from the column, fx) were analyzed by Western immunoblotting (IB) with an anti-HRP avidin reagent after nonreducing SDS-PAGE. A (Small panel), A fraction of whole-cell lysate (WCL) from quiescent SMCs was separated by nonreducing SDS-PAGE and ß3 visualized after Western immunoblotting with an anti-ß3 antibody. B, Aliquots of the starting material, flow through and fractions containing proteins that eluted specifically from the Vn HBD affinity column were analyzed by Western immunoblotting with a ß3 antibody after reducing (+) or nonreducing (–) SDS-PAGE. IP, Immunoprecipitation; DTT, dithiothreitol.

View larger version (27K): [in this window] [in a new window]   Fig. 2. The HBD of Vn Enhances ß3 Phosphorylation A, Quiescent SMCs were preincubated with increasing concentrations of Vn HBD peptide (1–10 µg/ml) or the HBD BP-5K208A (BP5M) peptide (10 µg/ml) for 2 h at 37 C before lysis and immunoprecipitation (IP) with the anti ß3 antibody raised to the cytoplasmic tail of ß3 (c-tail). The extent of ß3 phosphorylation was determined by immunoblotting with an anti phosphotyrosine antibody (p-Tyr). To control for differences in protein levels equal amounts of total protein from each sample were immunoblotted directly with the anti ß3 antibody (c-tail).***, P < 0.005 when ß3 phosphorylation in the presence of the Vn HBD is compared with ß3 phosphorylation in its absence. B, Quiescent SMCs were incubated with either the Vn HBD peptide (10 µg/ml) or whole Vn (2 µg/ml) before lysis and immunoprecipitation with the anti ß3 antibody raised to the cytoplasmic tail of ß3 (c-tail). The extent of ß3 phosphorylation was determined by immunoblotting with an antiphosphotyrosine antibody (p-Tyr). To control for differences in protein levels equal amounts of total protein from each sample were immunoblotted directly with the anti ß3 antibody (c-tail). ***, P < 0.005 when phosphorylation in the presence of either Vn HBD or Vn is compared with phosphorylation in their absence. C, Cells were treated as above except for a pretreatment with either the C-loop ß3 antibody or a control antibody (4 h at 37 C; 1 µg/ml) before treatment with the Vn HBD (10 µg/ml for 1 h at 37 C). ***, P < 0.005 when phosphorylation in the presence of the C-loop antibody is compared with phosphorylation in its absence. IB, Immunoblotting.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Deletion of the HBD of Vn Blocks the Ability of Vn to Enhance IGF-I Signaling A, Migration: Cells were grown to confluency in six-well dishes before treatment with either serum purified Vn (Wt), recombinant wild-type Vn (rWt), HBD deletion mutant Vn (HBD) or the RGD-RGE mutant Vn (RGE), all at a concentration of 2 µg/ml, and IGF-I (100 ng/ml) for 48 h. The number of cells migrating past the wound line into at least 10 predetermined 1-mm2 areas was then counted. The results shown are the mean ± SEM of three independent experiments. *, P < 0.05 when the number of cells migrating in response to IGF-I in the presence of Vn is compared with the number of cells migrating in SFM alone. B, Proliferation: 2 x 104 cells were plated in each well of a 24-well plate before treatment with either WtVn, rWT, VnHBD, or the RGD-RGE mutant Vn (Vn RGE) all at a concentration of 2 µg/ml and IGF-I (100 ng/ml). After 48 h, cell number was determined by trypan blue staining and counting. The results shown are the mean from three independent experiments. *, P < 0.05 when the increase in cell number in response to IGF-I in the presence of Vn is compared with cell number in the presence of IGF-I alone.

Vn HBD Enhancement of IGF-I-Stimulated Effects Requires Binding to the Cysteine Loop Region of ß3
Having determined that the HBD of Vn bound to the cysteine loop region of ß3 and that it could enhance IGF-I signaling in a manner similar to that seen with whole Vn, we next wanted to determine whether its effects were mediated through the interaction with the C-loop region of the ß3 subunit. To this end, we compared the effects of the HBD peptide in the presence or absence of the C-loop ß3 antibody. In Fig. 4, it can be seen that the positive effects of the HBD peptide on both cell migration and proliferation were blocked in the presence of the C-loop antibody but not in the presence of control IgG.

