(Received for publication, December 20, 1994; and in revised form, April 7, 1995)
From the
To define the role of the N-terminal region of insulin-like
growth factor-II (IGF-II) in its binding to insulin and IGF receptors,
deletion mutants des-(1-5)-, des-(1-7)-, and
des-(1-8)-recombinant (r) IGF-II, and the Gly Insulin-like growth factor (IGF) Insulin,
IGF-I, and IGF-II bind with high affinity to their own specific
receptors, i.e. insulin, IGF-I, and IGF-II/CIM6-P receptors.
IGF-II, unlike IGF-I and insulin, also binds with moderate to high
affinity to the IGF-I and insulin
receptors(4, 5, 6) . Because of its high
affinity receptor binding properties, our laboratories have undertaken
to identify the residues that are critical for IGF-II binding to the
insulin, IGF-I, and IGF-II/CIM6-P receptors using site-directed
mutagenic procedures. In our initial study, we observed that
Tyr The importance of the N-terminal pentapeptide
of IGF-I for their functions has been reported(8) . A naturally
occurring truncated form of IGF-I, des-(1-3)-IGF-I, with enhanced
mitogenic activity is present in human fetal and adult
brain(9, 10) , bovine colostrum(11) , and
porcine uterus(12) . The increase in mitogenic potency was
postulated to be the result of decreases in the affinity of
des-(1-3)-rIGF-I for IGFBPs with the consequence that more
unbound growth factor would be available to interact with IGF-I
receptors(13, 14) . To date, the isolation of a
naturally occurring truncated form of IGF-II has not been reported.
However, several deletion mutants of the N-terminal sequence of rIGF-II
have been prepared by recombinant procedures, i.e. des-(1-6)- and des-(1-8)-rIGF-II(15) . It was
shown that des-(1-6)-rIGF-II had approximately 10-fold reduced
affinity for IGFBP-3 but retained an equal affinity for the IGF-I
receptor, whereas the relative affinity of des-(1-8)-rIGF-II for
IGF receptors and IGFBP-3 was
Insulin and IGF-I receptors were purified to apparent homogeneity
from Triton X-100-solubilized human placental membrane as described
previously(18, 19) . The IGF-II/CIM6-P receptor was
partially purified from Oligonucleotides were synthesized by an Applied Biosystems model
380A synthesizer, purified as described previously(22) , and
sequences confirmed by the dideoxynucleotide chain termination
method(23) .
Figure 1:
DNA sequence of the
5` region and the N-terminal amino acid sequence of human IGF-II. The
amino acid of each rIGF-II mutant that has been changed is shown in bold letters. The synthetic oligonucleotides that were used to
construct the mutant IGF-II genes are also
indicated.
Proper folding of the
purified rIGF-II mutants except for [Gly
The
affinities of the mutants for IGF-II/CIM6-P receptor were measured by
using partially purified rat placental membrane-derived receptors
prepared as described previously(20) . Briefly, 1.0-1.5
µg of partially purified receptor preparations were incubated in a
final volume of 0.4 ml of 100 mM HEPES buffer (pH 8.0)
containing 120 mM NaCl, 1 mM EDTA, 1.2 mM
MgSO The data from two to four experiments were
plotted as the B/B
Figure 5:
Stimulation of DNA synthesis in Balb/c 3T3
cells by rIGF-II and the five rIGF-II mutants.
Figure 2:
Schematic illustration of human IGF-II and
the substitutions introduced into the five rIGF-II mutants. The three
disulfide bonds are connected by lines and the positions of the
cysteine residues are numbered.
