From the Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina 29425
Received for publication, August 17, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Elevated insulin-like growth factor (IGF)-1
levels are prognostic for the development of prostate and breast
cancers and exacerbate the complications of diabetes. In each case,
perturbation of the balance between IGF-1/2, the IGF-1 receptor, and
the IGF-binding proteins (IGFBPs) leads to elevated IGF-1
sensitivity. Blockade of IGF action in these diseases would be
clinically significant. Unfortunately, effective IGF antagonists are
currently unavailable. The IGFBPs exhibit high affinity and specificity
for the IGFs and serve as natural IGF antagonists, limiting their
mitogenic/anti-apoptotic effects. As an initial step in
designing IGFBP-based agents that antagonize IGF action, we have begun
to analyze the structure of the IGF-binding site on IGFBP-2. To this
end, two IGF-1 photoprobes, N Insulin-like growth factor
(IGF)1-1 and IGF-2 play
central roles in a number of cellular processes, including growth,
proliferation, differentiation, survival, transformation, and
metastasis (1, 2). Enhanced activity of the IGFs has been implicated in
diabetic complications and cancer. These effects are mediated by the
IGF-1 receptor (IGF-1R), a member of the receptor tyrosine kinase
family of cell-surface receptors. The IGF-2 receptor, which lacks
signaling activity, plays a role in clearing IGF-2 from the cell
surface (3, 4). The IGFs are regulated at the extracellular level by
a family of six IGF-binding proteins (IGFBPs), designated IGFBP-1-6 (5-7). These six proteins exhibit higher affinities for the IGFs than
the IGF-1R, while having negligible affinity for insulin.
Renewed interest in the function of the IGF system stems from the
observations that IGF-1 and IGF-2, acting through the IGF-1R, increase
the tumorigenic potential of breast and prostate cancer cells (8).
Accordingly, increased serum IGF-1 levels have been shown to be
prognostic for the development of prostate and breast cancers (9, 10).
Alterations in IGFBP expression may also contribute to disease states.
For example, IGFBP-3 is a target of the p53 tumor suppressor, and a
common p53 mutation results in decreased IGFBP-3 secretion (11, 12),
which is likely to cause an increased proliferative response to IGF-1.
Also, reduced IGFBP-2 expression resulting from the hyperglycemia of
diabetes was recently shown to enhance the sensitivity of renal
mesangial cells to the growth and secretory effects of IGF-1, pushing
the cells toward a glomerulosclerotic phenotype (13). Because IGF-1 can
suppress apoptosis, cells lacking IGF-1 receptors, cells with compromised IGF-1R signaling pathways, or cells treated with the IGFBPs
may selectively die by apoptosis (8). Taken together, these findings
suggest that the IGFBPs serve a role as natural IGF antagonists.
IGF-1 and IGF-2 are homologous protein hormones of 70 and 67 amino
acids in length, respectively (14). Based on studies of chemically
modified and mutated IGF-1, a number of residues have been identified
as being part of the IGF-1R contact site, in particular the aromatic
residues at positions 23-25 (15). Cooke et al. (16) used
NMR and restrained molecular dynamics to elicit the solution structure
of IGF-1; this model clearly illustrates an IGFBP-interacting domain on
the surface of IGF-1 and its lack of overlap with the receptor-docking
site (see Fig. 1). Specifically, this site consists of the N-terminal
tripeptide Gly-Pro-Glu (17) and residues 49-51 (18). These two regions come together to form an independent binding domain (see Fig. 1) (16).
The analog des-1-3-IGF-1 binds to the IGF-1R with high affinity, but
has dramatically reduced affinity for the IGFBPs, underscoring the
importance of the N-terminal contact site on IGF-1 for binding protein
specificity (17). In addition, mutations of Glu3 or
residues 49-51 result in a ligand with severely reduced binding activity (19). In good agreement with these findings, insulin lacks
these residues common in the IGFBP-binding region and thus does
not bind to the IGFBPs with high affinity.
The IGFBPs are globular proteins containing 18 spatially conserved
cysteine residues participating in the formation of nine disulfide
bonds. They range in size from 200 to 300 amino acids and, based on
their high degree of homology, can be divided into three distinct
domains, each constituting about one-third of the protein (20). The N-
and C-terminal regions, designated domains 1 and 3, respectively, share
the highest homology (20, 21), whereas the intervening region (domain
2) is highly variable (<30% homology) (6). Domains 1 and 3 contain 12 and 6 spatially conserved cysteine residues, respectively,
except in the case of IGFBP-6, which is missing 2 cysteine residues in
domain 1 (5); IGFBP-4 has 2 additional cysteine residues in domain 2. Because of the high homology of these proteins in domains 1 and 3 (~70%) (6), the IGF-binding domain has been proposed to reside
within one of these regions. Support for this notion is based on
studies in which N- or C-terminal IGFBP fragments were found to retain high affinity binding activity for IGF-1 and/or IGF-2 (19). IGFBP-related protein-1 and -2, also known as IGFBP-7/Mac25 and IGFBP-8/connective tissue growth factor, respectively, have homologies within their N termini to IGFBP-1-6 (23). These proteins have lower
affinities for the IGFs compared with the IGFBPs and have been reported
to interact with insulin (24). The precise mechanism by which the
IGFBPs inhibit IGF-1 and IGF-2 action is presently unknown. It is
thought to involve high affinity binding of the IGFs by the IGFBPs,
thereby limiting their access to the IGF-1R. A complete understanding
of this inhibitory action will come with the solution of the
three-dimensional structure of the IGFBPs. As of yet, the IGF-binding
domain on the IGFBPs has not been defined.
To date, extensive use of molecular techniques has been applied to
assess the binding domain on the IGFBPs. However, only sparse
structural information has been obtained. Currently, a debate exists as
to which domain(s) of the IGFBPs are most crucial for IGF binding. This
is further confounded by the paucity of precise structural information
about the IGFBPs as recently reviewed by Baxter (25). To more precisely
identify the points of contact between IGF-1 and IGFBP-2, we chose to
pursue a photoaffinity labeling approach, which has been used to
identify sites of interaction between a number of interacting proteins.