View larger version (16K): [in this window] [in a new window]   Fig. 4. The Positive Effect of the Vn HBD Is Blocked in the Presence of the C-Loop ß3 Antibody A, Cells were grown to confluency in six-well dishes before treatment with or without the C-loop ß3 (1 µg/ml) or control antibody (Con IgG) then the HBD of Vn at a concentration of 10 µg/ml and IGF-I (100 ng/ml) for 48 h. The number of cells migrating past the wound line into at least 10 predetermined 1-mm2 areas was then counted. The results shown are the mean ± SEM of five independent experiments. **, P < 0.01 when the number of cells migrating in response to IGF-I in the presence of Vn HBD is compared with the number of cells migrating in response to IGF-I alone. B, Cells (2 x 104) were plated in each well of a 24-well plate before exposure to Vn HBD (10 µg/ml), C-loop ß3 (1 µg/ml), or the same concentration of control antibody (Con IgG) and IGF-I (50 ng/ml) in DMEM-H + 0.2% PPP. Forty-eight hours after the addition of IGF-I (50 ng/ml), cell number was determined by trypan blue staining and counting. The results shown are the mean ± SEM of three independent experiments. **, P < 0.01 when the increase in cell number in response to IGF-I in the presence of Vn HBD is compared with cell number in the presence of IGF-I alone.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Vn HBD Enhances IGF-I-Stimulated Shc Phosphorylation and MAPK Activation Quiescent pSMCs were treated with or without either the Vn HBD peptide (10 µg/ml) or whole Vn (2 µg/ml) for 1 h before exposure to IGF-I (100 ng/ml) for the times indicated. A and B, The extent of Shc phosphorylation was determined by immunoprecipitating (IP) cell lysates with an anti-Shc antibody, then immunoblotting (IB) with an antiphosphotyrosine antibody (p-Tyr). As a control, aliquots of cell lysate were removed before immunoprecipitation and immunoblotted directly with an anti-Shc antibody. The upper panel shows the results of three similar experiments expressed fold increase in phosphorylation of Shc compared with basal. *, P < 0.05 when phosphorylation in the presence of either Vn HBD or Vn plus IGF-I is compared with phosphorylation in the presence of IGF-I alone. C and D, The ability of IGF-I (100 ng/ml) to activate MAPK in quiescent pSMCs, as measured by the extent of phosphorylation of ERK1/2 (pERK1/2) is shown. After treatment, cells were lysed, then clarified lysates were separated by SDS-PAGE and ERK1/2 visualized using a phosphospecific antibody. To control for loading equivalent amounts of lysate were analyzed simultaneously but immunoblotting for total ERK1/2 protein. The upper panel shows the results of three similar experiments expressed as the fold increase in phosphorylation of ERK1/2 over basal. *, P < 0.05 when phosphorylation in the presence of Vn HBD or Vn plus IGF-I is compared with phosphorylation in the presence of IGF-I alone.

Effect of the C-Loop ß3 Antibody on the Ability of the Vn HBD to Enhance IGF-I Signaling via Its Ability to Enhance Shc Phosphorylation and MAPK Activation
Because we had demonstrated that the positive effects of Vn HBD on IGF-I-stimulated migration and proliferation required Vn binding to the C-loop region of ß3, we next determined whether the effects of the Vn HBD Shc phosphorylation and MAPK activation were also mediated through its interaction with ß3. When with the C-loop ß3 antibody was added before the addition of Vn HBD and IGF-I after a 10-min stimulation with IGF-I, Shc phosphorylation was reduced by 87.3 ± 0.5% (mean ± SEM; n = 3) compared with cultures that were treated with a control IgG (Fig. 6B). In the presence of the Vn HBD after a 10-min exposure to IGF-I, phosphorylation of ERK1/2 was reduced to 43.5 ± 2.5% (mean ± SEM; n = 3) compared with ERK 1/2 phosphorylation in the presence of the control IgG (Fig. 6C).