Figure 3:
Comparison of
Figure 4:
Characterization of the binding of rIGF-II
mutants to insulin, IGF-I, and IGF-II/CIM6-P receptors. Competitive
inhibition of
The deletion of the first five, then six, and finally seven amino
acids from the N-terminal end of the B-domain caused, with one
exception, i.e. the IGF-I receptor data of
des-(1-6)-rIGF-II, a progressive decrease in the relative
affinity of the rIGF-II mutants for the insulin and IGF-I receptors (Fig. 4, A and B). With seven amino acids
removed, des-(1-7)-rIGF-II bound to the insulin and IGF-I
receptors with 11 and 3.3%, respectively, of the affinity of rIGF-II (Table 1). In sharp contrast, the sequential deletion, i.e. residues 5 to 7, had the opposite effect on the binding of the
IGF-II mutants to the IGF-II/CIM6-P receptor. For example, the relative
affinities of des-(1-5)-, des-(1-6)-, and
des-(1-7)-rIGF-II for the rat placental membrane IGF-II/CIM6-P
receptor were 350, 700, and 1,200% of that of rIGF-II. This unexpected
result prompted us to examine the effect of N-terminal amino acid
deletions of IGF-II on its binding to bovine liver IGF receptors. In
this study, the K The greatest adverse effect on the
binding of the rIGF-II to all three receptors occurred when Gly was
substituted for Leu
In our previous studies with rIGF-II mutants, it was
established that Phe Beginning with des-(1-5)-rIGF-II, the
relative affinity of the N-terminal amino acid deletion mutants of
rIGF-II for the IGF-I and insulin receptors decreased as this
octapeptide decreased in length (Fig. 4, A and B, and Table 1). When the eight amino acids were
removed, the relative affinity of the des-(1-8) mutant was <1%
of that of rIGF-II. The decrease in the relative affinity of the
deletion mutants for the IGF-I receptor with a decrease in the length
of their N-terminal octapeptide sequence was paralleled by the decrease
in their ability to stimulate thymidine incorporation into cultured
Balb/c 3T3 cells (Fig. 5). These results are similar in general
to what was observed by Lthi et al.(15) in studies of the binding of des-(1-6)- and
des-(1-8)-rIGF-II to IGF-I receptors and their biological
activities on NIH 3T3 cells. For the comparison, a summary of relative
affinities for analogous mutants of insulin and IGF-I from other
laboratories is tabulated (Table 2). According to
Lthi et al.(15) , the relative
affinity of des-(1-8)-rIGF-II for IGF receptors was reduced to
Des-(1-7)-rIGF-II bound to the IGF-I and insulin receptors
with 3 and 11% of the relative affinity, respectively, of that of
rIGF-II. The relative affinity of des-(1-7)-rIGF-II for a
partially purified preparation of rat placental IGF-II/CIM6-P
receptors, however, was increased 12-fold (Fig. 4C and Table 1). Des-(1-5)- and des-(1-6)-rIGF-II also
showed increased affinity for the IGF-II/CIM6-P receptors. These
observations were generally confirmed in competitive ligand binding
studies with purified bovine liver IGF-II/CIM6-P receptors and
demonstrate the pivotal role that the first seven amino acids in IGF-II
serve in defining its specificity for IGF-I and insulin receptors versus the IGF-II/CIM6-P receptors. Three-dimensional
structural analysis of IGF-II by NMR that was carried out recently by
us (17) showed that Thr
Figure 6:
Three-dimensional structure of rIGF-II.
Schematic ribbon drawing of rIGF-II that had been subjected to NMR
spectroscopy. Side chains of the amino acids that are important for
this study are shown and labeled, Glu
The decrease in affinity of
des-(1-8)-rIGF-II for the IGF-I, insulin, and IGF-II/CIM6-P
receptors was not the consequence of simply removing the N-terminal
octapeptide because [Gly
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
for Leu
substitution mutant of rIGF-II were prepared by site-directed
mutagenesis, expressed in Escherichia coli, and purified. The
binding affinity and mitogenic activity of these rIGF-II mutants as
well as commercially available des-(1-6)-rIGF-II were analyzed.
While the relative affinity of des-(1-5)- and
des-(1-6)-rIGF-II for purified human insulin and IGF-I receptors
remained at
50% levels of that of rIGF-II, the affinity of
des-(1-7)-rIGF-II decreased to
10% and
3%,
respectively, of that of rIGF-II. When the octapeptide including
Leu
was removed prior to the Cys
-Cys
intrachain bond, the relative affinity of this deletion mutant,
des-(1-8)-rIGF-II, for these receptors dramatically decreased to
<1% of that of rIGF-II. Substituting Gly
for Leu in
rIGF-II decreased the affinity of this mutant for the IGF-I and insulin
receptors to about the same extent. These results suggest that the side
chains of Thr
and Leu
may play an important
role in retaining all of the IGF-II functions. Decreases in the
relative affinity for binding of the mutants to these receptors
paralleled the decreases in their mitogenic potency for cultured Balb/c
3T3 cells. Although the relative affinity of des-(1-8)- or
[Gly
]rIGF-II for rat IGF-II/CIM6-P
(cation-independent mannose 6-phosphate) receptors was also <1% of
that of rIGF-II, the relative affinities of des-(1-5)-,
des-(1-6)-, and des-(1-7)-rIGF-II for these receptors was
significantly greater than that of rIGF-II. These results clearly
demonstrate that Thr
and Leu
are important for
binding to insulin and IGF-I receptors and Leu
is critical
for expression of all IGF-II functions.