On this basis, we derivatized the Materials--
Recombinant human IGF-1 was provided by
Genentech, Inc. (South San Francisco, CA). HSAB was synthesized from
p-aminobenzoic acid, sodium azide (Sigma),and
dicyclohexylcarbodiimide (Pierce) and purified according to the method
of Galardy et al. (26). HPLC columns were from Vydac
Instruments (Hesperia, CA). SBED, UltraLinkTM monomeric
avidin-agarose, NeutrAvidinTM-peroxidase, and
tris(2-carboxyethylyl)phosphine hydrochloride were obtained from
Pierce. Methotrexate was from Immunex Corp. (Seattle, WA), and fetal
bovine serum was from Summit Biotechnology (Fort Collins, CO). cDNA
encoding human IGFBP-2 (27) was obtained from Dr. Jörg Landwehr
(Hoffmann-La Roche, Basel, Switzerland). All other materials were of
reagent grade or higher.
Synthesis and Purification of abG1IGF-1 and
bedG1IGF-1--
All derivatizations and handling of
photoprobes were carried out under subdued lighting or under a red
safety light. abG1IGF-1 was synthesized by reaction of
recombinant human IGF-1 (1 mg, 130 nmol) with a 10-fold molar excess of
HSAB (0.34 mg, 1.3 µmol) for 1 h at 23 °C. Unreacted ester
was quenched by the addition of 30 µl of ethanolamine for 30 min, and
the entire reaction mixture was lyophilized. The lyophilized reaction
mixture was dissolved in 0.5 M acetic acid and 10 µl of
trifluoroacetic acid and injected onto a C18 column
equilibrated in 0.1% trifluoroacetic acid and 24% acetonitrile at a
flow rate of 1 ml/min. After 20 min, a linear gradient of 24-60%
acetonitrile was developed over 60 min to elute IGF-1 and the reaction
products. Peak 4 was collected, dried in vacuo in a SpeedVac
concentrator (Savant Instruments, Inc., Farmingdale, NY), reinjected
onto the C18 column equilibrated in 50 mM
triethanolamine phosphate (pH 3.0) containing 27.5% acetonitrile, and
eluted with a gradient of 27.5-38% acetonitrile over 60 min. The
major peak was collected, dried, and further analyzed. Synthesis and
purification of bedG1IGF-1 was carried out essentially as
described for abG1IGF-1 using a 1:1 ratio of SBED to protein.
Pepsin Digestion of abG1IGF-1 and
bedG1IGF-1--
IGF-1 and the photoprobes were digested
with pepsin at an enzyme/substrate ratio of 1:20 in 0.01 M
HCl for 5 h at 23 °C (28). Digests were then injected onto a
C18 column equilibrated in 0.1% trifluoroacetic acid, and
the fragments were eluted using a linear gradient of 0-60%
acetonitrile over 90 min. For each photoprobe, the HPLC fractions
containing fragments with potentially modified residues (AE, CG, and D
fragments) (see Fig. 3) (28) were analyzed by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS).
MALDI-TOF-MS--
Dried HPLC fractions were dissolved in 0.1%
trifluoroacetic acid containing 70% acetonitrile. 0.5-µl aliquots of
each fraction were mixed with 1 µl of 50 mM
Purification of rhIGFBP-2--
rhIGFBP-2 was purified from
DHFR IGF-1-Agarose Affinity Chromatography--
2 ml of Affi-Gel
10-activated immunoaffinity support (Bio-Rad) were washed four times
with 3 volumes of cold water, followed by the addition of 2 mg of IGF-1
in 100 mM HEPES (pH 7.4). The mixture was agitated
overnight at 4 °C, followed by two washes with 100 mM
HEPES (pH 8.0). Unreacted sites were then quenched with 2 ml of 1 M Tris-HCl (pH 7.9) for 1 h at ambient temperature. The column was then washed with three cycles of alternating low pH/high
pH salt washes (0.5 M NaCl and 0.1 M sodium
acetate (pH 4.0) and 0.5 M NaCl and 0.1 M Tris
(pH 8.0), respectively). Finally, the column was washed and stored in
10 ml of 150 mM NaCl and 50 mM HEPES (pH 7.4)
with 0.05% sodium azide.
Dialyzed and lyophilized conditioned medium was dissolved in 40-45 ml
of 50 mM HEPES (pH 7.4) containing 150 mM NaCl
(Buffer A). Insoluble material was removed by centrifugation at
3000 × g for 20 min. 2 ml of IGF-1-agarose in Buffer A
were then added, and the slurry was incubated overnight at 4 °C with
gentle agitation. The column was subsequently washed with 100 ml of
Buffer A, followed by 50 ml of 10% Buffer A. Proteins bound to the
column were then eluted with 15 ml of 0.5 M acetic acid,
and the column was washed with 20 ml of Buffer A plus 1 ml of 1 M HEPES (pH 7.4), followed by 40 ml of Buffer A. The eluate
was dried in vacuo using a SpeedVac concentrator. The dried
eluate was stored at IGFBP-2 Binding Assay--
Soluble IGFBP-2 binding assays were
carried out using polyethylene glycol precipitation and centrifugation
(31). 1 ng of rhIGFBP-2 was combined with various concentrations of
IGF-1, abG1IGF-1, or bedG1IGF-1 ranging from 30 fM to 100 nM in binding assay buffer (100 mM HEPES (pH 7.4), 44 mM NaHCO3,
0.01% bovine serum albumin, 0.01% Triton X-100, and 0.02%
NaN3), followed by the addition of 10 nCi of
125I-IGF-1 (Amersham Pharmacia Biotech). After a 4-h
incubation at room temperature, 250 µl of 0.5% bovine Photoaffinity Labeling--
Equimolar quantities of
abG1IGF-1 or bedG1IGF-1 and rhIGFBP-2 were
allowed to attain equilibrium binding by co-incubation for 4 h at
23 °C in 100 mM HEPES (pH 7.4) containing 44 mM NaHCO3 and 0.01% Triton X-100. The sample
was then placed in ice water and irradiated for 2 h with a
prewarmed Fotodyne hand-held, single-wavelength UV lamp (2 × 4-watt 300-nm bulbs) at a distance of 2 cm. The mixture was
dried in vacuo, and the proteins were reduced and alkylated using tris(2-carboxyethylyl)phosphine (32) and 4-vinylpyridine (33).
abG1IGF-1- and bedG1IGF-1-photolabeled
IGFBP-2 proteins were separated from unreacted IGFBP-2 by
reversed-phase HPLC and trypsinized as described below.
