From the Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Among the well defined insulin-like growth factor
(IGF)-binding proteins (IGFBPs), IGFBP-3 is characterized by its
interaction with an acid-labile glycoprotein (ALS) in the presence of
IGFs. To identify the structural determinants on IGFBP-3 required for ligand binding and cell association, five recombinant human IGFBP-3 variants were expressed in Chinese hamster ovary cells: deletions of
amino acids 89-264, 89-184, and 185-264, and site-specific mutations
228KGRKR MDGEA and
253KED
RGD. The basic carboxyl-terminal region of
IGFBP-3 was required for binding to heparin. The deletion variants had
greatly decreased IGF binding ability as assessed by ligand blotting
and solution binding assays; affinity cross-linking indicated at least
a 20-fold decrease in IGF affinity. The RGD mutant had a 4-6-fold
reduced affinity for both IGFs, but the MDGEA mutant bound IGF-I with near normal affinity and IGF-II with a 3-fold reduction in affinity. The three deletion variants were incapable of binding ALS; but of the
site-specific variants, the MDGEA mutant bound ALS with 90% lower
affinity (Ka = 2.5 ± 0.9 liters/nmol) than seen for rhIGFBP-3 (Ka = 24.3 ± 5.2 liters/nmol), whereas the RGD mutation had no effect on ALS affinity
(Ka = 21.7 ± 4.5 liters/nmol). The ability of
IGFBP-3 to associate with the cell surface was lost in variants lacking
residues 185-264 and in the 228KGRKR
MDGEA mutant.
We conclude that residues 228-232 of IGFBP-3 are essential for cell
association and are required for normal ALS binding affinity.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The insulin-like growth factor-binding proteins (IGFBPs)1 are a family of at least six related proteins involved in regulating the bioavailability of insulin-like growth factors, IGF-I and IGF-II. The IGFBP structure may be divided into cysteine-rich amino- and carboxyl-terminal domains, which show considerable structural conservation among IGFBP-1 to -6, and a central domain that is unique for each IGFBP. IGFBP-3, a glycoprotein of 40-45 kDa, is characterized by its ability to bind to another glycoprotein, the 85-kDa acid-labile subunit (ALS), in the presence of either IGF-I or IGF-II to form a ternary complex of 150 kDa (1). Because the majority of serum IGFs are bound in this form (2, 3), the ternary complex acts essentially as a circulating reservoir of IGFs and regulates the delivery of the IGFs to target tissues.
IGFBP-3 has been implicated at the cellular level as a modulator of IGF-I action (4, 5). Soluble IGFBP-3 can inhibit IGF-I activity by sequestering the peptide and consequently preventing interaction with its receptor. In contrast, potentiation of IGF-I action has been attributed to cell surface-associated IGFBP-3 (5). Several studies have also suggested that IGFBP-3 may modulate cell growth independently of IGFs (6-8). This IGF-independent growth-inhibitory effect was recently shown to be mediated by the direct induction of apoptosis by IGFBP-3 (9).
This multifaceted role of IGFBP-3 has led to extensive studies on its regulation, expression, distribution, and function in cultured cells and in animal models (3, 10-12). To date however, there have been few studies aimed at elucidating the structural determinants involved in the protein-protein and protein-cell interactions required for IGFBP-3 function. In this study we describe the generation and expression of cDNAs encoding the natural form, three deletion mutants, and two site-specific mutants of human IGFBP-3. The resulting recombinant proteins have allowed the delineation of domains involved in IGF-I and ALS binding and in cell surface association.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents--
All radiolabeled proteins used were prepared as
described previously (13, 14). Restriction enzymes were from Promega
Corp. (Madison, WI). T7 DNA polymerase was from Pharmacia Biotech Inc. (Uppsala, Sweden). Pfu DNA polymerase was from Stratagene
(La Jolla, CA). Hexadimethrine bromide (Polybrene), dexamethasone, hypoxanthine, xanthine, thymidine, and mycophenolic acid were purchased
from Sigma Chemical Co. (St. Louis, MO) and aminopterin from Life
Technologies Inc. (Gaithersburg, MD). Nucleoside-free -modified
Eagle's medium (
-MEM) and fetal calf serum were from Cytosystems
(North Ryde, NSW, Australia).
cDNA Constructs--
A 1,080-base pair
EcoRI-PvuII fragment, excised from ibp.118 (15),
containing the full coding sequence of hIGFBP-3 (provided by Dr. W. I.