View larger version (15K): [in this window] [in a new window]   Fig. 6. C-Loop ß3 Antibody Blocks the Effect of the Vn HBD on IGF-I-Stimulated Shc Phosphorylation and MAPK Activation A and B, The effect of the C-loop antibody on Shc phosphorylation and MAPK activation in the presence of the Vn HBD and IGF-I was determined as for Fig. 5, except that treatment with the Vn HBD was preceded by treatment with either control IgG or the C-loop Ab (1 µg/ml for 4 h at 37 C). The upper panels show the results expressed as percent phosphorylation in the presence of the antibody compared with phosphorylation in the absence of the antibody and are derived from scanning units obtained from three independent experiments. **, P < 0.01 when phosphorylation in the presence of the C-loop ß3 antibody is compared with phosphorylation in the presence of the control IgG.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

The HBD of Vn has been shown to bind various cell surface binding sites including the Vß5 integrin (23) and syndecans (24), thus raising the question as to whether the effects of the HBD of Vn on IGF-I action are mediated solely through Vß3. pSMCs do not express a significant amount of Vß5; therefore, because our previous studies had shown that ligand occupancy of Vß3 regulated IGF-I signaling, it seemed highly likely that the effect of the HBD peptide was mediated through Vß3. This conclusion is supported by the data in which we show that both binding and the physiological effect of the HBD peptide are blocked with the anti-Vß3 antibody prepared against cysteine loop region of ß3. Therefore, our results extend previous studies by demonstrating that, in addition to binding to cell surface proteins including Vß5, urokinase-type plasminogen activator receptor, and syndecans, the HBD of Vn binds to the cysteine loop region of Vß3 and regulates IGF-IR mediated signaling via its interaction with this site.

Takagi et al. (25) reported that the short cysteine loop between residues 177 and 184 of ß3 conferred ß3 ligand specificity. When they substituted this sequence for the comparable sequence in ß1, the Vß1 mutant showed a marked increase in its affinity for Vß3 ligands (e.g. fibrinogen, von Willebrand factor, and Vn). Conversely, when they swapped the ß1 sequence for the ß3 sequence in Vß3, Vß3 had greatly reduced affinity for these ligands. They proposed that this cysteine loop was critical for integrin ligand specificity. Our recent studies demonstrate that ligand binding to this region is required for the positive effect of Vn on IGF-I signaling (16). Our antibody, targeted against the cysteine loop sequence, which does not occur in any other cell surface expressed proteins, and which binds specifically to ß3 (16), inhibited both HBD peptide binding and the ability of both the peptide and intact Vn to enhance multiple IGF-I actions. This supports our conclusion that the HBD of Vn binds to this specific region of ß3 and that the interaction between the HBD of Vn and the cysteine loop is responsible for the positive effect of Vn on IGF-I signaling.

The addition of both intact Vn and the HBD of Vn enhanced ß3 phosphorylation, and this is consistent with our recent studies that showed blocking ligand binding to this region blocked ß3 phosphorylation. The effect of the HBD and whole Vn on ß3 phosphorylation is likely to be due to a conformational change induced after binding to the C-loop region (26). It seems likely that the IGFBP-5 K208A mutant peptide that prevented Vn HBD from binding to the cell surface but did not induce ß3 phosphorylation was unable to induce the conformational change despite its apparent ability to bind to ß3.

Exactly how binding of the HBD of Vn to the 177–184 region of ß3 regulates IGF-I signaling remains to be determined. Vn HBD enhanced the IGF-I-stimulated Shc phosphorylation response, a response that is required for optimal IGF-IR signaling (21). We showed recently that blocking Vn binding to the C-loop region of ß3 impaired IGF-I signaling. Because stimulation of ß3 phosphorylation leads to enhanced Shc recruitment to SHP-substrate-1 (21), we hypothesize that this is at least one of the mechanism by which Vn HBD binding to Vß3 leads to enhancement of IGF-I actions.

