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Original Studies |
Departments of Physiology (Y.G., L.J.M.) and Internal Medicine (L.J.M.), University of Manitoba, Winnipeg, Canada R3E 0W3
Address all correspondence and requests for reprints to: L. J. Murphy, M.B., Ph.D., Department of Physiology, University of Manitoba, Winnipeg, Canada R3E 0W3. E-mail: ljmurph{at}cc.umanitoba.ca
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Abstract |
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Introduction |
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As an approach to understanding the mechanisms underlying the IGF-independent effects of IGFBP-3 we used the yeast two-hybrid system to identify binding partners for this protein. One potential binding partner identified in this screen was fibronectin (FN), a ubiquitous protein present in plasma and extracellular matrix. Here we demonstrate that IGFBP-3 does indeed interact with FN at the protein level and that IGF-I/IGFBP-3/FN complexes are present in plasma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human plasma-derived FN together with 
-chymotrypsin-digested
40- and 120-kDa FN fragments were purchased from Life Technologies, Inc. (Burlington, Canada). Bovine and rat FN were
obtained from the same source. Human recombinant glycosylated IGFBP-3,
nonglycosylated N109D IGFBP-3, IGF-I, IGFBP-5, and rabbit anti-IGFBP-3
polyclonal antiserum were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Amniotic fluid-derived IGFBP-1 was
obtained from Calbiochem (San Diego, CA). Iodinated
IGFBP-3 (45 µCi/µg) was purchased from Diagnostics Systems Laboratories, Inc. (Webster, TX), and
[125I]IGF-I was obtained from NEN Life Science Products (Boston, MA). Human plasma was prepared from
blood collected from four healthy laboratory personnel in
ethylenediamine tetraacetate-containing tubes. All other reagents were
obtained from Sigma-Aldrich Corp. (Oakville, Canada)
unless otherwise stated.
DNA constructs and library screening
A full-length human IGFBP-3 complementary DNA (cDNA) was ligated into the blunted BamHI site of vector pAS1, resulting in an in-frame fusion of human IGFBP-3 cDNA downstream of the GAL4 DNA-binding domain. The correct reading frame was confirmed by sequence analysis. The expression of the fusion protein with a Mr of 48 kDa was confirmed by Western blot of lysates from CG1945 yeast cells transfected with this construct.
A human placental cDNA library in the pACT2 plasmid (CLONTECH Laboratories, Inc., Palo Alto, CA) was screened with the bait construct, pAS1/hBP-3, using the large scale sequential polyethylene glycol/lithium acetate transformation method according to the manufacturer’s instructions. Yeast cells containing pAS1/hBP-3 were transformed with 20–40 µg library DNA/transformation. The transformed cells were spread on SD/Trp/Leu/His plates containing 5 mmol/L 3-amino-1,24-triazole (3-AT) and incubated 5 days at 30 C. Plasmid DNAs were extracted from yeast cells cultured in SD/Trp/Leu/His/3-AT medium. Separation of the pAS1/BP-3 plasmid from the AD/library plasmids was carried out by transformation of Escherichia coli HB101 carrying a Leu B mutation. The E. coli cells were grown on M9 agar medium containing 50 µg/mL ampicillin, 40 µg/mL proline, and 1 mmol/L thiamine-HCl. The interaction of IGFBP-3 with the positive cDNA clones was verified by mating yeast. Yeast Y187 cells were transformed with pAS1/BP-3 and the yeast CG1945 cells were transformed with AD-positive plasmids prepared from E. coli HB101. These two yeast cells were mated and plated on SD/Trp/Leu/His/5 mmol/L 3-AT medium. Plasmids were sequenced on an ABI automated DNA sequencer with a dye terminator kit (PE Applied Biosystems, Foster City, CA), and comparison of the DNA sequence with those in GenBank was made using the BLAST search and Antheprot software.
Biotinylation of IGFBP-3
Nonglycosylated, E. coli-derived IGFBP-3 was dissolved in 400 µL PBS (pH 7.4) at a concentration of 1 µg/µl and incubated with 10 µL D-biotinoyl-aminocaproic acid-N-hydroxysuccinimide ester (Roche Molecular Biochemicals, Mannheim, Germany) for 2 h at room temperature. The cross-linking reaction was terminated by separating the free and bound biotin ester on a disposable Sephadex G-25 column, preequilibrated with 1 mL blocking solution (Roche Molecular Biochemicals). The sample was eluted with PBS and collected in 0.2-mL fractions. The protein concentration was measured using the Bradford protein assay (Bio-Rad Laboratories, Inc., Mississauga, Canada). The biotinylated IGFBP-3 was stored frozen at -80 C until use.
