From Insulin Research, Novo Nordisk A/S, 2880 Bagsvaerd, Denmark
Received for publication, October 16, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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The insulin receptor (IR) is a dimeric
receptor, and its activation is thought to involve cross-linking
between monomers initiated by binding of a single insulin molecule to
separate epitopes on each monomer. We have previously shown that a
minimized insulin receptor consisting of the first three domains of the
human IR fused to 16 amino acids from the C-terminal of the Insulin mediates its effects by binding to tyrosine kinase
receptors in the plasma membrane of targets cells. The primary sequence
of the IR1 was cloned in 1985 (1, 2), and the exon-intron structure of the IR gene was described in
1989 by Seino et al. (3). The IR protein is a dimer of two
identical -subunit
was monomeric and bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Schäffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780-17786). To
investigate the insulin binding properties of dimerized
-subunits,
we have reintroduced the domains containing
-
disulfide bonds
into this minireceptor. When inserting either the first fibronectin
type III domain or the full-length sequence of exon 10, the receptor
fragments were predominantly secreted as disulfide-linked dimers that
both had nanomolar affinity for insulin, similar to the affinity found
for the minireceptor. However, when both these domains were included we
obtained a soluble dimeric receptor that bound insulin with 1000-fold
higher affinity (4-8 pM) similar to what was obtained for
the solubilized holoreceptor (14-24 pM). Moreover,
dissociation of labeled insulin from this receptor was accelerated in
the presence of unlabeled insulin, demonstrating another characteristic
feature of the holoreceptor. This is the first direct demonstration
showing that the
-subunit of IR contains all the epitopes required
for binding insulin with full holoreceptor affinity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
monomers covalently linked by disulfide bonds (4).
Each monomer is synthesized as a heavily glycosylated single chain
proreceptor, which is proteolytically cleaved by furin yielding
and
subunits. The subunits are disulfide linked in a
-
-
-
conformation (Fig. 1). The IR structure
function has recently been reviewed (5). The extracellular
domain is composed of the entire
-subunit (residues
1-719)2 and the first 194 residues of the
-subunit. Predictions of the tertiary structure of
the IR ectodomain have been based on sequence alignments with
homologous domains in other receptors (reviewed by Marino-Buslje
et al. in Ref. 6). The consensus from these alignments is
that the first 468 residues (exons 2-6) of the
-subunit contain two
large homologous domains L1 and L2 separated by a cysteine-rich (Fig.
1, L1/CYS/L2) region (7, 8). A crystal structure of the
L1/CYS/L2 region of the homologous IGFI receptor has been solved
confirming this domain structure (9). The remainder of the IR
ectodomain has been predicted to contain three fibronectin type III
(Fn) domains (10-13). The first fibronectin domain, Fn0 (exons 7 and
8) contains Cys524 that is involved in an
-
disulfide
bridge (14). The second fibronectin domain Fn1 (exons 9-12) is
involved in
-
contact via Cys647 that is disulfide
linked to Cys860, located in the third fibronectin domain
Fn2 (exons 13 and 14) (4). Fn1 is predicted to contain an insertion
domain of ~125 amino acids encoded by exons 10-11 and the first half
of exon 12 including the tetrabasic cleavage site of
-
junction
in the proreceptor. Exon 10 contains the triplet of cysteines at
positions 682, 683, and 685, which are involved in the
-
disulfide linkage (4). Exon 10 also contains residues 704-717 that are
essential for insulin binding (15, 16). A schematic diagram showing the
relative arrangement of subunits and domains of IR is shown in Fig.
1.
View larger version (29K):
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Fig. 1.
Insulin receptor constructs. A,
structure of the insulin receptor gene and the relative spans of exons
and encoded domains. Domains are the homologous L1 and L2 domains
separated by the cysteine-rich domain (CYS) followed by
three fibronectin type III domains (Fn0, Fn1 and
Fn2), the transmembrane domain (TM), and the
intracellular kinase domain. Notably, nearly all domain boundaries
correspond to boundaries between exons. The L1/CYS/L2 domains are
encoded by exons 2-6, Fn0 is encoded by exons 7-8, Fn2 is encoded by
exons 13-14, TM is encoded by exon 15, and the kinase domain is
encoded by exons 16-22. Fn1 is encoded by exon 9 in the -subunit,
and the C-terminal half of exon 12 in the
-subunit and contains an
insert domain that can be subdivided into its
-subunit component
encoded by exon 10 (and 11) and its
-subunit component encoded by
the N-terminal half of exon 12. B, organization of domains
within the insulin receptor constructs. The holoreceptor
(hIR) is depicted as a dimer inserted into the plasma
membrane (PM) showing the approximate location of the
-
and
-
disulfide bounds. The length of the soluble
ectodomain, sIR, is shown at the left. mIR.Ex10, mIR.Fn0, and
mIR.Fn0/Ex10 are depicted in their dimeric forms showing the expected
arrangement of disulfide bounds between inserted domains. Ex10
comprises the last 70 residues (residues 650-719) of the
-subunit
encoded by exon 10 and residues 718-719, the first two residues
encoded by exon 12. C-terminal FLAG-epitope tags are shown as
open triangles.