Wood, Genentech, South San Francisco, CA) was inserted into pSELECT
(Promega). Using this recombinant plasmid as cDNA template and
pairs of oligonucleotides (see below), fragments containing full or
partial hIGFBP-3 coding sequences were amplified by Pfu DNA
polymerase in polymerase chain reactions and cloned into the expression
vector pMSG (Pharmacia). This generated expression plasmids rhIGFBP-3,
rhIGFBP-3[89-264], rhIGFBP-3[
185-264], and rhIGFBP-3[
89-184] (Fig.
1A). Site-directed mutagenesis
(16), employing oligonucleotides and the pSELECT-hIGFBP-3 plasmid as the mutagenesis vector, was carried out to introduce specific mutations
in vitro. cDNA fragments amplified from these mutated plasmids were cloned into pMSG to generate expression plasmids rhIGFBP-3[253KED
RGD] and
rhIGFBP-3[228KGRKR
MDGEA], each carrying the
IGFBP-1 sequence analogous to the mutated region (Fig. 1A).
The hIGFBP-3 coding sequences of each construct were verified by
plasmid DNA sequencing (17).
|
Cell Culture and Transfection--
CHO cells were grown in
-MEM supplemented with 10% (v/v) fetal calf serum at 37 °C. For
transfection, the cells were plated out at 6 × 105
cells/75-cm2 flask, incubated for 24 h, and then
transfected with 20 µg of DNA of each rhIGFBP-3 expression plasmid or
pMSG in the presence of 100 µg of Polybrene (18). The plasmids
contain the guanine phosphoribosyltransferase (gpt) gene,
which confers resistance to mycophenolic acid. The transfected cells
were cultured in GPT selection medium for 21 days to select for stable
transfectants. The medium for GPT selection consists of
-MEM
supplemented with 10% (v/v) fetal calf serum, 250 µg/ml xanthine, 25 µg/ml mycophenolic acid, 2 µg/ml aminopterin, 10 µg/ml thymidine,
and 15 µg/ml hypoxanthine. Expression of the recombinant proteins is
driven by the mouse mammary tumor virus long terminal repeat promoter
on pMSG which has a glucocorticoid-responsive element, hence expression
is inducible by dexamethasone. Following the selection period, the
mixed population of each transfected cell line was grown to confluence
and the media changed to serum-free
-MEM supplemented with 0.1%
(w/v) bovine serum albumin (BSA) and 10 µM dexamethasone.
After 72 h, the conditioned media were collected and stored at
15 °C before assaying for rhIGFBP-3 by a radioimmunoassay (RIA)
specific for hIGFBP-3 (13).
Purification of IGFBP-3 Variants--
Serum-free media
conditioned for 48-72 h by each of the transfected CHO populations
were collected and clarified by centrifugation at 15,300 × g for 20 min. A mixture of protease inhibitors (500 units/ml
aprotinin, 5 µg/ml 2-macroglobulin, 0.5 µg/ml
leupeptin, 0.5 mg/ml Na2EDTA) was added to the medium.
Immuno- and Ligand Blotting--
Conditioned media were
concentrated by centrifugation through either Centricon-3 or
Centricon-10 microconcentrators (Amicon Inc., Beverley, MA) and the
IGFBP-3 concentration in each sample determined by RIA. Each protein
(approximately 50 ng) was reconstituted in 50 µl of Laemmli sample
buffer, heated at 95 °C for 5 min, and fractionated under
nonreducing conditions on a 12% SDS-polyacrylamide gel overnight at
100 V (20). The proteins were then transferred to Hybond-C Extra
supported nitrocellulose (Amersham, Bucks, UK) by electroblotting using
a Multiphor II Novablot unit (Pharmacia). After transfer, the blot was
incubated at 37 °C for 3 h in Tris-buffered saline (TBS, 10 mM Tris, 150 mM NaCl, pH 7.4) containing 1%
(w/v) BSA and then probed with anti-hIGFBP-3 antiserum at a final
concentration of 1:10,000 (prepared in TBS containing 1% (w/v) BSA and
0.05% (v/v) Nonidet P-40). The blot was washed three times in TBS,
once in TBS containing 0.05% Nonidet P-40, and a further three times in TBS and then incubated for 2 h at 22 °C in
125I-protein A (1 × 106 cpm/50 ml of TBS
containing 1% (w/v) BSA and 0.05% (v/v) Nonidet P-40). Following the
wash regime used above, the dried blot was placed against Hyperfilm MP
autoradiographic film (Amersham) for 1-3 days at 70 °C.