Trypsinization of IGF-1, IGFBP-2, and Photoaffinity-labeled
Complexes--
Trypsinization of reduced and alkylated proteins was
performed according to Honegger and Humbel (34). Proteins (20 µM) were dissolved in 100 mM
N-ethylmorpholine acetate (pH 8.5) to which was added
sufficient trypsin (1 µg/µl) to achieve a 1:50 enzyme/substrate ratio. Mixtures were then incubated for 2 h at 37 °C, followed by a second addition of trypsin. After 2 additional h at 37 °C, the
reaction was stopped by the addition of an equal volume of 0.1%
trifluoroacetic acid. The mixture was dried in vacuo or
applied directly to a C18 reversed-phase HPLC column.
Avidin Chromatography of bedG1IGF-1·IGFBP-2 Tryptic
Peptides--
Tryptic peptides generated from the
bedG1IGF-1·IGFBP-2 complex were applied to an
UltraLinkTM monomeric avidin column. Flow-through fractions
were collected and pooled. The column was eluted with low pH buffer,
and the eluate fractions were pooled. The flow-through and eluate
fractions were dried and further analyzed by reversed-phase HPLC on a
C18 column equilibrated in 0.1% trifluoroacetic acid and
eluted with a linear gradient of acetonitrile. Eluted peaks were
analyzed by MALDI-TOF-MS.
Immunoblots--
Samples were dissolved in SDS sample buffer
with or without dithiothreitol and resolved on a 10 or 12.5%
SDS-polyacrylamide gel according to the procedure of Laemmli (35) using
a Hoefer Scientific Instruments apparatus. The proteins were
transferred to nitrocellulose and immunoblotted using a commercial
antiserum against intact bovine IGFBP-2 (Upstate Biotechnology, Inc.,
Lake Placid, NY). Blots were developed with horseradish
peroxidase-labeled secondary antibody (Chemicon International, Inc.,
Temecula, CA) and an enhanced chemiluminescence kit (Amersham Pharmacia
Biotech). For sequential anti-IGF-1/anti-IGFBP-2 antibody analysis of
the same blot, the membrane was stripped for 20 min at 60 °C in 1 M Tris (pH 6.7) containing 10% SDS and 0.1 M
Synthesis and Purification of abG1IGF-1 and
bedG1IGF-1--
As shown in Fig.
1, the three-dimensional structure of
IGF-1 reveals the presence of distinct binding domains for the IGFBPs and the IGF-1R, which do not overlap. IGF-1 contains four primary amines (the
As shown in Fig. 2, reaction of HSAB and SBED with IGF-1 as described
under "Experimental Procedures" resulted in production of three
major products (peaks 2-4) in addition to unreacted IGF-1 (peak 1).
Analysis of the HSAB reaction products by acidic polyacrylamide gel
electrophoresis in 8 M urea and by MALDI-TOF-MS revealed
that these three peaks were monoderivatized forms of IGF-1 (data not shown). Peak 2 had previously been shown to represent the elution position of abK27IGF-1 (where K27 is
Lys27) (36). Peaks 3 and 4 had been tentatively identified
as abK65IGF-1 and abG1IGF-1, respectively,
based on amino acid sequencing analysis (36). No abK68IGF-1
was detected. The three product peaks were further purified by
chromatography on a C18 column equilibrated in
triethanolamine phosphate and acetonitrile. Structural assignments were
confirmed using pepsin digestion as described below. After
derivatization with SBED, peaks 2-4 were shown to similarly represent
bedK27IGF-1, bedK65IGF-1, and
bedG1IGF-1 (Fig. 2B).
Characterization of abG1IGF-1 and
bedG1IGF-1--
To verify that the "ab" and "bed"
moieties were covalently bound to the
For each photoprobe, HPLC purification of the pepsin digestion products
revealed that the retention times for the derivatized AE fragments were
significantly increased compared with the underivatized AE fragment
(data not shown). This was predicted based on the added hydrophobicity
of the additional functional groups. Fig. 4A shows the MALDI-TOF-MS of
abG1AE. This fragment had the correct mass (predicted
average mass of 2419.7 Da; observed mass of 2419.8 Da) and exhibited
loss of nitrogen as a result of photoactivation by the 337-nm laser, to yield a second peak at 2394.3 Da. The identities of the CG and D
fragments, containing Lys65/Lys68 and
Lys27, respectively, were also confirmed by MALDI-TOF-MS
and were in good agreement with those of Forsberg et al.
(28). Similar observations were made for bedG1IGF-1. As
shown in Fig. 5, a peak at 2956.2 Da
(predicted average mass of 2955.9 Da) was observed along with a second
peak at 2929.4 Da, reflecting the loss of nitrogen resulting from
photolysis. Again, the CG and D fragments exhibited masses consistent
with a lack of derivatization. As an alternate method of validation of
the derivatization, electrospray ionization MS3 was
also carried out (data not shown).
Having demonstrated the structure and purity of abG1IGF-1
and bedG1IGF-1, it was necessary to ensure that each probe
retained high affinity binding for IGFBP-2 similar to that of native
IGF-1. The results of IGFBP-2 competition binding assays are shown in Fig. 6. The IC50 values for
IGF-1, abG1IGF-1, and bedG1IGF-1 were 173, 131, and 168 pM, respectively, indicating that the photoprobes
and IGF-1 had identical affinity for IGFBP-2. These data indicate that
the addition of the photoreactive group does not significantly hinder
the IGF-1/IGFBP-2 interaction.
Photoaffinity Labeling of IGFBP-2--
Photoaffinity labeling of
IGFBP-2 with abG1IGF-1 and bedG1IGF-1 was
carried out as described under "Experimental Procedures." In a
small-scale photolabeling study with abG1IGF-1, an aliquot
of the reaction mixture was subjected to immunoblot analysis with
anti- IGFBP-2 and anti-IGF-1 antisera under reducing conditions (Fig.
