(Received for publication, June 7, 1995; and in revised form, December 27, 1995)
From the
Heparin binding to insulin-like growth factor (IGF)-binding
protein 5 (IGFBP-5) leads to a 17-fold decrease in its affinity for
IGF-I, and a region that contains several basic amino acids
(Arg-Arg
) may be involved in this
affinity shift. In the present study, mutagenesis was used to analyze
the effect of substitutions for basic amino acids in the
Arg
-Arg
region of IGFBP-5 on
heparin-binding and the heparin-induced affinity shift. Nine mutant
forms were prepared. Their association constants (K
) for IGF-I were similar to native
IGFBP-5. When 10 µg/ml of heparin was added, the K
of native IGFBP-5 decreased 17-fold,
and the K
of the K134A/R136A mutant
decreased 16-fold. In contrast, substitutions for specific basic amino
acids in the Arg
-Arg
region decrease
the affinity shift to 1.1-3.2-fold. Lys
was
especially important. When a mutant containing that single substitution
was tested, heparin caused only a 2.5-fold reduction in IGF-I affinity.
Affinity cross-linking studies showed that heparin was equipotent in
inhibiting the formation of
I-IGF-I
K134A/R136A
mutant complexes compared to native IGFBP-5. In contrast, heparin had
minimal effects on the formation of complexes between
I-IGF-I and the other mutants. The heparin-binding
activity of each mutant was determined. Four mutants, R201A/K202N,
K202A/K206A/R207A, R201A/K202N/K206N/K208N, and
K211N/R214A/K217A/R218A, had reduced heparin binding compared to native
IGFBP-5. The other five mutants, including the K211N mutant, showed no
change in heparin binding. The four mutants with reduced heparin
binding could be dissociated from heparin-Sepharose with much lower
NaCl concentrations, indicating that they had reduced affinity. These
findings suggest that Arg
, Lys
,
Lys
, and Arg
are important for heparin
binding. In contrast, Lys
is not important for the
binding of IGFBP-5 to heparin, but substitution for it reduced the
heparin-induced affinity shift.
Insulin-like growth factors (IGFs) ()in extracellular
fluids are bound to insulin-like growth factor-binding proteins
(IGFBPs), and IGFBPs are important regulators of IGF's biological
actions(1) . When IGFBPs are present in a soluble, high
affinity state they reduce the amount of IGF-I or -II that is available
for receptor interaction and inhibit IGF
bioactivity(2, 3, 4) . However, IGFBP-5,
unlike IGFBP-1, -2, and -4, binds to both cell surfaces and
extracellular matrix (ECM). IGFBP-5 binding to ECM results in a
reduction in its affinity for IGF-I and enhancement of IGF-I's
biologic actions(5, 6) . Therefore, it is important to
determine the specific amino acids in IGFBP-5 that account for ECM
binding and for the reduction in its affinity. Glycosaminoglycans are
abundant components in ECM that can modulate cell and protein
attachment. That IGFBP-5 may bind to glycosaminoglycans, such as
heparin and heparan sulfate, is suggested by the observation that
incubation of IGFBP-5 with glycosaminoglycans results in a 17-fold
decrease in the affinity of IGFBP-5 for IGF-I(7) . Peptide
competition studies have suggested that a basic amino acid-rich region
(Arg
-Arg
) of IGFBP-5 contains the
amino acids that are necessary for this reduction to occur. The
reduction of the affinity has been proposed to be due to a
conformational change of IGFBP-5, which is induced by heparin binding,
since the heparin and IGF-I binding sites of IGFBP-5 are
distinct(7) .
The Arg-Arg
region of IGFBP-5 contains 10 basic amino acids including a
putative heparin binding domain containing a BBBXXB motif
where B is a basic amino acid and X is a neutral one. Since
heparin and heparan sulfate are composed of repeating disaccharides and
are highly sulfated(8) , they are strongly anionic. These
groups are believed to align with the basic residues in heparin-binding
proteins. The heparin-induced affinity shift of IGFBP-5 for IGF-I has
been proposed to be a two-step process, heparin-binding followed by a
conformational change of IGFBP-5 that results in a decrease in its
affinity. The purpose of this study was to determine the effect of
substitutions for basic amino acids on the binding of IGFBP-5 to
heparin and on the heparin-induced reduction in affinity for IGF-I. We
prepared nine mutants of IGFBP-5 in which basic amino acids were
substituted by neutral ones. Their heparin-binding activities and
affinity shifts in response to heparin were compared.