Western blotting
FN and its various fragments were resolved on 7% or 10% SDS-polyacrylamide gel and then transferred to nitrocellulose membrane (Micron Separation, Inc., Westborough, MA). The membranes were briefly washed with TBST (Tris-buffered saline pH 7.6, and 0.1% Tween-20) and then blocked with TBST containing 1% BSA for 1 h at room temperature. The membranes were incubated overnight with IGFBP-3 (50 ng/mL) or biotinylated IGFBP-3 (100 ng/mL) in TBST at cold room. After washing in TBST (5 min, three times), the membrane was incubated with anti-IGFBP-3 rabbit polyclonal antiserum, diluted 1:500, for 2 h, then with biotinylated goat antirabbit IgG (diluted 1:5000; Bio-Rad Laboratories, Inc.) for 1 h at room temperature. After washing in TBST, these membranes, and membranes probed directly with biotinylated IGFBP-3 were incubated with streptavidin-horseradish peroxidase conjugate diluted 1:3000 (Life Technologies, Inc.) for 1 h at room temperature and washed as described above. The membranes were then analyzed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Baie d’Urfe, Canada) and exposed to Kodak BioMax x-ray film (Eastman Kodak Co., Rochester, NY) for 2–5 min.
Immunoprecipitation of IGFBP-FN complexes from serum
For immunoprecipitation, protein A-Sepharose 6MB (Amersham Pharmacia Biotech) was initially washed twice in 50 mmol/L Tris-HCl, pH 7.4. Anti-FN monoclonal antibody (Pierce Chemical Co., Rockford, IL; 2 µL) was incubated with 100 µL 20% washed protein A-Sepharose 6MB for 4 h at 4 C. The complex was centrifuged and washed, then incubated overnight at 4 C with 0.5 mL normal human plasma. On some occasions the complex was preincubated with [125I]IGF-I (10,000–50,000 cpm) at room temperature for 6 h. The resulting pellets were washed three times, then boiled with 60 µL Laemmli buffer for 5 min. The samples were clarified by centrifugation, analyzed by SDS-PAGE, and transferred to nitrocellulose membrane. The membranes were incubated with either mouse antihuman FN or biotinylated goat antihuman IGFBP-3 antibody (Diagnostics Systems Laboratories, Inc.) in a 1:500 dilution. Detection of immune complexes was achieved with secondary antibody or streptavidin-horseradish peroxidase followed by enhanced chemiluminescence Western blotting reagent. To detect the presence of IGF-I in the FN-IGFBP-3 complexes, the gel was dried and directly exposed to x-ray film at -70 C for 3 days.
Solid phase binding assays
An immobilized ligand-based assay system was used to characterize binding of [125I]IGFBP-3 to FN. Human FN and its different fragments were coated on 96-well Maxisorp immunological plates (InterMed, Nunc, Denmark) in 0.1 mol/L Na2CO3, pH 9.8, overnight at 4 C. The plates were rinsed with 200 µL 10 mmol/L sodium phosphate (pH 7.4) and 150 mmol/L NaCl and blocked with 200 µL 10 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween-20, 1% BSA, and 0.02% NaN3, pH 7.5, for 1 h at 37 C. Plates were rinsed twice with 200 µL 10 mmol/L sodium phosphate (pH 7.4) and 150 mmol/L NaCl and once with assay buffer [30 mmol/L Tris acetate (pH 7.4), 10 mmol/L sodium phosphate, 0.1% Tween-20, and 0.2% NaN3].
For the IGFBP-3 binding assay, [125I]IGFBP-3 (20,000 cpm/well) was incubated together with various concentrations of unlabeled IGFBP-3, or other competitor in 100 µL assay buffer for 1 h at 37 C. Unbound radioactivity was removed by rinsing the wells twice with 200 µL ice-cold assay buffer.
For the IGF-I binding assay, the 40-kDa fragment of FN was coated onto 96-well plates as described above. IGFBP-3 (10 ng) was added to each well in 100 µL assay buffer for 1 h at 37 C. Unbound IGFBP-3 was removed by washing, [125I]IGF-I (25,000 cpm/well) was added together with various concentrations of unlabeled IGF-I in 100 µL assay buffer, and the incubation was continued for 1 h at 37 C. As a control, IGFBP-3 (10 ng/well) was directly coated onto the wells, and the incubation with [125I]IGF-I was performed in an identical fashion. Unbound radioactivity was removed by rinsing the wells twice with 200 µL ice-cold assay buffer. Bound radioactivity was solubilized with 200 µL 1 mol/L NaOH, then transferred to plastic test tubes and counted for radioactivity.
