From the
We have recently characterized a mutant insulin receptor
(HIR
Insulin binding to the extracellular
Recently, Yamamoto
et al.(8) have characterized a mutant insulin
receptor, deleting the COOH-terminal 83 amino acids. In the report,
Chinese hamster ovary cells overexpressing the truncated insulin
receptor exhibited enhanced insulin sensitivity in spite of defective
kinase activity of the truncated receptor. Indeed, in earlier studies
(9) , we have shown that Rat1 fibroblasts overexpressing a
truncated insulin receptor lacking 365 amino acids from the COOH
terminus also demonstrated enhanced biological actions of insulin,
although the truncated receptor itself lacked tyrosine kinase activity.
A common feature of these mutant cell lines is normal IRS-1
phosphorylation, although kinase activity of the transfected receptors
per se is markedly decreased
(8, 9) . Based on
our earlier studies, we suggested that the enhanced insulin action in
HIR
We have previously shown that overexpression of truncated
Insulin stimulates
the transcription of a series of early response genes, an example of
which is c- fos(19, 20) . To quantitatively
examine this biologic effect, we have utilized the reverse
transcriptasePCR method to measure insulin-induced induction of
c- fos mRNA and have compared the dose response effects of
insulin in HIRc cells, HIR
To
understand the mechanism of this enhanced insulin sensitivity, it is
important to determine whether the increased biologic signaling is
specific for insulin. For example, if overexpression of HIR
We have previously shown that insulin-stimulated IRS-1
phosphorylation is greater in HIR
We also assessed the functional
importance of IRS-I in the augmented insulin effects by performing
signal cell microinjection studies in which anti-IRS-I antibodies were
directly introduced into living HIR
It is known that overexpression of insulin receptors in
fibroblasts can lead to the formation of hybrid receptors consisting of
insulin receptor
In summary, the
current studies demonstrate that the expression of truncated
HIR
We thank Dr. Alan R. Saltiel for p85-SH2 GST fusion
protein and anti-IRS-1 antibody, Dr. Kenneth Siddle for anti-insulin
receptor antibody, and Dr. Byung H. Jhun for advice on the c-fos induction studies. We are grateful to Elizabeth Hansen for her
assistance in the preparation of this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
978) in which the insulin receptor
-subunit was truncated
at amino acid residue 978. Compared with parental Rat1 cells, the cells
expressing the truncated receptor exhibited enhanced sensitivity to
insulin's biologic actions. All of these effects are now extended
to transcriptional events, since we now show enhanced sensitivity to
insulin stimulation of c -fos mRNA expression. These effects
were insulin-specific, since insulin-like growth factor-1 stimulation
of glucose incorporation into glycogen,
-aminoisobutyric acid
uptake, and thymidine incorporation into DNA were normal. In addition,
the truncated receptor exhibited enhanced sensitivity only in
vivo, but not in vitro, since the kinase activity of
wheat germ agglutinin-purified receptor preparations was comparable
between HIR
978 and parental Rat1 insulin receptors. Parental rat
endogenous insulin-like growth factor-1 receptors and transfected human
insulin receptors form hybrid receptors as well as homologous
tetrameric receptors. The normal heterotetrameric receptors possess
kinase activity in vivo leading to tyrosine phosphorylation of
insulin receptor substrate-1 (IRS-1) and its association with the p85
regulatory subunit of phosphatidyl inositol 3-kinase. Interestingly,
preincubation with human-specific anti-insulin receptor antibody
abolished the increased insulin sensitivity in glucose incorporation
into glycogen in HIR
978 cells. Furthermore, microinjection of
anti-IRS-1 antibody into HIR
978 cells inhibited insulin
stimulation of DNA synthesis. In summary: 1) truncated receptors on the
cell surface confer enhanced insulin sensitivity in vivo; 2)
the normal heterotetrameric receptors are functionally active and
couple to IRS-1 efficiently; and 3) IRS-1 is an important molecule
transmitting insulin's biological signals in HIR
978 cells.
-subunits activates
the receptor tyrosine kinase properties of the cytoplasmic portion of
the
-subunit of the insulin receptor
(1) . The receptor
undergoes autophosphorylation, which enables the receptor kinase to
phosphorylate various substrates. One of the cellular substrates for
the insulin receptor tyrosine kinase is IRS-1
(
)(2, 3) . IRS-1 undergoes tyrosine
phosphorylation in response to insulin and acts as a multisite docking
protein to interact with Src homology 2 (SH2) domains of various
signal-transducing molecules to propagate the insulin signal downstream
(3) . A variety of studies have indicated that activation of the
receptor kinase is necessary for IRS-1 phosphorylation leading to the
biological actions of insulin
(4, 5, 6, 7) .
978 cells was directly mediated by the endogenous rodent
insulin receptors, rather than the HIR
978 receptors, and that the
mutant receptors increased the coupling efficiency between endogenous
insulin receptors and downstream signaling molecules. In the current
report, we have further explored the cellular mechanisms underlying the
enhanced insulin sensitivity of HIR
978 cells.
Cell Lines and Materials
The generation of
stable clonal cell lines expressing wild type insulin receptors (HIRc)
and truncated mutant insulin receptors (HIR978), in which the
insulin receptor
-subunit was truncated at amino acid residue 978
according to the numbering system by Ullrich et al.(1) , and cell cultures have been previously described
(9, 10) . HIRc cells expressing 1.5
10
insulin receptors and HIR
978 cells expressing
1.2
10
truncated insulin receptors were used for this
study, while Rat1 fibroblasts expressed
2000 endogenous insulin
receptors.