-I (
)and
IGF-II are single chain polypeptides of 70 and 67 residues,
respectively, that have amino acid sequence homology with each other
and with proinsulin(1) . On the basis of this homology, the
structure of IGF-I and IGF-II has been divided into four domains
designated B, C, A, and D beginning at the N terminus. The B- and
A-domains of IGF-II like IGF-I and proinsulin are connected by three
intrachain disulfide bonds between Cys
-Cys
,
Cys
-Cys
, and Cys
-Cys
that determine the proper folding and receptor binding
specificities of the molecule(2, 3) .
, Phe
, and Val
in the B- and
A-domains were essential residues for IGF-II binding to the insulin and
IGF-I receptors, whereas Phe
, Arg
,
Ser
, Ala
, and Leu
in the
A-domain are residues that were required for its binding to
IGF-II/CIM6-P receptors(6) . Using these mutants, we showed
evidence that IGF-II stimulated DNA synthesis in Balb/c 3T3 cells and
glycogen synthesis in HepG2 cells via the IGF-I receptor(6) .
Finally in a more recent study, we established that residues
Phe
, Arg
, and Ser
were essential
for the binding of IGF-II to IGF-binding proteins (IGFBPs)
1-6(7) .
20% of that of rIGF-II. Our
preliminary observations with des-(1-8)-rIGF-II and
[Gly
]rIGF-II indicated, however, that the
relative affinity of both mutants for IGF and insulin receptors are
<1% of that of rIGF-II(16) . The solution structure of
rIGF-II that recently was determined by us (17) revealed that
the N-terminal Ala
-Glu
region is not well
defined, i.e. the B-domain N terminus is flexible because it
lacks distance constraints. In this study, we prepared a series of
deletion mutants and a substitution mutant in the N-terminal first
eight amino acids to more precisely define the contribution this region
of the B-domain makes to the binding of rIGF-II to insulin, IGF-I, and
IGF-II/CIM6-P receptors.
Materials
Restriction endonucleases and Klenow
large fragment were purchased from Toyobo (Tokyo, Japan).
Des-(1-6)-rIGF-II was purchased from Gropep Pty. Ltd. (Adelaide,
Australia). I-human rIGF-I (2,000 Ci/mmol) and
I-human rIGF-II (2,000 Ci/mmol) were purchased from
Amersham Co. (Buckinghamshire, United Kingdom) with the exception of
I-human rIGF-II (2,000 Ci/mmol) that was used to measure
the affinity of binding of IGF-II to bovine liver IGF-II/CIM6-P
receptors (see below). This material was prepared by the Daiichi
Radioisotope Laboratory (Tokyo, Japan).
I-Human insulin
(2,000 Ci/mmol) and [methyl-
H]thymidine
(20 Ci/mmol) were from DuPont NEN. Pepsin was purchased from Sigma. All
other chemicals were of the highest quality commercially available.
-octyl glucoside-solubilized rat placenta
as described previously(20) . The IGF-II/CIM6-P receptor was
purified from bovine liver according to the previously published
procedure (21) with some modifications. Briefly, bovine liver
acetone powder was prepared from fresh bovine liver. The acetone powder
was solubilized with 2% (w/v)
-octyl glucoside, followed by wheat
germ agglutinin-agarose, rIGF-II-coupled affinity resin, and
[Leu
]rIGF-II
(
)-coupled
affinity resin column chromatography. The purity of the IGF-II/CIM6-P
receptor preparation was confirmed by SDS-polyacrylamide gel
electrophoresis followed by silver staining (data not shown).