7A). In addition to rhIGFBP-2
migrating at ~32 kDa and detected by anti-IGFBP-2 antiserum, a second
protein species migrating at ~40 kDa was detected. This band reacted
with both anti-IGF-1 and anti-IGFBP-2 antibodies, indicating that the
photoaffinity labeling reaction had successfully generated
abG1IGF-1·IGFBP-2 complexes. These data confirm that
photolabeling of IGFBP-2 with abG1IGF-1 results in a stable
covalent IGF-1·IGFBP-2 complex that is not disrupted by
reduction.
A similar analysis was carried out for bedG1IGF-1 (Fig.
7B). As shown in lane 1, two bands were detected
with anti- IGFBP-2 antiserum: IGFBP-2 itself and the
bedG1IGF-1·IGFBP-2 complex. This complex bound to and
was specifically eluted from an avidin-agarose column, whereas IGFBP-2
did not bind at all (lane 2). Under nonreducing conditions,
NeutrAvidinTM-peroxidase was unable to detect the
bedG1IGF-1·IGFBP-2 complex (lane 3). In
lanes 4-6, the bedG1IGF-1·IGFBP-2 complex was
first reduced and alkylated with tris(2-carboxyethylyl)phosphine and
4-vinylpyridine prior to electrophoresis. As discussed above, reduction
and alkylation of bedG1IGF-1· IGFBP-2 should yield
IGFBP-2 biotinylated at the site of photoincorporation. As shown in
lanes 4-6, two bands were again detected with reduction and
alkylation of the samples. The upper band had the same electrophoretic
mobility as uncross-linked, reduced, and alkylated IGFBP-2 and
was detectable with anti-IGFBP-2 antibodies. However, roughly one-half
of the content of this band was retained on the avidin-agarose column
(lane 5) and reacted with
NeutrAvidinTM-peroxidase (lane 6), indicating
that the band in lane 4 contains both unreacted and
biotinylated IGFBP-2. The lower band reacted with anti-IGFBP-2
antibodies, was retained by the avidin-agarose column (lane
5), and could be labeled with NeutrAvidinTM-peroxidase
(lane 6), indicating that it represents a biotinylated fragment of IGFBP-2. These results indicate that reduction of the
bedG1IGF-1·IGFBP-2 complex allowed greater access of
avidin-peroxidase to the biotin moiety on photolabeled IGFBP-2.
Identification of the abG1IGF-1 Photoincorporation
Site--
To identify the site of photoincorporation of
abG1IGF-1, the abG1IGF-1·IGFBP-2 complex was
isolated by HPLC following reduction and alkylation of the photolysis
reaction mixture. Owing to the similar retention times of the free and
cross-linked species, the two components overlapped significantly (Fig.
8A). Subsequent re-chromatography of each component provided sufficient resolution to
attain a high level of purification of the
abG1IGF-1·IGFBP-2 complex (Fig. 8B).
Immunoblot analysis of the column fractions indicated that >40%
photoincorporation was achieved.
Fractions containing purified abG1IGF-1·IGFBP-2 were
pooled, dried, and trypsinized. It was anticipated that tryptic
digestion of the abG1IGF-1·IGFBP-2 complex would yield a
cross-linked peptide containing a tryptic fragment of IGFBP-2 (BP2T)
and the N-terminal tryptic fragment of abG1IGF-1 (abIGF1T).
Since abIGF1T alone (residues 1-21) is 2521 Da, this represents the
minimum mass for the resulting cross-linked peptide (BP2T-abIGF1T). The
tryptic digest was applied to a C18 column and eluted with
a shallow gradient of acetonitrile to obtain optimal separation of the
tryptic peptides (data not shown). MALDI-TOF-MS was then performed on
each fraction to locate BP2T-abIGF1T. The MALDI mass spectrum of the
identified fraction is shown in Fig. 9A. Although not pure, we
obtained a significant enrichment of BP2T-abIGF1T in this fraction with
an observed mass of 5295.6 Da. These results suggest that abIGF1T
(predicted mass of 2521 Da) was covalently incorporated into the
C-terminal tryptic peptide of IGFBP-2 corresponding to residues
266-287 (BP2T predicted mass of 2772 Da), yielding a complex with a
predicted mass of 5293 Da. To further validate this assignment, the
fraction containing this complex was subdigested with
Staphylococcus aureus V8 protease and analyzed by
MALDI-TOF-MS. As illustrated in Fig. 9 (B and C),
MALDI-TOF-MS of this fraction identified four distinct peptides consistent with the proposed structure. These data indicate that when
attached to the Identification of the bedG1IGF-1 Photoincorporation
Site--
To determine the site of photoincorporation of
bedG1IGF-1 into IGFBP-2, the photolyzed reaction mixture
obtained as described under "Experimental Procedures" was reduced
and alkylated with tris(2-carboxyethylyl)phosphine and 4-vinylpyridine,
resulting in cleavage of IGF-1 from the complex and biotinylation of
IGFBP-2 at the site of photoincorporation. The mixture was then
desalted on a C4 column, dried in vacuo, and
digested with trypsin. The tryptic peptides were loaded onto an
UltraLinkTM monomeric avidin column, which was washed with
6 column volumes of phosphate-buffered saline. To release retained
biotinylated tryptic peptides, the column was eluted with regeneration
buffer (0.2 M glycine (pH 2.8)). The flow-through/washes
and eluate fractions were pooled separately and dried, and the peptides
present in each were resolved by HPLC on a C18 column. As
expected, the majority of peptides eluted in the flow-through fraction
of the avidin column (Fig.
10A), resembling a
representative combined tryptic digest of uncross-linked IGF-1 and
IGFBP-2 (data not shown). Two major peaks were observed in the low pH
elution of the column. The first peak exhibited a mass of 2595.7 Da
when analyzed by MALDI-TOF-MS (Fig. 10B). When corrected for
the mass of the biotin and remaining cross-linker residues from the bed
moiety (653.3 Da), this corresponded to a BP2T fragment of 1942.3 Da
(Fig. 10B). This coincides with tryptic peptide 212-227
(predicted mass of 1942.2 Da) from the C terminus of IGFBP-2.
