From the Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
Received for publication, September 12, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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Insulin-like growth factor-binding protein-1
(IGFBP-1) binds to insulin-like growth factors (IGFs) and has been
shown to inhibit or stimulate cellular responses to IGF-I in
vitro. This capacity of IGFBP-1 to inhibit or stimulate IGF-I
actions correlates with its ability to form stable high molecular
weight multimers. Since the ability of some proteins to polymerize is
dependent upon transglutamination, we determined if tissue
transglutaminase could catalyze this reaction and the effect of
polymerization of IGFBP-1 upon IGF-I action. Following incubation with
pure tissue transglutaminase (Tg), IGFBP-1 formed covalently linked
multimers that were stable during SDS-polyacrylamide gel
electrophoresis using reducing conditions. Dephosphorylated IGFBP-1
polymerized more rapidly and to a greater extent compared with native
(phosphorylated) IGFBP-1. Exposure to IGF-I stimulated transglutamination of IGFBP-1 in vitro. An IGFBP-1 mutant
in which Gln66-Gln67 had been altered to
Ala66-Ala67 (Q66A/Q67A) was relatively
resistant to polymerization by Tg compared with native IGFBP-1. Tg
localized in fibroblast membranes was also shown to catalyze the
formation of native IGFBP-1 multimers, however, Q66A/Q67A IGFBP-1
failed to polymerize. Although the mutant IGFBP-1 potently inhibited
IGF-I stimulated protein synthesis in pSMC cultures, the same
concentration of native IGFBP-1 had no inhibitory effect. The addition
of higher concentrations of native IGFBP-1 did inhibit the protein
synthesis response, and this degree of inhibition correlated with the
amount of monomeric IGFBP-1 that was present. In conclusion, IGFBP-1 is
a substrate for tissue transglutaminase and Tg leads to the formation
of high molecular weight covalently linked multimers. Polymerization is an important post-translational modification of IGFBP-1 that regulates cellular responses to IGF-I.
Insulin-like growth factors (IGF-I and
IGF-II)1 are associated with
their specific binding proteins (IGFBPs) in extracellular fluids, and
these binding proteins regulate IGF actions (1, 2). The mechanisms
responsible for IGFBP-induced changes are not well defined. Several
post-translational modifications have been shown to alter the affinity
of IGFBPs for IGFs, including phosphorylation (3, 4), proteolysis (5,
6), and polymerization (7, 8). Dephosphorylation of human IGFBP-1
lowers its affinity for IGF-I by 6-fold (9), and the addition of
dephosphorylated IGFBP-1 to cells in culture has been shown to enhance
the capacity of IGF-I to stimulate DNA synthesis (1, 10). In contrast, phosphorylated IGFBP-1 inhibits IGF-I actions (2, 10). Our laboratory
has previously demonstrated that polymerized forms of IGFBP-1 were
present in human amniotic fluid and were abundant in the
chromatographic fractions of IGFBP-1 purified from amniotic fluid that
had the capacity to potentiate IGF-I-stimulated thymidine incorporation
into porcine smooth muscle cells (pSMC) (1, 7, 10). In contrast,
chromatographic fractions of purified IGFBP-1 that inhibited IGF-I
actions had no detectable multimers (10). The mechanism(s) accounting
for the appearance of these multimers have not been determined.
Tissue transglutaminase (transglutaminase type II) is a
calcium-dependent enzyme that catalyzes the formation of
isopeptide cross-links between glutamine and lysine residues and also
can attach primary amines to peptide-bonded glutamines. The isopeptide cross-links are stable and resistant to proteolysis, thereby increasing resistance to chemical, enzymatic, or mechanical disruption. Tissue transglutaminase (Tg) activity is widely distributed in many tissues and organs, and it has been localized to the cytoplasm (11), cell
surface (12), and extracellular matrix (13). The ability of some
proteins to polymerize and form covalently linked multimers is
dependent upon this activity (14, 15). Therefore, we speculated that
IGFBP-1 might be a substrate for tissue transglutaminase and that
polymerization of IGFBP-1 might change its ability to modulate IGF-I
actions in vitro. The current studies were undertaken to
determine whether tissue transglutaminase could catalyze the polymerization of IGFBP-1, to identify the factors that enhanced or
inhibited polymerization, and to analyze the effect of polymerization upon IGF-I actions.
Materials--
pSMC were obtained from aortic explants
and maintained in our laboratory as previously described (16). Human
fetal fibroblasts (GM 10) were purchased from the Human Mutant Genetic
Cell Repository (Camden, NJ). Calf serum was purchased from Colorado
Laboratories Inc. (Logan, UT). [35S]Methionine was from
ICN Biomedical Inc. (Costa Mesa, CA). Tissue culture media, fetal calf
serum, penicillin, streptomycin, and Geneticin (G418) were from Life
Technologies, Inc. IGF-I was a gift from Genentech (South San
Francisco, CA). Tissue transglutaminase and cystamine were purchased
from Sigma. Native IGFBP-1 was obtained from media conditioned by CHO
cells that had been transfected with an expression plasmid that
contained the human IGFBP-1 cDNA (CHOBP1-D6) as previously
described (3). Polyclonal rabbit antiserum for human IGFBP-1 was
prepared as previously described (17). A monoclonal antibody to tissue
transglutaminase was purchased from NeoMarker (Fremont, CA).
