| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Vß3
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 INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Vß3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-Vß3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177–184 (177CYDMKTTC184) within the extracellular domain of ß3 have been proposed to confer the ligand specificity of Vß3; therefore, we hypothesized that ligand binding to the 177–184 cysteine loop of ß3 may be an important regulator of the cross talk between Vß3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the ß3 subunit of Vß3 (i.e. amino acids 177–184) blocked Vn binding to the ß3 subunit of Vß3 and correspondingly ß3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of ß3 in which three critical residues within the 177–184 sequence were altered ß3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177–184 sequence of ß3 is necessary for Vn binding to Vß3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The role of integrins goes beyond mediating cellular attachment. Most normal cells are anchorage dependent for growth, i.e. they do not respond to mitogens unless they are stably attached via integrin-ECM interactions. The response of smooth muscle cells (SMCs) to IGF-I requires ligand occupancy of Vß3 (3, 4). Increasing ligand occupancy of Vß3 by the addition of soluble forms of ECM proteins such as vitronectin (Vn) enhances IGF-I actions (3). Blocking Vß3 ligand occupancy with the RGD containing disintegrin, echistatin, or the Vß3 monoclonal antibody, LM609, blocks IGF-I signaling without affecting cell attachment (3).
An RGD peptide has been shown to bind to a region of Vß3 contained within amino acids 109–171 (5). Because the RGD containing peptide echistatin inhibits IGF-I actions (3, 4), this suggests that the interaction of ligands with this site alone is not sufficient to mediate the positive effect of Vn or other Vß3 ligands on IGF-I signaling. The amino acids 177–184 of the extracellular domain of the ß3 subunit, which form a cysteine loop (177CYDMKTTC184), have been shown to confer ligand specificity to Vß3 (6). When this sequence was substituted using the corresponding sequence from the ß1 subunit, Vß3 lost its ability to bind Vn. Correspondingly, when amino acids 177–184 of ß3 were inserted into ß1, the ß1 integrin acquired Vn binding capacity (7). The functional significance of this sequence is further suggested by the fact that an llbß3 function blocking monoclonal antibody (7E3) that inhibits fibrinogen enhanced platelet aggregation has been shown to bind to the 177–184 cysteine loop (7). Although the significance of this site has been suggested by these previous studies, to our knowledge there are no studies that specifically address the functional role of this region of the ß3 subunit in the cross talk between Vß3 and growth factor signaling pathways. We hypothesized that ligand binding to this region may play an important role in the ability of Vß3 to regulate IGF-I signaling in SMCs.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
, the antibody raised against the C terminus of ß3 (C-tail) immunoprecipitated two bands that had electrophoretic mobilities that corresponded to ß3 (97 and 103 kDa). The C-loop ß3 antibody immunoprecipitated the same two bands. ß3 migrates as two different size bands, when separated by nonreducing SDS-PAGE, depending upon its conformation (8), and this accounts for the two distinct bands detected by each of the two ß3 antibodies as shown in Fig. 1A. All of the other bands that were detected were also present when the control IgG was used for immunoprecipitation and therefore are either nonspecific or lower molecular weight fragments of ß3 that precipitate nonspecifically. There were no other proteins that were specifically immunoprecipitated with either of the two antibodies. To further determine its ability to bind ß3, we immunoprecipitated cell lysate with the C-tail ß3 antibody and then immunoblotted the resulting precipitate with the C-loop ß3 antibody. Again, two distinct bands corresponding to ß3 were detected (Fig. 1B).
|
, it can be seen that the C-loop ß3 antibody specifically recognized a band that had a relative molecular mass that corresponded to that of ß3, whereas no band was detected if the antibody had been preincubated with the peptide immunogen.