MALDI-TOF-MS analysis of the second peak eluted from the avidin column
revealed the presence of a major signal at 1667.4 Da. When corrected
for cross-linker/biotin, the BP2T fragment had a mass of 1014.1 Da,
which did not correspond to any tryptic peptides present in rhIGFBP-2
(Fig. 10B). However, this mass did correspond to the first 9 residues of the BP2T fragment already identified (residues 212-220).
These findings suggest that photoaffinity labeling with the bed reagent
resulted in partial peptide bond cleavage at the site of covalent
insertion. The potential for peptide bond cleavage during photoaffinity
labeling has previously been suggested in the literature (37). This
conclusion is further supported by the data presented in Fig.
7B (lanes 4-6), where we detected an IGFBP-2
fragment that was reactive with NeutrAvidinTM-peroxidase.
Taken together, these results suggest that the site of insertion may be
residue 220 of IGFBP-2 (Fig.
11A).
In this report, we present evidence for a C-terminal contact site
on IGFBP-2 for IGF-1 based on direct photoaffinity labeling studies
with two unique N-terminally modified photoaffinity derivatives of
IGF-1. We interpret these findings as an indication of the presence of
a high affinity binding site for IGF-1 within the C terminus of
IGFBP-2. Consistent with this finding are a number of reports
describing the C terminus of IGFBP-2 as the domain containing the high
affinity IGF-binding site. Wang et al. (38) and Ho and
Baxter (39) each identified C-terminal fragments of IGFBP-2 with high
affinity IGF-binding activity. In preliminary studies, we have
characterized a 15.8-kDa C-terminal fragment of rhIGFBP-2 from our
transfected Chinese hamster ovary cell cultures that exhibits an
affinity for IGF-1 identical to that of intact rhIGFBP-2.2 This fragment
lacks N-terminal and mid-region epitopes based on tryptic peptide
mapping studies. Additional support for the C terminus as the site of
IGF binding stems from studies of Brinkman et al. (40), who
deleted the last 20 amino acids from the C terminus of IGFBP-1 and
thereby abolished IGF-1-binding activity. Similarly, Forbes et
al. (41) generated a series of four sequential C-terminal
truncation mutants of bovine IGFBP-2 and concluded that residues
222-236 are required for high affinity IGF interactions. Finally,
Schuller et al. (42) prepared monoclonal antibodies to
residues 188-196 and 222-227 of IGFBP-1 that blocked IGF-1 binding.
These investigators concluded that the regions surrounding these
epitopes were important for IGF binding.
Introduction of photoactivable aryl azide moieties within the
IGFBP-binding domain on IGF-1 (residues 1-3 and 49-51) resulted in
the selective photoaffinity labeling of two separate sites in the C
terminus of IGFBP-2, within tryptic peptides 266-287 (abG1IGF-1) and 212-227 (bedG1IGF-1) (Fig.
11A). The labeling of these different sites can be attributed to the different side chain lengths of the two IGF-1 photoprobes. abG1IGF-1 contains an azidobenzoyl moiety and
thus lacks an appreciable side chain between the Based on chemical modifications, site-directed mutagenesis studies, and
the identification of fragments with IGF-binding activity, the N
terminus of the IGFBPs has also been described as the site of the
IGF-binding domain. Iodination studies on bovine IGFBP-2 in isolation
or as a complex bound to IGF-2 resulted in efficient iodination of Tyr
residues at positions 71, 98, 213, 226, and 269; Tyr60 was
protected from iodination by bound IGF-2 (44). Huhtala et
al. (45) isolated a 21-kDa N-terminal fragment of IGFBP-1 that
retained some IGF-1-binding activity. More recently, Kalus et
al. (46) reported that N-terminal fragments of IGFBP-5
representing residues 1-104 and 40-92 exhibit weak IGF-binding
activity. Based on NMR studies, they defined a hydrophobic patch
comprising residues 49, 50, 62, and 68-75 that potentially represents
the primary IGF-binding site on IGFBP-5. This hypothesis was
subsequently tested by the construction of full-length IGFBP-5 and
IGFBP-3 mutants in which combined substitutions at residues 68, 69, 70, 73, and 74 resulted in a >1000-fold reduction in binding affinity (47). Contrary to these mutagenesis results, Ständker et
al. (48) reported that the C terminus of IGFBP-5 contains the
IGF-binding domain based on the isolation of a naturally occurring,
biologically active truncation of IGFBP-5. The observation by Yamanaka
et al. (24) that insulin can bind to (and be
chemically cross-linked into) the N terminus of IGFBP-related protein-1
may provide further insight into the mechanism of IGF/IGFBP
interactions, suggesting the notion that the N- and C-terminal domains
may both interact with IGF-1, but with differing specificity and
function (49).
In the context of the present findings, we propose the following model
(Fig. 12) based on the idea that two
binding sites for IGF-1 exist on the IGFBPs. The first is a high
affinity, high specificity site (C terminus) responsible for binding
selectivity for IGF-1 via its IGFBP-binding domain. The second low
affinity site (N terminus) binds to IGF-1 via its IGF-1R-binding domain and thus plays a role in blocking IGF-1 binding to the IGF-1R. This
model would likely require a conformational change to take place within
the N terminus following IGF binding to the C terminus. Evidence to
support this comes from NMR analyses of IGF·IGFBP complexes (50, 51).
It is possible to speculate that such conformational changes may
reflect the ability of the IGFBP, following initial binding to the N
terminus (IGFBP-binding domain) of IGF-1, to interact with the IGF-1R
domain of IGF-1. This could provide a steric mechanism through which
inhibition of IGF-1R activation is accomplished. This in turn explains
low affinity insulin binding to the N terminus of IGFBP-related
protein-1 (9). In addition, a conformational change explains why
des-1-3-IGF-1 does not bind the IGFBPs well, even though it still
contains an intact IGF-1R-binding domain.