Construction of Mutant rIGF-II Genes for Expression in
Escherichia coli
The recombinant human IGF-II gene that was used
in this study was synthesized from the known sequence of human IGF-II
cDNA as described previously(22) . The N-terminal amino acid
substitution or deletion mutants of rIGF-II were constructed by
standard site-directed mutagenesis procedures using the synthetic
oligonucleotides that are illustrated in Fig. 1. The
substitution mutant [Gly]rIGF-II was prepared
based on the results of a study (24) that found that the mutant
where Leu
was replaced by Gly
decreased its
affinity to the insulin receptor markedly. In the preparation of the
N-terminal deletion mutants, des-(1-5)-, des-(1-7)-, and
des-(1-8)-rIGF-II, methionine was introduced at N-terminal
residues 4, 6, or 7, so that their respective N termini can be removed
by cleavage with cyanogen bromide (CNBr) during purification as
described below.
Expression, Preparation, and Chemical Characterization of
Recombinant IGF-II Mutants
rIGF-II mutants were expressed in E. coli MC1061 as fusion proteins with rat interleukin-1 (6) . Purified preparations of these fusion proteins were
cleaved by CNBr treatment. The IGF-II mutant proteins were refolded,
intrachain disulfide bonds formed, and the native molecules were
purified by reverse phase-high performance liquid chromatography
(rp-HPLC) as described previously(6) .]rIGF-II
was determined by a previously described peptide mapping procedures
that involved pepsin digestion, rp-HPLC, and amino acid composition
analysis and N-terminal amino acid sequence analysis of the isolated
peptide fragments(2, 3, 6) .
[Gly
]rIGF-II could not be characterized in the
same way because the amino acid, i.e. Leu
, which
is usually cleaved by pepsin becomes more resistant. Therefore,
peptides corresponding to those generated when
[Gly
]rIGF-II was digested were synthesized by an
ABI model 431A peptide synthesizer using the selective S-S formation
procedures(24) . These were then compared with those obtained
when [Gly
]rIGF-II was digested for their
retention time during analytical rp-HPLC (data not shown).
NMR Measurements
Wild type rIGF-II and
[Gly]rIGF-II at concentrations of 2.4 mM in 85% H
O, 10% D
O, 5%
CD
CO
D, and des-(1-7)- and
des-(1-8)-rIGF-II at 0.6 mM in the same solvent were
adjusted to a pH of 2.6, and their
H NMR spectra were
recorded on a UNITY plus 600 spectrometer operating at
H
600 MHz and 50 °C with a spectral width of 7,000 Hz. Chemical
shifts were referenced relative to the internal standard
2,2-dimethyl-2-silapentane-5-sulfonate.
Characterization of Binding of rIGF-II Mutants to
Insulin, IGF-I, and IGF-II/CIM6-P Receptors
Competition for the
binding of I-insulin and
I-IGF-I to insulin
and IGF-I receptors by insulin, IGF-I, rIGF-II, and rIGF-II mutants was
carried out using human placental receptors that were purified as
described previously(18, 19) . Briefly, duplicate
aliquots of 12-32 fmol of receptor were incubated at 4 °C in
0.3 ml of 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% (w/v)
bovine serum albumin, 0.075% (v/v) Triton X-100, 20,000 cpm of
I-insulin or
I-IGF-I, and competing ligands
at concentrations of 0.04-1.106 nM. Following an
overnight incubation at 4 °C, ligand-receptor complexes were
precipitated by the addition of 0.1 ml of 0.4% (w/v)
-globulin and
0.5 ml of 25% (w/v) polyethylene glycol. Pellets were washed once and
counted in a Pharmacia model 1272 Clini Gamma Counter.