I-Insulin and
I-IGF-1 were
generously provided by the Lilly Co. p85-SH2 glutathione S-transferase
(GST) fusion protein and an anti-IRS-1 antibody were a kind gift from
Dr. Alan R. Saltiel (Parke-Davis Pharmaceutical Co., Ann Arbor, MI). An
anti-insulin receptor antibody ) was kindly provided by Dr. Kenneth
Siddle (University of Cambridge, United Kingdom)
(11) .
D-[
C]Glucose (287 mCi/mmol),
-[methyl-
H]aminoisobutyric acid (AIB),
[
H]thymidine (83 Ci/mmol),
[
-
P]dCTP (3000 Ci/mmol), and
[
-
P]ATP (6000 Ci/mmol) were purchased from
DuPont NEN. cDNA cycle kit was from Invitrogen (San Diego, CA).
10
PCR amplification buffer and Taq-polymerase were from
Perkin-Elmer Cetus. Bromodeoxyuridine (BrdU), a monoclonal anti-BrdU
antibody, and enhanced chemiluminescence reagents were from Amersham
Corp.. A monoclonal anti-phosphotyrosine antibody (pY20) was from ICN
(Costa Mesa, CA). Rabbit immunoglobulin G (IgG), fluorescein
isothiocyanate- or rhodamine-conjugated anti-mouse and anti-rabbit IgG
antibodies were from Jackson Laboratories (West Grove, NY).
Electrophoresis reagents were from Bio-Rad. All other reagents were
purchased from Sigma.
c-fos Expression
Cells were grown to confluence
and starved for 24 h with serum-free Dulbecco's modified
Eagle's medium (DMEM). After stimulation with various
concentrations of insulin for 30 min at 37 °C, total cellular RNA
was isolated from the cells by the modified method of single-step
guanidinium thiocyanate-chloroform-phenol RNA extraction
(12) .
cDNA from the total cellular RNA was synthesized by using a cDNA cycle
kit and the manufacturer's instructions (Invitrogen). Specific
cDNA as defined by the c -fos primers was PCR-amplified with
L30 primers as an internal standard in the presence of
[P]dCTP. PCR products were electrophoresed and
visualized by autoradiography. The relative amounts of c -fosversusL30 were quantitated by measuring the
incorporated radioactivity on the gels with a Molecular Dynamics
PhosphorImager (Sunnyvale, CA)
(9) .
Glucose Incorporation into Glycogen
Confluent cell
monolayers were incubated in glucose-free DMEM for 3 h. Cells were
stimulated with hormones for 2 h at 37 °C with 5 mM
glucose and D-[C]glucose. The
monolayers were washed with phosphate-buffered saline and solubilized
with 30% KOH solution for 30 min at 37 °C. After boiling the
samples for 30 min with carrier glycogen, glycogen was precipitated by
the addition of ethanol, and radioactivity of the precipitates was
counted
(13) .
AIB Uptake
Confluent cell monolayers were
incubated with Earle's balanced salt solution supplemented with
25 mM NaHCO and 0.1% bovine serum albumin for 2 h
at 37 °C. After cells were stimulated with various concentrations
of IGF-1 for 3 h,
-[methyl-
H]AIB and 0.8
µM unlabeled AIB were added for 12 min. AIB uptake was
terminated by washing three times with phosphate-buffered saline, and
radioactivity was determined
(14) .
Thymidine Incorporation
Confluent cell monolayers
were incubated in DMEM containing 0.05% fetal calf serum (FCS) for 24
h, then various concentrations of IGF-1 were added. Twenty h later, the
cells were pulsed for 4 h with [H]thymidine. The
cells were washed twice with phosphate-buffered saline, twice with 10%
trichloroacetic acid, and once with 95% ethanol, and radioactivity was
measured
(15) .
Cross-linking
Cells were incubated with
[I]B2(2-nitro-4-azidophenylacetyl)-des-Phe
-insulin
(NAPA-insulin) for 2 h at 12 °C. UV cross-linking of the insulin to
its receptor was then carried out as described previously
(16) .
The cells were solubilized in a buffer containing 25 mM HEPES,
120 mM NaCl, 5 mM KCl, 1 mM
MgCl
, 1 mM CaCl
, 1% Triton X-100, 8
mM EDTA, 2 mM dichloroacetic acid, 0.5 mg/ml
bacitracin, 15 mM benzamidine, 10 µg/ml aprotinin, 2
mM phenylmethylsulfonyl fluoride, 10% (w/v) glycerol, pH 7.4.
After removal of insoluble material by centrifugation, the supernatants
were immunoprecipitated with specified antibody and analyzed by
SDS-polyacrylamide gel electrophoresis under non-reducing conditions,
followed by autoradiography.
Autophosphorylation of Partially Purified IGF-1
Receptors
Autophosphorylation of lectin-purified IGF-1 receptors
was measured in vitro. The IGF-1 receptor preparations were
incubated with various concentrations of IGF-1 for 16 h at 4 °C.