Our conclusion that the HBD of Vn is sufficient for the optimal response to IGF-I does not exclude a role for activation of the Vß3 after binding to the RGD sequence within Vn to ß3 during attachment. The results show, however, that no additional interaction between the RGD sequence of Vn and Vß3 is required for Vn to enhance IGF-I signaling in stably attached cells. This conclusion is supported by the data showing that the addition of the mutant form of Vn in which the RGD sequence had been modified to RGE was equally potent in enhancing IGF-I-stimulated migration and receptor phosphorylation as compared with wild-type Vn. However, a basal level of continuously synthesized Vn would still be bound to Vß3 through the RGD domain. In other studies, we have shown that small peptides containing the RGD domain or peptide mimetics that bind to the same site on ß3 do not enhance IGF-I signaling or actions (27). Therefore, although it is unlikely that binding through this domain contributes to the ability of added native Vn to enhance IGF-I actions, we cannot exclude the possibility that Vn in the ECM to which the SMCs originally attach plays a role in IGF-I-stimulated effects. Therefore, the findings support the conclusion that the HBD of Vn is the predominant determinant of the effects of Vn on IGF-I actions. Other Vß3 ligands, including thrombospondin and osteopontin, also contain HBD sequences, and because we have shown previously that these proteins, like Vn, can enhance IGF-I signaling (3), it is possible that their positive effect is also mediated through interaction with Vß3 with their HBDs.

By identifying a specific sequence within Vn and its binding site on ß3, the results of our study expand our understanding of the molecular nature of integrin-growth factor receptor cross-talk. Furthermore studies that focus specifically on the molecular events triggered by binding of the HBD of Vn and how they impinge on IGF-IR-mediated signaling events will further enhance our understanding of the relationship between these two signaling pathways.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Human IGF-I was a gift from Genentech (South San Francisco, CA); Polyvinyl difluoride membranes (Immobilon P) were purchased from Millipore Corp. (Billerica, MA). Autoradiographic film was obtained from Pierce (Rockford, IL). Fetal bovine serum, DMEM, penicillin, and streptomycin were purchased from Life Technologies (Grand Island, NY). The IGF-IR ß chain polyclonal and the monoclonal phosphotyrosine antibody (PY99) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Vß3 monoclonal antibody, LM609, was purchased from Chemicon International (Temecula, CA). The ß3 polyclonal antibody was prepared by injecting rabbits with two peptides, one containing amino acids 60–85 and the other 653–680 of porcine ß3 sequence that had been conjugated to KLH. The ß3 antibody specific for the cytoplasmic tail of ß3 prepared by injecting rabbits with a peptide containing amino acids 742–762 of ß3 (c-tail ß3) that had been conjugated to keyhole limpet hemocyanin (KLH). The Shc, ERK1/2 antibodies were purchased from BD Transduction Laboratories (Lexington, KY). The method to prepare the Vn antibody was described previously (3). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.

Porcine SMCs (pSMCs) were isolated and maintained as previously described (28).

Synthetic Peptide Synthesis
A synthetic peptide corresponding to the HBD (amino acids 365–381) of human Vn (29) (LAKKQRFRHRNRKGYRS), a peptide corresponding to the HBD of human IGFBP-5 (²⁰¹RKGFYKRKQCKPS²¹³) and a peptide corresponding to the HBD of IGFBP-5 but with a one amino acid substitution (K²⁰⁸-A) (RKGFYKRAQCKPS) were synthesized by the peptide synthesis facility at UNC School of Medicine. The peptides were purified by HPLC and that the sequences were correct was verified by mass spectrometry.

Generation and Purification of the Antibody against HBD of ß3 (C-Loop ß3 Antibody)
An antibody against the amino acid sequence (¹⁷⁷CYDMKTTC¹⁸⁴) of human ß3 was generated as we have previously described (16). Briefly, a KLH-peptide (CYDMKTTC) conjugate was used to immunize a rabbit. The antibody fraction was purified from rabbit serum by protein G Sepharose affinity chromatography. The material eluting from the protein G column was then purified over an affinity column prepared by coupling the synthetic peptide to Sepharose according to the manufacturer’s instructions (Pierce). The control Ig was prepared by protein G affinity chromatography of nonimmune rabbit serum.