, 2.5 mM KCl, 10 mM glucose, 0.5%
(w/v)
-octyl glucoside, 0.1% (w/v) bovine serum albumin, 20,000
cpm of
I-IGF-II, and competing ligands at concentrations
of 0.0075-200 nM. The affinities of the mutants for this
receptor were also measured using purified bovine liver IGF-II/CIM6-P
receptors. Purified bovine receptors (0.15 µg) were incubated in a
final volume of 0.3 ml of 50 mM sodium phosphate buffer (pH
7.4) containing 0.1% (w/v) bovine serum albumin, 0.2% (w/v)
-octyl
glucoside, 1 mM phenylmethanesulfonyl fluoride, 20,000 cpm of
I-IGF-II, and competing ligands at concentrations of
0.004-32 nM. Receptor-ligand complexes were separated
from unbound ligands by the polyethylene glycol procedure. Pellets were
washed once and counted.
where B is
the quantity of
I-labeled ligand bound in the presence of
a given concentration of competing ligand, and B
is the quantity of
I-labeled ligand bound in the
absence of unlabeled ligand. The apparent equilibrium dissociation
constants, K
, for the
binding of these ligands to the receptors were determined from the
nanomoler concentrations of ligand that displaced 50% of specifically
bound ligand.
Characterization of the Biological Activities of rIGF-II
Mutants
The biological activities of rIGF-II mutants were
measured by the incorporation of [H]thymidine
into DNA as described previously(6, 25) . In brief,
subconfluent cultures of Balb/c 3T3 cells that were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
(v/v) fetal calf serum were plated in 96-well microtiter plates
(collagen-coated cell wells; Corning) at a density of 1
10
cells/well. rIGF-II or rIGF-II mutants at concentrations
designated in Fig. 5were added to the cells in Dulbecco's
modified Eagle's medium for 24 h at 37 °C. At the end of this
incubation, the cells were incubated at 37 °C for 2 h with 0.5
µCi/well of [
H]thymidine and unincorporated
radioactivity removed by washing. The magnitude of
[
H]thymidine incorporation into cellular DNA was
quantified by liquid scintillation counting.
, rIGF-II;
, [Gly
]rIGF-II;
,
des-(1-5)-rIGF-II;
, des-(1-6)-rIGF-II;
,
des-(1-7)-rIGF-II; and
, des-(1-8)-rIGF-II. The data
are expressed as the mean ± S.D. of four
determinations.
Preparation of rIGF-II Mutants
The N-terminal
deletion mutants, des-(1-5)-, des-(1-7)-, and
des-(1-8)-rIGF-II, and the amino acid substitution mutant
[Gly]rIGF-II were prepared as described under
``Experimental Procedures'' and their purity established by
SDS-polyacrylamide gel electrophoresis, analytical rp-HPLC, and amino
acid composition analysis (data not shown). The position of the three
intrachain disulfide bonds between Cys
and
Cys
, Cys
and Cys
, and Cys
and Cys
in the three deletion mutants were confirmed
by peptide mapping procedures and the
[Gly
]rIGF-II mutant was found to be properly
folded. The N-terminal amino acids for des-(1-5)-,
des-(1-7)-, and des-(1-8)-rIGF-II were determined by
protein sequencing to be Glu
, Leu
, and
Cys
, respectively. The composition of the three disulfide
bonds and mutation sites of these mutants are illustrated in Fig. 2. One-dimensional NMR spectra of des-(1-7)-,
des-(1-8)-, and [Gly
]rIGF-II mutants were
compared together with that of the wild type rIGF-II in Fig. 3.
The spectra were similar with each other, supporting that
three-dimensional structure of the mutants were not significantly
different from that of the wild type rIGF-II.
H NMR spectrum
between rIGF-II and rIGF-II mutants. Amide and aromatic proton regions
of 600 MHz
H NMR spectra of rIGF-II (A),
des-(1-7)-rIGF-II (B), des-(1-8) rIGF-II (C), and [Gly
]rIGF-II (D) were
measured in 85% H
O, 10% D
O, 5%
CD
CO
D at 50 °C as described under
``Experimental Procedures''.
Relative Affinity of rIGF-II Mutants for Binding to
Insulin, IGF-I, and IGF-II/CIM6-P Receptors
The K values for the binding
of insulin and rIGF-II to human placental membrane insulin receptors
were 0.12 and 0.9 nM, respectively (Fig. 4A).
The K
values for the
binding of rIGF-I and rIGF-II to human IGF-I receptors were 0.12 and
0.33 nM, respectively (Fig. 4B). These values
are similar to those observed in an earlier study (6) for these
ligands. The K
of
0.1-0.4 nM that was calculated for rIGF-II binding to
the rat placental IGF-II/CIM6-P receptors (Fig. 4C) is
3-10 times higher than was observed in our initial study in which
the K
was 0.04
nM(6) . This difference probably reflects the use of
two to three times more of the partially purified receptors than was
used previously in order to obtain the same percent of binding. A
relative affinity of binding of each mutant to the three receptors was
calculated from their displacement curves shown in Fig. 4and
compared with that of rIGF-II, which was set to 100% (Table 1).