Autophosphorylation of the IGF-1 receptor was measured after incubation
with 50 µM [-
P]ATP for 10 min
at 4 °C. Phosphorylated receptors were immunoprecipitated with
anti-insulin receptor antibody to remove insulin receptors and insulin
receptor-IGF-1 receptor hybrids. The remaining supernatants including
homologous tetrameric IGF-1 receptors were subjected to 7.5% SDS-PAGE
under reducing condition
(9) .
Kinase Activity of Partially Purified Receptor
Preparations
Kinase activity to phosphorylate
Glu:Tyr
polypeptides was measured in
vitro. The lectin-purified receptor preparations were incubated
with various concentrations of hormones for 16 h at 4 °C. Kinase
activity was measured by incubation with Glu
:Tyr
polypeptides and 50 µM
[
-
P]ATP for 30 min at 4 °C. The
reaction was stopped by spotting on Whatman 3MM paper, and washed five
times with 10% trichloroacetic acid and counted by
-counter
(6, 15) .
Western Blotting Studies
Cell monolayers were
starved for 24 h in DMEM containing 0.05% FCS. The cells were then
treated with various concentrations of IGF-1 for 1 min at 37 °C.
Cells were then solubilized directly in hot Laemmli buffer, and lysates
were separated by SDS-PAGE. For affinity precipitation with p85-SH2 GST
fusion protein, after stimulation with hormones, the cells were
solubilized in a buffer containing 50 mM HEPES, 150
mM NaCl, 10 mM EDTA, 1% Triton X-100, 4 mM
NaVO, 200 mM NaF, 20 mM
Na
P
PO
, 10 µg/ml aprotinin, 2
mM phenylmethylsulfonyl fluoride, 10% (w/v) glycerol, pH 7.4.
The lysates were incubated with 5 µg of p85-SH2 GST fusion protein
and glutathione-Sepharose beads for 90 min at 4 °C. The
precipitates were subjected to SDS-PAGE under non-reducing condition.
The proteins were then transferred onto Immobilon-P by electroblotting.
For immunoblotting, membranes were blocked and probed with
anti-phosphotyrosine antibody. Blots were then incubated with
horseradish peroxidase-linked second antibody followed by enhanced
chemiluminescence detection, according to the manufacturer's
instructions (Amersham Corp.)
(6, 9) .
Microinjection
Cells were grown on glass
coverslips and rendered quiescent by starvation for 24 h in serum-free
DMEM. Antibodies, which were solubilized in microinjection buffer
consisting of 5 mM NaPO and 100 mM KCl,
pH 7.4., were then microinjected using glass capillary needles.
Approximately 1
10
liters of the buffer
were introduced into each cell. The injection included about 1
10
molecules of IgG. Two h after microinjection, cells were
incubated with BrdU plus growth factors for 16 h at 37 °C. The
cells were fixed with acid alcohol (90% ethanol, 5% acetic acid) for 20
min at 22 °C and then incubated with mouse monoclonal anti-BrdU
antibody for 1 h at 22 °C. The cells were then stained by
incubation with rhodamine-labeled donkey anti-mouse IgG antibody and
fluorescein isothiocyanate-labeled donkey anti-rabbit IgG antibody for
1 h at 22 °C. After the coverslips were mounted, the cells were
analyzed and photographed with an Axiphot fluorescence microscope (Carl
Zeiss). Microinjected cell numbers were 250-300/coverslip.
Immunofluorescent staining of the injected cells has indicated that
about 75% of the cells were successfully microinjected
(17, 18) .
Induction of c-fos mRNA
We have previously
reported that the ability of insulin to stimulate glucose incorporation
into glycogen, AIB uptake, thymidine incorporation, and S6 kinase
activity is enhanced in HIR978 cells compared to parental Rat1
cells
(9) . Another action of insulin is stimulation of gene
transcription, and insulin is known to increase induction of c- fos mRNA
(19, 20) . As can be seen in Fig. 1,
insulin stimulates c- fos mRNA induction in Rat1 cells with an
ED
of
15 nM. In contrast, the dose-response
curve is left-shifted to the same degree in HIR
978 and HIRc cells
(ED
1.5 nM). The relative amount of
c- fos induction was greater in HIR
978 cells than in Rat1
cells, and this further extends our previous observation that
HIR
978 cells display enhanced insulin signaling although >90%
of the HIR
978 receptor cytoplasmic
-subunit domain is
deleted.
Figure 1:
Dose-response of c-fos mRNA
expression after insulin stimulation. A, cells were
serum-starved for 24 h and then stimulated with indicated
concentrations of insulin for 30 min at 37 °C. Total cellular RNA
was isolated, and cDNA was synthesized from the RNA. Specific cDNA as
defined by the c- fos primers was subjected to PCR
amplification with L30 primers as an internal standard in the
presence of [P]dCTP. PCR products were resolved
by polyacrylamide gel electrophoresis and visualized by
autoradiography. B, the relative amounts of c- fosversusL30 from HIRc (
), HIR
978
(
), and Rat1 (
) cells were quantitated by measuring the
incorporated radioactivity on the gel with a PhosphorImager. Results
are the mean of two separate experiments and are expressed as
percentage over basal response.