Gel permeation chromatography of human plasma
Radiolabeled IGFBP-3 (
200,000 cpm) was incubated with 1 mL
human plasma overnight at 4 C. This mixture was then applied to a
1 x 40-cm Sephacryl S-200 column (Amersham Pharmacia Biotech). Fractions of 0.4 mL were collected and counted in a
-counter. A 100-µL aliquot of each fraction was applied to
nitrocellulose using a dot-blot apparatus, and FN was visualized using
anti-FN antibody as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 5 x 106 tryptophan and leucine auxotropic transformants were screened, resulting in the identification of 53 positive colonies. Colonies were chosen at random for further study. The plasmids were rescued from these colonies, expanded in E. coli, and reintroduced into yeast cells to confirm the interaction with the pAS1/hBP-3 bait plasmid. The inserts present in the AD-positive plasmids studied to date were sequenced and found to be identical to the previously reported sequence of human FN (7), TAP-1 (transporter associated with antigen processing) (8), and a disintegrin metalloprotease-12 (9). The interaction of IGFBP-3 with a disintegrin metalloprotease-12 was subsequently demonstrated using recombinantly expressed proteins (10).
The human FN insert was approximately 3.2 kb and contained sequence
starting from nucleotide 4436 of the reported DNA sequence
(7). The insert encoded the carboxyl-terminal fragment of
FN including the cell binding site, the ED region, the
heparin-binding domain, and the fibrin-binding domain (Fig. 1
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Although the interaction of IGFBP-3 with extracellular matrix has
been documented (6), the interaction with FN has not
previously been reported. Human FN together with its 40- and 120-kDa
fragments generated by -chymotrypsin digestion were separated by
PAGE and transferred to nitrocellulose. Glycosylated and
nonglycosylated IGFBP-3 were used to probe the nitrocellulose filters
(Fig. 2). Both glycosylated and
nonglycosylated IGFBP-3 bound to intact human FN and to the 40-kDa
carboxyl-terminal fragment. In contrast, there was no binding of
IGFBP-3 to the 120-kDa fragment of FN, which lacks a heparin-binding
domain. Using the glycosylated IGFBP-3, binding of IGFBP-3 to an
approximately 30-kDa fragment was also observed in the 40-kDa FN and
120-kDa FN fragments. The nature of this 30-kDa fragment is unclear.
Its detection using glycosylated IGFBP-3, but not biotinylated,
nonglycosylated IGFBP-3, probably represents differences in the
sensitivities of the two methodologies used. Alternatively, the 30-kDa
FN fragment may have a decreased affinity for nonglycosylated IGFBP-3.
IGFBP-3 binding to bovine and rat FN was also observed (data not
shown).
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). Various amounts of
FN or the 40-kDa fragment were coated onto polystyrene tubes and
incubated with radiolabeled IGFBP-3. When equimolar amounts of FN and
the 40-kDa fragment were compared, similar IGFBP-3 binding was
observed.
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. The displacement curve for
nonglycosylated, E. coli-derived IGFBP-3 was shifted to the
left compared with that of glycosylated Chinese hamster ovary
cell-derived IGFBP-3. Half-maximal displacement required a
concentration of about 7 ng/mL nonglycosylated IGFBP-3 compared with 20
ng/mL glycosylated IGFBP-3. The dissociation constant for the
interaction of glycosylated IGFBP-3 with FN calculated by Scatchard
analysis was approximately 0.31 nmol/L.
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). Similarly,
IGFBP-1 did not compete with IGFBP-3 for binding to FN. The closely
related binding protein, IGFBP-5, was able to compete with
[125I]IGFBP-3 for binding to FN with similar or
greater potency than unlabeled IGFBP-3. Binding of IGFBP-5 to FN as
well as other components of extracellular matrix has previously been
reported (6).
As IGFBP-3 binding was localized to the heparin-binding domain of FN,
we examined the effects of heparin on the interaction of IGFBP-3 with
FN. Competition between heparin and
[125I]IGFBP-3 was observed, with half-maximal
binding seen with 0.2 µg/mL heparin (Fig. 4).