Gly1-(4-azidobenzoyl)-IGF-1
(abG1IGF-1) and
N
Gly1-([2-6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionoyl)-IGF-1 (bedG1IGF-1), selective for the IGFBPs were synthesized by
derivatization of the
-amino group of Gly1, known to be
part of the IGFBP-binding domain. Mass spectrometric analysis of the
reduced, alkylated, and trypsin-digested
abG1IGF-1·recombinant human IGFBP-2 (rhIGFBP-2)
complex indicated photoincorporation near the carboxyl terminus of
rhIGFBP-2, between residues 266 and 287. Mass spectrometric analysis of
avidin-purified tryptic peptides of the
bedG1IGF-1·rhIGFBP-2 complex revealed photoincorporation
within residues 212-227. Taken together, these data indicate that the
IGFBP-binding domain on IGF-1 contacts the distal third of IGFBP-2,
providing evidence that the IGF-1-binding domain is located within the
C terminus of IGFBP-2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group of the N-terminal
glycine of IGF-1 with two different photoaffinity reagents,
N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and
sulfosuccinimidyl
[2-6-(biotinamido)-2-(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionate (SBED), to generate IGF-1 photoprobes capable of selectively
labeling the IGF-binding domain on IGFBP-2. The advantage of this
approach is that the photoreactive group is inserted within the region shown to be essential for high affinity binding of IGF-1 to the IGFBPs.
Since IGF-2, which has a 3-residue N-terminal extension, exhibits a
higher affinity for IGFBP-2 than IGF-1, reduced binding affinity of
IGF-1 for IGFBP-2 resulting from N-terminal substitutions was not
anticipated. In this study, we describe the synthesis and
characterization of
N
Gly1-(4-azidobenzoyl)-IGF-1
(abG1IGF-1) and
N
Gly1-([2-6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionoyl)IGF-1 (bedG1IGF-1) and their successful application to
photoaffinity label rhIGFBP-2. Based on these direct photoaffinity
labeling analyses, our results indicate that the C terminus of
IGFBP-2 contains the IGF-binding domain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyano-4-hydroxycinnamic acid (Sigma) in 0.1% trifluoroacetic acid
and 70% acetonitrile. The mixture was spotted onto a gold-coated,
stainless steel sample plate and air-dried. The samples were analyzed
using a PerSeptive Biosystems Voyager-DE MALDI-TOF mass spectrometer
equipped with a 337-nm nitrogen laser. A delayed extraction source was
operated in linear mode (1.2-m ion flight path, 20-kV
accelerating voltage), yielding an instrumental resolution of ~700
(full width at half-maximum) at m/z 1297.5. One mass
spectrum was based on 256 averaged mass scans. External mass
calibration was performed using angiotensin I (1297.5 Da) and
bovine insulin (5734.54 Da) as standards. Mass accuracy was
±0.1%.
Chinese hamster ovary cells (29) stably transfected
with 30 µg of pCMV-hIGFBP-2 and 2 µg of pMT2 containing
murine dihydrofolate reductase (30) using calcium phosphate.
Cells were grown to confluence in roller bottles and subjected to a
weekly cycle of 3 days growth in serum-containing medium, followed by 4 days in serum-free medium. Pooled conditioned medium (400-600 ml) was
acidified to pH 3-4 with glacial acetic acid and dialyzed against 10 mM acetic acid in Spectrapor 4 membranes (12,000-14,000
Mr cutoff; Spectrum, Laguna Hills, CA) to remove salt and IGFs. After lyophilization, IGFBP-2 was purified by sequential IGF-1-agarose affinity chromatography and reversed-phase HPLC on
a C4 column equilibrated in 0.1% trifluoroacetic acid.
IGFBP-2 eluted between 30 and 40% acetonitrile.
20 °C until further purification as described above.
-globulin
were added, followed by 500 µl of 25% polyethylene glycol (average
Mr of 8000; Sigma). The samples were incubated
for 10 min at room temperature and centrifuged for 3 min at 15,000 × g. The pellets were washed with 1 ml of 6.25%
polyethylene glycol, and bound radioactivity was quantified in a
Compugamma spectrometer (LKB-Wallac, Turku, Finland). Counts
bound in the presence of 1 µM or 100 nM IGF-1 (nonspecific binding) were subtracted to obtain specific binding. IC50 values were calculated using the equation
B = Bmax/(1 + [ligand]/IC50), where B is the concentration
of bound ligand and Bmax is the maximal binding
observed. The Microsoft Excel 97 Solver was used to minimize the sum of
the squares of the differences from the mean IC50 values for each IGF-1 concentration by optimizing B restrained by
the above equation. The calculated IC50 values were used to
generate smooth curves.
-mercaptoethanol. The stripped membrane was washed twice with
Tris-buffered saline containing Tween for 10 min at 23 °C, followed
by blocking for 1 h with 5% nonfat dry milk and reprobed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino groups of 3 lysyl residues and the
-amino group of Gly1), all of which are reactive with HSAB and
SBED. Fig. 2 shows the structures of
these reagents and highlights the relative lengths of the spacer arms
from the site of covalent linkage to IGF-1 to the photoreactive azide
group. In addition to a significantly longer spacer arm, SBED also
contains a reduction-sensitive disulfide linkage and a biotin moiety.
As a result, although we anticipated that use of HSAB as a
photocross-linking agent would yield a reduction-stable covalent
IGF-1·IGFBP-2 complex, use of SBED was expected to produce a
reduction-sensitive IGF-1·IGFBP-2 complex that, after reduction, would result in biotinylation of IGFBP-2 at the site of
photoincorporation.
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Fig. 1.
IGFBP-binding site on IGF-1.
Left, the N-terminal residues Gly1,
Pro2, and Glu3 combine with Phe49,
Arg50, and Ser51 to form the IGFBP-binding
domain (6). Note that the Gly1 -NH2 group is
the site chosen for derivatization with aryl azide-based photoaffinity
reagents. Right, the IGF-1R-binding domain on IGF-1. The
image was obtained by RasMol Version 2.6 using coordinates (6) from the
Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University (New Brunswick, NJ).
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Fig. 2.
Synthesis of
N Gly1-derivatized
IGF-1 photoprobes. Shown above each chromatogram are the
structures of the photoreagents used to derivatize IGF-1. Below are the
elution profiles of the reaction products obtained on a C18
reversed-phase column. Typically, four major products were obtained in
each reaction. For each probe, peak 4 was collected from each of two
separate synthesis reactions pooled, ,dried and rechromatographed on a
C18 column as described under "Experimental
Procedures." A, derivatization with HSAB; B,
derivatization with SBED. Each HPLC chromatogram shown is
representative of four independent synthesis reactions.