IGF-I Signaling
To investigate whether enhanced
insulin signaling is mediated by IGF-1 receptors in HIR978 cells,
and whether the enhanced signaling is specific for insulin, we studied
IGF-1 stimulation of glucose incorporation into glycogen, AIB uptake,
and thymidine incorporation. The dose-response curves for IGF-1
stimulation of these biologic effects are all comparable among HIRc,
HIR
978, and Rat1 cell lines (Fig. 2). It should be noted
that all cell lines express similar numbers of IGF-I receptors
(1.2-1.5
10
receptors/cell). Since IGF-I
receptors utilize the same downstream signaling pathways as insulin
receptors, including phosphorylation of IRS-I, these results indicate
that the enhanced insulin action in HIR
978 cells is due to
increased signaling at the level of the insulin receptor per
se.
Figure 2:
Biologic actions of IGF-1 in parental and
transfected fibroblasts. A, glucose incorporation into
glycogen. Absolute values for basal ( b) and maximal
( m) stimulation were as follows: HIRc, b =
12.1 nmol/200 µg of protein/2 h and m = 19.8;
HIR978, b = 12.8 and m = 18.2;
Rat1, b = 13.7 and m = 21.2.
B, AIB uptake. Absolute values for basal ( b) and
maximal ( m) stimulations were as follows: HIRc, b = 0.61 nmol/200 µg of protein/12 min and m = 0.92; HIR
978, b = 0.58 and m = 0.88; Rat1, b = 0.54 and m = 0.83. C, thymidine incorporation. Absolute
counts of basal levels ( b) and maximal stimulations
( m) were as follows: HIRc, b = 14,850
disintegrations/min and m = 1.83-fold; HIR
978,
b = 14,342 and m = 1.98; Rat1, 12,898
and m = 2.02. All results are the mean of three
separate experiments and are presented as the percentage of maximal
IGF-1 stimulation of HIRc (
), HIR
978 (
), and Rat1
(
) cells.
Autophosphorylation-Kinase Activity
To further
explore the function of IGF-1 receptors, autophosphorylation was
assessed in lectin affinity receptor prepartions. To exclude the
influence of truncated insulin receptor-IGF-1 receptor hybrids, a
human-specific monoclonal antibody was used to immunodeplete the
hybrids from IGF-1-stimulated preparations, and the supernatants were
analyzed. As seen in Fig. 3 A, IGF-1 stimulated
autophosphorylation was similar in HIRc and HIR978 receptor
preparations. IGF-1 stimulation of receptor autophosphorylation was
also comparable in Rat1 receptors (data not shown). IGF-1 stimulation
of endogenous substrate phosphorylation was also assessed in these cell
lines. Cells were incubated with various concentrations of IGF-1, and
cell lysates were subjected to Western blotting analysis using
anti-phosphotyrosine antibodies. As shown in Fig. 3 B,
three prominent IGF-1-stimulated phosphorylated bands can be
visualized. The 95 and 105 kDa bands correspond to the
-subunit of
the IGF-1 receptor, whereas the 185 kDa band represents an endogenous
substrate (IRS-1). IGF-1 stimulation of IRS-1 phosphorylation was
comparable among HIRc, HIR
978, and Rat1 cells. Thus, the enhanced
signaling in HIR
978 cells is restricted to insulin stimulation.
Figure 3:
IGF-1 stimulation of autophosphorylation
and endogenous substrate phosphorylation. A,
autophosphorylation in vitro. The WGA-purified receptor
preparations from HIRc ( lanes 1-5) and HIR978
( lanes 6-10) cells were incubated with 0 ( lanes 1 and 6), 0.13 ( lanes 2 and 7), 1.3
( lanes 3 and 8), 13 ( lanes 4 and
9), and 67 ( lanes 5 and 10) nM
IGF-1 for 16 h at 4 °C. The receptors were then autophosphorylated.
Insulin receptors and insulin receptor-IGF-1 receptor hybrids are
immunodepleted by insulin receptor-specific antibody and the remaining
sample representing IGF-1 receptors were analyzed by SDS-PAGE under
reducing condition. A representative autoradiogram is shown.
B, tyrosine phosphorylation of endogenous substrates.
Serum-starved HIRc ( lanes 1-4), HIR
978 ( lanes
5-8), and Rat1 ( lanes 9-12) cells were
stimulated with 0 ( lanes 1, 5, and 9), 1.3
( lanes 2, 6, and 10), 13 ( lanes 3,
7, and 11), and 67 ( lanes 4, 8, and
12) nM IGF-1 for 1 min at 37 °C. The cells were
then lysed in Laemmli buffer. An equivalent number of cells were
processed for immunoblot analysis using pY20. A representative
autoradiogram is shown. Molecular masses of IRS-1 (185 kDa), p120 (120
kDa), and IGF-1 receptor
-subunits (95 and 105 kDa) are shown by
arrows.
Insulin stimulation of IRS-1 phosphorylation is enhanced in
HIR978 cells. To test the possibility that this might occur by
slower dephosphorylation of IRS-1, we examined the time course of IRS-1
phosphorylation. Insulin stimulated rapid tyrosine phosphorylation of
IRS-1, with peak phosphorylation by 1 min, declining thereafter in all
three cell lines. The amount of insulin-stimulated IRS-1
phosphorylation was similar in HIRc and HIR
978 cells and far
greater than that seen in Rat1 cells. However, the time course of IRS-1
dephosphorylation was somewhat faster in HIR
978 cells compared to
HIRc cells and was comparable to Rat1 cells. Clearly, the mechanism of
enhanced IRS-1 phosphorylation is not due to impaired dephosphorylation
in HIR
978 cells (Fig. 4).