Several groups have shown that mammalian cells expressing the IR
ectodomain secrete a soluble and properly processed dimeric receptor
(sIR) that binds insulin in the nanomolar range (17-20). However, this
affinity is considerably lower than that typically found with the
full-length holoreceptor (hIR) (18, 21-23). In addition to its high
affinity for insulin, hIR exhibits nonclassical receptor binding
properties suggestive of negative cooperativity or site-site
interactions between the two receptor halves, namely accelerated
dissociation of labeled insulin in the presence of unlabeled insulin
and curvilinear Scatchard plots (24). In contrast, sIR binds two
molecules of insulin with low affinity and displays linear Scatchard
plots (25). To account for these ligand binding properties, it has been
suggested that high affinity binding involves one molecule of insulin
that cross-links to distinct sites on opposite -subunits (23, 24)
and that sIR lacks this communication between
-subunits (23).
There have been some reports on soluble IR ectodomains attaining better
than nanomolar affinity, for insulin, either by certain purification
procedures (26) or by expressing sIR as a fusion with self-associating
proteins such as the immunoglobulin Fc and domains (27) or a
leucine zipper (28) placed at the C terminus of the truncated
-subunit.
In the present study we have made dimeric fragments of the -subunit
by reintroducing the Fn0 domain and/or the entire exon10 into the
minimized IR (16, 29). We demonstrate that introduction of both these
domains generates a soluble dimeric
-subunit fragment that binds
insulin with very high affinity in the low picomolar range similar to
that obtained with the full-length holoreceptor.
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EXPERIMENTAL PROCEDURES |
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Miscellaneous-- Insulin and [125I-TyrA14]insulin were from Novo Nordisk. DNA restriction enzymes and T4 DNA ligase were from New England BioLabs, Pwo polymerase was from Roche Molecular Biochemicals. Preparation of plasmid DNA and agarose gel electrophoresis were performed according to standard methods. For DNA minipreps QIAprep 8 kit was used (Qiagen). DSS (Disuccinimidyl suberate) was from Pierce Chemical Co., and other chemicals were from Sigma. The insulin receptor monoclonal antibodies F12 and F26 were raised and kindly donated by Jes Thorn Clausen, Novo Nordisk. Binding data were fitted using nonlinear regression algorithm in GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA).
Construction and Expression of Insulin Receptor
Fragments--
An overview of the receptor constructs and the
abbreviations used is shown in Fig. 1 and Table I. hIR is the
full-length human insulin receptor lacking exon 11, sIR is the soluble
ectodomain of IR (exon 11 minus form), and mIR is the minimized
-subunit comprising the first 3 domains of IR (residues 1-468)
fused to a 16-amino acid peptide from the C-terminal of the
-subunit
(residues 704-719) followed by the FLAG epitope (DYKDDDDK) (29). These three receptors have been described previously (18, 21, 29).
All DNA constructs were inserted in the pZem expression vector (21) and
stably expressed in baby hamster kidney (BHK) cells. Cells were grown
in Dulbecco's modified Eagle`s medium (Life Technologies Inc.). Cell
transfection procedures and culture conditions were described in detail
previously (29). For the present study, samples of hIR were made by
solubilizing transfected BHK cells overexpressing hIR in ice-cold lysis
buffer (50 mM Hepes pH 8.0, 150 mM NaCl, 1%
Triton X-100, 2 mM EDTA, 10% glycerol, 10 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride) (4 ml
lysis buffer/1 × 108 cells). The lysate was cleared
by centrifugation for 15 min at 35,000 × g,
concentrated three-fold on Microcon-100 (Millipore) and stored at
80 °C. Unless stated otherwise, samples of all other receptors
fragments were culture supernatants from BHK cells expressing the
receptor constructs.