Conditioned medium
containing the IGFBP-5 mutants was collected and centrifuged at 10,000
g for 20 min to remove cellular debris. The mutants
were purified as described previously(12) . The amount of each
mutant IGFBP-5 was quantified by comparing their high performance
liquid chromatography peak areas to an IGFBP-5 standard. The protein
concentration of the standard was determined by amino acid composition
analysis. To further ensure that a heparin-induced change in affinity
could be validly estimated for each mutant, Scatchard analysis was used
to calculate the affinity of each mutant for IGF-I, and the results
were compared to native IGFBP-5 (see Table 1).
To determine the region of IGFBP-5 that contained the heparin binding site, competition studies were carried out as described previously(7) . IGFBP-5 (80 nM) was incubated with 0.025 µl of heparin-Sepharose beads in the presence of various concentrations (0, 0.27, 2.7, or 27 µM) of peptide A or B. After an overnight incubation, the samples were centrifuged as described previously, and both the IGFBP-5 that bound to heparin-Sepharose beads and that remained in the supernatant were analyzed by immunoblotting.
Figure 1: A, effect of soluble heparin on IGFBP-5 binding to heparin-Sepharose beads. IGFBP-5 (80 nM) was incubated with 5 µl of Sepharose or heparin-Sepharose beads in 50 µl of EMEM, supplemented with 20 mM, HEPES, 0.1% Tween 20, and 20 mM EDTA. Some tubes received the indicated concentrations of soluble heparin in the buffer. After an overnight incubation, the samples were centrifuged. The IGFBP-5 in both the pellets and the supernatants of Sepharose beads or heparin-Sepharose beads was analyzed by ligand blotting as described under ``Experimental Procedures.'' The arrow denotes the position of the unbound IGFBP-5 in the supernatant, and the arrowhead denotes the bound IGFBP-5 in the pellet. Lane 1, Sepharose beads; lanes 2-6, heparin-Sepharose beads. Lanes 1 and 2, no heparin; lane 3, heparin 0.02 mg/ml; lane 4, 0.05 mg/ml; lane 5, 0.25 mg/ml; lane 6, 0.5 mg/ml. B, specificity of IGFBP-5 binding to heparin. The experiment was performed as described in A except that the incubation buffer included: lanes 1 and 2, no glycosaminoglycan; lane 3, heparin, 0.2 mg/ml; lane 4, heparan sulfate, 0.5 mg/ml; lane 5, chondroitin sulfate A, 0.5 mg/ml; lane 6, dermatan sulfate, 0.5 mg/ml. Lane 1, Sepharose beads, lanes 2-6, 0.025 µl of heparin-Sepharose beads.
Native IGFBP-5 binding increased when increasing amounts of heparin-Sepharose beads were used. 0.01 µl of heparin-Sepharose beads bound nearly 50% of the native IGFBP-5 (Fig. 2, lane 2), and 99% of the material was pelleted when 0.025 µl was used (Fig. 2, lane 3). Therefore 0.025 µl of heparin-Sepharose beads was selected as the minimum volume to be used in any experiment.
Figure 2: IGFBP-5 binding to heparin-Sepharose beads. Native IGFBP-5 (80 nM) was incubated with Sepharose beads or the indicated volume of heparin-Sepharose beads in 50 µl of EMEM, supplemented with 20 mM HEPES, 0.1% Tween 20, and 20 mM EDTA. After an overnight incubation, the samples were centrifuged. The IGFBP-5 in both the pellets and the supernatants was analyzed by ligand blotting as described under ``Experimental Procedures.'' The arrow denotes unbound IGFBP-5 in the supernatant, and the arrowhead denotes bound IGFBP-5 in the pellet. Lane 1, Sepharose beads; lane 2, heparin-Sepharose beads, 0.01 µl; lane 3, 0.025 µl; lane 4, 0.05 µl; and lane 5, 0.1 µl.
Figure 3:
Competition binding of IGFBP-5 to
heparin-Sepharose beads. Native IGFBP-5 (80 nM) was added in
50 µl of EMEM supplemented with 20 mM HEPES, 0.1% Tween
20, and 20 mM EDTA and incubated with 0.025 µl of
heparin-Sepharose beads or Sepharose beads in the presence of the
indicated concentrations of Arg-Arg
peptide, or Ala
-Thr
peptide.