Polyvinylidene difluoride transfer membranes were obtained from
Millipore Corp. (Bedford, MA).
Tissue Culture--
Human fetal fibroblast (GM 10) cells were
maintained in Eagle's minimum essential medium (EMEM) (Life
Technologies, Inc., Gaithersburg, MD) supplemented with 10% (v/v) calf
serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). pSMC
were cultured in Dulbecco's modified Eagle's medium (DMEM) (Life
Technologies) supplemented with 10% fetal calf serum and antibiotics
(100 units/ml penicillin and 100 µg/ml streptomycin). The medium was
changed on the third day after plating, and the cultures reached 80%
confluency in another 3 days, at which time they were used for
experiments. GM-10 cells were used between passages 8 and 16, and pSMC
were used between passages 4 and 8.
Polymerization of IGFBP-1 with Tissue
Transglutaminase--
IGFBP-1 (50 ng) was incubated with 0.5 microunits of pure tissue transglutaminase (purified from guinea pig
liver) in reaction buffer containing 100 mM Tris-HCl, 20 mM dithiothreitol, 2.5 mM CaCl2,
10% (v/v) glycerol, pH 8.5, for the indicated times at 37 °C (final
assay volume 30 µl). The reactions were terminated by the addition of
4× Laemmli buffer containing 400 mM dithiothreitol and
boiling for 10 min. The proteins were analyzed by 10% SDS-PAGE with
immunoblotting for IGFBP-1 (7-9). The immune complexes were visualized
using an alkaline phosphatase-conjugated anti-rabbit IgG and a
phosphatase-dependent color development system as
previously described (18). For Western ligand blotting, the proteins
were transferred to polyvinylidene difluoride membranes; probed in 4.0 ml of 10 mM Tris-HCl, 150 mM NaCl, 0.05%
sodium azide, 1% bovine serum albumin (pH 7.4) containing
125I-IGF-I (100,000 cpm/ml), specific activity 125 µCi/µg; and then washed and visualized by autoradiography as
described previously (19). Densitometric quantification of the bands
was performed by scanning the film and analyzing the band intensities
using NIH Image.
Polymerization of IGFBP-1 in Human Fetal Fibroblast in
Cultures--
GM 10 cells were grown to 80% confluency on 24-well
culture plates (Falcon Labware, division of Becton Dickinson, Franklin, NJ; catalog no. 3047). The cultures were rinsed once with
serum-free EMEM and incubated for 5 h in 250 µl of same medium.
After the incubation, medium was replaced with serum-free EMEM
containing 1 µg/ml of either native or dephosphorylated IGFBP-1 and
incubated for 20-40 min at 37 °C, and then 30 µl of each sample
was collected. The reactions were terminated by the addition of 4×
Laemmli buffer containing 400 mM dithiothreitol. The
reaction products were analyzed by SDS-PAGE with Western immunoblotting
for IGFBP-1.
Preparation of Membrane Extract from Human Fibroblasts--
GM
10 cells were grown to confluency on 10-cm tissue culture dishes
(Falcon; catalog no. 3003). The cultures were rinsed with serum-free EMEM and incubated overnight in the same medium in the presence or absence of IGF-I (30 ng/ml). The cells were scraped from the dishes with a cell scraper and washed three times with ice-cold Ca2+- and Mg2+-free phosphate-buffered
saline. The washed cells were sonicated in sonication buffer (20 mM Hepes, pH 7.4, 100 mM NaCl, 5 mM
KCl, 0.3 mM Na2HPO4, 1 mM
NaHCO3, 250 mM sucrose, 1 mM
phenylmethanesulfonyl fluoride, 1 µg/ml pepstatin, 1 µg/ml
leupeptin). The cell lysate was centrifuged at 16,000 × g for 15 min, and the pellet was washed with the sonication
buffer twice, solubilized in the same buffer containing 10 mM CHAPS, and centrifuged at 16,000 × g
for 15 min. The transglutaminase activity in the detergent-soluble
membrane extracts was determined by adding 10.5 µg of membrane
protein to 30 µl of the assay buffer described previously except that the tubes also contained 25 ng of IGFBP-1. To determine whether polymerization was being catalyzed by Tg, some tubes contained cystamine (20 mM), a specific inhibitor of Tg activity
(20).
Plasmid Construction for Expression of the IGFBP-1 Mutant--
A
full-length human IGFBP-1 cDNA was cloned into the
HindIII and XbaI sites of a mammalian expression
vector pRcRSV (pRcRSV-hBP-1) that was prepared from the plasmid pRcCMV
(Invitrogen, La Jolla, CA). The pRcRSV-hBP-1 contains a bacteriophage
origin of replication that allows production of plasmid DNA in a
single-stranded form suitable for site-directed mutagenesis. The mutant
IGFBP-1 cDNA was generated using site-directed mutagenesis (21).