C-Loop ß3 Antibody Has a Direct Effect on Vß3 Ligand Binding and ß3 Activation
To determine whether the anti-177–184 cysteine loop antibody could inhibit Vn binding to ß3 we incubated confluent monolayers of porcine SMCs (pSMCs) with the C-loop antibody before immunoprecipitating with the C-tail ß3 antibody and immunoblotting with an antibody that recognizes Vn. After ß3 immunoprecipitation with the C-tail ß3 antibody, Vn associated with ß3 was detected (Fig. 2A). However, when cells were preincubated with the C-loop ß3 antibody there was a significant reduction in the amount of Vn associated with ß3 compared with cells treated with control IgG (a 52 ± 12% reduction, mean ± SEM, n = 3, P < 0.05). Because the cells were grown for 7 d in medium containing fetal bovine serum (FBS) before the initiation of the experiment, it seems reasonable to assume that this is the source of the Vn, although there is likely to be some contribution from Vn that is secreted from the cells.
|
Vß3 with the C-loop ß3 antibody on ß3 phosphorylation (8, 9). In Fig. 2B, it can be seen that ß3 phosphorylation was significantly reduced in the presence of the C-loop antibody compared with cells treated with the control antibody (an 85 ± 15% reduction, mean ± SEM, n = 3, P < 0.05). Of the two bands of ß3 that are detected by immunoblotting, the band with the lower apparent molecular weight is the one that is phosphorylated. It is possible that either the upper band is in a slightly different conformation and therefore cannot be detected by the phosphotyrosine antibody or that this form of ß3 is not phosphorylated.
Blocking ß3 Ligand Occupancy with the C-Loop Antibody Inhibits IGF-I Receptor-Mediated Signaling
We have shown previously that ß3 phosphorylation is required for both Shc phosphorylation and MAPK activation response to IGF-I and that the phosphorylation of Shc was required for MAPK activation (10). In our previous studies, we determined that IGF-I predominantly stimulated an increase in p52 Shc (8, 10). Therefore, we next examined the effect of the C-loop ß3 antibody on IGF-I-stimulated Shc phosphorylation and MAPK activation. It can be seen in Fig. 3A that the ability of IGF-I to stimulate p52 Shc phosphorylation was significantly impaired in the presence of the C-loop ß3 antibody compared with cultures exposed to a control antibody. Specifically, IGF-I-stimulated p52 Shc phosphorylation was reduced by 83 ± 18% after both 10 and 20 min stimulation with IGF-I compared with cells treated with a control antibody (mean ± SEM, n = 3, P < 0.05). Similarly, the ability of IGF-I to activate ERK1/2 was also impaired (Fig. 3B). In the presence of the C-loop antibody, IGF-I-stimulated ERK1/2 phosphorylation was reduced by 82 ± 15 and 56 ± 14% after 10 and 20 min treatment with IGF-I, respectively, compared with cells treated with control IgG (mean ± SEM, n = 3, P < 0.05). In contrast, the C-loop ß3 antibody had no effect on IGF-I-stimulated receptor phosphorylation and therefore its inhibitory effects on Shc phosphorylation and MAPK activation is not due to reduced receptor activation (Fig. 3C).
|
, it can be seen that IGF-I-stimulated migration of pSMCs was completely inhibited in the presence of the C-loop ß3 antibody; importantly, there were no significant effects on cell shape or attachment (data not shown). Furthermore, the ability of IGF-I to stimulate cell proliferation was also completely inhibited by the C-loop ß3 antibody (Fig. 4B).
|
(upper panel), the expression level of flag-tagged wild-type ß3 and the ß3NNA mutant protein in pSMCs that had been transduced with the respective constructs can be seen. The total level of ß3 is seen in the lower panel of Fig. 5A and shows that the total amount of ß3 is the same between the two cell lines.