After an overnight incubation, the samples were centrifuged. Both the
IGFBP-5 that bound to heparin-Sepharose beads and remained in the
supernatant were analyzed by immunoblotting as described under
``Experimental Procedures.'' The arrow denotes
unbound IGFBP-5 in the supernatants, and the arrowhead denotes
bound IGFBP-5 in the pellets. Lane 1, Sepharose beads; lanes 2-8, heparin-Sepharose beads. Lanes 1 and 2, no peptide; lanes 3, 4, and 5,
Arg
-Arg
peptide, 0.27, 2.7, and 27
uM, respectively; lanes 6, 7, and 8, Ala
-Thr
peptide, 0.27,
2.7, and 27 µM, respectively.
Figure 4: Effect of heparinase on ECM and tenascin binding of IGFBP-5. ECM was prepared or purified tenascin was layered on to 35-mm plastic tissue culture plates. The ECM and tenascin were exposed to heparinase (0.1 unit/ml) for 2 h at 37 °C. IGFBP-5 (3.4 nM) was incubated with the ECM or tenascin, and the amount of bound material was determined by immunoblotting. Lane 1, ECM control; lane 2, ECM after heparinase; lane 3, tenascin control; lane 4, tenascin after heparinase.
To identify the
basic amino acids in the Arg-Arg
region that are involved in heparin binding, we compared the
amounts of native and of each IGFBP-5 mutant that bound to 0.025 µl
of heparin-Sepharose beads. Native IGFBP-5 (Fig. 5A, lanes 4-7) and the K211N mutant (Fig. 5A, lanes 11-14) bound to
heparin-Sepharose beads dose dependently. Scanning densitometry showed
that the heparin binding activity of the K211N mutant was equal to
native IGFBP-5. The binding ratio defined as a percentage of IGFBP-5
that binds the heparin-Sepharose beads divided by the total detectible
IGFBP-5 (the amount bound in the pellet plus the supernatant) was
calculated. The binding ratios of 1.67, 3.33, and 6.66 pmol of native
IGFBP-5 to heparin-Sepharose beads were 96, 93, and 88%, respectively (Table 2), and for the K211N mutant they were 97, 99, and 96%,
respectively (Table 2). Similarly, the K134A/R136A/K211N mutant (Fig. 5C), the R207A/K211N mutant (Fig. 5E), and the K217A/R218A mutant (Fig. 5G) bound as well to heparin-Sepharose beads as
native IGFBP-5 (Table 2). In contrast, heparin-binding activity
of the K202A/K206A/R207A mutant (Fig. 5B), the
R201A/K202N/K206N/K208N mutant (Fig. 5D), the
K211N/R214A/K217A/R218A mutant (Fig. 5F), and the
R201A/K202N (Fig. 5H) mutant were decreased. When 1.6
pmol of each of these mutants were added, only 37, 41, 46, and 62% of
each of these mutants, respectively, bound to the heparin-Sepharose
beads, compared to 96% for native IGFBP-5 (Table 2). Nonspecific
binding was very low, since native IGFBP-5 and each mutant bound only
minimally (<7%) to Sepharose beads. Since the NaCl concentration
that is necessary to inhibit the binding of proteins to heparin is
inversely proportional to the K
value, we
quantified the binding of native IGFBP-5 and the mutants to
heparin-Sepharose using NaCl concentrations between 150 and 500
mM. As shown in Table 3, the maximal decrease in binding
of native IGFBP-5 binding to heparin occurred when NaCl concentrations
between 300 and 350 mM were added. Similarly the K134A/R136A,
K134A/R136A/K211N, K211N, R207A/K211N, and K217A/R218A mutants showed
maximal decreases between 300 and 350 mM. In contrast, the
R201A/K202N mutant showed the greatest change between 200 and 250
mM NaCl, and the K202A/K206A/R207A,R201A/K202N/K206N/K208N,
and K211N/R214A/K217A/R218A mutants had maximum reductions between 150
and 200 mM NaCl. This indicates that they have an affinity for
heparin that is considerably less than native IGFBP-5.