Single-stranded phagemid DNA was generated from pRcRSV-hBP-1, and the
substitutions were introduced using synthetic oligonucleotide as a
substrate for antisense DNA synthesis. The sequence of complementary
oligonucleotide was 3'-CGTGCAGAGGTGCCGCCTCCCCCGGCA-5'. This encoded
conversions of Gln66 to Ala and Gln67 to Ala
(designated pRcRSV-hBP-1Q66A/Q67A). The cDNA was sequenced, and the
clones containing correct sequence were amplified. The plasmid DNA was
purified using silica gel anion exchange resin chromatography (Qiagen,
Chatsworth, MA).
Transfection of Chinese Hamster Ovary Cells--
CHO-K1 cells
were obtained from Lineberger Comprehensive Center Tissue Culture
Facility (University of North Carolina). Cells were maintained in
Protein Purification--
The Q66A/Q67A mutant IGFBP-1 was
purified from conditioned medium of the
pRcRSVhBP-1Q66A/Q67A-transfected CHO cells using procedures identical
to those used to purify native IGFBP-1 as described previously (3).
Twenty micrograms of native IGFBP-1 was dephosphorylated by incubating
it with 50 units of calf intestinal alkaline phosphatase at 37 °C in
50 mM Tris-HCl, 0.1 mM EDTA (pH 8.5) for
16 h and repurified by reverse phase high pressure liquid chromatography. The purity of each form of IGFBP-1 was determined by
SDS-PAGE with silver staining. Analysis of each form revealed a single
monomeric band.
[35S]Methionine Incorporation into pSMC--
pSMC
were grown to 80% confluency on 24-well culture plates. The cultures
were rinsed once with serum-free DMEM and incubated overnight in the
same media. After the incubation, media were replaced with 0.25 ml of
methionine-free DMEM supplemented with 2 mM
CaCl2 for 4 h in the presence of 0.05 µCi/well
[35S]methionine (specific activity, 1, 206 Ci/mmol),
IGF-I (0 or 50 ng/ml), and various concentrations of either native or
mutant IGFBP-1 (0-2,500 ng/ml). The plates were placed on ice, washed with ice-cold phosphate-buffered saline twice, and incubated with 10%
trichloroacetic acid for 10 min. The trichloroacetic acid-precipitable radioactivity was solubilized in 1% SDS, 0.1 N NaOH and
scintillation mixture (ScintiSafeTM Econo2; Fischer, Fair
Lawn, NJ) and counted in a liquid scintillation counter (Beckman
Instruments). Statistical comparisons were performed using a paired
Student's t test.
IGFBP-1 Is a Substrate for Tissue Transglutaminase--
Following
exposure to pure tissue Tg, IGFBP-1 formed covalent multimers that were
stable during SDS-PAGE and were not altered with reducing agents (Fig.
1A). Incubation with tissue
transglutaminase was associated with a time-dependent
increase in dimer and multimer formation (Fig. 1A). Scanning
densitometry showed that 57% of the total IGFBP-1 was present as
polymerized forms after 5 h of incubation. When increasing
concentrations of Tg were added, there was a progressive increase in
dimer formation (Fig. 1B).
Dephosphorylated IGFBP-1 Polymerizes More Readily Compared with
Native IGFBP-1--
To determine whether the phosphorylation status of
IGFBP-1 affects its ability to polymerize, dephosphorylated IGFBP-1 (50 ng) was incubated with increasing concentrations of tissue Tg (0-1.0
microunits/tube) for 30 min at 37 °C, and the products of the
reaction were analyzed by SDS-PAGE using reduced conditions (Fig.
2). Dephosphorylated IGFBP-1 polymerized
after exposure to 0.1 microunits of tissue Tg, whereas native IGFBP-1
required 0.4 microunits (Fig. 1B) or 0.5 microunits (Fig. 2)
of Tg to detect polymerization. Dephosphorylated IGFBP-1 polymerized to
a greater extent with each concentration of tissue Tg that was used.
Therefore, dephosphorylation of IGFBP-1 enhances its susceptibility to
Tg-catalyzed polymerization in vitro.
Alteration of Residues Gln66 and
Gln67 in IGFBP-1 Inhibits Its Polymerization by
Tissue Transglutaminase--
To determine whether the adjacent
glutamine residues, Gln66 and Gln67, were
involved in the formation of Tg catalyzed polymerization, the Q66A/Q67A
IGFBP-1 mutant was incubated with various concentrations of tissue Tg
for 30 min at 37 °C, and the products of the reaction were analyzed
by SDS-PAGE using reducing conditions. The Q66A/Q67A mutant IGFBP-1
required 1.0 microunit of Tg to polymerize (Fig. 3), and only minimal amounts of dimer
were detected. In contrast, the same concentration of native IGFBP-1
was polymerized to a much greater extent by 0.5 microunits of Tg. This
reveals that substitution for Gln66-Gln67 in
IGFBP-1 inhibits transglutamination and that
Gln66-Gln67 is probably one of the
cross-linking sites in IGFBP-1.