I-human IGF-II binding to purified human
placental insulin receptors (A), purified human placental
IGF-I receptors (B), and partially purified rat placental
IGF-II/CIM6-P receptors (C) by insulin (⊡), IGF-I
(
), rIGF-II (
), [Gly
]rIGF-II
(
), des-(1-5)-rIGF-II (
), des-(1-6)-rIGF-II
(
), des-(1-7)-rIGF-II (
); and des-(1-8)-rIGF-II
(
). The results are the mean of two to four experiments and are
plotted as B/B
, where B is the
quantity of
I-labeled ligand bound in the presence of a
given concentration of competing ligand and B
is
the quantity of
I-labeled ligand bound in its
absence.
for the
binding of rIGF-II to bovine liver IGF-II/CIM6-P receptors was 0.06
nM. This K
value
was lower than that of the rat placental IGF-II/CIM6-P receptor
preparation used in the current study but was similar to the value
obtained in the previous study(6) . The affinities of
des-(1-5)- and des-(1-7)-rIGF-II for the bovine receptors
were 300 and 200%, respectively, of that of rIGF-II (Table 1).
The affinity of the latter for the bovine receptor was much less than
it was for the rat placental IGF-II/CIM6-P receptor, i.e. for
1,200%. However, the affinity of des-(1-5)-rIGF-II was about the
same for both sources of IGF-II/CIM6-P receptor. Thus, the results of
binding of the IGF-II mutants to both rat placental and bovine liver
IGF-II/CIM6-P receptors indicated that deletion of the first seven
N-terminal amino acids increased the affinity of the IGF-II mutants for
the IGF-II/CIM6-P receptors.
or the N-terminal sequence was deleted
to Cys
. The binding of [Gly
]rIGF-II
and des-(1-8)-rIGF-II was <2.0% of that of rIGF-II. In these
analyses, [Gly
]rIGF-II tended to have relatively
higher affinities for all three receptors when compared with
des-(1-8)-rIGF-II. The effect of a single amino acid removal from
IGF-II on its ability to bind to the IGF-II/CIM6-P receptors was
clearly evident when deleting Leu
from
des-(1-7)-rIGF-II decreased the binding affinity of the new
mutant by 3 logs. Finally, this result could not be due to drastically
altered three-dimensional structure of the mutants, since the overall
structure and proper disulfide bond formation of
[Gly
]rIGF-II or des-(1-8)-rIGF-II have been
confirmed by one-dimensional NMR and peptide mapping analysis,
respectively.
Characterization of the Biological Activities of rIGF-II
Mutants
[H]Thymidine incorporation into
the DNA of Balb/c 3T3 cells was used in our previous study to show that
the amino acids Phe
, Tyr
, and Val
that are required for the binding of IGF-II to IGF-I receptors
were also required for the stimulation of DNA synthesis(6) .
Using the same approach, the magnitude of incorporation of
[
H]thymidine into Balb/c 3T3 cells treated with
varying concentrations of the N-terminal amino acid deletion mutants of
rIGF-II decreased as the number of the amino acids deleted increased.
For example, des-(1-5)-rIGF-II was equipotent with rIGF-II and
des-(1-6)- and des-(1-7)-rIGF-II were proportionally less
potent (Fig. 5). Des-(1-8)- and
[Gly
]rIGF-II at concentrations of 200
nM, that produced a near-maximum response with rIGF-II, were
10-fold less potent as stimulators of DNA synthesis. Among the three
receptors tested (Table 1), the relationship between mitogenic
potency and N-terminal amino acid length to Thr
of the
deletion mutants was correlated the best with their ability to bind to
IGF-I receptors.
, Tyr
, and Val
were essential for IGF-II binding to insulin and IGF-I receptors
and that Phe
, Arg
, Ser
,
Ala
, and Leu
were essential for its
interaction with IGF-II/CIM6-P receptors(6, 16) .