Figure 5: A-H, heparin binding activity of IGFBP-5. Native IGFBP-5 or each mutant was incubated with Sepharose beads or heparin-Sepharose beads (0.025 µl) in 50 µl of EMEM, supplemented with 20 mM HEPES, 0.1% Tween 20, and 20 mM EDTA. After an overnight incubation, the samples were centrifuged. The amount of each form of IGFBP-5 in both the pellet and the supernatant was determined by ligand blotting and scanning densitometry as described under ``Experimental Procedures.'' The results were confirmed by immunoblotting (data not shown). Lanes 1-7 of each panel contain native IGFBP-5, and lanes 8-14 contain each mutant; these include A, K211N; C, K134A/R136A/K211N; E, R207A/K211N; B, K202A/K206A/R207A; D, R201A/K202N/K206N/K208N; F, K211N/R214A/K217A/R218A; G, K217A/R218A; H, R201A/K202N. Lanes 1-3 and 8-10, Sepharose beads; lanes 4-7 and 11-14, heparin-Sepharose beads. Lanes 4 and 11, IGFBP-5 (3.33 nM); lanes 1, 5, 8, and 12, IGFBP-5 (6.66 nM); lanes 2, 6, 9, and 13, IGFBP-5 (13.3 nM); and lanes 3, 7, 10, and 14, IGFBP-5 (26.6 nM). The arrows denote the position of unbound IGFBP-5 that remained in the supernatant, and the arrowheads denote the bound IGFBP-5 that remained in the pellet.
Taken
together the results show that five basic amino acids are potentially
required to maintain the heparin binding activity of IGFBP-5. These
include positions 201, 202, 206, 208, and 214. Although mutants
containing single amino acid substitutions will be required to
determine the necessity of each of these residues, some preliminary
conclusions can be inferred. The K211N/R214A/K217A/R218A mutant had
markedly reduced heparin binding. Since the K211N, K217A, and R218A
substitutions had no effect, this suggests that Arg may
be a critical determinant of heparin binding or that some combination
of Arg
with the other three basic amino acids may be
necessary. We also noted a substantial reduction in binding of the
K202A/K206A/R207A mutant to heparin. Arg
is probably not
important, since the R207A/K211N mutant bound heparin normally,
although the effect of altering Arg
alone was not
determined.
The degree of change in affinity of each mutant for IGF-I in
response to heparin was also determined using cross-linking studies.
Coincubation with heparin inhibited I-IGF-I-native
IGFBP-5 complex formation in a dose-dependent manner (Fig. 6A and Table 4). The
I-IGF-I
K134A/R136A mutant complex formation was
inhibited by heparin, and the inhibition was comparable to its effect
on the
I-IGF-I-native IGFBP-5 complexes (Fig. 6B) (Table 4). In contrast, the
responsiveness of the other eight IGFBP-5 mutants to heparin was
decreased compared to native IGFBP-5. When
I-IGF-I was
cross-linked to the other eight mutants (Fig. 6, C-J) in the presence of heparin, the band intensities of
the complexes were greater at all heparin concentrations tested
compared to the
I-IGF-I-native IGFBP-5 or to the
I-IGF-I
K134A/R136A mutant complex. These results
confirm that the basic amino acids in the
Arg
-Arg
region are responsible for
the heparin-induced affinity shift. Similar responsiveness to heparin
was found between the K134A/R136A/K211N mutant (Fig. 6C) and the K211N mutant (Fig. 6D), further suggesting that Lys
and Arg
do not contribute to the affinity shift in
response to heparin.
Figure 6:
A-J, inhibitory effect of heparin on
forming IGF-IIGFBP-5 complexes.
I-IGF-I (30,000
cpm/tube) was incubated with each form of IGFBP-5 (4 nM) in
100 µl of EMEM supplemented with 20 mM HEPES, pH 7.3, in
the presence of the indicated concentrations of heparin. After a 1-h
incubation at room temperature, the samples were cross-linked using 0.5
mM disuccinimidyl suberate and the reaction was stopped by
addition of 10 µl of 0.5 M Tris, pH 7.4. The samples were
subjected to SDS-PAGE under reducing conditions (0.1 M dithiothreitol). A gel was fixed, dried, and autoradiographed as
described under ``Experimental Procedures.'' Lanes
1-6, IGFBP-5 (4 nM). Lanes 1 and 6, no heparin; lane 2, 0.1 µg/ml heparin; lane 3, 1 µg/ml heparin; lane 4, 10 µg/ml
heparin; lane 5, 100 µg/ml heparin. Lane 6, IGF-I
(13.3 nM). A, native IGFBP-5; B,
K134A/R136A; C, K134A/R136A/K211N; D, K211N; E, R207A/K211N; F, K202A/K206A/R207A; G,
R201A/K202N/K206N/K208N; H, K211N/R214A/K217A/R218A; I, R201A/R202N; J,
R217A/R218A.