mIR.Ex10 that consists of residues 1-468 and 650-719 fused to the FLAG epitope was made by PCR amplification of exon 10 using DNA-encoding wild-type human IR as template with the sense primer 5'-GACAAGGCTAGCTGTGGGCTGAAGCTGCCC-3' (NheI site underlined) and antisense primer 5'-TTTTCCTAGGGACGAAAACCACG-3' (AvrII site underlined). The PCR fragment was digested with NheI and AvrII and ligated into the corresponding sites in the plasmid encoding mIR (29). For making mIR.Fn0 that consist of IR residues 1-601 and 704-719 fused to the FLAG epitope, two complementary oligonucleotides, 5'-GATCCAACGTTTGAGGATTACCTGCACAACGTGGTTTTCGTCC-3' and 5'-CTAGGGACGAAAACCACGTTGTGCAGGTAATCCTCAAACGTTG-3' (both with phosphorylated 5'-ends) were hybridized to produce a double-stranded fragment encoding residues 704-717 with BamHI and AvrII compatible ends. The fragment was ligated with a 3.5-kilobase AvrII/MfeI vector fragment from plasmid encoding mIR, which has the C-terminal FLAG sequence downstream from the AvrII site, and a 3.2-kilobase MfeI/BamHI fragment from the IRwt construct described previously (16) having the N-terminal IR residues 1-601 upstream from the BamHI site. Finally, mIR.Fn0/Ex10 consisting of residues 1-601 and 650-719 fused to the FLAG epitope, was made by PCR amplification of exon 10 using the mIR.Ex10 construct as template with the sense primer 5'-CGCTATCGCGGATCCAGGGCTGAAGCTGCCCTC-3' (BamHI site underlined) and an antisense primer down stream from the FLAG epitope encoding sequence and its flanking XbaI site. The PCR fragment was digested with BamHI and XbaI and ligated into the corresponding sites in the plasmid encoding mIR.Fn0.
Receptor Competition Binding Assays-- Two types of competition binding assays were used, a polyethylene glycol (PEG) precipitation assay and a microtiter plate assay. For both assays the concentration of receptor was adjusted to yield ~10% binding of tracer when no competing insulin was added. This corresponds to receptor concentrations ~10-fold lower than the IC50 obtained for the given receptor. Moreover, the amount of tracer and duration of incubation was adjusted depending on the affinity of the receptor, so that low concentrations of tracer (~3 pM) and longer incubation times (>48 h) were used for receptors with high affinity binding (picomolar range).
For the PEG assay a suitable dilution of receptor sample was incubated
for 16-60 h at 4 °C in a total volume of 200 µl with 3-12
pM of [125I-A14]insulin and various
concentrations of unlabeled insulin in binding buffer (100 mM Hepes, pH 8.0, 100 mM NaCl, 10 mM MgCl2, 0.25% (w/v) BSA, 0.025% (w/v)
Triton X-100). Subsequently bound counts were recovered by
precipitation with 0.2% -globulin and 740 µl of 30% (w/v)
polyethylene glycol Mr 8000. Bound
125I-labeled insulin was counted in a
-counter.
The microtiter plate assay was performed essentially as described in
the literature (30). For immobilization of receptor fragments, an
insulin receptor specific antibody, F12, was used. F12 was raised
against purified minireceptor IR703 (16) and shown to recognize an
epitope within residues 1-468 (data not shown). First, microtiter
plates (Lockwell C8, maxisorp from Nunc) were coated with goat
anti-mouse IgG antibody (Pierce); for each well was used 50 µl of a
20 µg/ml solution in TBS (0.01 M Tris, pH 7.5, 100 mM NaCl). Plates were incubated for 1 h at room
temperature before washing two times with TBS and blocking with 250 µl of Superblock (Pierce). Then 50 µl of affinity-purified F12
antibody (1.2 µg/ml) was added to each well. Plates were incubated
for 2 h at room temperature before washing three times with
binding buffer followed by the addition of 50 µl of a suitable
dilution of receptor sample. After a 2-h incubation at room
temperature, plates were washed three times, and binding experiments
were performed by adding a total volume of 150 µl of binding buffer
with 3-10 pM of 125I-labeled insulin and
varying concentrations of insulin. After 16-60 h at 4 °C, unbound
ligand was removed by washing once with cold binding buffer, and the
tracer bound in each well was counted in a
-counter.