Binding of the IGF-I to the IGFBP-3/FN binary complex
As stated above, IGF-I had no effect on binding of IGFBP-3 to
immobilized FN. We next determined whether IGF-I was able to bind to
IGFBP-3 immobilized on FN. [125I]IGF-I was
incubated with IGFBP-3 that had been bound to FN-coated wells (Fig. 5A). As controls,
[125I]IGF-I was also incubated with uncoated
wells or wells that had been coated with FN only. Negligible binding of
[125I]IGF-I was observed to uncoated wells or
to wells where FN alone was present on the well. BSA-coated wells that
were subsequently incubated with IGFBP-3 bound 2.7% of the added
radioactivity, whereas FN-coated wells that were incubated with IGFBP-3
bound 12% of the added [125I]IGF-I (Fig. 5A).
The affinity of IGF-I for IGFBP-3 bound to FN (Kd
= 0.6 nmol/L) was similar to that for IGFBP-3 immobilized on
polystyrene wells (Kd = 0.8 nmol/L; Fig. 5, B and
C).
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To determine whether the interaction of IGFBP-3 with FN occurred
in the circulating plasma, [125I]IGFBP-3 was
incubated with human plasma, and the resulting mixture was analyzed by
gel permeation chromatography. A number of distinct peaks of
radioactivity were apparent (Fig. 6). The
first two peaks to elute corresponded to high molecular mass IGFBP-3
complexes. Immunoblotting revealed that FN was also present in these
early peaks. FN was most abundant in the second peak (fractions
86–100). The third peak, which contained the majority of the
radiolabeled IGFBP-3, was devoid of FN. The fourth peak (fractions
119–135) corresponded to where free IGFBP-3 would elute and was also
devoid of FN. Fractions 170–200 probably represent radioiodinated
IGFBP-3 degradation fragments. The peak of radioactivity starting at
fraction 240, corresponding to the total volume of the column,
represents free radioiodine and iodinated amino acids.
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, upper panel, lanes 5–7).
A small amount of IGFBP-3 was also apparent when Sepharose beads coated
with mouse Ig were used for immunoprecipitation in some experiments
(Fig. 7A, lane 4). However, this was consistently less than that seen
with anti-FN antibody-coated beads (Fig. 7A, lower panel,
lane 4). Preincubation of [125I]IGF-I with the
human serum resulted in the formation of IGF-I/IGFBP-3/FN ternary
complexes. These complexes were demonstrated by immunoprecipitation
with anti-FN antibody (Fig. 7B). The presence of
[125I]IGF-I was detectable by autoradiography
in immunoprecipitates of human plasma with anti-FN-coated Sepharose
beads (lanes 3–6). When mouse IgG-coated Sepharose beads were used as
a control, no [125I]IGF-I was detectable (lane
2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are data from experiments with various cell systems to suggest that IGFBP-3 may have additional IGF-independent actions. These effects, which included induction of apoptosis and inhibition of proliferation, are demonstrable with IGFBP-3 fragments that have reduced IGF-I binding affinity and using cell lines that either lack IGF-I receptors or are unresponsive to IGF-I (3, 4, 5). Cross-linking experiments indicate that IGFBP-3 is able to bind to a variety of cell surface proteins, including the type V transforming growth factor-ß receptor (3, 14). However, physiological relevance of these cell surface binding sites and their role in the IGF-independent actions of IGFBP-3 have yet to be determined.
In an attempt to identified binding partners that may be involved in
the IGF-independent actions of IGFBP-3, we have used IGFBP-3 as bait in
yeast 2-hybrid assay system. Here we report the identification of 1 of
about 50 positive colonies in a screen of a placental cDNA library. The
colony contained a plasmid that expressed the carboxyl-terminal
fragment of FN. We chose to study the interaction of IGFBP-3 with FN in
detail, because FN is a relatively abundant protein in plasma and
extracellular matrix, and interestingly, FN has both proapoptotic
activity in monocytes and antiapoptotic activity in a variety of
attached cell lines, including osteoblasts and melanocytes
(15, 16, 17). In addition, FN can be associated with the cell
surface membrane via the
5ß1 integrin receptor,
and this may be important in the antiapoptotic actions of FN
(18). The ability of FN to associate with the cell
membrane has led us to speculate that FN may be one of the cell surface
binding sites for IGFBP-3 previously identified in cross-linking
studies.
FN is glycoprotein of approximately 220,000 Mr present in both plasma and extracellular matrix. In plasma it circulates predominantly as a dimer (19), whereas in extracellular matrix FN is present as multimers (20). FN is thought to function as a structural and adhesive protein, tethering cells to the substratum. It has an important role in the maintenance of normal cell morphology, cell migration, metastasis, wound healing, and, as discussed above, cell survival (20). Binding of FN to collagen (21), glycosaminoglycans (22), and fibrin (23) in addition to cell membranes has been reported.