Affinity Labeling--
10 ng of each rhIGFBP-3 variant was
incubated with 125I-IGF-I or 125I-IGF-II
(0.4 × 106 cpm) in the presence of unlabeled IGF-I or
IGF-II, respectively, at concentrations over the range of
1011 to 10
6 M. The reactions
were made up to a final volume of 45 µl with 50 mM sodium
phosphate (pH 6.5) containing 0.05% (w/v) BSA. The complexes were
cross-linked with 0.25 mM disuccinimidyl suberate (Pierce,
Rockford, IL), and after 30 min incubation at 4 °C, the reactions
were terminated by the addition of 2 µl of 1.0 M Tris base. Reactions were heated to 95 °C before electrophoresis on either 10% or 12% SDS-PAGE. The gels were processed for
autoradiography, and radiolabeled protein bands were quantified by
densitometry (Video Densitometer model 620, Bio-Rad).
Binding Assays-- Binary and ternary complex formation in the presence of rhIGFBP-3 or variants was measured essentially as described previously (21). Briefly, reactions containing 125I-IGF-I or 125I-IGF-II (10,000 cpm) and rhIGFBP-3 analogs over the concentration range of 0-5 ng in a total volume of 0.3 ml of 50 mM sodium phosphate containing 1% (w/v) BSA, were incubated at 22 °C for 2 h. The binary complexes were immunoprecipitated with 0.5 µl of IGFBP-3 antibody and 2.5 µl of goat anti-rabbit immunoglobulin in the presence of a 4% final concentration of polyethylene glycol. Ternary complex formation was measured by incubating 125I-ALS (10,000 cpm) with mixtures of IGF-I (50 ng) and varying concentrations of rhIGFBP-3 or variants (over the range of 0-20 ng) in a total volume of 0.3 ml of 50 mM sodium phosphate containing 1% (w/v) BSA at 22 °C for 2 h. Ternary complexes were then separated from unbound tracer as described above.
The affinity of IGF binding to rhIGFBP-3 analogs was measured essentially as described above except that the concentration of rhIGFBP-3 analogs was held constant at 0.5 ng. Unlabeled IGF was added over the concentration range of 0.0025-1 ng in a total volume of 0.3 ml. Complexes were separated from unbound tracer by immunoprecipitation as described above. The affinity of ALS binding to the rhIGFBP-3·IGF-I binary complex was determined as described previously (14) except that the concentrations of rhIGFBP-3 analogs (0.5 ng) and IGF-I (50 ng) were held constant while unlabeled ALS was added over the concentration range of 2.5-200 ng in a total volume of 0.3 ml. Bound tracer was separated from free tracer, as described above. Scatchard analysis was as described previously (14).Detection of Cell-associated IGFBP-3--
Cell surface
association of rhIGFBP-3 produced endogenously by the transfected cell
lines was measured by an immunological assay described previously (22).
Briefly, cells were plated at 2 × 104 cells/well in
24-well plates for 48 h. Cultures were changed to serum-free media
supplemented with 0.1% (w/v) BSA and incubated for a further 48 h. The cell monolayers were then washed and incubated with either
hIGFBP-3 antibody (R-100) or normal rabbit serum (as control for
nonspecific effects) diluted 1:5,000 in 0.5 ml of medium. After a 16-h
incubation at 22 °C, the cell monolayers were washed again before
incubation with 125I-labeled protein A (20,000 cpm in 0.5 ml of medium) for 2 h. Unbound tracer was removed by washing, and
the cells were solubilized with 0.5% (w/v) SDS. The cell lysates were
collected, and radioactivity was determined in a -counter.
Statistical Analysis-- Statistical analysis was carried out using StatView 4.02 (Abacus Concepts Inc., Berkeley, CA). Differences between groups were evaluated by Fisher's Protected Least Significant Difference test after analysis of variance, and a significant difference was defined as p < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
rhIGFBP-3 proteins, detectable by RIA, were secreted by each of the transfected cell lines, and the expression of these proteins was inducible up to 7-fold by dexamethasone (Fig. 1B). The differences in levels of stimulation by dexamethasone are probably due to the heterogeneous population of transfectants in each cell line. There was no detectable hIGFBP-3 in media conditioned by CHO cells transfected with pMSG as a control. The polyclonal IGFBP-3 antibody recognized all variant proteins including the deletion variants, indicating that an antigenic epitope is present in the amino-terminal portion of the protein, the only region common to all variants. In the RIA, the full-length analogs and rhIGFBP-3 yielded displacement curves that were parallel to pure serum-derived IGFBP-3. In contrast, the deletion variants displayed parallelism up to 1-2 ng, but the displacement curves became nonparallel at higher concentrations (data not shown). All samples were assayed under conditions where parallelism was observed. Under these conditions, the RIA was considered the best method available for quantifying low amounts of these proteins.
Immunoblots of the five variants are shown in Fig.
