Department of Internal Medicine, Diabetes and Endocrinology Research Center, Veterans Administration Medical Center, The University of Iowa, Iowa City, Iowa 52246
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ABSTRACT |
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Insulin-like growth factor-binding protein (IGFBP)-3 contains a highly basic COOH-terminal heparin-binding region, the P3 region, which is thought to be important in the binding of IGFBP-3 to endothelial cells. IGFBP-3 and IGFBP-4, and their chimeras IGFBP-34 and IGFBP-43, were treated with plasmin and with thrombin, proteases known to cleave IGFBP-3. IGFBP-3 was highly susceptible to plasmin, whereas IGFBP-4 was less so. Substitution of the P3 region for the P4 region in IGFBP-4 (IGFBP-43) increased the ability of the protease to digest IGFBP-43; substitution of the P4 region for the P3 region in IGFBP-3 (IGFBP-34) decreased the digestion of IGFBP-34. When 125I-labeled IGFBP-3 or 125I-IGFBP-43 was first bound to vascular endothelial cells, subsequent proteolysis by either plasmin or thrombin was substantially inhibited. Proteolysis of 125I-IGFBP-34 was not inhibited in the presence of endothelial cells. The P3 peptide was cleaved by plasmin but not by thrombin. We conclude that the P3 region is central to proteolysis of IGFBP-3 by plasmin and thrombin, processes which were inhibited by association of IGFBP-3 with endothelial cells.
IGFBP proteases
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INTRODUCTION |
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THE INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS (IGFBPs) constitute a family of six high-affinity insulin-like growth factor (IGF)-binding proteins found in the circulation and produced by several types of cells. Of these six proteins, IGFBP-3 is the most prevalent, circulating primarily as a 140-kDa ternary complex with IGF-I or IGF-II and an acid-labile subunit (ALS) (3, 14). Several proteases have been shown to cleave IGFBP-3 (24), including plasmin (1, 4, 6, 9, 13, 20), thrombin (6, 26), kallikrein (13), prostate-specific antigen (11), matrix metalloproteases (16, 19), and cathepsin D (10, 12).
The position of a number of plasmin and thrombin cleavage sites on IGFBP-3 has been determined (6). In addition to these sites, additional plasmin cleavage sites must be present, because further digestion with plasmin led to the disappearance of both intact IGFBP-3 and IGFBP-3 digestion products (6).
IGFBP-3 binds to several cell types, including cultured bovine microvessel endothelial cells (5, 15, 23). The affinity of IGFBP-3 for IGF is greater than the affinity of the type 1 IGF receptor, whereas binding of IGFBP-3 to cells appears to decrease the affinity of IGFBP-3 for IGF (3, 23). It has been suggested that this decrease in affinity of cell-bound IGFBP-3 for IGF increases the release of IGF from cell-surface IGFBP-3, facilitating IGF binding to IGF receptors. (3, 23).
IGFBP-3 contains a highly basic heparin-binding area, the P3 region, in its COOH-terminal portion (5). This region contains the cell-surface recognition site for IGFBP-3 (18), a nuclear localization sequence (17, 21, 24), and is important in ALS binding (15, 25). A similar region, P5, is found in IGFBP-5 and appears to serve a comparable function (5, 8, 24, 25). Recent studies have shown that the P3 region of IGFBP-3, or the P5 region in IGFBP-5, serves as a binding site for the proenzymes plasminogen and prekallikrein (9, 13). Activation of plasminogen to plasmin or prekallikrein to kallikrein resulted in proteolysis of IGFBP-3. Proteolysis of IGFBP-3 by plasmin presumably decreases its association with IGF, freeing IGF to interact with its receptor (4, 9, 13, 20).
Chimeras of IGFBP-3 and IGFBP-4, IGFBP-34 and IGFBP-43, were constructed; the P3 region in IGFBP-3 was replaced by the equivalent region of IGFBP-4 and vice versa (18). These chimeras show changes in cellular binding properties; although IGFBP-4 does not bind to endothelial cells, cellular binding of IGFBP-43 is equal to or greater than cellular binding of IGFBP-3, whereas binding of IGFBP-34 is minimal (18). The purpose of the present study was to further investigate, using these IGFBP chimeras, the effect of the P3 region on protease sensitivity to plasmin and thrombin. The chimeras IGFBP-34 and IGFBP-43 show altered plasmin and thrombin sensitivity relative to IGFBP-3 or IGFBP-4. The apparent importance of the P3 region was not restricted to binding of a proenzyme followed by activation but was demonstrated using the active protease. Perhaps most significantly, we demonstrate that binding of IGFBP-3 or IGFBP-43 to endothelial cells protects these proteins from degradation by plasmin or thrombin.