|
(upper panel), it can be seen that, after immunoprecipitation with an antiphosphotyrosine antibody and immunoblotting with an anti-FLAG antibody, the robust ß3 phosphorylation that is detected in cells expressing wild-type ß3 was reduced by 97 ± 0.8% when the ß3NNA mutant cells were analyzed (P < 0.05 when ß3 phosphorylation in the ß3 WT cells is compared with ß3 phosphorylation in the ß3NNA cells). When the total ß3 phosphorylation was examined by immunoprecipitating with an anti-ß3 antibody followed by immunoblotting with an antiphosphotyrosine antibody, it can be seen that there was no phosphorylation of ß3 detected in cells expressing the ß3NNA construct. This suggested that the ß3NNA is acting in a dominant-negative manner. Consistent with our previous reports the lack of ß3 phosphorylation was associated with a complete inhibition in the ability of IGF-I to stimulate an increase in Shc phosphorylation (Fig. 5D). Furthermore, when cells expressing the mutant form of ß3 were analyzed there was no increase in cell proliferation in response to IGF-I (Fig. 5E). Cells expressing wild-type ß3 responded to IGF-I with a 2.1 ± 0.08-fold increase in cell number in contrast to the 1.1 ± 0.07-fold increase in cells expressing ß3NNA (mean ± SEM, n = 3, P < 0.005 when the response of ß3 WT cells is compared with ß3NNA cells). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Vß3 and is reflected by an increase in cell migration and proliferation, as well as enhancement of phosphorylation of IGF-IR and downstream signaling elements. Our results show that binding of Vn to the region of the ß3 subunit (i.e. amino acids 177–184) that has been proposed previously to confer ligand specificity is essential for SMCs to respond optimally to IGF-I. We demonstrated this in two ways: first, by showing that blocking ligand occupancy of this site with an anti-ß3 antibody inhibited IGF-I signaling, and by showing that mutation of residues within this region of ß3, which disrupts ligand binding was also associated with impaired IGF-I signaling.
Takagi et al. (6) reported that the 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 comparable region within the ß1 sequence for the ß3 sequence in Vß3, Vß3 had greatly reduced affinity for these ligands. Our results would suggest that, in addition to regulating the specificity of ligand binding to ß3, binding of ligands to this region is also required Vß3 for mediated actions that regulate IGF-I signaling and responsiveness. Elucidation of the region of Vn that interacts with this site will be essential for fully understanding the role of ligand binding to this region of ß3.
Our previous studies have shown that ligand occupancy of Vß3 by Vn or ECM proteins is required for optimal IGF-I stimulation of cellular responses such as cell division and migration. These studies suggested that the regulation of ß3 phosphorylation is one component of the mechanism by which Vß3 ligand occupancy regulates IGF-I signaling (8). Specifically, we have shown that ß3 phosphorylation is necessary to generate a specific binding site for DOK-1 (downstream of kinase-1), which in turn recruits the tyrosine phosphatase SHP-2 to the cell membrane (9). This recruitment of SHP-2 to the cell membrane is necessary for its subsequent transfer to SHP substrate-1 (SHPS-1), which occurs in response to SHPS-1 phosphorylation after activation of the IGF-I receptor kinase (8, 10). The transfer of SHP-2 is necessary for the recruitment of Shc to SHPS-1 and for the subsequent phosphorylation of Shc (10). The formation of the SHPS-1-SHP-2-Shc complex results in sustained Shc phosphorylation and MAPK activation and in an enhanced mitogenic response to IGF-I (10, 11). Our results from this present study would suggest that ligand binding of Vn specifically to the 177–184 region of ß3 contributes to this signaling pathway by enhancing ß3 phosphorylation the most proximal step in the regulation of IGF-I signaling by Vß3 ligand occupancy. This conclusion is supported by our findings that the antibody against this region as well as mutation of three residues within this region inhibited both ß3 and Shc phosphorylation as well as cell proliferation in response to IGF-I. These findings further our prior published observations (12, 14) that optimal stimulation of Shc and MAPK by IGF-I requires ß3 ligand occupancy and that inhibition of ß3 occupancy will attenuate not only these signaling responses but also the mitogenic and cell migration responses to IGF-I.
In order for Shc to couple growth factor receptors to downstream signaling pathways, it must first be recruited to the cell membrane where it is tyrosine phosphorylated, generating a binding site for the Grb2/SOS complex that then activates components of the MAPK pathway (11). In this study, we show that when ß3 ligand binding is blocked, Shc phosphorylation is impaired despite normal phosphorylation of IGF-IR. This would argue for a role for a kinase distinct from the intrinsic kinase activity of the IGF-IR in the phosphorylation of Shc. We have previously hypothesized that, in addition to the recruitment of Shc and SHP-2 to SHPS-1, the phosphorylation of Shc may also require the recruitment of a tyrosine kinase to SHPS-1 (10). Boney et al. (12) reported that Shc phosphorylation is mediated by a Src family kinase. Therefore, it is possible that ß3 phosphorylation may regulate its activation (13) or recruitment to SHPS-1 and thereby regulate Shc phosphorylation.