2A. rhIGFBP-3,
rhIGFBP-3[253KED RGD], and
rhIGFBP-3[228KGRKR
MDGEA] produced a 40-45-kDa
doublet as well as a 30-kDa form, essentially identical to
serum-derived hIGFBP-3. rhIGFBP-3[
185-264] migrated as a doublet
of 30-35 kDa. Adding an estimated mass of 10-15 kDa of carbohydrate
to the calculated 19-kDa core protein, the observed bands of
rhIGFBP-3[
185-264] corresponded well with the predicted size of
29-34 kDa. The doublet evident in rhIGFBP-3[
185-264] probably
represents different glycoforms of the protein consistent with the
current view that the 40-45-kDa doublet of IGFBP-3 consists of
glycoforms (23); IGFBP-3 contains three potential
N-glycosylation sites at 89NAS,
109NAS, and 172NFS (15).
rhIGFBP-3[
89-264] corresponded to an apparent size of
approximately 15 kDa, whereas rhIGFBP-3[
89-184] migrated as a
major band at 21 kDa and a minor band at 17 kDa. Because the deduced
molecular masses for the
89-264 and
89-184 variants are 9 and
18 kDa, respectively, it would appear that the proteins are migrating
aberrantly on SDS-PAGE. Presumably, the smaller 17-kDa form of
rhIGFBP-3[
89-184] represents a proteolyzed form of the
protein, comparable to the 30-kDa form of rhIGFBP-3,
rhIGFBP-3[253KED
RGD], and
rhIGFBP-3[228KGRKR
MDGEA].
|
The ability of the rhIGFBP-3 proteins to bind IGF-I was examined by
ligand blot using 125I-IGF-I. A preliminary experiment
revealed that there were two forms of IGF-I-binding protein present in
the media conditioned by cells transfected with the vector, pMSG (Fig.
2B). Based on the apparent sizes of these proteins
(approximately 23 and 28 kDa), we assume that they are glycoforms of
CHO-derived IGFBP-4. Immunoprecipitation of the sample with hIGFBP-3
antibody before electrophoresis removed these proteins (Fig.
2B). Ligand blotting of immunoprecipitated IGFBP-3 variant
proteins (Fig. 2C) indicated that
rhIGFBP-3[253KED RGD] and
rhIGFBP-3[228KGRKR
MDGEA] have an IGF-I binding
function. In contrast, none of the deletion variants showed detectable
IGF binding by this method.
To examine their binding properties in more detail, the full-length
rhIGFBP-3 analogs were purified from conditioned media by
heparin-Sepharose chromatography followed by reverse phase HPLC. The
wild-type protein was eluted from the heparin column as a single peak
by 0.75 M NaCl (Fig.
3A).
rhIGFBP-3[253KED RGD] showed a similar elution
profile (Fig. 3B). In contrast, rhIGFBP-3[228KGRKR
MDGEA] eluted at a concentration
of only 0.5 M (Fig. 3C), and
rhIGFBP-3[
89-184] eluted as two peaks at 0.5 and 0.75 M NaCl (Fig. 3D). Among the deletion analogs,
the
89-184 variant showed a marked reduction in binding activity,
but the other two analogs did not bind to heparin-Sepharose at all
(data not shown). Endogenous IGFBP-4 from CHO cells also bound to
heparin-Sepharose and eluted at 0.5 M NaCl (data not
shown), consistent with previous studies (24). The CHO-derived IGFBP-4
was separated from rhIGFBP-3[228KGRKR
MDGEA] on
reverse phase HPLC.
|
Because of the poor binding of the rhIGFBP-3 deletion analogs to heparin-Sepharose, these analogs were purified from an antibody affinity column followed by reverse phase HPLC, as described under "Materials and Methods." The proteins purified by either heparin or antibody affinity were analyzed by SDS-PAGE to check for protein integrity and purity. Dose-response curves for the binding of various IGFBP-3 analogs to either 125I-IGF-I (Fig. 4A) or 125I-IGF-II (Fig. 4B) were obtained from solution binding assays. In both instances, there was little or no detectable binding of the deletion analogs to either IGF ligand, consistent with the results obtained from ligand blotting (Fig. 2C). Competitive binding curves were generated for IGFBP-3 forms that showed IGF binding, to compare their relative affinities for IGF-I (Fig. 4C) and IGF-II (Fig. 4D). Data from these assays were analyzed by Scatchard plot, and the derived binding affinities are summarized in Table I. Consistent with the previously determined affinities for IGF-I and IGF-II binding by serum-derived IGFBP-3 (19, 25), rhIGFBP-3 displayed a higher affinity for IGF-II than for IGF-I. However, the difference in Ka values was not statistically significant (p = 0.06). This may result from different post-translational modifications on the IGFBP-3 from different sources. The RGD mutant had significant decreases in its affinities for both IGF-I and IGF-II (4- and 6-fold, respectively; Table I). The MDGEA mutant showed significant reduction in IGF-II affinity (3-fold), but its affinity to IGF-I was near normal (Table I).