Enhancement of Polymerization of IGFBP-1 by IGF-I--
Native
IGFBP-1 was incubated with IGF-I at room temperature for 30 min. The
IGF-I·IGFBP-1 complex was then incubated with 0.3 microunits
of tissue Tg for 1 h at 37 °C, and the reaction products were
analyzed by SDS-PAGE using reducing conditions (Fig. 4). The complexes containing IGF-I showed
enhanced polymerization of IGFBP-1 by Tg compared with IGFBP-1 that was
not exposed to IGF-I. Incubation with IGF-I and no Tg had no
effect.
Polymerization of IGFBP-1 by Exposure to Fetal Fibroblast
Cultures--
Polymerization of IGFBP-1 had been observed previously
in cell culture supernatants (7). Since tissue Tg is widely distributed in many tissues and organs, we determined if exposure of IGFBP-1 to
fibroblast cultures would allow polymerization. Native and dephosphorylated IGFBP-1 were polymerized within 20 min, when IGFBP-1
(1 µg/ml) was added to GM 10 fetal fibroblast cultures (Fig.
5). Dephosphorylated IGFBP-1 was more
readily polymerized compared with native IGFBP-1 by cell-associated
Tg.
Polymerization of IGFBP-1 Incubated with Fetal Fibroblast Membrane
Extract--
Since tissue Tg is not secreted into the cell culture
medium, we hypothesized that the polymerization of IGFBP-1 might be occurring on cell surfaces. To determine whether cellular membrane extracts could facilitate polymerization of IGFBP-1 and whether IGF-I
altered the extent of polymerization, IGFBP-1 was incubated with
fibroblast membrane extracts. Cell membrane extracts were prepared from
cells that had been incubated for 14 h, and the samples were
analyzed by SDS-PAGE under the reducing conditions (Fig.
6A). Exposure of the cultures
to IGF-I enhanced polymerization of both phosphorylated and
dephosphorylated IGFBP-1 compared with membrane extracts from control
cultures exposed only to serum-free medium. However, if IGF-I was added
in vitro directly to the membrane extract, the extent of
polymerization was much greater. When cystamine, a specific inhibitor
of Tg, was coincubated with IGFBP-1 and the membrane extracts, no
polymerization was observed (Fig. 6B). Fig. 6C
shows that these cellular membrane extracts contained equivalent concentrations of Tg.
Q66A/Q67A Mutant IGFBP-1 Inhibits IGF-I-stimulated Protein
Synthesis on pSMC in Culture--
pSMC were grown to 80% confluency
in 24-well culture plates and the ability of IGF to stimulate
[35S]methionine incorporation into protein was
determined. IGF-I stimulated the incorporation of
[35S]methionine into pSMC by 60% above the basal level.
The addition of native IGFBP-1 at concentrations as high as 1,000 ng/ml
reduced this response by 20%, although the decrease was not
significant (p = NS). Native IGFBP-1 (2,500 ng/ml) was
required to obtain complete inhibition. In contrast, the addition of
1,000 ng/ml Q66A/Q67A mutant IGFBP-1 resulted in complete inhibition of
protein synthesis that had been stimulated by IGF-I (p < 0.001) (Fig. 7). Analysis of the forms
of IGFBP-1 that were present in the media at the end of the incubation
showed that the native IGFBP-1 polymerized, but the Q66A/Q67A mutant
IGFBP-1 was present only in the monomeric form. The scanning
densitometric values of 1,000 ng/ml of mutant IGFBP-1 and 2500 ng/ml of
native IGFBP-1 showed that approximately the same amount of monomeric
IGFBP-1 was present. When the polyvinylidene difluoride membrane was
probed with radiolabeled IGF-I, the intensity of the native IGFBP-1
monomeric band was reduced compared with Q66A/Q76A mutant IGFBP-1
monomer. More importantly, the native IGFBP-1 polymeric form did not
bind to radiolabeled IGF-I, and no band could be detected.
The current studies demonstrate that IGFBP-1 is a substrate for
tissue Tg and that Gln66-Gln67 is a one of the
amine acceptor sites in IGFBP-1. This polymerization-resistant mutant
form of IGFBP-1 potently inhibits IGF-I-stimulated protein synthesis
into pSMC. In contrast, when polymerizable native IGFBP-1 was added,
much higher concentrations were required to inhibit IGF-I action. The
polymerization of IGFBP-1 by tissue Tg was also shown to be altered by
the phosphorylation state of IGFBP-1 and by the presence of IGF-I in
the incubation buffer.