Exactly how the binding of Vn to the CYDMKTTC region of ß3 regulates ß3 phosphorylation is as yet unknown; however, we would propose that ligand binding to this extracellular region confers a conformational change in the cytoplasmic domain leading to its phosphorylation. Nonligated integrins on the cell surface are generally considered to be in the inactive state. Ligand binding is associated with both changes in conformation and changes in activation state (14). The conformational changes associated with integrin activation have been documented in a number of ways including studies showing changes in antibody binding specificity (15) and crystallographic studies that demonstrate a requirement for hinge movement between the extracellular and cytoplasmic domains (16). A recent study has implicated ß3 phosphorylation as an important component that links the conformational change with changes in integrin activation. It was shown that ß3 phosphorylation correlated with the extent of integrin activation (17). Adhesion to Vn was completely blocked when the tyrosines in the cytoplasmic tail were mutated to phenylalanine (17). More recently, it was shown that the initial interaction of Vß3 with Vn is a low affinity interaction and that transition to a high-affinity interaction requires ß3 phosphorylation and changes in ß3 interaction with the cytoskeleton (18). Our results would suggest that the binding of Vn to the cysteine loop region of ß3 plays a significant role in activation of the integrin by virtue of its ability to enhance ß3 phosphorylation.
The potential significance of this cysteine loop region of ß3 has been suggested previously by two studies that demonstrated that the monoclonal antibody 7E3 and its humanized derivative, Abciximab, which blocks fibrinogen binding to IIbß3and inhibits platelet activation. This antibody binds to four epitopes on ß3 including the 177CYDMKTTC184 region (7). Artoni et al. (7) proposed that this antibody might work by sterically hindering the ability of the integrin ligand to access the RGD binding site because the crystal structure of Vß3 shows that the 177CYDMKTTC184 region of ß3 is in close proximity to the RGD binding site. However, because the RGD binding site is contained within a distinct region of ß3 between amino acids 109 and 171 (5), our findings raise the possibility that ligand binding to the 177–184 region of ß3 has direct effects not mediated via disruption of binding of the RGD sequence. Although neither of the studies discussed above identified the functional effect of specifically inhibiting ligand binding to the 177–184 sequence alone, the findings that the Abciximab antibody binds to 177CYDMKTTC 184 (as well as three other regions of ß3) and has a distinct effects in vivo (14, 19), supports our conclusion that the interaction between ligands, and their specific binding sites on ß3 is of major significance.
Several studies have demonstrated a direct interaction between growth factor receptors and integrins or between signaling components that are activated in response to ligand occupancy of the two receptor signaling systems. However, the signaling events that occur in response to Vß3 integrin ligand occupancy that act to enhance IGF-I receptor signaling have not been completely characterized. By identifying a specific binding site on ß3 that must be ligand occupied for IGF-I to stimulate downstream signaling events the results of our study expand our understanding of the molecular nature of integrin-growth factor receptor cross talk. Further studies that focus specifically on the proximal events triggered by ligand occupancy of this specific region of ß3 and how they function to regulate proximal signaling events such as ß3 phosphorylation, DOK-1-SHP-2 transfer to ß3 and SHP-2 transfer to SHPS-1 will enhance our understanding of the relationship between these two signaling pathways.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
pSMCs were isolated and maintained as previously described (20)
Generation and Purification of the C177-C184 ß3 Antibody (C-Loop ß3 Antibody)