|
|
To investigate the possibility that the deletion analogs may have low affinities for IGF which were undetectable by solution binding, 125I-IGF-I or 125I-IGF-II was covalently cross-linked to the analogs in the presence of competing unlabeled IGF-I or IGF-II, respectively. The samples were analyzed on SDS-PAGE, and the resulting band intensities were quantified by scanning densitometry, to generate displacement curves. Typical electrophoretograms for rhIGFBP-3 are shown (Fig. 5, panels A and F). By this technique, half-maximal displacement of bound IGF-I or IGF-II tracer from rhIGFBP-3 (Fig. 5, B and G, respectively) was seen at unlabeled ligand concentrations similar to those in the solution binding assays (Fig. 4, C and D), confirming the validity of the affinity labeling technique.
|
As shown in Fig. 5, all three deletion analogs were cross-linked to
either 125I-IGF-I or 125I-IGF-II, and the
tracers were displaceable by unlabeled IGF-I or IGF-II. However, the
affinities were 20-60-fold lower than that of rhIGFBP-3. The
concentrations of IGF-I required to displace 50% of tracer binding to
the various analogs were 0.12 ± 0.02 nM (rhIGFBP-3),
2.02 ± 0.96 nM (185-264), 4.42 ± 1.19 nM (
89-184), and 7.00 ± 1.22 nM
(
89-264). The relative concentrations of IGF-II required to
displace 50% of tracer binding to the various analogs were 0.06 ± 0.03 nM (rhIGFBP-3), 2.43 ± 0.61 nM
(
185-264), 2.14 ± 0.59 nM (
89-184), and
2.50 ± 0.94 nM (
89-264).
Fig. 6A shows the
dose-response curves of the various IGFBP-3 analogs for binding to
ALS in the presence of excess (50 ng) IGF-I, i.e. for
ternary complex formation. As predicted by the lack of binding in IGF-I
solution binding assays above (Fig. 4A), the deletion
variants also displayed barely detectable levels of ALS binding.
Similar results were obtained when the binding assays were performed in
the presence of IGF-II (data not shown). On the other hand,
rhIGFBP-3[253KED RGD] exhibited a dose-response
curve parallel to that of rhIGFBP-3, but the curve had shifted to the
right, indicating that the ALS binding activity of this variant was
decreased compared with rhIGFBP-3. This may reflect its decreased
affinity for IGF-I (Table I). Although
rhIGFBP-3[228KGRKR
MDGEA] demonstrated a smaller,
nonsignificant decrease in affinity for IGF-I compared with
rhIGFBP-3[253KED
RGD] (Table I), its binding to ALS
was decreased markedly (Fig. 6A), suggesting that the
mutation in this analog predominantly affects its ALS binding
function.
|
To compare the relative affinities of ALS for the IGFBP-3 variants, we
determined the displacement of 125I-ALS from the ternary
complexes, formed in the presence of 50 ng of IGF-I and the IGF-binding
IGFBP-3 variants, by increasing concentrations of unlabeled ALS (data
not shown). Representative Scatchard plots derived from these
competition curves are shown in Fig. 6B and the binding
parameters summarized in Table I. Whereas
rhIGFBP-3[253KED RGD] had an affinity for ALS
(21.7 ± 4.5 × 109 liters/mol) similar to
rhIGFBP-3 (24.3 ± 5.2 × 109 liters/mol),
rhIGFBP-3[228KGRKR
MDGEA] showed a 90% reduction
in ALS affinity (2.5 ± 0.9 × 109 l/mol,
p < 0.05), indicating the importance of these basic
residues in ALS binding.