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MATERIALS AND METHODS |
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Recombinant IGFBP-3 and IGFBP-4, and their chimeras, IGFBP-34 and IGFBP-43, were isolated as secreted proteins in a baculovirus system and purified on IGF affinity columns, as previously described (18). Unlabeled IGFBP-3, IGFBP-34, IGFBP-4, and IGFBP-43 (20 µg/20 µl) were incubated with 0.05 µl (1×) or 0.1 µl (2×) plasmin, stock concentration 2 µg/µl (American Diagnostica, Greenwich, CT) for 20 min at 37°C in PBS, pH 7.3, or PBS containing 90 mM Tris, pH 7.5 (PBS-Tris). Plasmin was used at molar ratios of ~1:300 and ~1:600 plasmin/IGFBP. Incubation was stopped by the addition of phenylmethylsulfonyl fluoride (PMSF), final concentration 1 mM, and 2× SDS-PAGE sample buffer. Samples were immediately boiled for 5 min and electrophoresed on nonreducing 12% SDS gels. Gels were stained with Coomassie blue, scanned, and analyzed using Sigma Gel software (Jandel Scientific). The integrated area of the IGFBP band in the control (no protease) lane was compared with the integrated areas of the IGFBP bands from protease-treated samples. Unlabeled binding proteins, at 20 µg/20 µl, were also incubated with thrombin from bovine plasma (1,500-2,500 NIH units/mg protein; Sigma, St Louis, MO) at 37°C for 2 h in PBS-Tris buffer and analyzed as for plasmin.
IGFBPs were iodinated and purified on Sephadex G100 columns, as previously described (18). Concentrations of plasmin or thrombin were established that would achieve measurable 125I-IGFBP-3 degradation in solution after a 20-min incubation at 22°C in HLB (in mM: 100 HEPES, 120 NaCl, 1.7 MgSO4, 25 sodium acetate, 2.4 KCl, 0.8 EDTA, 10 glucose, and 10 mg/ml BSA, pH 7.8) or serum-free Medium 199 (M199; Life Technologies, Rockville, MD) plus 50 mM HEPES, pH 7.3. Aliquots were analyzed by SDS-PAGE, and dried gels were exposed to X-ray film (Eastman Kodak, Rochester, NY), scanned, and analyzed as described for stained gels. All experiments were performed at least twice, with duplicate samples in each experiment. When replicate samples were electrophoresed, the integrated areas of the 125I-IGFBP band in two or more lanes were averaged, and the standard deviation was calculated. Data were analyzed by ANOVA, with the Newman-Keuls post test, or by t-tests where appropriate, with the use of GraphPad Prism (GraphPad Software, San Diego, CA).
Microvessel endothelial cells were prepared from bovine heart adipose tissue and characterized as previously described (2). Cells were preincubated with 125I-IGFBPs for 1 h at 22°C in 12-well trays in HLB or M199. In some experiments, the supernatant was removed, and the cells were washed and then incubated with the appropriate protease. In other experiments using 125I-IGFBP-34 (which does not bind significantly to cells) after the preincubation, the proteases were added directly to the medium, and the supernatant was not removed. Parallel incubations were conducted in solution (i.e., in the absence of cells) for 20 min at 22°C. All reactions were performed in duplicate.
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RESULTS |
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Unlabeled IGFBP-3, IGFBP-34, IGFBP-4, and
IGFBP-43 (1 µg/µl) were incubated with plasmin
for 20 min at 37°C. Similar conditions were previously determined to
digest the majority, but not all, of the IGFBP-3 (6).