The FN molecule consists of various functional domains
(16). The major heparin-binding domain resides in the
carboxyl-terminal region, and it is this region that has an affinity
for binding IGFBP-3. A fibrin- and heparin-binding domain is also
present at the amino-terminal end of the molecule (16).
Further downstream there is a gelatin- and collagen-binding domain. The
120-kDa fragment generated by -chymotrypsin digestion is devoid of
the amino-terminal gelatin/collagen-binding domain and the
carboxyl-terminal heparin-binding domain, but contains the cell-binding
domain (16). This fragment did not bind IGFBP-3. Thus,
although it is unclear whether IGFBP-3 is able to bind to the
aminoterminal heparin-binding domain, we provide convincing
evidence using both the yeast system and purified proteins that IGFBP-3
binds to the carboxyl-terminal region containing the heparin-binding
domain and not to the central 120-kDa fragment. We also show that
heparin is able to disrupt the interaction of IGFBP-3 with FN. Although
heparin can disrupt the binding of IGFBP-3 to FN, it is not necessary
for heparin to be present for this interaction to occur. However, as
IGFBP-3 also contains a heparin-binding domain that appears to be
important in the interaction of IGFBP-3 with other proteins, such as
fibrinogen and plasminogen (11, 12), it is not clear
whether disruption of the IGFBP-3-FN interaction by heparin involves
the interaction of heparin with IGFBP-3 or FN.
The binding affinity of IGFBP-3 for FN was similar to that for the interaction of IGFBP-3 with fibrinogen and slightly lower than that with acid-labile subunit (ALS) and plasminogen (11, 12, 24). However, despite this lower affinity, immunoprecipitation studies with anti-FN antibody indicated that IGFBP-3/FN complexes are present in plasma.
When human plasma is analyzed by gel permeation chromatography, IGFBP-3 is found as both a large molecular weight component and a smaller molecular weight component. These components have been considered to represent the ternary complex, consisting of IGF-I, IGFBP-3 together with ALS, and the binary complex of IGF-I/IGFBP-3, respectively. Although the majority of the larger molecular weight components of IGFBP-3 probably represent the IGF-I/IGFBP-3/ALS ternary complex, other IGFBP-3-containing complexes may also be present in less abundance. Novel proteins associated with IGFBP-3 in human serum have been previously reported by Collett-Solberg and colleagues (25), although none of these would appear to have a molecular mass equivalent to that of FN.
FN immunoreactivity was present in the first and second peaks of radioactivity that eluted when plasma equilibrated with [125I]IGFBP-3 was analyzed on Sephacryl S-200. An IGF-I/IGFBP-3/FN ternary complex would have a molecular mass of about 260 kDa, considerably larger than the approximately 150-kDa IGF-I/IGFBP-3/ALS ternary complex. As FN may be present as a dimer, IGFBP-3/FN complexes with a molecular mass of approximately 500 kDa are also possible. The gel permeation chromatography system we used would not reliably distinguish between such large molecular mass complexes. However, it is clear that there was some overlap between the elution patterns of FN and [125I]IGFBP-3. Approximately 27% of the [125I]IGFBP-3 radioactivity was associated with elution peak of FN. Definitive evidence for the presence of IGFBP-3/FN complexes in plasma was provided by the immunoprecipitation studies. These complexes also contained [125I]IGF-I, indicating that the presence of tertiary complexes in the circulation where FN, rather than ALS, is present.
The data reported here clearly demonstrate that the IGFBP-3/FN interaction identified in the yeast two-hybrid system also occurs under in vitro conditions at the protein level and, more importantly, that IGFBP-3/FN and IGF-I/IGFBP-3/FN complexes are present in plasma. As FN is also present in extracellular matrix and on cell membranes, it is reasonable to speculate that some of the IGFBP-3 binding to extracellular matrix and cell membranes may be attributable at least in part to IGFBP-3 binding to FN. Further studies are required to investigate the functional importance of the interaction of IGFBP-3 with FN.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Medical Research Council Senior Scientist Award and
an endowed Research Professorship in Metabolic Diseases.
Received November 10, 2000.
Revised January 19, 2001.
Accepted February 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) is an active metalloprotease. J Biol Chem. 273:16993–16997.5ß1 integrin supports
survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA. 92:6161–6165.This article has been cited by other articles:
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