It has been shown previously that IGFBP-3 synthesized by fibroblasts
can associate with the cell surface and can be displaced by the
addition of IGF-I (22). The interaction between the various endogenously produced rhIGFBP-3 forms and the cell surface was examined
(Fig. 7). Cell surface-associated
rhIGFBP-3 proteins were only detected in cell lines transfected with
rhIGFBP-3, rhIGFBP-3[89-184], and
rhIGFBP-3[253KED
RGD]. The absence of
cell-associated forms of recombinant proteins in cells expressing the
89-264 and
185-264 variants indicates that the
carboxyl-terminal region of the protein is necessary for cell surface
association. Furthermore, the basic residues (228KGRKR) in
the carboxyl-terminal region, shown above to be important determinants
of affinity for ALS, are also integral to the cell association domain,
as mutation of these residues abolished the ability of
rhIGFBP-3[228KGRKR
MDGEA] to interact with the cell
surface. Consistent with previous evidence indicating that IGF-I
displaces cell surface-associated IGFBP-3 into the extracellular medium
(22), rhIGFBP-3- and
rhIGFBP-3[253KED
RGD]-transfected cells showed
a decrease (approximately 40 and 60%, respectively) in
cell-associated binding proteins when incubated with IGF-I (Fig. 7). On
the other hand, there was no difference in the levels of
cell-associated protein when cells transfected with
rhIGFBP-3[
89-184] were incubated in the presence or absence of
IGF-I. This is in accord with previous observations that displacement
of cell surface-associated IGFBP-3 by IGF-I requires a direct
interaction between the two proteins because rhIGFBP-3[
89-184]
has a greatly reduced affinity for IGF-I.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Comparison of the primary sequences of the six well characterized IGFBPs indicates that the strongest homology is in the amino- and carboxyl-terminal regions of the proteins, whereas the central regions are unique to each protein. The amino- and carboxyl-terminal regions contain 18 cysteine residues, at identical positions in the IGFBPs (IGFBP-6 lacks two of the residues), which probably confer similar constraints on the structure-function of these proteins (26). The IGF binding domain could therefore involve either or both of these regions. In this study we examined the role of the carboxyl-terminal conserved region in the binding of IGF-I, IGF-II, and ALS by generating three deletion variants of IGFBP-3.
The deletion of either the conserved carboxyl-terminal region
(rhIGFBP-3[185-264]) or the nonconserved central region
(rhIGFBP-3[
89-184]) or the combined deletion of both regions
(rhIGFBP-3[
89-264]) abolished IGF-I and IGF-II binding when
analyzed by either ligand blotting or solution binding. However, all
three deletion analogs showed some affinity for both IGF ligands in
affinity labeling experiments, in agreement with a previous preliminary
report, unsupported by data, that rhIGFBP-3[
89-264] and
rhIGFBP-3[
162-264] are capable of binding to IGF-I when analyzed
by a similar method (27). It was also reported that deletion of amino
acid residues 92-184 or 92-223 did not abolish IGF-I binding. When
competitive binding curves for IGF-I or IGF-II binding to rhIGFBP-3
were compared for the solution binding and affinity labeling methods,
similar affinity estimates (as determined by half-maximal displacement of radioligand) were obtained, providing some validation of the affinity labeling technique. However, the failure of binding assays to
detect IGF binding to the deletion mutants unless stabilized by
affinity cross-linking suggests that these relatively low affinity interactions must have quite rapid off-rates.
Deletion of the central region from IGFBP-3 (89-184) appears to
have decreased both IGF-I and IGF-II binding by approximately 40-fold.
Whether this nonconserved region is directly involved in the binding
interactions or simply helps to maintain the spatial configuration of
the amino- and carboxyl-terminal domains is unknown. Deletion of the
carboxyl-terminal region (
185-264) alone affected IGF-II binding
(~40-fold reduction) more than IGF-I binding (~20-fold reduction).
This region is likely to make a specific contribution to the IGF
binding site (particularly IGF-II) because carboxyl-terminal fragments
of IGFBP-2 have been shown to retain considerable affinity for IGF-II
but little for IGF-I (28). The amino-terminal domain must contribute
similarly to the IGF binding site because the
89-264 variant
retained some binding activity.
The specific mutations in rhIGFBP-3[253KED RGD] and
rhIGFBP-3[228KGRKR
MDGEA] did not abolish either
IGF-I or IGF-II binding as determined by solution binding assays. The
relative binding affinities of
rhIGFBP-3[253KED
RGD] for IGF-I and IGF-II were
decreased by approximately 4- and 6-fold, respectively. In both of the
site-specific IGFBP-3 variants, the altered amino acids were replaced
by analogous sequences from IGFBP-1. Interestingly, it has been
reported that when the RGD sequence in IGFBP-1 was replaced by KED (the
corresponding IGFBP-3 sequence), the IGFBP-1 variant lost its IGF-I
binding activity which was attributed to the formation of dimers by the IGFBP-1 variant. When the RGD sequence in IGFBP-1 was replaced by KGD,
however, the mutation did not abolish IGF-I binding (29). The mutation
in rhIGFBP-3[228KGRKR
MDGEA] decreased its affinity
for IGF-I and IGF-II by 2- and 3-fold, respectively (Table I). Taken
together, these results would suggest that these mutated sequences,
compared with the deletion analogs, make a relatively minor
contribution to the IGF-I and IGF-II binding sites. The
carboxyl-terminal region, where these specific mutations reside,
appears to be more important for IGF-II binding than IGF-I binding,
which is consistent with the relative IGF-I and IGF-II binding
abilities of the deletion analogs.