A peptide comprising 177CYDMKTTC184 was synthesized by the peptide synthesis facility at University of North Carolina (UNC) School of Medicine. The peptide was purified by HPLC and the sequence was verified by mass spectrometry. A total of 4.7 mg of peptide was conjugated sequentially to a total of 2 mg of maleimide-activated Mariculture Keyhole Limpet Hemocyanin (Pierce). After dialysis and lyophilization, intradermal injections of a total of 1.34 mg of the conjugated peptide (dissolved in water and Complete Freunds adjuvant, 1:1) over 15 sites was performed. This was followed by booster sc injections of 1 mg of conjugate dissolved in water and Incomplete Freunds adjuvant (1:1). At monthly intervals, the rabbit was bled. The IgG fraction from a pool of the rabbit antiserum was purified by Protein G Sepharose affinity chromatography. The material that was eluted from the Protein G column was dialyzed and then purified over a peptide affinity column. The affinity column was prepared by coupling the cysteine loop peptide to agarose using Sulfolink Coupling Gel according to the manufacturer’s instructions (Pierce). The Protein G purified IgG fraction was circulated over the peptide affinity column equilibrated in 25 mM sodium phosphate (pH 7.2) containing 50 mM sodium chloride. After sample loading the column was washed with 10-column volumes of loading buffer, and the specific antibody fraction was eluted with 0. 1 M glycine (pH 2.7). The antibody was immediately neutralized with 1 M Tris (pH 9) and stored at –20 C (at a final concentration of 300 µg/ml). Serum from a nonimmunized rabbit was purified using Protein G Sepharose, and it was used as a control at the same final concentrations. Optimum concentrations for immunoprecipitation, immunoblotting, and other experiments were determined empirically (data not shown). A concentration of 1 µg/ml was used for immunoprecipitation, 600 ng/ml was used for immunoblotting and when whole cells were exposed to antibody 1 µg/ml was used.
Generation of Plenti-Expression Vectors
pLenti-FLAG human ß3 (ß3WT) and pLenti-FLAG-ß3/D179A/T182N/T183A (ß3NNA).
Full-length human ß3 cDNA was generated by RT-PCR from mRNA that had been derived from human fibroblasts (GM10, Human Genetic Cell Repository, Camden, NJ) (21). The full-length ß3 sequence was PCR amplified using previously generated pcDNA-ß3 (22) as a template and cloned into the expression vector plenti6/V5-D-TOPO (Invitrogen, Carlsbad, CA). The forward and reverse primers used to generate the PCR product were: 5'-caccatgcgagcgcggccgcggccc-3' and 5'-TTTGTCGTCGTCGTCTTTGTAGTCagtgccccggtacgtgatattggtg-3'. The PCR product containing a Kozak sequence (CACC) at the 5' end of the ß3WT coding sequence and a FLAG (capitalized) sequence at the 3' end was cloned into the plenti6/V5-D-TOPO expression vector. The complete sequence was verified by DNA sequencing.
pLenti-FLAG ß3D179N, T182N, T183A was generated from the human ß3WT template. Human ß3WT cDNA was cloned in to pENTR/D-TOPO Gateway entry vector according to the manufacturer’s instructions (Invitrogen). Wild-type sequences were transferred to plentiCMV Gateway by LR clonase reaction and transformed into Stbl3 competent cells (Invitrogen). The bases encoding: the aspartic acid at position 177 and the two threonine at position 182 and 183 were changed to asparagine (177 and 182), and alanine (183) by double-stranded mutagenesis of pENTRß3WT. A total of 125 ng of complimentary oligonucleotides were annealed to 50 ng supercoiled plasmid and extended by linear PCR amplification using Pfx polymerase (Invitrogen). The oligonucleotide sequences were 5'-ccctcgaaaacccctgctatAatatgaagaAcGcctgcttgcccatgtttgg-3' forward and 5'-ccaaacatgggcaagcaggCgTtcttcatatTatagcaggggttttcgaggg-3' reverse, where the capitalized bases indicate the substitutions. The resulting plasmids were digested by DpnI (NEB, Beverly, MA) and transformed into TOP10 competent cells (Invitrogen). The cells were plated and colonies isolated. Correct incorporation of the changes was confirmed by DNA sequencing (UNC Genome Analysis Facility, Chapel Hill, NC).