AUFS, absorbance units at full scale.
-amino group of
Gly1 for each photoprobe, it was necessary to isolate
Gly1 from the 3 Lys residues. Since reduction and
alkylation might disrupt the azide moiety, we utilized pepsin digestion
of nonreduced abG1IGF-1 and bedG1IGF-1. As
reported by Forsberg et al. (28) and shown in Fig. 3, pepsin releases a disulfide-linked AE
fragment containing Gly1 and no Lys residues. Pepsin
digestion of abG1IGF-1 and bedG1IGF-1 was
carried out in the dark to avoid photoactivation of the probe, and
recombinant human IGF-1 was also digested in parallel to serve as a
control for subsequent HPLC and mass spectrometric analyses.
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Fig. 3.
Pepsin cleavage sites in IGF-1. To
verify derivatization of IGF-1 at its N-terminal glycine residue,
purified probes were digested with pepsin. The primary sequence of
IGF-1 is shown, with arrowheads indicating the primary sites
of pepsin cleavage (28). Letters A-G indicate the principal
peptides generated. The disulfide-linked AE fragment
(shaded) contains the N-terminal glycyl residue and none of
the lysyl residues.
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Fig. 4.
Characterization of abG1IGF-1 by
pepsin digestion. The AE (A), CG (B), and D
(C) fragments of abG1IGF-1 were purified by HPLC
and analyzed by MALDI-TOF-MS.
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Fig. 5.
Characterization of bedG1IGF-1 by
pepsin digestion. The AE (A), CG (B), and D
(C) fragments of bedG1IGF-1 were purified by
HPLC and analyzed by MALDI-TOF-MS.
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Fig. 6.
Competition binding analyses of
N Gly1-derivatized
photoprobes. The binding activities of the IGF-1 photoprobes were
analyzed with a polyethylene glycol precipitation-based assay using
IGF-1 (
), abG1IGF-1 (
), or
bedG1IGF-1(
) to compete with 125I-IGF-1 for
binding to IGFBP-2 (see "Experimental Procedures"). Data points
shown are average values of two independent experiments performed in
triplicate. Binding curves were generated as described under
"Experimental Procedures."
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Fig. 7.
Analysis of photoaffinity-labeled
complexes. A, abG1IGF-1-labeled
complexes were resolved on an SDS gel and transferred to
nitrocellulose. The blot was probed with anti-IGFBP-2 antibodies,
followed by stripping and reprobing with anti-IGF-1 antibodies.
B, products of photoaffinity labeling with
bedG1IGF-1 were immunoblotted with a polyclonal
anti-IGFBP-2 antibody or probed with
NeutrAvidinTM-peroxidase (Av-HRP) as indicated.
Some aliquots were applied to an UltraLinkTM avidin-agarose
(Av-Ag) column; the low pH eluate was run on the gel.
Red/Alk, reduction and alkylation.
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Fig. 8.
Purification of IGF-1·IGFBP-2
complexes. A, following reduction and alkylation, the
abG1IGF-1/IGFBP-2 photolysis reaction mixture was purified
on a C18 column equilibrated in 0.1% trifluoroacetic acid
and 24% acetonitrile. B, the major components
(a-c) of the elution profile obtained in A were
re-chromatographed separately using a shallow acetonitrile gradient.
Insets, fractions from each run were analyzed by
immunoblotting with anti-IGFBP-2 antibody. B, IGFBP-2;
BI, IGF-1·IGFBP-2 complex.
-amino group of Gly1, the
azidobenzoyl moiety contacts IGFBP-2 in its distal C-terminal end
within the tryptic peptide corresponding to residues 266-287 (see Fig.
11A).
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Fig. 9.
Analysis of
abG1IGF-1·IGFBP-2 photoaffinity-labeled complex.
A, shown is a MALDI-TOF mass spectrum of a unique peak
having a mass (5295.6 Da) that lacks correspondence to IGF-1 or IGFBP-2
tryptic peptides. B, the column fraction containing the
5295.6-Da fragment was dried and subdigested with S. aureus
V8 protease. Shown is the MALDI-TOF mass spectrum of the digestion
products. C, the peptides identified in the V8 digest in
B are indicated.
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Fig. 10.
Analysis of bedG1IGF-1·IGFBP-2
photoaffinity-labeled complex. A, elution profile of
the reduced, alkylated, and trypsin-digested photolysis reaction
mixture following chromatography on a column of monomeric avidin. Shown
are the HPLC column profiles of the column flow-through/washes and
eluate. B, MALDI-TOF mass spectrum of the peaks identified
by arrows in A. The first peak exhibited a mass
of 2595.7 Da. After correcting the observed mass for the associated
biotin and spacer, it was identified as tryptic peptide 212-227. The
second biotinylated component (second arrow) was identified
as having a mass of 1014.1 Da, which corresponds to a truncated form of
the same tryptic peptide (residues 212-220) and suggests rupture of
the peptide backbone of IGFBP-2 during photoincorporation. This finding
explains the composition of the smaller band observed in Fig.
7B (loss of ~7 kDa; residues 221-289). Z,
pyridylethylated cysteine.
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Fig. 11.
Sites of photoincorporation into
IGFBP-2. A, depiction of sites of photoincorporation of
abG1IGF-1 and bedG1IGF-1 into IGFBP-2.
White lines represent sites of trypsin cleavage.
B, detailed view of the C-terminal region (residues
210-289) showing the known disulfide bonding pattern (41).
Underlined peptides represent tryptic peptides identified as
sites of photoincorporation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group of
Gly1 and the aryl azide. In this case, the aryl azide
resides near the outer edge of the IGF-1-binding domain, defining the
site of contact for Gly1. The longer, more flexible side
chain present on the aryl azide in bedG1IGF-1 has the
potential to define a contact site within the vicinity of the other
residues of the IGFBP-binding domain, including Glu3, which
plays an essential role in maintaining high affinity binding to the
IGFBPs (43). Although we cannot rule out the possibility that this side
chain results in the labeling of regions of the IGFBPs outside the
IGF-binding domain, we believe that the likelihood of this occurring is
minimal based on the previous identifications of C-terminal binding
activity described above. Given the globular nature of the IGFBPs and
the disulfide bonding pattern in this domain (Fig. 11B)
(41), these two sites are likely to be closely apposed in
three-dimensional space. Although the two photoprobes labeled sites
within the C terminus of IGFBP-2 that are separated by ~40 amino
acids in the primary sequence, these sites are likely to be much closer
when the secondary structure of the protein is taken into consideration
(Fig. 11B). Indeed, the two labeled sites cannot be farther
apart than the total length of the two photoprobe spacer arms combined
(~300 nm).