Aliquots were analyzed by 12% SDS-PAGE and stained with Coomassie blue
(Fig. 1). In the control ("0") lanes,
there was no degradation of the binding proteins after 20 min at 37°C
when compared with the starting IGFBP. After digestion with 1×
plasmin, 58% of IGFBP-3 vs. 81% of IGFBP-34 remained
intact; after digestion with 2× plasmin, 18% of IGFBP-3 vs. 72% of
IGFBP-34 remained intact. IGFBP-4 was relatively
insensitive to plasmin. As predicted on the basis of the importance of
the P3 region as a plasmin-binding site, substitution of the P3 for the
P4 region in IGFBP-4 (IGFBP-43, Fig. 1) increased
susceptibility to plasmin. After digestion with 1× plasmin, 101% of
IGFBP-4 vs. 54% of IGFBP-43 remained intact, whereas after
digestion with 2× plasmin, 88% of IGFBP-4 vs. 58% of
IGFBP-43 remained intact. Although the presence of the P3
region increased plasmin digestion of IGFBP-43, presumably by serving as a binding site for plasmin, there was less digestion of
IGFBP-43 than of IGFBP-3 with 2× plasmin (58% intact for
IGFBP-43 vs. 18% intact for IGFBP-3). Similar results were
seen when IGFBP-3 and IGFBP-34 were treated with
either plasmin or thrombin (Fig. 2). In
the controls, there was no digestion after incubation at 37°C
(compare C with C0). After treatment with
plasmin, 17% of IGFBP-3 and 50% of IGFBP-34 remained
intact; after treatment with thrombin, 26% of IGFBP-3 and 44% of
IGFBP-34 remained intact.
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To determine whether the presence of cells could protect IGFBPs containing the P3 region from digestion, IGFBP-3, IGFBP-43, and IGFBP-34 were iodinated and exposed to plasmin and thrombin. For these studies, several preliminary experiments were performed. The concentrations of plasmin or thrombin that gave measurable digestion of 125I-IGFBP-3 or 125I-IGFBP-43 were determined. Next, studies were performed in HLB or M199 to determine conditions of preincubation to achieve near-maximal binding of IGFBPs to cells without substantial degradation in the control samples. After 1 h at 22°C in HLB, total binding was 17% of added 125I-IGFBP-43, and specific binding was 14%. After 1 h in M199, total binding was 11% of added 125I-IGFBP-43, and specific binding was 8%. We then determined whether plasmin or thrombin treatment of cells in HLB or M199 would affect cell morphology. Microvessel endothelial cells, in HLB or M199, were treated with appropriate concentrations of plasmin or thrombin and compared visually with untreated cells over a period of 5 h. No differences in cell morphology or attachment were observed between protease-treated and -untreated cells.
Endothelial cells were preincubated with
125I- IGFBP-3 or
125I-IGFBP-43 in HLB. The medium containing the
free binding protein was removed, the cells were washed once with HLB,
and then fresh HLB and the proteases were added. After 20 min, an
aliquot of the supernatant was saved for SDS-PAGE analysis, the rest
was discarded, and the cells were lysed in SDS-PAGE sample buffer.
Parallel incubations were conducted in solution. In solution, 9 ± 2% (n = 2, ±SD) of the 125I-IGFBP-3
remained intact after plasmin treatment, and 47 ± 2% remained
intact after thrombin treatment (Fig. 3).
As seen with use of the unlabeled binding proteins, in solution,
125I-IGFBP-43 was less sensitive to either
enzyme than 125I-IGFBP-3, with 20 ± 11% remaining
after plasmin, and 85 ± 12% remaining after thrombin treatment.
After incubation in the presence of endothelial cells, there was less
degradation of both cell-attached binding proteins with either enzyme.
For 125I-IGFBP-3, 53 ± 5% of cell-attached binding
protein remained intact after plasmin (vs. 9 ± 2% in solution),
and 70 ± 4% remained intact after thrombin (vs. 47 ± 2%
in solution). For 125I-IGFBP-43, 67 ± 2%
remained after plasmin (vs. 20 ± 11% in solution) and 104 ± 3% after thrombin (vs. 85 ± 12% in solution) (Fig. 3). After
20 min, there was some dissociation of the bound 125I-IGFBP
into the cell supernatant. The counts in the supernatant were similar
for untreated and protease-treated samples. These supernatants, i.e.,
the material dissociated from the cells, were analyzed (data not
shown). For plasmin, the percentage of intact binding protein in the
supernatant (125I-IGFBP-3, 13 ± 1% and
125I-IGFBP-43, 15 ± 0%) was similar to
that seen in the solution reaction (125I-IGFBP-3, 9 ± 2% and 125I-IGFBP-43, 20 ± 11%). For
thrombin, the material dissociated from the cells
(125I-IGFBP-3, 19 ± 2% and
125I-IGFBP-43, 27 ± 3%) was digested to
a greater extent than that seen in the solution reaction
(125I-IGFBP-3, 47 ± 2% and
125I-IGFBP-43, 85 ± 12%).