Figure 4:
Time course of insulin stimulation of
IRS-1 phosphorylation. A, serum-starved cells were treated
with 17 nM insulin for the indicated times. The cell lysates
containing an equivalent number of cells were analyzed by
immunoblotting with pY20. A representative autoradiogram is shown and
molecular mass of IRS-1 (185 kDa) is shown by an arrow. B,
quantitation of time course of IRS-1 phosphorylation. IRS-1 bands were
analyzed by densitometry, and the time course of IRS-1 phosphorylation
from HIRc (), HIR
978 (
), and Rat1 (
) cells is
summarized as percentage of maximal tyrosine phosphorylation. Results
are the mean of two separate experiments.
Kinase activity of the insulin
receptor was determined with Glu:Tyr
as
substrate. Using lectin affinity purified receptor preparations,
containing equal amounts of protein, insulin led to a 3.0 ±
0.1-, 2.9 ± 0.4-, and 2.9 ± 0.3-fold increase in
phosphorylation of the exogenous substrate Glu
:Tyr
with HIRc, HIR
978, and Rat1 receptors, respectively. On the
other hand, insulin sensitivity was markedly decreased with the mutant
receptor, with half-maximal stimulation of 0.8, 4.0, and 4.3
nM in HIRc, HIR
978, and Rat1 receptors, respectively
(Fig. 5 A). Kinase activity of the IGF-1 receptor was
also examined. IGF-1 stimulated a 2.9 ± 0.2-, 3.0 ± 0.3-,
and 3.0 ± 0.3-fold increase in phosphorylation of
Glu
:Tyr
with HIRc, HIR
978, and Rat1
receptors. Dose-response curves of IGF-1 stimulation of
Glu
:Tyr
phosphorylation were similar between
HIRc and Rat1 receptors and slightly decreased at lower concentrations
of IGF-1 in HIR
978 receptor preparations (Fig. 5 B).
Figure 5:
Kinase activity of WGA-agarose purified
receptor preparations. Kinase activity of insulin receptors ( panelA) and IGF-1 receptors ( panelB) in
vitro. The cells were solubilized in the presence of 1% Triton
X-100. The lysates were partially purified on WGA-agarose. The receptor
preparations with same amount of insulin ( panelA) or
IGF-1 ( panelB) binding capacity were incubated with
various concentrations of insulin ( panelA) or IGF-1
( panelB) for 16 h at 4 °C and were allowed to
phosphorylate Glu:Tyr
polypeptides in the
presence of [
P]ATP for 30 min at 4 °C as
described under ``Experimental Procedures.'' Absolute values
for basal ( b), maximal insulin ( m1), maximal IGF-1
( m2) stimulations were as follows: HIRc, b =
0.91 pmol/10 min, m1 = 2.70, and m2 =
2.65; HIR
978, b = 0.87, m1 = 2.56,
and m2 = 2.59; Rat1, b = 0.88, m = 2.51, and m2 = 2.60. Results are the mean
of three separate experiments and presented as the percent of maximal
hormone response for HIRc (
), HIR
978 (
), and Rat1
(
) cells.
In addition, WGA preparations were obtained from all three cell
types and immunoprecipitated with a human-specific monoclonal
anti-insulin receptor antibody. With this approach, the endogenous
rodent receptors remain in the supernatants, and the human receptors
are in the immunoprecipitates. Kinase activity toward
Glu:Tyr
was then measured in both supernatants
and precipitates (Fig. 6). Kinase activity was readily detected
in the precipitates from the HIRc cells, but not from HIR
978 or
Rat1 cells. In addition, kinase activity was detected in the
supernatants from all three cell lines, representing the contribution
of endogenous rodent receptors. Thus, a marked decrease in
autophosphorylation and kinase activity was noted with the mutant
receptor, and the kinase activity toward Glu
:Tyr
can be attributed to the contribution of the endogenous rodent
receptors in the HIR
978 cells in vitro.
Figure 6:
Immunodepletion of kinase activity by
anti-insulin receptor antibody. Effect of immunodepletion in HIRc
( panelA), HIR978 ( panelB),
and Rat1 ( panelC) cells. WGA-agarose-purified
receptor preparations were incubated with 83-14 (1:200 dilution)
for 5 h at 4 °C. The entire precipitates (
) and all of the
remaining supernatants (
) were incubated with various
concentrations of insulin for 16 h at 4 °C, and then the sample
were allowed to phosphorylate Glu
:Tyr
polypeptides in the presence of [
P]ATP for
30 min at 4 °C. The reaction was stopped by spotting on Whatman 3MM
filter paper, and washed five times with 10% trichloroacetic acid and
counted by
-counter. Results are the mean of three separate
experiments and presented as the percent of maximal insulin
response.
Autophosphorylation of Cross-linked
Receptors
Previously, we have shown that insulin stimulation of
-subunit phosphorylation is markedly decreased, but still
detectable, in HIR
978 cells
(9) . To examine which receptor
species are autophosphorylated after insulin stimulation, cells were
incubated with NAPA-[
I]insulin, a photoaffinity
insulin derivative, which was then cross-linked to the receptors.