Generation of Virus Stocks
Supernatant from 293FT cells (Invitrogen) transduced to generate modified lentivirus encapsulating each of the plenti constructs was generated as we have previously described (10). Briefly, cells, plated at 5 x 106 per 75 cm2 plate (Corning Inc., Corning, NY) the day before transfection were incubated with 5 ml of Opti-MEM-I (Invitrogen) without antibiotics or serum and a DNA-Lipofectamine 2000 complex for each transfection sample that had been prepared according to the manufacturer’s protocol (Invitrogen). The virus-containing supernatants were harvested 48–72 h after transfection and centrifuged at 3000 rpm for 15 min at 4 C to pellet the cell debris. The supernatants were filtered and stored as 1-ml aliquots at –80 C.
Establishment of SMCs Expressing the ß3Wt and pLenti-FLAG ß3D179N, T182N, T183A pLenti-Constructs
pSMCs (passages 4–5) were transduced with viral complexes generated for each as we have described previously (10). Briefly, virus from 1 ml of virus stock were pelleted by incubation with 1 µl of an 80 mg/ml solution of chondroitin sulfate (Sigma), followed by 1 µl of 80 mg/ml hexadimethrine bromide (Sigma). The mixture was centrifuged at 10,000 rpm for 5 min to pellet virus and the supernatant was removed. For transduction, the pellet was resuspended in 1 ml of growth medium and 1 µl of polybrene (40 mg/ml) was added then the mixture was incubated with the cells for 24 h. The virus-containing medium was removed and changed to 2 ml growth medium for another 24 h, then replaced with selection medium (growth medium containing 4 µg/ml blasticidin). The cultures were then grown to confluency.
The expression of FLAG-tagged ß3 protein was detected by immunoblotting with a 1:1000 dilution of an anti-FLAG antibody (Sigma) using 30 µl cell of lysate.
Cell Migration
Wounding of wild-type pSMC and pSMCs that had been transfected to over express the human ß3 constructs (ß3Wt and ß3NNA) was performed as previously described (3). The wounded monolayers were treated with the c-loop ß3 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
Normal and transduced pSMCs 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 + 0.2% platelet poor plasma. Twenty-four hours later, treatments C-loop ß3 IgG, or control IgG and IGF-I were added. The cells were incubated for a further 48 h, and cell number was determined after trypsinization, trypan blue staining and counting.
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 below.
Cell Lysis, Immunoprecipitation, and Western Immunoblotting
Cells were grown to confluency and then made quiescent as described previously (23). Cells were pretreated with either the affinity purified C-loop or control IgG (1 µg/ml for 2 h at 37 C). After appropriate treatments, cell monolayers were lysed 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, 1 µg/ml aprotinin (RIPA). The clarified lysates were then used for immunoprecipitation as described previously (23), 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 (IGF-IR, C-tail ß3, PY99, pERK1/2, ERK, Shc, FLAG, or Vn using a 1:500 dilution or purified C-loop ß3 or control IgG at 600 ng/ml). To visualize phosphorylation of ERK1/2, as a marker of MAPK activation, the RIPA 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.
Statistical Analysis
Chemiluminescent images obtained were scanned using a DuoScan T1200 (AGFA Brussels, Belgium) and band intensities of the scanned images were analyzed using NIH Image, 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.
|
FOOTNOTES |
|---|
First Published Online September 29, 2005
Abbreviations: ECM, Extracellular matrix; FBS, fetal bovine serum; pSMC, porcine SMC; RGD, Arg-Gly-Asp; SHPS-1, SHP substrate-1; SMC, smooth muscle cell; VN, vitronectin.