In this study we extended our previous observations (7) to report that site-directed mutagenesis of specific basic
residues in IGFBP-5 results in a reduction of the capacity of this
protein to associate with heparin. Since we had shown previously (7) that a peptide containing residues in the basic region
Arg-Arg
could nullify the effect of
heparin on the change in IGFBP-5 affinity for IGF-I, we reasoned that
basic amino acids in this region might be involved in heparin binding.
In the present study the possibility that this region contained amino
acids that formed the heparin binding site of IGFBP-5 was confirmed.
Coincubation of native IGFBP-5 with a peptide that contained the
Arg
-Arg
sequence inhibited native
IGFBP-5 binding to heparin-Sepharose beads. In contrast, a peptide
containing the Ala
-Thr
sequence in
IGFBP-5 that has a similar charge to mass ratio had no effect. We next
evaluated the contribution of specific basic amino acids in this region
to heparin binding using the IGFBP-5 mutants. Four of the mutants
showed a significant reduction in heparin binding, and binding of these
mutants to heparin was inhibited by lower NaCl concentrations than were
required to inhibit the binding of native IGFBP-5 to heparin. In
contrast, four other mutants that also contained substitutions or basic
amino acids within the Arg
-Arg
region
had no reduction in heparin binding, suggesting that the specific
positional locations of the basic residues may be important.
The degree of reduction in the affinity of the IGFBP-5 mutants for heparin is similar to that reported for the effects of specific amino substitutions on the affinity of plasminogen activator inhibitor I (PAI-1) binding to heparin(14) . In that study, the investigators reported that the wild type protein required 293-318 mM NaCl to disassociate PAI-1 from heparin-Sepharose whereas the PAI-1 mutants were dissociated with NaCl concentrations between 175 and 238 mM. Native IGFBP-5 required somewhat higher salt concentration for significant inhibition of heparin binding (e.g. 300-350 mM) but the binding of our mutants was inhibited using NaCl concentrations that were similar to those used to inhibit mutant PAI-1 binding (e.g. 150-200 mM). These results indicate that these substitutions for basic residues in IGFBP-5 had a significant effect on its affinity for heparin-Sepharose.
Substitution for two residues
within the linear BBBXXB motif (positions 207 and 211) (15) did not alter heparin binding. In contrast the
three-dimensional structure of antithrombin III (AT-III) a
heparin-binding protein, suggests that the basic amino acids in the
BBBXXB motif (positions 131-136) (15) are
located in or near the heparin binding
region(16, 17) . No natural or site-directed AT-III
mutant that has a substitution for the basic amino acids in positions
131-136 has been analyzed(18, 19) . In contrast,
mutation of basic amino acids outside the motif can result in major
reduction in heparin binding(18, 19) . Chemical
modification of Lys in AT-III suggests that it
contributes to low affinity heparin binding (17, 20, 21) , but Lys
, which
is outside the BBBXXB motif, is an important residue for high
affinity heparin binding. The positions of Arg
and
Lys
in AT-III correspond to Arg
and
Arg
in heparin cofactor II, and mutagenesis of these
residues results in decreased dermatan sulfate binding(22) .
Therefore it is possible that Lys
and Arg
in AT-III are important, but this has not been determined. In
summary, several basic amino acids in AT-III and IGFBP-5 that are
responsible for heparin binding are located outside the proposed
heparin binding BBBXXB motif, suggesting that for both
proteins the determinants of heparin binding in IGFBP-5 may be more
complex.
A reduction in heparin binding is not required to induce
the affinity shift since the K211N or Lys plus
Ala
substitutions alter the response to heparin
extensively. AT-III mutants that alter its function have been analyzed
extensively. However, studies that show mutations that have no effect
on heparin binding but alter the conformational change in AT-III that
occurs with heparin binding have not been reported. The ATIII position
that corresponds to the Lys
position within IGFBP-5, e.g. lysine 136, has not been analyzed in this manner.