Following cross-linking, the cell lysates were immunoprecipitated by
anti-insulin receptor antibody or phosphotyrosine antibody and analyzed
by SDS-PAGE under non-reducing conditions (Fig. 7). With this
approach, the intersubunit disulfide bonds were not disrupted and the
heterotetrameric
2
2 structure was maintained. In HIRc cells,
wild type insulin receptors were demonstrated by the band at 430 kDa
(Fig. 7, lane 1). The presence of a small number of
endogenous Rat1 insulin receptors was demonstrated by a faint band of
430 kDa (Fig. 7, lanes 3-6). Approximately 70% of
the wild type or endogenous insulin receptors were tyrosine
phosphorylated, as shown by anti-phosphotyrosine antibody precipitation
(Fig. 7, lanes 2 and 6). In HIR
978 cells,
three distinct bands migrating at 430, 380, and 330 kDa, molecular
masses which correspond to endogenous rodent receptors,
HIR
978:IGF-1 receptor hybrids, and HIR
978 homodimers,
respectively, were demonstrated (Fig. 7, lane 3). Among
these species, only endogenous insulin receptors were
autophosphorylated, as shown in the anti-phosphotyrosine
immunoprecipitates (Fig. 7, lane 4). Thus, in
HIR
978 cells, only intact heterotetrameric endogenous receptors
are functionally active in terms of autophosphorylation as well as
kinase activity.
Figure 7:
Insulin stimulation of autophosphorylation
in vivo. HIRc ( lanes 1 and 2), HIR978
( lanes 3 and 4), and Rat1 ( lanes 5 and
6) were serum-starved for 16 h. An equivalent number of cells
were incubated with [
I]NAPA-insulin for 2 h at
12 °C. UV cross-linking of the insulin to its receptor was carried
out, and the cells were solubilized. After removal of insoluble
material by centrifugation, the supernatants were immunoprecipitated
with anti-insulin receptor antibody ( lanes 1, 3, and
5) or anti-phosphotyrosine antibody ( lanes 2,
4, and 6). The precipitates were then analyzed by
5-12% SDS-PAGE under non-reducing condition. A representative
autoradiogram is shown.
Interaction with p85 Subunit of PI3K
Insulin
receptor and IRS-1 interact with the SH2 domain of the p85 regulatory
subunit of PI3-kinase
(21, 22) . Lysates from
insulin-stimulated cells were incubated with the
NH-terminal p85-SH2 domain expressed as a GST fusion
protein bound to glutathione-agarose beads. Associated proteins were
separated by SDS-PAGE under non-reducing conditions and analyzed by
immunoblotting with anti-phosphotyrosine antibody
(Fig. 8 A). Phosphorylated IRS-1 was affinity
precipitated by the p85-SH2 domain from all three cell types;
quantitatively the amount of IRS-I precipitated was HIRc >
HIR
978 > Rat1 cells. In addition, in HIRc and Rat1 cells, the
430 kDa band, corresponding to
2
2 heterotetrameric human or
rodent insulin receptors, respectively, was associated with the p85-SH2
domain. Interestingly, among the three types of receptors shown in
Fig. 7
( lane 3), only intact endogenous rodent receptors
were associated with the p85-SH2 domain in HIR
978 cells
(Fig. 8 A, lane 4). Furthermore, insulin
increased the association of a 120-kDa protein with the p85-SH2 domain,
although the identity of this 120-kDa protein is unknown. To more
directly determine which types of insulin receptors were associated
with the p85 SH2 domain, cells were incubated with
NAPA-[
I]insulin, and the ligand was
cross-linked. Cell lysates were affinity precipitated by the p85-SH2
domain GST fusion protein bound to glutathione-agarose beads and
analyzed by SDS-PAGE under non-reducing conditions. By this analysis,
the association of
2
2 heterotetrameric insulin receptors with
the p85-SH2 domain was visualized in both HIRc and Rat1 cells.
Importantly, of the three receptor species in the HIR
978 cells,
only the 430-kDa endogenous rodent receptors were associated with the
p85-SH2 domain (Fig. 8 B).
Figure 8:
Association of the insulin receptor with
p85 subunit of PI 3-kinase. A, association of p85 with
tyrosine-phosphorylated protein. HIRc ( lanes 1 and
2), HIR978 ( lanes 3 and 4), and Rat1
( lanes 5 and 6) were serum-starved for 16 h. An
equivalent number of cells were stimulated without ( lanes 1,
3, and 5) or with 17 nM ( lanes 2,
4, and 6) insulin for 1 min at 37 °C. The cells
were then solubilized and insoluble materials were removed by
centrifugation. The supernatants were affinity precipitated with
GST-p85 SH2 fusion protein. The precipitants were subjected to
5-12% SDS-PAGE under non-reducing condition and analyzed by
immunoblotting with anti-phosphotyrosine antibody. A representative
autoradiogram is shown. B, association of p85 with the insulin
receptor. After the cells were serum-starved, an equivalent number of
cells were incubated with 0.2 nM
[
I]NAPA-insulin for 2 h at 12 °C.
UVcross-linking of the insulin to its receptor was carried out, and the
cells were solubilized. The lysates were affinity precipitated with
GST-p85 SH2. The precipitates were then analyzed by 5-12%
SDS-PAGE. A representative autoradiogram is
shown.