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There are several possible explanations for the "cell- protective"
effect we observed: 1) protection of the protease-binding site (P3 region) on the binding proteins by cell attachment at that
site, thus preventing plasmin or thrombin attachment; 2) protection of the binding proteins by protein(s) secreted by the endothelial cells during the 20-min incubation; 3)
protection of the binding proteins by cell membrane proteins or
components unrelated to the putative IGFBP-3 basic cellular binding
region. To determine whether the protective effect was indeed due to
the cellular binding region (P3), we designed a series of similar experiments with 125I-IGFBP-34. Because only a
minimal amount of this protein binds specifically to endothelial cells
(<2% binding for 125I-IGFBP-34 compared with
14% for 125I-IGFBP-43), the cell-associated
fraction and the cell-supernatant fraction were analyzed after a 1-h
preincubation in HLB followed by incubation with the proteases. The
nonbound material was not removed after the preincubation; the enzymes
were added directly to the incubation after 1 h, and samples were
incubated for a further 20 min at 22°C.
125I-IGFBP-34 and 125I-IGFBP-3 were
both included in these experiments to show that, under these slightly
modified conditions, the 125I-IGFBP-3 was still protected
by the cells. Additionally, a longer (40-min) incubation with thrombin
(T2) was performed in an attempt to increase the degradation of
125I-IGFBP-34. One set of gels is shown in Fig.
4, which indicates the percentage of
125I-IGFBP-3 or 125I-IGFBP-34
remaining intact in the solution reaction, the percentage in the
supernatant of the cells (i.e., the media in which the cells were
incubated), and the percentage attached to the cells. As in the
previous experiments, 125I-IGFBP-3 was protected in the
presence of cells. However, 125I-IGFBP-34 was
not protected from plasmin digestion (Fig. 4). There was insufficient
thrombin digestion of 125I-IGFBP-34 in the
solution reaction to study the effect of the cells. The amount of IGFBP
digestion in the cell supernatant was intermediate to that seen in the
solution reaction and that seen for cell-associated binding protein
(Fig. 4).
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Because HLB contains 10 mg/ml BSA and 0.8 mM EDTA, which might affect
thrombin activity, the experiments were repeated using serum-free M199
(Fig. 5). Again, the cells protected
125I-IGFBP-3 from digestion by plasmin.
125I-IGFBP-3 was 1 ± 1% (mean ± SD,
n = 4) intact in solution, vs. cells 63 ± 24%
intact. With thrombin, although there was an apparent protective effect
of cells, it was not statistically significant, with the reaction in
solution 39 ± 19% intact vs. that in cells 62 ± 10%
intact. With 125I-IGFBP-34, which binds
minimally to cells, there was no significant protective effect of cells
for plasmin digestion (compare 21 ± 4 with 30 ± 19%). With
thrombin, however, there appeared to be increased degradation of the
small amount of the 125I-IGFBP-34 attached to
cells (compare 103 ± 11 with 47 ± 6%). The material in the
cell supernatant (predominantly unbound 125I-IGFBP rather
than dissociating bound 125I-IGFBP) was generally "not
protected" by the presence of cells.
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It is unclear whether the P3 region, as the putative plasmin (and perhaps thrombin) binding site, is itself being cut by either of these enzymes. In our previous studies (6, 7), we sequenced major plasmin and thrombin digestion products of IGFBP-3. We did not obtain an NH2-terminal sequence beginning in this region. However, these results do not mean that there is not a cleavage site within the P3 region, because the pieces may be too small to isolate on a 12% SDS gel. To determine whether plasmin or thrombin could cleave the P3 peptide, we incubated 20 µg of P3 (MW ~2,300) in M199 with several concentrations of plasmin and thrombin and assessed the degradation of P3 on a Coomassie-stained 16.5% Tris-Tricine gel. Plasmin digested the P3 peptide after 20 min at 37°C. Thrombin had little or no effect on the P3 peptide, even after 2 h of incubation at 37°C (data not shown).