Therefore a direct comparison is not possible. It is possible that the
conformational change that occurs in IGFBP-5 that alters its affinity
for IGF-I in response to heparin is based on a more simplified model
than AT-III or other serpins, and therefore its conformational change
in response to heparin binding may be altered more extensively by
single amino acid substitutions.
Our previous report (7) showing that IGFBP-1, IGFBP-2, and IGFBP-4 do not contain
the Arg-Arg
sequence and do not
undergo the heparin-induced affinity shift further suggests that this
sequence is important for either heparin binding and conformational
changes in affinity for IGF-I that are induced by heparin. IGFBP-3,
like IGFBP-5, contains 10 of 18 amino acids in the region corresponding
to Arg
-Arg
that are basic, and all of
these positions have been conserved(23) . However, we do not
note as great an affinity shift after heparin binding with IGFBP-3,
suggesting that, even though its affinity for heparin appears to be
similar to IGFBP-5(7, 24) , IGFBP-3 has other
structural determinants that limit its change in affinity in response
to heparin binding.
Mutagenesis did not induce significant changes
in the affinity of any of the IGFBP-5 mutants for IGF-I. Slight
increases in the affinity were detected, but all were less than
1.5-fold. These results suggest that these basic amino acids play a
minimal role in the binding of IGFBP-5 to IGF-I. This conclusion is
consistent with our previous results (7) showing that the
Arg-Arg
region does not directly
compete with IGFBP-5 binding to IGF-I and excludes the possibility that
both the affinity shift and heparin binding changes noted herein are
simply due to changes in the affinity of each mutant for IGF-I.
Recent evidence has been presented that the binding of IGFBP-3 and
IGFBP-5 to proteoglycans or glycosaminoglycans may play a significant
role in the regulation of cellular responses to IGF/IGFBP combinations.
Smith et al.(25) reported that IGFBP-3 is associated
with Leydig cell surface proteoglycans, and this association influences
IGFBP-3 clearance from conditioned medium. Martin et al.(26) reported that IGFBP-3 associated with the fibroblast
cell surface is displaced by the addition of heparin in conditioned
medium, suggesting that IGFBP-3 binds to cell surface proteoglycans. We
recently have shown that heparin binding to IGFBP-5 or IGFBP-3 leads to
a decrease in the binding affinity of IGFBP-5 or IGFBP-3 for
IGF-I(7) . Importantly IGFBP-3 contains a sequence that is
identical to the Arg-Arg
region of
IGFBP-5, and this region in IGFBP-3 has been proposed to mediate
glycosaminoglycan binding(22) . These findings have led
ourselves and others to hypothesize that IGF-I
IGFBP-5 or
IGF-I
IGFBP-3 complexes adhere to heparan sulfate proteoglycans on
cell surfaces or in ECM. Such adherence results in a shift in the IGFBP
affinity for IGF-I, allowing release from the complex and thus making
free IGF-I available to bind to receptors. This hypothesis is supported
by our previous reports (6, 27) showing the affinity
of IGFBP-3 for IGF-I in conditioned medium is 12-fold higher than the
affinity of IGFBP-3 associated with cell surface and that the affinity
of IGFBP-5 in the conditioned medium is 8-fold higher than for IGFBP-5
that is associated with ECM. More importantly the ability of IGFBP-3 or
IGFBP-5 to potentiate IGF-I action appears to require the affinity
shifts, since when these forms are present in solution they usually
inhibit IGF-I actions, whereas when they are associated with either ECM
or cell surface, they have been shown to potentiate IGF-I
actions(4, 5, 6) .
We previously reported that human fibroblasts secrete a serine protease that cleaves IGFBP-5(28) . Heparin binds to this protease and multiple glycosaminoglycans inhibit its activity(29) . Furthermore, the effect of heparin on this protease can be enhanced by AT-III or heparin cofactor II, suggesting that heparin binding may function to regulate IGFBP-5 abundance as well as its affinity for IGF-I(28) . Since extracellular matrix contains multiple proteoglycans, these proteoglycans in ECM and on cell surfaces may also serve to modulate the activity of this protease and therefore indirectly alter cellular responsiveness to the IGFs.
Proteoglycans in ECM represent an important potential reservoir for binding IGFBP-5 and thereby modulate its activity. They may provide an important means for controlling its affinity for IGF-I (6) and its cleavage by serine proteases(29) . The effect of these mutations on susceptibility to proteolysis and the responsiveness of fibroblasts to IGF-I deserves further analysis.