Generation of Recombinant Wild-Type and Mutant Forms of Vn
RT-PCR was used to generate the WtVn cDNA from mRNA prepared from porcine SMCs (30). The 5' primer sequence was identical with bases 75–96 of porcine Vn and the 3' sequence was complimentary to nucleotides 1430–1452. The PCR product was cloned into a pcDNA 3.1 vector. To generate a form of Vn in which the HBD had been deleted (VnHBD), i.e. deletion of amino acids 342–358 (this porcine sequence encompasses almost the entire human HBD Vn sequence between amino acids 365–381 contained in the HBD peptide and contains the amino acid sequence QPKMTKSARRSGKRYRS), a single primer that corresponded to the sequence immediately before and after the deletion site was used (bases 1062–1076 and 1120–1134) to perform double stranded mutagenesis using the full-length WtVn in pcDNA 3.1 vector as template (Invitrogen, Carlsbad CA). Vn WT and VnHBD cDNA were cloned from the pcDNA 3.1 vector in to a Gateway entry vector pENTR/D-TOPO (Invitrogen) and then into the insect expression vector pIB/V5-HisDEST using LR clonase according to the manufacturer’s instructions (Invitrogen).

The RGD⁶⁶E mutant pVn (the position of the RGD sequence is the same in both human and porcine Vn sequences) was generated from the WtVN pENTR/D-TOPO template. Two overlapping DNA fragments were generated. The first encoded bases 75–293 (i.e. amino acids 1–73) and the second encoded bases 255-1452 (amino acids 61–459). The 5' primer for the first fragment was 5'-caccATGGCACCCCTGAGGCCCCTTCT-3' (primer 1; the same 5' primer used to amplify wild-type Vn DNA) and the 3' primer sequence was 5'-ATCATCTGGCTGAAGGAATACtTCCCGCGAGTCACTGG-3' (primer 2). The D66E change was encoded by the lowercase base in the underlined codon. Primers 1 and 2 amplified a 219-bp fragment encoding the first 73 amino acids of pVN including the D66E. The primers used to generate the second fragment amplified a 1198-bp fragment encoding amino acids 61–459. The 5' primer used to synthesize the second fragment corresponded to nucleotides 255–293, i.e. the same sequence as the 3' fragment of the first fragment. The sequence used was 5'-CAAGTGACTCGCGGGGaaGTATTCCTTCAGCCAGATGAT-3' (primer 3); again, the D66E change was encoded by the lowercase base in the underlined codon. The 3' primer for the second fragment encoded nucleotides 1430–1452 (i.e. the same as the 3' primer to synthesize wild-type Vn) 5'-CTTCTGGTCTGGAACTGGGCAGC-3' (primer 4). The gel-purified fragments were combined and annealed to each other to form a full-length template containing the D66E substitution. This template was amplified in a second PCR using primers 1 (5') and 4 (3') to produce a full-length product. The PCR product was cloned into the pENTR/D-TOPO entry vector and transferred to the pIB/V5-HisDEST vector as described above. The protein expressed from this construct is referred to as RGE mutant.

Sf9 cells were transfected with each construct according to the manufacturer’s instructions (Invitrogen). Selected cells were pooled then transferred to 250-ml flasks at a density of 5 x 10⁵/ml. Flasks were incubated at room temperature with shaking until a density of 5–6 x 10⁶ /ml was reached. Medium was harvested by centrifugation and stored at –80 C.

Purification of Recombinant Vn
Conditioned medium was passed over a diethylaminoethyl Sepharose Fast Flow column (Amersham Biosciences, Piscataway, NJ) with 50 mM Tris (pH 7.6), containing 25 mM NaCl at 4 C. The proteins adhering to the column were eluted using increasing concentrations of NaCl (from 250 mM to 1 M). His tagged Vn from the Vn-positive fractions (as determined by immunoblotting) was purified using Ni-NTA Resin (Invitrogen) overnight at 4 C according to the manufacturer’s protocol. The Vn eluate was concentrated and simultaneously exchanged into a 25 mM Tris (pH 7.2) buffer containing 250 mM NaCl using centrifugal filter devices (Millipore Corp.).