Numerous proteins have been demonstrated as substrates for Tg; however,
a general consensus recognition sequence for transglutaminase has not
been determined. Two directly adjacent glutamine residues have
frequently been identified as amine-accepting sites. The
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-modified Eagle's minimum essential medium containing 10% fetal
calf serum, penicillin, and streptomycin. CHO cells were transfected
with the pRcRSV-hBP-1Q66A/Q67A using a standard calcium phosphate
precipitation procedure (22). The positive clones were selected with
800 µg/ml of G418 and maintained in a long term culture in 400 µg/ml G418.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Polymerization of IGFBP-1 with tissue
transglutaminase. Native IGFBP-1 (50 ng) was incubated with 0.5 microunits (µU)/tube of tissue transglutaminase
for the indicated time periods (A) or with increasing
concentrations (0-0.5 microunits/tube) of tissue transglutaminase for
30 min (B) at 37 °C. The reaction products were analyzed
by 10% SDS-PAGE followed by immunoblotting using human IGFBP-1
antiserum. The arrows denote monomer, dimer, and multimer
forms of IGFBP-1. For A, the intensities of all of the bands
were determined by scanning densitometry. The values shown represent
the percentage of total scanning units that was detected as the IGFBP-1
monomer.
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Fig. 2.
Polymerization of dephosphorylated IGFBP-1
with increasing concentrations of tissue transglutaminase.
Dephosphorylated IGFBP-1 was prepared as described under
"Experimental Procedures." To determine whether its phosphorylation
state would affect its polymerization, native IGFBP-1 (N) or
dephosphorylated IGFBP-1 (D) (50 ng) was incubated with
increasing concentrations (0.1-1.0 microunits
(µU)/tube) of tissue transglutaminase for 30 min at 37 °C. The reaction products were analyzed by 10% SDS-PAGE
with Western immunoblotting using human IGFBP-1 antiserum. The
bottom arrow denotes the monomeric form, and the
top arrow denotes the dimeric form of
IGFBP-1.
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Fig. 3.
Polymerization of Q66A/Q67A mutant IGFBP-1
with increasing concentrations of tissue transglutaminase. Native
IGFBP-1 (N) or Q66A/Q67A mutant IGFBP-1 (Q) (50 ng) was incubated with increasing concentrations (0.1-1.0 microunits
(µU)/tube) of tissue transglutaminase for 30 min at 37 °C. The reaction products were analyzed by 10% SDS-PAGE
with Western immunoblotting using human IGFBP-1 antiserum. The
bottom arrow denotes the monomeric form, and the
top arrow denotes the dimeric form of
IGFBP-1.
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Fig. 4.
Enhancement of polymerization of IGFBP-1 by
IGF-I. Native IGFBP-1 (50 ng) was incubated with 1 ng of IGF-I for
1 h at room temperature followed by the addition of 0.3 microunits/tube of tissue transglutaminase, and the incubation was
continued for 1 h at 37 °C. The reaction products were analyzed
by 10% SDS-PAGE with Western immunoblotting using human IGFBP-1
antiserum. The bottom arrow denotes the monomeric
form, and the top arrow denotes the dimeric form
of IGFBP-1.
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Fig. 5.
Polymerization of IGFBP-1 incubated with GM
10 fetal fibroblasts in culture. GM 10 cells were grown to 80%
confluency on 24-well tissue culture plates. Cells were incubated
with serum-free EMEM containing either native (N) or
dephosphorylated (D) IGFBP-1 (1 µg/ml) for the indicated
times. Thirty microliters of each sample was collected and analyzed by
SDS-PAGE with immunoblotting. The bottom arrow
denotes the monomeric form, and the top arrow
denotes the dimeric form of IGFBP-1.
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Fig. 6.
Polymerization of IGFBP-1 incubated with GM
10 fetal fibroblast membrane extracts. GM 10 cells were grown to
confluency on 10-cm tissue culture plates. In A, the cells
were incubated with serum-free EMEM in the absence (lanes
a and b) or presence (lane
c) of IGF-I (30 ng/ml). After the incubation, the cellular
membrane extracts were prepared as described under "Experimental
Procedures." Each membrane extract was incubated with either native
(N) or dephosphorylated IGFBP-1 (D) (25 ng) in
the absence (lanes a and c) or
presence (lane b) of IGF-I (1 ng/tube) for
14 h at 37 °C. The reactions were analyzed by SDS-PAGE with
immunoblotting for IGFBP-1 (A). The bottom
arrow denotes the monomeric form, and the top
two arrows denote the dimeric and trimeric forms
of IGFBP-1, respectively. B, the membrane extract was
incubated with dephosphorylated IGFBP-1 (25 ng) in the presence and
absence of cystamine (20 mM) for 6 h at 37 °C. The
reactions were analyzed by SDS-PAGE with immunoblotting for IGFBP-1.
C, the same membrane extracts were analyzed by SDS-PAGE and
immunoblotted with anti-tissue transglutaminase antibody.
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Fig. 7.