Furthermore, Phe
, Arg
, and Ser
were also involved in IGF-II binding to IGFBPs 1-6 (7, 16) . The positions of these residues in the
IGF-II molecule were determined by NMR analysis which showed that these
residues lay on the surface of the molecule(17) . In the
present study, the contribution of the first eight N-terminal amino
acids, i.e. Ala
, Tyr
, Arg
,
Pro
, Ser
, Glu
, Thr
, and
Leu
, of IGF-II on its binding affinity for IGF and insulin
receptors were examined.
20% of that of rIGF-II. This result is different from our
observations that the same mutant has relative affinities for IGF and
insulin receptors that are <1% of that of rIGF-II. It could be due
to the sources of the receptors used for the different analyses. Our
results with des-(1-8)- and [Gly
]rIGF-II
are instead very similar to those observed with the analogous
corresponding insulin mutants, des-(1-6)- and
[Gly
]insulin (24) as well as a series of
IGF-I deletion mutants (Table 2). We did not test additional
shorter or longer deletion mutants, since in the first case,
des-(1-5)-rIGF-II bound to IGF-I receptors with an affinity that
was similar to rIGF-II (Table 1). While the deletion of the next
amino acid, i.e. Cys
, would eliminate the
disulfide bond between it and Cys
.
is located approximate to
the A-domain residues of Phe
and Ser
and the
disulfide bridge of Cys
-Cys
(Fig. 6).
Phe
and Ser
are part of the binding site that
interacts with the IGF-II/CIM6-P receptors (6) and the IGFBPs
1-6 (7) . The interaction between Thr
and
Phe
/Ser
and Cys
-Cys
may serve to stabilize the apparently flexible N-terminal
hexapeptide of IGF-II that is involved in the binding of the growth
factor to the IGFBPs and insulin and IGF-I receptors. However, the
apparent greater affinity with which des-(1-7)-rIGF-II binds to
IGF-II/CIM6-P receptors suggests that the N-terminal amino acid
sequence serves to structurally modulate the magnitude of IGF-II
binding. Alternatively, the removal of this sequence could result in
the establishment of a higher affinity binding site because of the
greater ability of the mutant to induce a fit between its more exposed
surface residues especially Phe
/Ser
and the
IGF-II/CIM6-P receptor. The resolution of these two possibilities
awaits further study.
in pink, Thr
in light green, Phe
in purple, Phe
in
orange, Arg
in blue, Ser
in green, Ala
in red, Leu
in light blue, and the three disulfide
bonds in yellow. The N and C termini of rIGF-II are labeled as N and C, respectively.
]rIGF-II decreased the
affinity to these receptors about the same extent (Table 1).
Finally, [Gly
]rIGF-II also had relative
affinities for IGFBPs 1-6 that were less than 1% of that of
rIGF-II(16) . The solution structural analysis of IGF-II
revealed that the side chain of Leu
was not likely to be
involved in a structural stabilization of IGF-II molecule, since
nuclear Overhauser effect signals between Leu
and other
regions were not detected. Nevertheless, Leu
and Leu
may serve pivotal roles in determining the biological activity of
IGF-II and presumably IGF-I, respectively. The evidence to suggest such
a role stems from the observation that the dramatic decreases in the
binding affinities of des-(1-8)- and
[Gly
]rIGF-II for the IGF and insulin receptors
are results that are very similar to what was observed for
des-(1-6)- and [Gly
]insulin in binding to
the insulin receptor(24) . As evidenced from one-dimensional
NMR analysis, the decrease in affinity of des-(1-8)- and
[Gly
]rIGF-II for the receptors and IGFBPs was not
due to the drastic alternation of their three-dimensional structure (Fig. 3). These results strongly suggested that the side chain
of Leu
of IGF-II are required for its binding to the IGF-I,
insulin, and especially IGF-II/CIM6-P receptors.
]rIGF-II binds
specifically to IGF-II/CIM6-P receptors but not to insulin and IGF-I
receptors (6).
We express our thanks to Dr. D. Kohda and Dr. H.
Hatanaka for many helpful discussions about the tertiary structure of
IGF-II. We are grateful to Dr. M. Furusawa for his valuable advice,
discussions, and continuous encouragement. We also thank Y. Kaibori, Y.
Arai, and K. Sato for their excellent technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.