In support of the participation of carboxyl-terminal residues in IGFBP binding activity, it has been reported that deletions of the carboxyl-terminal region of IGFBP-1 abolished IGF-I binding (29). Others have reported that natural proteolytic truncation of either the amino or carboxyl terminus of IGFBPs has adverse effects on IGF binding (30-33). Therefore it seems clear that both amino- and carboxyl-terminal regions of the IGFBPs participate in the formation of the ligand binding domain. Although the loss of IGF binding by the deletion variants may in part be the result of conformational changes caused by the large deletions, the structural disruption is apparently not excessive, as the epitopes on these proteins are still recognized by an hIGFBP-3 antibody. This antibody not only shows high specificity for hIGFBP-3 among the six hIGFBPs but also for IGFBP-3 from higher primates compared with other species (12).
The only deletion variant to associate with the cell surface was the
central domain deletion rhIGFBP-3[89-184], indicating that the
carboxyl-terminal region, but not the central region, is essential for
this function of IGFBP-3. Furthermore, the carbohydrate moieties on
IGFBP-3 are not required for cell surface association because all of
the potential N-linked glycosylation sites have been removed
from rhIGFBP-3[
89-184]. This is in agreement with a previous
study showing that nonglycosylated rhIGFBP-3 expressed in
Escherichia coli can associate with the cell surface of
bovine fibroblasts (5). The association of the central region deletion variant with the cell surface suggests that the structural integrity of
the carboxyl-terminal domain was retained despite the deletion of
one-third of the molecule.
In various studies, IGFBPs-1 to -5 have all been shown to bind to cells
(22, 24, 34-36). The RGD sequence within the carboxyl-terminal region
of IGFBP-1 and IGFBP-2 serves as a recognition motif on proteins that
adhere to the cell surface via integrin receptors. It has been shown
that IGFBP-1 associates with the 5
1
integrin and can stimulate the migration of transfected CHO cells (37). The RGD motif is not present in the sequence of native IGFBP-3. The
observation that cell-associated IGFBP-3 could be displaced from human
fibroblasts by heparin suggested that IGFBP-3 may interact with
proteoglycans or other negatively charged molecules on the cell surface
(22), possibly via the carboxyl-terminal portion of the protein that is
relatively abundant in basic amino acid residues. However, recent
evidence suggests that the heparin-inhibitable cell binding of IGFBP-3
may not be to heparan sulfate or chondroitin sulfate glycosaminoglycans
(38). This highly basic carboxyl-terminal region is also present in
IGFBP-5 and has been shown to be involved in the binding of
glycosaminoglycans (39).
There are two putative heparin binding motifs in IGFBP-3, located at
amino acids 148-153 and 219-226 in the central and carboxyl-terminal regions, respectively. A recent study (40) showed that synthetic peptides containing either one of these heparin binding consensus sequences bound heparin and that the peptide containing the
carboxyl-terminal motif had ~4-fold higher affinity for heparin. The
inability of rhIGFBP-3[185-264] to bind heparin-Sepharose would
suggest that the carboxyl-terminal region contributes structurally to
the major heparin binding site. This is supported by the finding that
rhIGFBP-3[
89-184] bound to heparin-Sepharose, although with less
avidity than rhIGFBP-3. Furthermore, heparin binding was affected when
amino acid residues adjacent to the carboxyl-terminal putative heparin
binding motif were mutated in
rhIGFBP-3[228KGRKR
MDGEA].
Partial replacement of the carboxyl-terminal basic region in hIGFBP-3
with the homologous acidic residues of hIGFBP-1
(rhIGFBP-3[228KGRKR MDGEA]) abolished its ability
to associate with the cell surface. On the other hand, the presence of
the analogous RGD sequence in IGFBP-3 did not appear to affect the
ability of the protein to bind to the cell surface. This suggests that
the amino acid residues 228KGRKR form a key part of the
cell surface association domain of IGFBP-3, lending further support to
the study in which synthetic peptides corresponding to the basic region
were shown to decrease IGFBP-3 and IGFBP-5 binding to the cell surface
(24).