Receptor Saturation Binding Assay--
For saturation binding
experiments, receptor samples were incubated in a total volume of 200 µl with various concentrations of [125I-A14]insulin in
binding buffer for at least 60 h at 4 °C. Subsequently, bound
counts were recovered by precipitation with 0.2% -globulin and 740 µl of 30% (w/v) polyethylene glycol Mr 8000. Bound 125I-labeled insulin was counted in a
-counter.
For each tracer concentration, nonspecific binding was determined by
measuring bound 125I-insulin in the presence of 1 µM of unlabeled insulin.
Immunoblotting--
The expressed receptor proteins were
detected by immunoblotting using the monoclonal antibody F26. This
antibody was raised against a peptide corresponding to residues 39-75
near the N terminus of the insulin receptor -subunit. For
immunoblotting, samples were mixed with 0.33 volumes of 4× LDS loading
buffer (Novex). Reduced samples were mixed with loading buffer
containing 100 mM dithiothreitol and incubated at 95 °C
for 5 min before loading 15 µl on a 4-12% polyacrylamide Bis-Tris
gel (NuPAGE, Novex). After electrophoresis in MOPS-running buffer
(Novex), proteins were blotted onto Immobilon-P membrane (Millipore).
The membrane was blocked by incubating with blocking buffer (2%
defatted skim milk, 1% BSA in TBS) for 1 h at room temperature.
The receptor antibody F26 (diluted in TBS, 1% BSA) was added to the
membrane and incubated for 16 h at 4 °C. The membrane was
washed with TBS before incubating with peroxidase-conjugated secondary
antibody (Dako, Denmark) diluted in 1% BSA in TBS. Finally the blot
was washed with TBS, and immunoreactive proteins were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) and visualized using the FujiFilm CCD camera, and the Image Gauge software
(Fuji Photo Film Co).
Chemical Cross-linking of125I-labeled Insulin to Receptors-- Chemical cross-linking was performed essentially as described (18, 31). Receptor samples were incubated for 60 min at room temperature with [125I-A14]insulin (0.2-0.3 nM) in the presence or absence of unlabeled insulin (1 µM). Disuccinimidyl suberate (DSS) in dimethyl sulfoxide was added from a 10 mM stock solution to a final concentration of 0.1 mM. After 15 min on ice, the reaction was stopped by adding 0.33 volume of 4× LDS loading buffer, and samples were separated by SDS electrophoresis as described for immunoblotting above. The gel was fixed in 10% acetic acid, 20% ethanol, and a phosphorimager screen was exposed with the dried gel.
Dissociation of 125I-labeled Insulin From
Receptors--
For investigating dissociation of labeled insulin,
receptors were immobilized in microtiter wells with the antibody F12 as described for the competition assay above. After immobilization of the
receptors, 150 µl of binding buffer containing labeled insulin (20 pM) was added and allowed to equilibrate by incubating for
2 h at room temperature, followed by washing with binding buffer.
Dissociation of tracer was followed under two conditions by adding
either binding buffer alone or binding buffer with 0.5 µM
unlabeled insulin. At various time points, wells were washed once with
ice-cold binding buffer, and the bound tracer was counted in a
-counter.
Purification and Gel Filtration of mIR.Fn0/Ex10--
Insulin
receptor fragments were purified from transfected BHK cell culture
supernatant by affinity chromatography using immobilized insulin as
described previously (25). After affinity purification, the dimeric
form of mIR.Fn0/Ex10 was separated from the monomer using a Sepharyl
S300 High Resolution column (Amersham Pharmacia Biotech: 2.5 × 89 cm) equilibrated in 0.2 M Tris, HCl buffer, pH 7.8. Chromatography was performed at a flow rate of 0.2 ml/min. Fractions
containing the dimer were pooled and concentrated on Centriprep-10
(Millipore). Fractions containing the monomer were pooled,
concentrated, and subsequently rerun over the column for further
purification, and a fraction was selected for characterization. The
purified receptor fragments were stored at 4 °C.