Received for publication June 20, 2005. Accepted for publication September 21, 2005.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-V-ß3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor. Proc Natl Acad Sci USA 93:2482–2487Vß3 integrin inhibits insulin-like growth factor I signaling in vascular smooth muscle cells. Proc Natl Acad Sci USA 95:11217–11222vß1 and vß3 integrins by swapping a short diverse sequence of the ß subunit. J Biol Chem 272:19794–19800Iibß3 activation. Proc Natl Acad Sci USA 101:13114–13120Vß3 integrin is required for membrane association of the tyrosine phosphatase SHP-2 and its further recruitment to the insulin-like growth factor I receptor. Mol Endocrinol 17:1824–1833Vß3 integrin and thereby regulates insulin-like growth factor I signaling in cultured vascular smooth muscle cells. J Biol Chem 280:3151–3158Vß3 integrin linked signaling pathways: cellular responses to changes in multiple signaling inputs. Mol Endocrinol 19:1–11 IIbß 3 based on electron cryomicroscopy and x-ray crystallography. Proc Natl Acad Sci USA 99:14059–14064vß3 avidity. J Biol Chem 272:28757–28761(v)ß3-vitronectin binding is a multistage process in which increases in bond strength are dependent on Y747 and Y759 in the cytoplasmic domain of ß3. Mol Biol Cell 12:1227–1237 V ß 3 integrin in IGF-I signaling. J Cell Sci 114:1417–1425[Abstract]This article has been cited by other articles:
 |
|
 |
|
  J. Saegusa, S. Yamaji, K. Ieguchi, C.-Y. Wu, K. S. Lam, F.-T. Liu, Y. K. Takada, and Y. Takada The Direct Binding of Insulin-like Growth Factor-1 (IGF-1) to Integrin {alpha}v{beta}3 Is Involved in IGF-1 Signaling J. Biol. Chem., September 4, 2009; 284(36): 24106 - 24114. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Mikelis, E. Sfaelou, M. Koutsioumpa, N. Kieffer, and E. Papadimitriou Integrin {alpha}{nu}{beta}3 is a pleiotrophin receptor required for pleiotrophin-induced endothelial cell migration through receptor protein tyrosine phosphatase {beta}/{zeta} FASEB J, May 1, 2009; 23(5): 1459 - 1469. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. B. Allen, B. E. Capps, E. C. Miller, D. R. Clemmons, and L. A. Maile Glucose-Oxidized Low-Density Lipoproteins Enhance Insulin-Like Growth Factor I-Stimulated Smooth Muscle Cell Proliferation by Inhibiting Integrin-Associated Protein Cleavage Endocrinology, March 1, 2009; 150(3): 1321 - 1329. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R. Legate, S. A. Wickstrom, and R. Fassler Genetic and cell biological analysis of integrin outside-in signaling Genes & Dev., February 15, 2009; 23(4): 397 - 418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Xi, X. Shen, and D. R. Clemmons p66shc Negatively Regulates Insulin-Like Growth Factor I Signal Transduction via Inhibition of p52shc Binding to Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 Leading to Impaired Growth Factor Receptor-Bound Protein-2 Membrane Recruitment Mol. Endocrinol., September 1, 2008; 22(9): 2162 - 2175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. G. Hollier, J. A. Kricker, D. R. Van Lonkhuyzen, D. I. Leavesley, and Z. Upton Substrate-Bound Insulin-Like Growth Factor (IGF)-I-IGF Binding Protein-Vitronectin-Stimulated Breast Cell Migration Is Enhanced by Coactivation of the Phosphatidylinositide 3-Kinase/AKT Pathway by {alpha}v-Integrins and the IGF-I Receptor Endocrinology, March 1, 2008; 149(3): 1075 - 1090. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. C. Miller, B. E. Capps, R. R. Sanghani, D. R. Clemmons, and L. A. Maile Regulation of IGF-I Signaling in Retinal Endothelial Cells by Hyperglycemia Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3878 - 3887. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Maile, B. E. Capps, Y. Ling, G. Xi, and D. R. Clemmons Hyperglycemia Alters the Responsiveness of Smooth Muscle Cells to Insulin-Like Growth Factor-I Endocrinology, May 1, 2007; 148(5): 2435 - 2443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Maile, W. H. Busby, K. Sitko, B. E. Capps, T. Sergent, J. Badley-Clarke, Y. Ling, and D. R. Clemmons The Heparin Binding Domain of Vitronectin Is the Region that Is Required to Enhance Insulin-Like Growth Factor-I Signaling Mol. Endocrinol., April 1, 2006; 20(4): 881 - 892. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