Previous studies from this laboratory have shown by several independent
methods, gel permeation chromatography, affinity labeling, and solution
binding assays with immunoprecipitation of complexes, that little or no
specific binding of human ALS to hIGFBP-3 occurs in the absence of
IGF-I or IGF-II. This concept has been challenged recently on the basis
of studies with rat ALS and a partially proteolyzed form of rat IGFBP-3
(41) and a study using human nonglycosylated IGFBP-3 (42), both of
which were interpreted to show that ALS may bind to IGFBP-3 in the
absence of IGFs. Whatever the explanation for these conflicting
results, our studies with natural human ALS and IGFBP-3 consistently
support the notion that the binary IGF·IGFBP-3 complex, rather than
IGFBP-3 itself, forms a high affinity binding site for ALS. Indeed, in
the presence of IGF-I variants (for example, with substitutions in the
B domain) with low affinity for IGFBP-3, the total binding of ALS is
low, because little binary (IGF·IGFBP-3) complex forms, but the
affinity of ALS for this complex is not reduced (14). In the presence of IGF-I, the deletion variants that had low affinities for IGF-I (decreased by 20-60-fold) showed very low or no binding to ALS, which is consistent with the requirement of an IGF·IGFBP-3
complex for ALS binding. rhIGFBP-3[253KED RGD],
which had a 4-fold decrease in IGF-I affinity, displayed normal ALS
binding affinity compared with rhIGFBP-3. In contrast, rhIGFBP-3[228KGRKR
MDGEA] showed markedly
impaired ALS binding function, attributable to a loss of affinity for
ALS, even though its IGF-I binding is relatively normal. These results
specifically implicate IGFBP-3 residues 228-232, but not 253-255, in
the interaction with ALS.
The two mutations, therefore, have quite distinct effects on the
protein-protein interactions within the ternary complex. The
KED RGD mutation has altered the capacity of IGFBP-3 to bind
to IGF-I without affecting the affinity of ALS. This implies that when
the binary complex between
rhIGFBP-3[253KED
RGD] and IGF-I has formed, the
mutation has no bearing on the structural integrity of the ALS binding
site, because the affinity of ALS remains unchanged. In contrast, the
KGRKR
MDGEA mutation appears to disrupt the ALS binding site, as
ALS affinity for this variant was decreased. ALS binding is known to be
sensitive to increasing ionic strength (25, 43), and the
KGRKR
MDGEA mutation introduces a significant charge reversal in
this region of IGFBP-3, suggesting that interaction between key charged
residues may be important. Although binding determinants in the ALS
structure have not been elucidated, there is a region of acidic
residues in the amino-terminal region of human ALS
(23DDDADE) (44) which might interact with the highly basic
region in IGFBP-3, and preliminary molecular modeling studies
suggest that within the leucine-rich repeating region of ALS, there may be a surface with an accumulation of negative
charge.2
In summary, this study has shown that the protein-protein and protein-cell interactions of IGFBP-3 are complex and involve distinct domains of the protein. The structural integrity of the IGF-I binding site is disrupted significantly by deletion of either the central or carboxyl-terminal region of IGFBP-3, but more specific mutations of the carboxyl-terminal region can reduce IGF-I binding. The IGF-I and ALS binding sites are functionally distinct as shown by contrasting the binding characteristics of the 253RGD variant, with decreased IGF-I binding but normal ALS affinity, and the 228MDGEA variant, with near normal IGF binding and greatly reduced ALS affinity. Finally, although gross deletions affected the ability of IGFBP-3 to bind to IGF-I and consequently ALS, the deletion of amino acid residues 89-184 did not alter its interaction with the cell surface. We therefore conclude that the carboxyl-terminal region and in particular, 228KGRKR, is essential for this function. The availability of IGFBP-3 mutants with selective reduction in the affinity for IGFs, on the one hand, and reduced binding to ALS and the cell surface on the other, will provide powerful tools to help elucidate further the dual roles of IGFBP-3 as a transporter of IGFs and a regulator of cell function.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the technical assistance of P. D. Fink in the preliminary experiments of this study and the Leo & Jenny Leukaemia and Cancer Foundation for the purchase of HPLC equipment.
![]() |
FOOTNOTES |
---|
* This study was supported by the National Health and Medical Research Council, Australia.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. Tel.: 61-2-9926-8486;
Fax: 61-2-9926-8484; E-mail: sfirth{at}med.usyd.edu.au.
1
The abbreviations used are: IGFBP(s),
insulin-like growth factor (IGF)-binding protein(s); ALS, acid-labile
subunit; h, human; rh, recombinant human; -MEM,
-modified
Eagle's medium; CHO, Chinese hamster ovary; BSA, bovine serum albumin;
RIA, radioimmunoassay; HPLC, high pressure liquid chromatography; TBS,
Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis.
2 J. Janosi and P. Ramsland, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|