Endogenous Rat Receptors Mediate the Insulin Signaling in
HIR
In previous studies with HIR978 Cells
978 cells, we
have found evidence that the enhanced insulin signaling is conveyed
from the endogenous rat receptors and that the overexpression of
HIR
978 receptors, in some way, enhances the coupling efficiency of
the rodent receptors
(9) . Thus, when cells were acutely
stimulated with an agonistic human specific monoclonal antibody, we
found that the antibody had marked agonistic effects in HIRc cells, but
was without biologic effect in HIR
978 cells, just as it was in
Rat1 cells
(9) . This indicated that insulin binds to both human
and rodent receptors in the HIR
978 cells, but that biologic
signaling occurs only through the rodent receptor. To further explore
this concept, we sought to determine whether binding of insulin to the
truncated HIR
978 receptors at the cell surface was necessary to
confer enhanced signaling properties to the endogenous receptors. To
answer this question, we incubated HIR
978 cells with a saturating
concentration of the human-specific monoclonal antibody, which competes
with insulin for receptor binding, for 60 min at 37 °C. This led to
antibody occupancy of the human insulin receptors, and
80% of them
remained at the cell surface, whereas
20% internalized (data not
shown). Similar experiments were also performed withHIRc cells and Rat1
fibroblasts. When the antibody preincubated cells were subsequently
stimulated with insulin and glucose incorporation into glycogen was
assayed (Fig. 9), the dose-response curves for both the HIRc
cells and HIR
978 cells were right-shifted, whereas no effect was
observed in Rat1 cells. The insulin sensitivity of the antibody
preincubated HIR
978 cells was comparable to that seen in parental
Rat1 fibroblasts. This suggested that inhibiting the binding of insulin
to the truncated receptors, or clearing the truncated human receptor
from the cell surface, prevented their ability to enhance the signaling
properties of the endogenous receptors.
Figure 9:
Effect
of anti-insulin receptor antibody on glucose incorporation into
glycogen. Serum- and glucose-starved HIRc ( panel A),
HIR978 ( panelB), and Rat1 ( panelC) cells were incubated without (
) or with (
)
83-14 (1:500 dilution) for 2 h at 37 °C. Glucose
incorporation into glycogen was then measured as described under
``Experimental Procedures.'' Results are the mean ±
S.E. of three separate experiments and are presented as percent of
maximal insulin stimulation. Absolute values for basal ( b) and
maximal ( m) stimulation were as follows: without
incubation; HIRc, b = 12.0 nmol/200 µg of
protein/2 h and m = 20.1; HIR
978, b = 12.7 and m = 19.2; Rat1, b = 13.4 and m = 21.0: with incubation; HIRc, b = 12.7 and m = 19.5;
HIR
978, b = 13.4 and m = 18.9;
Rat1, b = 13.6 and m =
21.3.
Microinjection of IRS-I Antibody
To evaluate the
functional role of IRS-1 in HIR978 cells, in transmembrane
signaling by insulin and IGF-1, we performed single cell microinjection
studies in which affinity purified anti-IRS-1 antibody was introduced
into HIR
978 cells. Following this, the cells were stimulated with
insulin or IGF-I and cell cycle progression was monitored by measuring
BrdU incorporation into newly synthesized DNA. In the basal state, 34.0
± 2.1% of cells incorporated BrdU. Insulin, IGF-1, and FCS
stimulated BrdU incorporation into 71.2 ± 1.9, 67.8 ±
1.8, and 75.0 ± 2.2% of cells, respectively. Microinjection of
preimmune control IgG did not alter this stimulatory effect (data not
shown). In contrast, microinjection of anti-IRS-1 antibody markedly
inhibited the ability of insulin and IGF-1 to induce DNA synthesis by
80 and 76%, respectively, whereas, the stimulatory effects of FCS were
not impaired (Fig. 10).
Figure 10:
Inhibition of DNA synthesis by
microinjection of anti-IRS-1 antibody in HIR978 cells.
Serum-starved cells were microinjected with anti-IRS-1 antibody. BrdU
incorporation in the injected cells ( open bars) and
uninjected cells ( hatched bars) on the same coverslip was
determined as described under ``Experimental Procedures.''
Cumulative data are shown and results are the mean ± S.E. of
three separate experiments.
978 insulin receptors confers enhanced insulin sensitivity in Rat1
fibroblasts
(9) . This is the case despite the fact that the
truncated receptor has no intrinsic kinase activity and is missing
90% of the intracellular cytoplasmic domain. Our previous studies
indicated that although the truncated insulin receptors do not directly
mediate insulin action by themselves, they increase the signaling
efficiency of the endogenous insulin receptors. Thus, the increased
insulin sensitivity in HIR
978 cells is due to enhanced signaling
properties of the endogenous insulin receptors
(9) . We have
termed this phenomenon dominant/positive potentiation, and in the
current studies we have extended these observations and have further
explored the mechanisms underlying this process.
978 cells, and untransfected parental
Rat1 fibroblasts. The results indicated that insulin leads to a
dose-responsive increase in c- fos mRNA in all cell lines, but
that the dose-response curves are comparably, and markedly,
left-shifted in both the HIRc and HIR
978 cells. Thus, expression
of the kinase inactive truncated insulin receptor leads to enhanced
insulin sensitivity for this biologic effect of insulin.