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Fig. 12.
Model of IGF-1/IGF-1R binding inhibition by
IGFBP-2. Based on the data presented, the IGFBP-binding domain on
IGF-1 interacts with the C terminus of IGFBP-2. Once this complex is
formed, the IGF-1R-binding domain on IGF-1 then binds to the N terminus
of IGFBP-2. IGFBP-rps, IGFBP-related proteins.
This scheme may serve to explain how N-terminal fragments and truncation mutants of the IGFBPs are able to bind to IGF-1. This would also support a steric hindrance-based model of IGFBP inhibition of IGF-1 action at the IGF-1R. Following high affinity binding between the IGFBP-binding domain on IGF-1 and its contact site within the C terminus of IGFBP-2, the ensuing conformational rearrangement enables high affinity interaction of the N terminus of IGFBP-2 with the IGF-1R binding domain on IGF-1. Obviously, this is one of several potential models one might propose; confirmation of any such model will require a more definitive understanding of the three-dimensional structure of the IGFBPs. Future photolabeling studies with abK27IGF-1 and bedK27IGF-1 may aid in addressing the issue of whether there are N-terminal contact sites on the IGFBPs for the IGF-1R-binding domain on IGF-1.
Based on their ability to block IGF actions, we have chosen to pursue
the design of IGF antagonists based on the structure of the IGF-binding
domain on the IGFBPs. This clearly requires more detailed information
than is currently available concerning IGFBP structure. We have
employed a photoaffinity labeling approach as a means of identifying
the IGF-1 site of contact on IGFBP-2. To this end, a photoreactive
derivative of IGF-1 was prepared, exploiting the reactivity of primary
amines in the protein to covalently attach an aryl azide moiety within
the IGFBP-binding domain. This approach has been used for many
photoaffinity labeling studies, including the insulin (52) and IGF-1
(36) receptors. It has the drawback of incorporating a bulky group
into the ligand, which may alter its binding characteristics. However,
in the case of abG1IGF-1 and bedG1IGF-1, we did
not observe a significant alteration in binding affinity for IGFBP-2.
An alternative approach would be to utilize an intrinsically labeled
IGF-1 derivative containing a photoactivable moiety at an aromatic
amino acid. This has been reported for the identification of an insulin
contact point on the insulin receptor (22). To accomplish this,
PheB25 in the receptor-binding domain of insulin was
replaced with p-azidophenylalanine to generate
(B25-p-azidophenylalanine--carboxamide)-insulin. To apply
a similar strategy to studies with IGF-1 would require considerable
peptide semi-syntheses. Instead, we chose the alternative of
incorporating an extrinsic photoactivable moiety into the
-amino group of Gly1, a residue that constitutes part of the
IGFBP-binding domain on IGF-1. Of the two photoprobes employed, the use
of SBED to biotinylate the site of IGF-1 photoincorporation provided a
more direct approach to the identification of the photocross-linked tryptic peptide. This represents the first report of the successful use
of this reagent in photolabeling a ligand-binding site.
In conclusion, this study represents the first report defining a
contact site between IGF-1 and IGFBP-2. We propose to exploit this
structural information to develop unique antagonists of the IGFs as
important therapeutic adjuncts in the prevention/treatment of various
cancers and the complications of diabetes. Small peptides comprising
the proposed IGF-binding site would serve as stable, protease-resistant
analogs of the IGFBPs (IGFBP mimetics). These compounds will be
designed to block IGF action and will serve as important templates for
the future design of peptidomimetics. To this end, IGF-1 antagonists
should provide important therapeutic adjuncts in the
prevention/treatment of these diseases.
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ACKNOWLEDGEMENTS |
---|
We thank Genentech, Inc. for generously providing recombinant human IGF-1; Dr. Jörg Landwehr for human IGFBP-2 cDNA; and Dr. David T. Kurtz (Medical University of South Carolina) for pMT2 cDNA, DG44 cells, and advice on the generation of human IGFBP-2-expressing Chinese hamster ovary cells. We especially thank Helga Hsu and Dr. Cecil C. Yip (University of Toronto) for many productive discussions. We also thank Dr. Kevin Schey (Medical University of South Carolina) for critical comments on the interpretation of MALDI-TOF-MS and electrospray ionization mass spectrometric data, Dr. Erika Büllesbach (Medical University of South Carolina) for helpful comments concerning synthesis conditions, and Dr. John Oatis (Medical University of South Carolina) for reduction and alkylation protocols. Finally, we thank the other members of the Rosenzweig laboratory for many helpful comments.
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FOOTNOTES |
---|
* This work was supported in part by Grant CA-78887 from the National Institutes of Health and Grant-in-aid S9868S from the American Heart Association, South Carolina Affiliate (to S. A. R.). A portion of this work was presented at the 82nd Annual Meeting of the Endocrine Society, June 21-24, 2000, Toronto, Ontario, Canada.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 Cell and
Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 843-792-5841; Fax: 843-792-2475; E-mail:
rosenzsa@musc.edu.
Published, JBC Papers in Press, November 3, 2000, DOI 10.1074/jbc.M007526200
2 M. J. Horney and S. A. Rosenzweig, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
IGF, insulin-like growth factor;
IGF-1R, insulin-like growth factor-1
receptor;
IGFBP, insulin-like growth factor-binding protein;
rhIGFBP, recombinant human insulin-like growth factor-binding protein;
HSAB, N-hydroxysuccinimidyl 4-azidobenzoate;
SBED, sulfosuccinimidyl
[2-6-(biotinamido)-2-(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionate;
abG1IGF-1, NGly1(4-azidobenzoyl)-IGF-1;
bedG1IGF-1, N
Gly1-([2-6-(biotinamido)2-(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionoyl)-IGF-1;
HPLC, high performance liquid chromatography;
MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry.
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