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DISCUSSION |
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The presence of the P3 region in IGFBP-3, when introduced into IGFBP-4 (i.e., IGFBP-43), increased the digestion of the IGFBP by plasmin and thrombin. However, although IGFBP-43 was more sensitive to digestion than IGFBP-4, it remained less sensitive to plasmin than IGFBP-3. When 125I-IGFBPs were used, the presence of the P3 region similarly increased digestion of the binding protein "in solution." Perhaps of greatest importance, IGFBP-3 and IGFBP-43, in the presence of endothelial cells, were protected from digestion. IGFBP-34, which does not contain the P3 region and which binds minimally to endothelial cells (18), was not protected from plasmin or thrombin proteolysis. There was some dissociation of cell-bound 125I-IGFBP-3 or 125I-IGFBP-43 during the 20-min incubation. The plasmin digestion of this material was similar to that seen for the samples in solution, but the thrombin digestion was greater than that seen for the samples in solution.
While associated with the endothelial cell surface, binding proteins containing the P3 region were significantly protected from digestion by plasmin and thrombin. Protection of endothelial cell-associated IGFBP-3 is likely to be important, because both plasmin and thrombin are found in the endothelial milieu, especially in the case of injury to the vascular endothelium. Several studies have indicated that NH2-terminal and possibly COOH-terminal proteolytic fragments of IGFBP-3 have biological activity unrelated to their IGF binding (1, 4, 7). Cytoprotection would decrease the generation of these fragments and their activity and potentially alter the pattern of fragments produced. It should be noted that the cytoprotection was not complete; some of the labeled binding protein in the cell fraction was not intact. A portion of this may represent cleavage of loosely or nonspecifically bound IGFBP-3.
The highly basic P3 region of IGFBP-3 serves a multitude of purposes. It contains a heparin-binding sequence (3, 5), serves as a cellular binding domain (5, 18, 23), contains a nuclear localization signal sequence (17, 21, 24), is important for ALS binding (15, 25), and binds the precursors of the serine proteases kallikrien and plasmin (8, 9, 13). On the basis of the current results, the P3 region may interact with the active proteases plasmin and thrombin, as well as with the proenzyme plasminogen. Although this region appears to be important in digestion of IGFBPs by plasmin and thrombin, several major protease cleavage sites are outside of the P3 region (6, 7). Cleavage of IGFBP-3 can take place in the absence of plasmin interaction with the P3 region; otherwise, plasmin would be totally unable to cleave IGFBP-34. One possibility is that major plasmin or thrombin cleavage sites exist within or at the periphery of the P3 region and that these cleavage sites are blocked by IGFBP-3 association with endothelial cells. Cleavage at other sites might be inhibited, but not blocked, by IGFBP-3 association with cells. Adherent IGFBP-3 cleaved in the P3 region would lose its ability to interact specifically with the cell surface while binding protein cleaved in other regions would remain cell associated. The P3 peptide was digested by plasmin, suggesting that there are one or more plasmin cleavage sites within the P3 region of IGFBP-3. Thrombin did not cleave the P3 peptide, which contains no Arg-Gly sequences. However, because the final residue of P3 is an Arg, followed by a Gly, IGFBP-3 may be cleaved by thrombin at the COOH terminus of the P3 region. It is important that the protease be able to detach from the BP binding site (i.e., the P3 region) to proceed on to the cleavage of further molecules. One mechanism for detachment would be cleavage of the binding protein within the P3 region. It is also possible that these serine proteases interact with the P3 region and then cleave the binding protein at other sites. Supporting this possibility is the relative insensitivity of IGFBP-4 to plasmin, the increased sensitivity of IGFBP-43, and the cytoprotective effect of endothelial cell binding of IGFBP-43 Specifically, the addition of the P3 region provides a site for plasmin interaction, increasing digestion, whereas blockage of the P3 region by cell attachment eliminates this site, and digestion is decreased. However, it should be noted that, although plasmin digestion of IGFBP-3 or IGFBP-43 is decreased in the presence of cells, it is not eliminated. Thus plasmin interaction with the P3 region is not essential for all of plasmin's action on IGFBP-3. Digestion is decreased but not eliminated by the exchange of the P3 region in IGFBP-3 for the P4 region (IGFBP-34). Cells do not protect IGFBP-34 from digestion. In fact, when experiments were conducted in M199, thrombin digestion of cell-associated IGFBP-34 was actually increased, suggesting that the small amount of cell-associated IGFBP-34 may be in proximity to thrombin concentrated on or near cell surface thrombin receptors.