[35S]methionine incorporation
into pSMC protein synthesis was measured using
[35S]methionine incorporation into protein as described
under "Experimental Procedures." pSMC were grown to 80%
confluency on 24-well tissue culture plates. The cultures were
incubated with serum-free DMEM for overnight, and then media were
replaced with methionine-free DMEM containing 0 or 50 ng/ml of IGF-I,
different concentrations of either native ( ) or Q66A/Q67A mutant
IGFBP-1 (
), and [35S]methionine for 4 h. The
amount of [35S]methionine activity incorporated into
cellular protein was measured. The results are expressed as a
percentage of increase over control cultures that were incubated with
methionine-free DMEM without IGF-I or IGFBP-1. Each value is mean ± S.E. of triplicate determinations. The inset shows a
Western ligand blot and immunoblot of the media samples obtained at the
end of the incubation from the cultures exposed to 20 µl of native or
Q66A/Q67A IGFBP-1. The scanning units that were detected in the
monomeric band (lower arrow) are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain of
fibrinogen (23) and fibronectin (24), which contain adjacent
glutaminase residues, are substrates for factor XIIIa. Plasminogen
activator inhibitor-2 (25),
A3-crystallin (26), and BM-40
(osteonectin) (27) are substrates for tissue Tg, and they have two
directly adjacent glutamine residues that have been identified as amine
acceptor sites. In addition, positively or negatively charged amino
acid residues are often found near amine acceptor sites in the sequence
of these proteins. Analysis of the sequences following
Gln66-Gln67 in IGFBP-1 showed that both
positively (Glu65) and negatively (His70)
charged residues are present (Fig. 8).
The positions of charged residues in several other substrates for
tissue Tg are shown for comparison. Because of these similarities, we
speculated that the adjacent glutamine residues in IGFBP-1
(Gln66-Gln67) could be amine acceptor sites.
The Q66A/Q67A mutant form of IGFBP-1 that was generated was resistant
to polymerization by pure tissue Tg and following exposure to cell
cultures that contained tissue Tg in their membrane extracts. These
findings strongly support the conclusion that tissue Tg is using these
residues as amine acceptor sites for cross-linking in IGFBP-1.
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Fig. 8.
Comparison of amino acid sequences flanking
the two adjacent glutamine residues that have been identified as amine
acceptor sites. Two adjacent glutamine residues (amine acceptor
site) are underlined, and charged residues are in
boldface type for IGFBP-1 and other proteins that
have been noted to undergo transglutamination.
IGFBP-1, -2, -3, and -5 have been shown to stimulate or inhibit IGF-I actions in vitro (28). Most studies have shown that excess concentrations of IGFBP-1 inhibit the IGF-I actions in vitro, particularly if high concentrations of IGFBP-1 are added using serum-free conditions (2, 10, 29-31). When platelet-poor plasma is added with IGF-I and dephosphorylated IGFBP-1, IGFBP-1 has been shown to potentiate the IGF-I-stimulated DNA synthesis in cultured fibroblasts and pSMC (1). The phosphorylation state of IGFBP-1 was also shown to be an important determinant of this response, and dephosphorylated IGFBP-1 was much more effective than phosphorylated IGFBP-1 (1, 9, 32). Analysis of the cell culture supernatants at the end of these incubations showed that the added IGFBP-1 had polymerized. Therefore, the current studies were undertaken to determine whether IGFBP-1 polymerized as a result of exposure to transglutaminase, if its phosphorylation status of IGF-I affected its capacity to polymerize, and if polymerization would be associated with a decrease in the ability to inhibit IGF-I-stimulated protein synthesis.
IGFBP-1 is a phosphoprotein that undergoes serine phosphorylation (9). Several phosphoisoforms of IGFBP-1 have been detected in serum, and the presence of highly phosphorylated forms of IGFBP-1 in physiologic fluids has been associated with conditions in which the target tissue actions of IGF-I are being inhibited (32, 33). Separation of phosphoisoforms of IGFBP-1 by anion exchange chromatography has shown that chromatographic fractions that contain phosphorylated forms inhibit IGF-I actions, whereas those that are enriched in nonphosphorylated forms did not inhibit IGF-I actions (9, 10, 28). In this study, dephosphorylated IGFBP-1 polymerized to a much greater extent compared with phosphorylated IGFBP-1. Since the ability of IGFBP-1 to polymerize has also been shown to be related to the loss of its capacity to inhibit IGF-I actions, these results suggest that this may be one mechanism by which phosphorylation of IGFBP-1 may enhance its capacity to inhibit IGF-I actions.
The results of several studies have shown that the loss of the ability of IGFBP-1, IGFBP-3, and IGFBP-5 to inhibit IGF-I actions is associated with a reduction in their affinities for IGF-I (9, 34-38). IGFBPs have high affinity for IGF-I, and their affinities are greater than that of the type-I IGF receptor. Therefore, factors such as dephosphorylation (3) or adherence to cell surfaces (18), which lower their affinity for IGF-I, will reduce their capacity to inhibit IGF-I actions, and in some cases this has been associated with stimulation of IGF-I actions (1). Our results showed that the multimeric form of IGFBP-1 (Fig. 7) that occurred following transglutamination had very low binding capacity for IGF-I and that polymerization was associated with a loss of IGFBP-1's inhibitory effect. Immunoblotting analysis at the end of incubation revealed that the concentration of monomeric IGFBP-1 correlated with the extent of inhibition of IGF-I action. This result suggests that polymerization is also an important mechanism by which the inhibitory effect of IGFBP-1 is attenuated.