Purity of the Vn preparations was determined by silver staining after SDS-PAGE using the PhastGel Silver kit (Amersham Biosciences) according to the manufacturer’s instructions. Quantification of each preparation was made using a BCA protein assay (Pierce) using dilutions of a known amount of Vn previously purified from porcine serum (3) as a standard. Because folding of the protein may affect its activity, we analyzed all of the recombinant Vn preps by SDS-PAGE. Polymerization of Vn is believed to be necessary for its biological activity, and polymerization of Vn can be detected by the apparent aggregation of Vn when it is separated by nonreducing SDS-PAGE (31). These aggregates are lost when separated under reducing conditions. When equal amounts of the three preparations of Vn were analyzed in this manner, there was an equal percent in each that appeared to be polymerized (data not shown).

Cell Migration
Wounding of wild-type pSMC was performed as previously described (2). The wounded monolayers were treated with Vn HBD peptide (10 µg/ml), intact Vn (2 µg/ml) or the C-loop ß3 antibody (1 µg/ml) or control IgG (1 µg/ml) and then exposed to 100 ng/ml IGF-I for 48 h at 37 C. The cells were fixed and stained (Diff Quick, Dade Behring, Inc., Newark, DE) and the number of cells migrating into the wound area was counted. At least five of the previously selected 1-mM areas at the edge of the wound were counted for each data point.

Cell Proliferation
Cells were plated at 2 x 10⁴ cells per well in each well of a 24-well plate in DMEM-H + 0.2% FBS. Cells were left to attach overnight before the medium was replaced with DMEM-H. Twenty-four hours later, treatments were added (as for the migration assay), and cells were incubated for a further 48 h. Cell number was determined after trypsinization, trypan blue staining, and counting.

Cell Lysis, Immunoprecipitation, and Western Immunoblotting
Cells were grown to confluency and then made quiescent as described previously (32). Cells were treated as appropriate: Vn HBD (10 µg/ml) or intact Vn (2µg/ml) for 1 h; C-loop ß3 antibody (1 µg/ml) or control IgG (1 µg/ml) for 4 h followed by IGF-I (100 ng/ml) for times as indicated. Cell monolayers were lyzed in 1.5 ml of ice-cold lysis buffer: 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM EGTA plus 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1 µg/ml aprotinin (radioimmunoprecipitation assay buffer). The clarified lysates were then used for immunoprecipitation as described previously (32) or 30-µl aliquots of each lysate were removed and proteins were separated by SDS-PAGE and then visualized by Western immunoblotting with appropriate antibody [ß3 (C-loop or c-tail), IGF-IR, PY99, pERK1/2, ERK, Shc using a 1:500 dilution]. To visualize phosphorylation of ERK1/2, as a marker of MAPK activation, the radioimmunoprecipitation assay buffer-insoluble pellet representing nuclear and cytoskeletal material was solubilized in 45 µl of reducing Laemmelli sample buffer. After boiling and centrifugation, the proteins were separated by SDS-PAGE and phosphorylation of ERK1/2 was visualized by Western immunoblotting.

Characterization of the Vn HBD Peptide Cell Surface Binding
Vn HBD peptide was labeled with ¹²⁵I using a Chloramine-T iodination method. The specific activity of the preparation was 31.2 µCi/µg. Wild-type pSMCs were plated at 5 x 10⁵ in each well of a 24-well plate and grown to 100% confluency. The characteristics of Vn HBD peptide cell surface binding were analyzed as follows:

Equilibrium/Time Dependency.
Confluent monolayers were then incubated with a range of radiolabeled peptide concentrations (0.4–20 nM or 10,000–500,000 cpm/well) in 0.5 ml of SFM plus 0.1% BSA and 50 mM HEPES (pH 7.4) at 4 C. After incubation, the monolayers were washed twice with PBS then cells were solublized in 500 µl of NaOH. The solubilized cell suspension was then counted. Equilibrium was reached after a 1-h incubation and sustained for at least 6 h over the range of peptide concentrations used (data not shown).