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RESULTS |
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Cloning and Expression of Receptor Constructs--
We previously
expressed a minimized insulin receptor (mIR) consisting of the first
three domains of the human insulin receptor (L1/CYS/L2, residues
1-468) fused directly to residues 704-719 from the C-terminal of the
-subunit and the FLAG epitope (29). In the present study three new
constructs were made on the basis of mIR as described under
"Experimental Procedures." All three constructs contained all
residues of mIR and in addition either the first fibronectin type III
domain, Fn0 (mIR.Fn0), the remaining residues of exon 10 (mIR.Ex10), or
both these regions (mIR.Fn0/Ex10). All constructs were stably expressed
in BHK cells, and the culture supernatants were used for the various
assays, except for hIR for which samples were made from solubilized
cells. An overview of all receptor constructs used in the present study
is shown in Fig. 1 and in Table I.
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Binding of Insulin Competition Assays--
The affinities of the
recombinant receptors for insulin were determined in two competition
binding assays: a soluble assay where receptors were precipitated with
polyethylene glycol and a microtiter plate assay in which receptors
were immobilized using a receptor-specific monoclonal antibody.
Representative binding curves for the competition assays are shown in
Fig. 2. In all cases, the binding curves
were fitted to a one-site binding model from which the binding
affinities (IC50) were determined. An overview of all
binding affinities is presented in Table I. In the PEG assay the
control receptors mIR, sIR, and hIR yielded affinities similar to what
has been found in previous studies; that is 5-7 nM for mIR
and sIR whereas the affinity for hIR was 0.017 nM. For the
new receptors, there was a slight increase in affinity compared with
mIR when inserting either the Fn0 domain or the full-length sequence of
exon 10, mIR.Fn0 and mIR.Ex10 yielding affinities of 3.5 and 3.0 nM respectively. However, the insertion of both these
domains led to a 1000-fold increase in binding affinity; the affinity
of mIR.Fn0/Ex10 was 8 pM, which is slightly better than the
17 pM found for the holoreceptor (Table I).
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The affinities obtained with the immobilized assay were similar to those found in the soluble PEG assay (Table I), demonstrating that the F12 antibody binds the various receptor fragments without affecting binding affinity for insulin.
Saturation Binding of Labeled Insulin--
The high insulin
binding affinity of mIR.Fn0/Ex10 allowed us to do saturation binding
experiments with labeled insulin only. Fig.
3 shows representative saturation binding
curves for mIR.Fn0/Ex10 and hIR. Both of these curves fitted to a
one-site binding model giving an equilibrium dissociation constant
(Kd) of 0.004 ± 0.002 nM for
mIR.Fn0/Ex10, which is slightly better than found for the holoreceptor,
hIR, 0.014 ± 0.006 nM. The saturation binding experiment clearly confirms the high affinities that were obtained for
mIR.Fn0/Ex10 in the competition binding experiments (Table I). Thus, in
all binding assays mIR.Fn0/Ex10 resembles hIR in having a binding
affinity for insulin in the low picomolar range.
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Detecting Recombinant Receptors by Immunoblotting--
An
antibody, F26, that recognizes the N terminus of the insulin receptor
-subunit, and thus recognizes all receptors in the present study,
was used to detect soluble insulin receptors secreted into culture
medium from transfected BHK cells. For comparison detergent lysates of
cells expressing hIR were also analyzed. Immunoblotting was performed
on reduced as well as nonreduced samples of each receptor. The
immunoblots are shown in Fig. 4.
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On the reduced gel (Fig. 4B) the antibody detects the
full-length -subunit of 130 kDa in the samples of sIR and hIR
(lanes 5-6), whereas the truncated
-subunit of mIR
(lane 1) shows an apparent mass of 80 kDa in accordance with
previously published data (16). When inserting domains into mIR we
obtain larger
-subunit fragments and accordingly mIR.Ex10
(lane 2), mIR.Fn0 (lane 3), and mIR.Fn0/Ex10
(lane 4) showed apparent masses of 95-105 kDa on the gels.
On the nonreduced gel (Fig. 4A), the size of mIR was found
to be the same as for the reduced sample (80 kDa) in accordance with
the minireceptor being a monomer, whereas sIR and hIR (lanes 5-6) gave high molecular mass bands of more than 220 kDa
corresponding to the dimers of disulfide-linked - and
-subunits.
In samples of mIR.Ex10, mIR.Fn0, and mIR.Fn0/Ex10 (lanes
2-4), two bands were recognized by the antibody. The prominent
upper bands in these samples had an apparent mass corresponding to a
dimeric receptor (190-210 kDa) consistent with the introduction of
regions containing the cysteine residues that are involved in the
-
disulfide connections in these receptor constructs (Fig. 1).
The relative intensities of the dimer band versus monomer
band were between 1:1 and 1.8:1.