The N-terminal domain of IGFBP-3 and -5 contain a binding site for IGF-I (39). In IGFBP-5, the IGF-I binding site is contained within amino acids 49-74, and in IGFBP-3 it is between residues 50 and 75. For IGFBP-5, the charged residue Lys68 and the hydrophobic residues Pro69, Leu70, Leu73, and Leu74 are required for binding. In the IGFBP-1, residues Pro68, Leu69, and Leu70 are homologous, suggesting that this is also an important site in IGFBP-1 for IGF-I binding. Our results demonstrate that following transglutamination, which occurs through Gln66-Gln67, the affinity of IGFBP-1 is markedly reduced. This suggests that cross-linking through these sites alters the conformation of the adjacent IGF-I binding site such that IGF-I has reduced access.
Our results also show that IGF-I stimulates the polymerization of IGFBP-1 by pure tissue Tg and by the Tg that was contained in fibroblast membrane extracts. The effect is direct and does not require the presence of some other component. Since IGF-I is probably binding to the region of IGFBP-1 that contains the amine acceptor sites, its presence may induce a conformational change in IGFBP-1 that stabilizes the conformation of the multimeric forms and facilitates cross-linking by tissue Tg.
Recently, tissue Tg has been shown to associate with 1
and
3 integrins, and it has been estimated that 5-40%
of
1 integrins on the cell surface are complexed with
tissue transglutaminase. Tissue Tg on the cell surface has been shown
to increase cell adhesion and spreading on fibronectin (40). IGFBP-1
has been shown to bind to the
5
1 integrin
and stimulate cell migration independently of IGF-I (41). Ligand
occupancy of
5
1 by IGFBP-1 has been shown
to induce focal adhesion kinase and PI 3-kinase. Therefore, IGFBP-1
could form an IGFBP-1-tissue Tg complex with the
1
integrin, and transglutamination of IGFBP-1 could potentially modulate
the
5
1 integrin-linked signaling.
Elucidation of the interaction between IGFBP-1 and tissue Tg will be a
great help in further analysis of the biological significance of
multiple IGFBP-1/IGF-I interactions that occur in vivo.
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ACKNOWLEDGEMENT |
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We thank George Mosley for help in preparing the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL-56580.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.
To whom correspondence should be addressed: Dept. of Medicine,
CB7170, University of North Carolina, School of Medicine, Chapel Hill, NC 27599. Tel.: 919-966-4735; Fax: 919-966-6025; E-mail: endo@med.unc.edu.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M008359200
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ABBREVIATIONS |
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The abbreviations used are: IGF, insulin-like growth factor; IGFBP-1, insulin-like growth factor-binding protein; Tg, tissue transglutaminase; pSMC, porcine smooth muscle cells; CHO, Chinese hamster ovary; EMEM, Eagle's minimum essential medium; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Elgin, R. D., Busby, W. H., and Clemmons, D. R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3254-3258[Abstract] |
2. | Ritvos, O., Ranta, T., Jalkanen, J., Suikkari, A.-M, Voutilainen, R., Bohn, H., and Rutanen, E.-M. (1988) Endocrinology 122, 2150-2157[Abstract] |
3. | Jones, J. I., D'Ercole, A. J., Camacho-Hubner, C., and Clemmons, D. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7481-7485[Abstract] |
4. | Hoeck, W. G., and Mukku, V. R. (1994) J. Cell. Biochem. 56, 262-273[Medline] [Order article via Infotrieve] |
5. | Kanzaki, S., Hilliker, S., Baylink, D. J., and Mohan, S. (1994) Endocrinology 134, 1254-1262[Abstract] |
6. | Sakai, K., Iwashita, M., and Takeda, Y. (1997) Endocr. J. 44, 409-417[Medline] [Order article via Infotrieve] |
7. | Busby, W. H., Hossenlopp, P., and Clemmons, D. R. (1989) Endocrinology 125, 773-777[Abstract] |
8. | Brinkman, A., Kortleve, D. J., Zwarthoff, E. C., and Drop, S. L. S. (1991) Mol. Endocrinol. 5, 987-994[Abstract] |
9. |
Jones, J. I.,
Busby, W. H.,
Wright, G.,
Smith, C. E.,
Kimack, N. M.,
and Clemmons, D. R.
(1993)
J. Biol. Chem.
268,
1125-1131 |
10. |
Busby, W. H.,
Klapper, D. G.,
and Clemmons, D. R.
(1988)
J. Biol. Chem.
263,
14203-14210 |
11. |
Greenberg, C. S.,
Birckbichler, P. J.,
and Rice, R. H.
(1991)
FASEB. J.
5,
3071-3077 |
12. | Gaudry, C. A., Verderio, E., Jones, R. A., Smith, C., and Griffin, M. (1999) Exp. Cell Res. 252, 104-113[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Barsigian, C.,
Stern, A. M.,
and Martinez, J.