Proteolysis of IGFBP-3 decreases its affinity for IGF, allowing the freed IGF to interact with the IGF receptor (9, 13, 20). IGFBP-3 proteolysis could thus lead to an increase in the effects of IGF or to increased transendothelial transport of IGF. Proteolysis of IGFBP-3 may also affect transport and action of IGFBP-3 fragments. IGFBP-3 containing the P3 region was detected by both fluorescence labeling and immunologic techniques in the nuclei of cells (17, 21, 24). Additionally, the physiological role of the ALS is not well defined. It has been presumed that the larger ALS molecule prevents entry of IGFBP-3 or IGFBP-3-IGF complex into subendothelial tissue. Because the P3 region is also important in ALS binding, the ternary complex may have decreased protease sensitivity. Thus the amount and positions of proteolytic cleavage of IGFBP-3 occurring at or near the endothelial surface may influence both IGF and IGFBP-3 actions.
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ACKNOWLEDGEMENTS |
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This work was supported by funds from Veterans Affairs research and by the National Institute of Diabetes and Digestive and Kidney Disease Grants DK-25421 and DK-25295.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. S. Bar, The Univ. of Iowa, Dept. of Internal Medicine, ENDO-3E19 VA Medical Center, Iowa City, IA 52246 (E-mail: rbar{at}icva.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 August 2001; accepted in final form 5 September 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Angelloz-Nicoud, P,
Harle L,
and
Binoux M.
Recombinant human insulin-like growth factor binding protein-3 stimulates prostate carcincoma cell proliferation via an IGF-dependent mechanism. Role of serine proteases.
Growth Regul
6:
130-136,
1996[ISI][Medline].
2.
Bar, RS,
Dolash S,
Dake BL,
and
Boes M.
Cultured capillary endothelial cells from bovine adipose tissue: a model for insulin binding and action in microvascular endothelium.
Metabolism
35:
317-322,
1986[ISI][Medline].
3.
Baxter, RC.
Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities.
Am J Physiol Endocrinol Metab
278:
E967-E976,
2000
4.
Blat, C,
Villaudy J,
and
Binoux M.
In vivo proteolysis of serum insulin-like growth factor (IGF) binding protein-3 results in increased availability of IGF to target cells.
J Clin Invest
93:
2286-2290,
1994[ISI][Medline].
5.
Booth, BA,
Boes M,
Andress DL,
Dake BL,
Kiefer MC,
Maack C,
Linhardt RJ,
Bar K,
Caldwell EEO,
Weiler J,
and
Bar RS.
IGFBP-3 and IGFBP-5 association with endothelial cells: role of C-terminal heparin binding domain.
Growth Regul
5:
1-17,
1995[ISI][Medline].
6.
Booth, BA,
Boes M,
and
Bar RS.
IGFBP-3 proteolysis by plasmin, thrombin, serum: heparin binding, IGF binding, and structure of fragments.
Am J Physiol Endocrinol Metab
271:
E465-E470,
1996
7.
Booth, BA,
Boes M,
Dake BL,
and
Bar RS.
Isolation and charcterization of plasmin-generated bioactive fragments of IGFBP-3.
Am J Physiol Endocrinol Metab
276:
E450-E454,
1999
8.
Campbell, PG,
and
Andress DL.
Plasmin degradation of insulin-like growth factor-binding protein-5 (IGFBP-5): regulation by IGFBP-5-(201-218).
Am J Physiol Endocrinol Metab
273:
E996-E1004,
1997
9.
Campbell, PG,
Durham SK,
Suwanichkul A,
Hayes JD,
and
Powell DR.
Plasminogen binds the heparin-binding domain of insulin-like growth factor-binding protein-3.
Am J Physiol Endocrinol Metab
275:
E321-E331,
1998[Abstract].
10.
Clausse, M,
Kubler B,
Wendland M,
Neifer K,
Schmidt B,
Zapf J,
and
Braulke T.
Proteolysis of insulin-like growth factors (IGF) and IGF binding proteins by cathepsin D.