Chemical Cross-linking of 125I-Insulin to
Receptors--
Labeled insulin was chemically cross-linked to
recombinant receptors using DSS and then separated by SDS-gel
electrophoresis under reducing and nonreducing conditions (Fig.
5). All receptor fragments detected by
immunoblotting could cross-link labeled insulin, and the cross-linking
gels show a pattern that is similar to the immunoblotting patterns
(Fig. 4). The only exception is that the 105-kDa band corresponding to
the monomeric form of mIR.Fn0/Ex10 is not detected by cross-linking in
the nonreduced gel (Fig. 5A, lane 7). This
observation indicates that the high affinity binding property of
mIR.Fn0/Ex10 is associated with the dimeric form only. For mIR.Ex10 and
mIR.Fn0 both dimeric and monomeric forms were cross-linked (Fig.
5A, lanes 3 and 5), showing that both
these forms bind insulin. Because all binding curves for these two
receptors fit to a one-site model, we presume that the monomeric and
dimeric forms of these receptors bind insulin with similar affinities, implying that binding of insulin to mIR.Ex10 or mIR.Fn0 is not influenced by -
disulfide contact(s). On the reduced gel (Fig. 5B), faint bands corresponding to the size of the dimers are
seen for all samples except for mIR. These are most likely caused by cross-linking between the two
-subunits in the dimeric receptor fragments.
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Dissociation of Labeled Insulin from Receptors; Effect of Unlabeled
Insulin--
One of the characteristic features of the insulin
holoreceptor hIR is that dissociation of bound tracer is markedly
accelerated in the presence of unlabeled insulin in the buffer (32). To examine mIR.Fn0/Ex10, we investigated the dissociation of insulin from
receptors immobilized with the F12 antibody. The resulting dissociation
curves of hIR, mIR.Fn0/Ex10, and sIR are shown in Fig.
6. The curves for hIR and the new
high-affinity receptor mIR.Fn0/Ex10 clearly show accelerated
dissociation in the presence of 0.5 µM unlabeled insulin.
After 20 min, about 85% of initially bound tracer is still bound in
the absence of unlabeled insulin whereas less than 40% is bound in the
presence of unlabeled insulin. In contrast, no accelerating affect of
unlabeled insulin is seen for the soluble receptor sIR. When comparing
the dissociation curves in the absence of unlabeled insulin, the
dissociation is clearly faster from sIR than from hIR or mIR.Fn0/Ex10,
probably reflecting the lower affinity of sIR.
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Characterizing Monomeric versus Dimeric Forms of
mIR.Fn0/Ex10--
mIR.Fn0/Ex10 was purified by affinity
chromatography, and subsequently the two forms of the receptor were
separated by gel filtration. The immunoblot in Fig.
7A shows that the mixture of the two forms found in the BHK cell culture supernatant (lane 1) have been separated into dimeric (lane 2) and
monomeric (lane 3) forms. The sample of purified dimer (Fig.
7A, lane 2) appears to contain an additional band
migrating slightly faster than the dimer band present in the culture
supernatant (Fig. 7A, lane 1). At present we do
not know what this receptor band represents, but it is also seen as a
weak band in the monomer fraction (Fig. 7A, lane
3) indicating that the monomer sample still contains traces of
higher molecular weight proteins. Chemical cross-linking of labeled
insulin to the same three samples (Fig. 7B) shows only one
band of ~200 kDa in the cell supernatant (lane 1) and in
the dimer sample (lane 3). The monomer fraction (Fig.
7B, lane 5) shows two bands of which the 100-kDa band
corresponds to the monomer, whereas the weaker 200-kDa band probably
represents a trace of the dimeric form of mIR.Fn0/Ex10.
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Insulin binding experiments with the two purified forms of mIR.Fn0/Ex10
(Fig. 7C) clearly show that the dimer has a much higher affinity (IC50 of 0.010 ± 0.002 nM) than
the monomer (IC50 of 0.9 ± 0.3 nM). The
poor fit of binding data for the purified monomer at the top of the
binding curve indicates that there is a small fraction of high affinity
receptor present in the sample. This high affinity receptor is probably
related to the presence of small amounts of dimer that was also seen in
the cross-linking experiment (Fig. 7B, lane
5).