(1991)
J. Biol. Chem.
266,
22501-22509 |
14. | Folk, J. E. (1980) Annu. Rev. Biochem. 49, 517-531[CrossRef][Medline] [Order article via Infotrieve] |
15. | Aeschlimann, D., and Paulsson, M. (1994) Thromb Haemost. 71, 402-415[Medline] [Order article via Infotrieve] |
16. | Gockerman, A., Prevette, T., Jones, J. I., and Clemmons, D. R. (1995) Endocrinology 136, 4168-4173[Abstract] |
17. | Busby, W. H., Snyder, D. K., and Clemmons, D. R. (1988) J. Clin. Endocrinol. Metab. 67, 1225-1230[Abstract] |
18. | McCusker, R. H., and Clemmons, D. R. (1988) J. Cell. Physiol. 137, 505-512[Medline] [Order article via Infotrieve] |
19. | Hossenlopp, P., Seurin, D., Segoria-Quinson, B., Hardouin, S., and Binoux, M. (1986) Anal. Biochem. 154, 138-143[Medline] [Order article via Infotrieve] |
20. | Siefring, G. E., Jr., Apostol, A. B., Velasco, P. T., and Lorand, L. (1978) Biochemistry 17, 2598-2604[Medline] [Order article via Infotrieve] |
21. |
Parker, A.,
Clarke, J. B.,
Busby, W. H.,
and Clemmons, D. R.
(1996)
J. Biol. Chem.
271,
13523-13529 |
22. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology , pp. 9.1.1-9.1.4, Wiley-Interscience, New York |
23. | Jones, J. I., and Clemmons, D. R. (1995) Endocr. Rev. 16, 3-34[Medline] [Order article via Infotrieve] |
24. | Chen, R., and Doolittle, R. F. (1971) Biochemistry 10, 4486-4491 |
25. | McDonagh, R. P., McDonagh, J., Petersen, T. E., Thogersen, H. C., Skorstengaard, K., Sottrup-Jensen, L., Magnusson, S., Dell, A., and Morris, H. R. (1981) FEBS Lett. 127, 174-178[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Jensen, P.,
Schuler, E.,
Woodrow, G.,
Richardson, M.,
Goss, N.,
Hojrup, P.,
Petersen, T. E.,
and Rasmussen, L. K.
(1994)
J. Biol. Chem.
269,
15394-15398 |
27. | Berbers, G. A. M., Feenstra, R. W., Van den Bos, R., Hoekman, W. A., Bloemendal, H., and DeJong, W. W. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7017-7020[Abstract] |
28. |
Hochenadl, C.,
Mann, K.,
Mayer, U.,
Timpl, R.,
Paulsson, M.,
and Aeschlimann, D.
(1995)
J. Biol. Chem.
270,
23415-23420 |
29. | Frauman, A. G., Tsuzaki, S., and Moses, A. C. (1989) Endocrinology 124, 2289-2296[Abstract] |
30. | Burch, W. W., Correa, J., Shaveley, J. E., and Powell, D. R. (1990) J. Clin. Endocrinol. Metab. 70, 173-180[Abstract] |
31. | Cambell, P. G., and Novack, J. F. (1991) J. Cell. Physiol. 149, 293-300[Medline] [Order article via Infotrieve] |
32. |
Frost, J. P.,
and Tseng, L. T.
(1991)
J. Biol. Chem.
266,
18082-18088 |
33. |
Westwood, M.,
Gibson, J. M.,
and White, A.
(1997)
Endocrinology
138,
1130-1136 |
34. | Jones, J. I., Gockerman, A., Busby, W. H., Camacho-Hubner, C., and Clemmons, D. R. (1993) J. Cell Biol. 121, 679-687[Abstract] |
35. | DeMellow, J. S. M., and Baxter, R. C. (1988) Biochem. Biophys. Res. Commun. 156, 199-204[Medline] [Order article via Infotrieve] |
36. | McCusker, R. H., Camacho-Hubner, C., Bayne, M. L., Cascieri, M. A., and Clemmons, D. R. (1990) J. Cell. Physiol. 144, 244-253[Medline] [Order article via Infotrieve] |
37. | Tsuboi, R., Shi, C. M., Sato, C., Cox, G. N., and Ogawa, H. (1995) J. Invest. Dermatol. 104, 199-203[Abstract] |
38. |
Galiano, R. D.,
Zhao, L.,
Clemmons, D. R.,
Roth, S. I.,
Lin, X.,
and Mustoe, T. A.
(1996)
J. Clin. Invest.
98,
2462-2468 |
39. |
Imai, Y.,
Moralez, A.,
Andog, U.,
Clarke, J. B.,
Busby, W. H.,
and Clemmons, D. R.
(2000)
J. Biol. Chem.
275,
18188-18194 |
40. |
Akimov, S. S.,
Krylov, D.,
Fleischman, L. F.,
and Belkin, A. M.
(2000)
J. Cell Biol.
148,
825-838 |
41. |
Frost, R. A.,
and Lang, C. H.
(1999)
Endocrinology
140,
3962-3970 |