Endocrinology
138:
3797-3803,
1997
11.
Cohen, P,
Peehl DM,
Graves HCB,
and
Rosenfeld RG.
Biological effects of prostate-specific antigen as an insulin-like growth factor binding protein-3 protease.
J Endocrinol
142:
407-415,
1994[Abstract].
12.
Conover, CA,
Perry JE,
and
Tindall DJ.
Endogenous cathepsin D-mediated hydrolysis of insulin-like growth factor-binding proteins in cultured human prostatic carcinoma cells.
J Clin Endocrinol Metab
80:
987-993,
1995[Abstract].
13.
Durham, SK,
Suwanichkul A,
Hayes JD,
Herington AC,
Powell DR,
and
Campbell PG.
The heparin binding domain of insulin-like growth factor binding protein (IGFBP)-3 increases susceptibility of IGFBP-3 to proteolysis.
Horm Metab Res
31:
216-225,
1999[ISI][Medline].
14.
Ferry, RJ, Jr,
Cerri RW,
and
Cohen P.
Insulin-like growth factor binding proteins: new proteins, new functions.
Horm Res (Basel)
51:
53-67,
1999[ISI][Medline].
15.
Firth, SM,
Ganeshprasad U,
and
Baxter RC.
Structural determinants of ligand and cell-surface-binding on insulin-like growth factor binding protein-3.
J Biol Chem
273:
2631-2638,
1998
16.
Fowlkes, JL,
Suzuki K,
Nagase H,
and
Thrailkill KM.
Proteolysis of insulin-like growth factor binding protein-3 during rat pregnancy: a role for matrix metalloproteinases.
Endocrinology
135:
2810-2813,
1994[Abstract].
17.
Jaques, G,
Noll K,
Wegmeann B,
Witten S,
Kogan E,
Radulescu RT,
and
Havemann K.
Nuclear localization of insulin-like growth factor binding protein-3 in a lung cancer cell line.
Endocrinology
138:
1767-1770,
1997
18.
Knudtson, KL,
Boes M,
Sandra A,
Dake BL,
Booth BA,
and
Bar RS.
Distribution of chimeric IGF binding protein (IGFBP)-3 and IGFBP-4 in the rat heart: importance of C-terminal basic region.
Endocrinology
142:
3749-3755,
2001
19.
Kubler, B,
Cowell S,
Zapf J,
and
Braulke T.
Proteolysis of insulin-like growth factor binding proteins by a novel 50-kilodalton metalloproteinase in human pregnancy serum.
Endocrinology
139:
1556-1563,
1998
20.
Lalou, C,
Silve C,
Rosato R,
Segovia B,
and
Binoux M.
Interactions between insulin-like growth factor-I (IGF-I) and the system of plasminogen activators and their inhibitors in the control of IGF-binding protein-3 production and proteolysis in human osteosarcoma cells.
Endocrinology
135:
2318-2326,
1994[Abstract].
21.
Li, W,
Fawcett J,
Widmer HR,
Fielder PJ,
Rabkin R,
and
Keller GA.
Nuclear transport of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in opossum kidney cells.
Endocrinology
138:
1763-1766,
1997
22.
Maile, LA,
and
Holly JMP
Insulin-like growth factor binding protein (IGFBP) proteolysis: occurrence, identification, role and regulation.
Growth Hormone IGF Res
9:
85-95,
1999[ISI][Medline].
23.
McCusker, RH,
and
Clemmons DR.
Use of lanthanum to accurately quantify insulin-like growth factor binding to proteins on cell surfaces.
J Cell Biochem
66:
256-267,
1997[ISI][Medline].
24.
Schedlich, LJ,
Young TF,
Firth SM,
and
Baxter RC.
Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 share a common nuclear transport pathway in T47D human breast carcinoma cells.
J Biol Chem
273:
18347-18352,
1998
25.
Twigg, SM,
Kiefer MC,
Zapf J,
and
Baxter RC.
Insulin-like growth factor-binding protein 5 complexes with the acid labile subunit: role of the carboxy-terminal domain.
J Biol Chem
273:
28791-28798,
1998
26.
Zheng, B,
Clarke JB,
Busby WH,
and
Clemmons DR.
Insulin-like growth factor-binding protein-5 is cleaved by physiological concentrations of thrombin.
Endocrinology
139:
1708-1714,
1998
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