Saturation of Specific Cell Surface Binding Sites.
Cells were incubated with a range of radiolabeled peptide concentrations (0.4–20 nM or 10,000–500,000 cpm/well) for 1 h, and saturation was achieved with 7.8 nM of peptide (data not shown).

Specificity.
Cells prepared as described above were incubated with excess (1, 5, and 10 µM) Vn HBD peptide for 1 h (5% of counts added were recovered from the cell surface) and also an irrelevant peptide that had no sequence homology to the Vn HBD peptide (also at 10 µM) before addition of the radiolabeled Vn HBD peptide (at 4 nM = 50% saturation). Under these conditions, there was no significant change in the amount of radiolabeled peptide that was bound when the irrelevant peptide was used, whereas binding of the radiolabeled peptide was reduced by 97% in the presence of 10 µM concentration of the unlabeled Vn HBD peptide (data not shown).

To determine whether the Vn HBD peptide was binding to the cysteine loop region of ß3, pSMCs were grown to confluency as described above. Cells were then incubated with increasing concentrations of the C-loop ß3 antibody (3 ng to 3 µg/ml) or an irrelevant control IgG before the addition of ¹²⁵I-labeled Vn HBD.

Prepartion of the Vn HBD Affinity Column
The Vn HBD peptide was cross-linked to agarose using SulfoLink Coupling Gel (Pierce). Briefly, 0.7 mg of peptide in 1 ml of coupling buffer [50 mM Tris (pH 8.5) containing 5 mM EDTA] was added to coupling gel and incubated at room temperature for 30 min. This was repeated with 0.8, 2.0, and then 3.2 mg of peptide added to the same gel. After 3 h, the gel was gently spun and 50 mM of cysteine in coupling buffer added to the coupling gel for 1 h to block unreacted sites.

Biotinylation of Cell Surface Proteins
Confluent monolayers of cells were incubated with 0.5 mg/ml EZ-link sulfo NHS SS biotin (Pierce) at 4 C for 30 min before lysis, immunoprecipitation, and immunoblotting as described above.

Affinity Purification of Cell Surface Proteins that Bind to the Vn HBD Peptide
Both biotinylated or nonbiotinylated lysates were passed over the Vn HBD affinity column that had been preequilibrated in 50 mM Tris, 50 mM NaCl (pH 7.2). The lysate was recirculated over the column for 24 h, and then the column washed with 10 vol of 50 mM Tris (pH 7.2). The proteins that had adhered to the Vn HBD column were eluted with 0.1 M glycine and immediately neutralized with 1 M Tris (pH 9.0). The starting material, flow through, and eluted proteins were then separated by SDS-PAGE (8%) and then detected with either avidin HRP or Western immunoblotting with an anti-ß3 antibody and enhanced chemiluminescence as described above.

Statistical Analysis
Chemiluminescent images obtained were scanned using a DuoScan T1200 (AGFA Brussels, Belgium), and band intensities of the scanned images were analyzed using the National Institutes of Health Image program, version 1.61. The Student’s t test was used to compare differences between treatments. The results that are shown in all experiments are representative of at least three separate experiments.

	ACKNOWLEDGMENTS

 
We thank Aleksandra Tenenbaum for technical assistance.

	FOOTNOTES

 
This work was supported by National Institutes of Health Grant AG02331 (to D.R.C.) and an American Heart Association Mid Atlantic Affiliate Beginning Grant in Aid (0465462U) (to L.A.M.).

Author Disclosure Summary: L.A.M, W.H.B., K.S., T.S., J.C. and Y.L. have nothing to declare. D.R.C received consulting and lecture fees from Pfizer and Consulting Fees from Lilly.

First Published Online December 1, 2005

Abbreviations: HBD, Heparin binding domain; HRP, horseradish peroxidase; IGFBP, IGF binding protein; IGF-IR, IGF-I receptor; KLH, keyhole limpet hemocyanin; ns, not significant; pSMC, porcine SMC; RGD, Arg-Gly-Asp; RGE, Arg-Gly-Glu; SFM, serum-free medium; Shc, Src homology 2 domain-containing transforming protein C; SMC, smooth muscle cell; Vn, vitronectin.

Received for publication September 16, 2005. Accepted for publication November 22, 2005.

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