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DISCUSSION |
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We have previously identified a minimized IR -subunit (mIR)
that binds insulin with the same nM affinity as the soluble
ectodomain, sIR (16, 29). The aim of the present study was to
investigate the insulin binding properties of dimeric
-subunit
fragments. We have employed a strategy of reintroducing domains that
contain the cysteines involved in
-
disulfide bonds into the
minimized insulin receptor. These domains are the first fibronectin
type III domain Fn0 that contains Cys524 and the region
encoded by exon 10 that contains Cys682,
Cys683, and Cys685.
When inserting only one of these domains we obtained mIR.Fn0 and
mIR.Ex10, which were both predominantly secreted as dimers (Fig.
4A). A receptor fragment similar to mIR.Fn0 was also
reported by Molina et al. (33), who found that a fragment
comprising the first four domains (L1/CYS/L2/Fn0) was predominantly
secreted as a dimer. However, this fragment did not bind insulin,
probably because of the lack of essential residues from the C-terminal of the -subunit (16). We found that mIR.Fn0 and mIR.Ex10 bound insulin with affinities in the low nanomolar range. This is slightly better than what is found for the monomeric minireceptor, mIR, and the
soluble ectodomain, sIR (Table I). When both domains were inserted into
mIR, we obtained mIR.Fn0/Ex10 that bound insulin with an affinity that
was 1000-fold higher than that found for mIR or the receptors with only
one domain inserted (Table I). This high affinity in the low picomolar
range was similar to what was found for the solubilized holoreceptor.
In the chemical cross-linking experiments insulin was found to be
associated with the dimeric form of mIR.Fn0/Ex10 only (Fig. 5A), whereas immunoblotting showed that the BHK cells
secreted a mixture of monomers and dimers (Fig. 4A). This
indicates that the picomolar affinity binding property is associated
only with the dimeric structure of the -subunits. This was confirmed
by characterizing the two receptor forms individually after separating them by gel filtration (Fig. 7). The binding data clearly showed that
the purified dimer bound insulin with high affinity (~10 pM) whereas the monomer bound with an affinity of ~1
nM. Schäffer (23) and De Meyts (24) have proposed
models for the interaction between insulin and its receptor involving
an initial binding of insulin to one site on one
-subunit followed
by cross-linking of the insulin molecule to a distinct site on the
other
-subunit. The initial binding event involves binding of
insulin with nanomolar affinity (sIR binding mode) whereas the high
affinity is obtained only when the insulin molecule also binds to the
second site on the opposite
-subunit resulting in the cross-linking
effect. Thus, according to these cross-linking models, the formation of dimers is a prerequisite for high affinity binding of insulin. Supporting these models are stochiometric studies showing that hIR
binds only one insulin molecule with high-affinity (34-36), whereas
monomers formed by controlled reduction bind one insulin molecule per monomer with lower affinity (nM) (34, 35, 37). The present
data on the receptor fragments containing either the Fn0 domain
(mIR.Fn0) or the Ex10 domain (mIR.Ex10) show that dimerization of mIR
per se does not increase binding affinity dramatically and
therefore suggest that both these domains are important for high
affinity binding of insulin.
The binding results for mIR.Fn0/Ex10 demonstrate for the first time
that the -subunit of IR contain all epitopes required for picomolar
affinity binding of insulin. It is possible that a second insulin
contact site has been restored by the introduction of one or both of
the Fn0 and exon 10 domains into mIR. For instance, putative contact
sites have been proposed to reside in the region of the L2/Fn0 junction
that is disrupted in mIR. This has been suggested by photoaffinity
labeling (residues 390-490, Ref. 38), chimeric receptors (residues
325-524, Ref. 39) and by antibodies that inhibit insulin binding
(residues 450-601, Ref. 40) or activates the insulin receptor
(residues 469-592, Ref. 41). Thus, this putative site could represent
the second binding site necessary for high affinity binding as proposed
by cross-linking binding models. However, the fact that this putative
contact site is also restored in mIR.Fn0 that bind insulin with low
affinity, indicates that properties of the additional sequences of exon 10 are also needed for the generation of high affinity receptors.
Several groups have used electron microscopy techniques to study the structure of the insulin receptor and its domain organization. These studies have suggested either extended T- or Y-shaped receptor conformations (11, 42, 43) or more globular conformations (44-46) but no clear consensus has emerged. The high affinity obtained with mIR.Fn0/Ex10 suggests that the first four domains, L1/Cys/L2/Fn0 and the Ex10 domain are in close proximity in the IR structure and that these most likely are the only domains interacting with the insulin molecule. The model based on electron microscopy and three-dimensional reconstruction suggested by Ottensmeyer et al. (46) is in accordance with our observations in having a single insulin molecule bound to a distinct central core composed of the two set of contiguous domains from L1 to Fn0 and additional residues encoded by exon 10.