978
receptors leads to augmentation of the insulin signaling pathway at a
post-receptor site, then one might anticipate increased signaling
properties of other growth factors. This is particularly true in the
case of IGF-I, which has a similar range of biologic effects as
insulin, and, beyond the receptor itself, appears to engage the same
set of signaling molecules, including IRS-I. Consequently, we measured
a variety of biologic effects of IGF-I in the various cell types and
found that the dose-response curves for IGF-I stimulation of glucose
incorporation into glycogen, AIB uptake, and DNA synthesis, were
comparable across the cell lines. Thus, HIR
978 receptors did not
lead to enhanced IGF-I signaling, in marked contrast to its effects on
insulin action. Since IGF-I and insulin signaling pathways are thought
to converge immediately after receptor activation, these results
strongly argue that the augmented insulin action in HIR
978 cells
is due to increased signaling properties of the insulin receptors
themselves. We have previously shown that homodimeric HIR
978
receptors are incapable of directly transmitting biologic signals,
since a human-specific agonistic monoclonal antibody had no stimulatory
effect in these cells, whereas, it led to marked stimulation of insulin
action in cells expressing intact human insulin receptors
(9) .
Taken together, these results indicate that the HIR
978 receptors
confer increased signaling properties to the endogenous rodent insulin
receptors in these cells. The current results also indicate that the
continuing presence of occupied HIR
978 receptors at the cell
surface is necessary to sustain this effect. Thus, HIR
978 cells
were incubated with a human-specific monoclonal antibody directed
against the insulin receptor ectodomain, and the cells were
subsequently stimulated with insulin. Receptors bound to the antibody
no longer can bind insulin (data not shown), and the truncated
receptors became occupied and
20% of them internalized. In
contrast, the endogenous rodent receptors did not interact with the
antibody and remained at the cell surface. Blockade of subsequent
insulin binding to receptors on the cell surface prior to insulin
stimulation abbrogated the enhanced insulin signaling properties of
these cells.
978 cells compared to Rat1
fibroblasts and is comparable to the degree of IRS-1 phosphorylation
observed in HIRc cells. Since IRS-1 is an immediate substrate of the
insulin receptor
(3) , it seems likely that enhanced
transduction of IRS-1 phosphorylation by endogenous insulin receptors
explains the increased insulin action observed in these cells. These
results are fully consistent with the data presented in
Fig. 4
which show greater IRS-1 phosphorylation in HIR
978
cells compared to Rat1 fibroblasts, and comparable peak IRS-1
phosphorylation between HIR
978 cells and HIRc cells. This line of
reasoning is strengthened by the current results on IGF-I signaling.
Thus, we find that IGF-I receptor kinase activity, and, more
importantly, stimulation of IRS-I phosphorylation is comparable across
the three cell lines. Thus, augmented stimulation of IRS-I
phosphorylation is only observed for the insulin signaling system. This
further points to the coupling step between endogenous insulin
receptors and IRS-I phosphorylation as the site of the enhanced
signaling efficiency in HIR
978 cells. Along these lines, the net
amount of tyrosine phosphorylated IRS-I is a function of insulin
receptor kinase activity as well as the rate of tyrosine
dephosphorylation. Thus, it seemed possible that increased IRS-I
phosphorylation in HIR
978 cells might be due to decreased IRS-I
dephosphorylation. To assess this, we performed time course studies to
determine whether dephosphorylation of IRS-I was delayed or decreased
in HIR
978 cells. The results showed that, if anything, the
dephosphorylation of IRS-I was slightly greater in HIR
978 cells
compared to HIRc cells. Although inferential, these results indicate
that increased dephosphorylation is not the mechanism for the increase
in the IRS-I phosphorylation state.
978 cells, followed by
stimulation with insulin and measurement of DNA synthesis. The results
showed that intracellular blockade of IRS-I led to an 80% inhibition of
insulin action, demonstrating the critical role of IRS-I
phosphorylation in the biological signaling properties of insulin in
these cells.
and IGF-I receptor
halves
(13) . Since the insulin receptor in HIR
978 cells contains
a large truncation, this allows us to visualize the different
homodimeric and hybrid receptor species which are formed in these
cells. Immunoprecipitation of
I-NAPA cross-linked insulin
receptors followed by SDS-PAGE on non-reducing gels revealed the three
distinct insulin binding species which exist in these cells. These
include the homodimeric endogenous insulin receptors (430 kDa), the
hybrid receptors (380 kDa), and the homodimeric HIR
978 receptors
(330 kDa). Only the intact endogenous insulin receptors are capable of
autophosphorylation, and both the homodimeric HIR
978 and hybrid
receptors were kinase inactive. Since the hybrid receptors contain an
intact
IGF-I receptor half, these results indicate that the
major intramolecular signal transduction is trans, rather than
cis, in mediating autophoshorylation.
978 insulin receptors at the cell surface imparts enhanced
signaling properties to the endogenous rodent insulin receptors. This
effect is specific for the insulin receptor system, since IGF-I action
is not increased in these cells. The site of this positive-dominant
effect appears to involve an increased coupling efficiency between the
endogenous rodent receptors and IRS-I, leading to a generalized
increase in insulin's biologic effects.
-[methyl-
H]aminoisobutyric acid; BrdU,
bromodeoxyuridine; DMEM, Dulbecco's modified Eagle's
medium; PCR, polymerase chain reaction; FCS, fetal calf serum; PAGE,
polyacrylamide gel electrophoresis; WGA, wheat germ agarose.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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Molecular and Cellular Proteomics
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