Previous attempts to characterize the full-length IR -subunit have
been difficult because of very low yields of secreted material (20,
47). In contrast, mIR.Fn0/Ex10 was found to be efficiently secreted
although this fragment only differs in having a 48-residue deletion of
the exon 9 region. In the native receptor, the deleted region is in
direct contact with the
-domain via the Cys647 that is
disulfide-linked to Cys860 in the
-subunit (4).
Furthermore this region contains the first five of the seven strands of
the predicted
-sandwich structure of Fn1, the other strands being
located in the
-subunit (11). Thus, it may be that the full-length
-subunit is poorly expressed because of problems with proper folding
of the exon 9 region that lacks its counterparts from the
-subunit.
There have been some reports on soluble insulin receptors that had
higher affinity for insulin than sIR but in all cases these included
the entire extracellular domain including the N-terminal part of the
-subunit (27, 28). The fact that sIR when purified under certain
conditions can obtain picomolar affinity (26) indicates that the IR
ectodomain in itself has the full potential for high affinity binding
of insulin. It has been reported that ectodomain constructs can be
stabilized in a high affinity conformation via a C-terminal
self-associating fusion partner. Bass et al. (27) fused sIR
to immunoglobulin domains, whereas Hoyne et al. (28) used a
33-amino acid leucine zipper sequence. In contrast to the fusion
proteins that had molecular masses of 300-400 kDa, our approach
yielded a much smaller high affinity
-subunit construct with an
apparent mass of only 200 kDa.
In conjunction with the property of high affinity binding of insulin, hIR is characterized by having nonclassical binding features that suggest negative cooperativity or site-site interaction between separate binding sites. Negative cooperativity has been demonstrated directly by accelerated dissociation of labeled insulin from the receptor when unlabeled insulin is present, or indirectly by Scatchard analysis yielding curvilinear plots (24, 32). In the present study, we demonstrated accelerated dissociation of bound labeled insulin in the presence of unlabeled insulin both for the soluble high affinity receptor mIR.Fn0/Ex10 and for hIR (Fig. 6). In contrast the dissociation of labeled insulin from the soluble ectodomain, sIR was unaffected by the presence of unlabeled insulin. To our knowledge this is the first time that dissociation experiments have been reported for this receptor. In the present study the binding curves for hIR and mIR.Fn0/Ex10 (Fig. 2) were best fitted to a one-site binding model and accordingly the Scatchard transformations resulted in linear plots. Summarizing all the binding data, we conclude that the soluble high affinity receptor mIR.Fn0/Ex10 has the same ligand binding properties as the holoreceptor hIR, in all aspects of ligand binding analyzed here.
All recombinant insulin receptors previously reported to bind insulin
with high affinity (pM) have included the entire
extracellular part of the -subunit. Here we demonstrate that a
dimeric fragment of the
-subunit binds insulin with full
holoreceptor affinity and also exhibits the dissociation properties of
the holoreceptor, making this minimized soluble insulin receptor an
attractive candidate for detailed structural analysis.
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ACKNOWLEDGEMENTS |
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We thank Inge Merete Hansen and Else Jost Jensen for excellent technical assistance and Lene Drube for purification and gel filtration of mIR.Fn0/Ex10.
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FOOTNOTES |
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* 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: Insulin Research, Novo
Nordisk A/S, Novo Allé 6B1.74, 2880 Bagsvaerd, Denmark. Tel.: 45 4442 3605; Fax: 45 4444 4250; E-mail: jakb@novonordisk.com.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M009402200
2 Numbering of IR residues is according to Ullrich et al. (1).
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ABBREVIATIONS |
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The abbreviations used are: IR, insulin receptor; BHK, baby hamster kidney; BSA, bovine serum albumin; DSS, disuccinimidyl suberate; IGFI, insulin-like growth factor I; LDS, Lithium dodecyl sulfate; mIR, minimized IR (IR residues 1-468 + 704-719 + FLAG epitope); PCR, polymerase chain reaction; sIR, soluble IR ectodomain; hIR, IR holoreceptor; PEG, polyethylene glycol; MOPS, 4-morpholinepropanesulfonic acid.
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