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INTRODUCTION |
A complete understanding of insulin action requires the
identification of the intracellular pathways that regulate
insulin-stimulated growth, development, and metabolism. Molecular
analysis of structures within the insulin receptor cytoplasmic domain
that are mediators of insulin-stimulated signal transduction pathways
has provided one strategy for the study of insulin-sensitive
intracellular signaling. The usual approach has been to create
mutations within specific receptor sequences that result in the
selective disruption of some insulin-regulated pathways, while leaving
others intact. Deletion mutagenesis of the insulin receptor cytoplasmic
domain has generated insulin receptors with altered biological
properties (1-4) and led to the suggestion that different regions of
the insulin receptor cytoplasmic domain play distinct roles in
modulating the biological effects of insulin. Most mutations of the
insulin receptor cytoplasmic domain that altered autophosphorylation
sites within the cytoplasmic domain (5-8) furthered the notion that tyrosine phosphorylation and the tyrosine kinase encoded within the
receptor
subunit are essential components of normal insulin action.
Previously, structure/function analysis required the introduction of
mutated insulin receptors into cells expressing low levels of
endogenous insulin receptors, e.g. Chinese hamster ovary or Rat-1 fibroblast cell lines (for a review, see Ref. 9) to minimize the
background signal generated by insulin activation of endogenous receptors. However, the relative insensitivity of fibroblast cell lines
to insulin treatment has made them less than ideal models for studying
the action of insulin on intermediary metabolism.
Conversely, insulin-responsive cell types are excellent systems in
which to study insulin action, but the fact that they express large
numbers of endogenous insulin receptors is an impediment to the
expression and analysis of mutated recombinant insulin receptors. To
circumvent this concern, the extracellular ligand binding domain of the
human CSF-11 receptor (10)
was spliced to the transmembrane and cytoplasmic domains of the human
insulin receptor (11-13) to construct a chimeric receptor (CSF1R/IR).
Recently, we showed that the CSF1R/IR has CSF-1-dependent
enzymatic and biological properties expected of the insulin receptor
tyrosine kinase in insulin-responsive cells (14). Deletion of 12 amino
acids within the juxtamembrane domain of the CSF1R/IR inhibits
CSF-1-stimulated phosphorylation of IRS-1 and Shc (14) and blocks
CSF1R/IR-mediated glucose uptake (15) but does not block the ability of
the chimeric receptor to stimulate the differentiation of 3T3-L1 cells
into adipocytes (14) or promote cell survival in the presence of an
apoptotic stimulus (15). In the experiments presented here we examined
the signaling properties of a CSF1R/IR construct bearing a single
mutation of the tyrosine at the position corresponding to
Tyr960 of the normal human insulin receptor to alanine. Our
data demonstrate that the Ala960 mutation partially
inhibits the ability of the CSF1R/IR to stimulate glucose uptake and
blocks the ability of the chimera to stimulate the phosphorylation of
IRS-1 but not IRS-2. Despite the ability of the CSF1R/IRA960 to
phosphorylate IRS-2, co-precipitation studies indicate that the
Ala960 mutation decreases the ability of the insulin
receptor cytoplasmic domain to interact with IRS-2.
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EXPERIMENTAL PROCEDURES |
3T3-L1 cells expressing the CSF1R/IR were described previously
(14). Antibodies Ca-1 and CT-1 directed against the cytoplasmic domain
of the human insulin receptor (17) were a gift from Ken Siddle,
Cambridge, UK. Antibodies to IRS-1 were a gift from L. Sweet, West
Haven, CT. Antibodies to IRS-2 were described previously (18).
Anti-phosphotyrosine (RC20H) antibodies were from Transduction Laboratories. Dulbecco's modified Eagle medium (DMEM), penicillin, streptomycin and neomycin antibiotics, G418, restriction endonucleases and LipofectAMINE were purchased from Life Technologies, Inc. Fetal
bovine serum was purchased from Atlanta Biologicals. Porcine insulin
was from Calbiochem. Dexamethasone and 3-isobutyl-1-methylxanthine were
from Sigma. Human recombinant CSF-1 was a gift from Genetics Institute.
Construction and Expression of the CSF1R/IR and the
CSF1R/IRA960--
Construction of the CSF1R/IR was described
previously (14). The CSF1R/IR chimera containing an alanine
substitution for Tyr960 in the insulin receptor cytoplasmic
domain (CSF1R/IRA960) was constructed by replacing the
SpeI/BglII fragment from the CSF1R/IR with the
corresponding fragment from pSGHIRcA960 (19). Expression of the
CSF1R/IR and the CSF1R/IRA960 cDNAs was accomplished by subcloning
the chimera cDNAs into the vector pEF-1 (14). Transfection of
3T3-L1 preadipocytes was performed with LipofectAMINE according to the
manufacturer's instructions. Cells expressing the CSF1R/IR were
selected with 800 µg/ml G418 and isolated by flow cytometry with a
monoclonal antibody against the extracellular domain of the human CSF-1
receptor (20) (Oncogene Science) and fluorescent secondary antibody.
Flow cytometry was performed by the University of Nebraska Medical
Center Flow Cytometry Core Laboratory.
Cell Culture--
3T3-L1 preadipocytes were maintained in DMEM
supplemented with 10% fetal bovine serum and antibiotics. All cells
were incubated at 37 °C in 5% CO2. Where indicated,
2 × 104 3T3-L1 preadipocytes were plated/35-mm dish
and grown to quiescence, and differentiation was induced with DMEM
containing 10% fetal bovine serum, 5 µg/ml insulin, 0.25 µM dexamethasone, and 0.5 mM
3-isobutyl-1-methylxanthine as described previously (14). Cells were
used 14 days after induction of differentiation.
2-[3H]Deoxy-D-glucose Uptake in 3T3-L1
Cells--
Glucose uptake in 3T3-L1 adipocytes was measured using a
modification of the method described previously (21). 3T3-L1 adipocytes were used 14 days after induction of differentiation, at which time
>90% of the cells expressed an adipocyte phenotype. Cell monolayers
were washed twice with buffer A (130 mM NaCl, 2.7 mM KCl, 1 mM KH2PO4,
8.1 mM Na2HPO4, 0.68 mM
CaCl2,0.49 mM MgCl2, pH 7.4) and
then incubated for 2 h at 37 °C in 1 ml of serum-free DMEM. To
determine nonspecific glucose uptake, the cells were treated for 10 min
in serum-free DMEM containing 20 µM cytochalasin B and
200 µM phloretin. Cell monolayers were washed once with 1 ml of Krebs-Ringer's phosphate buffer (130 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.3 mM MgSO4, 10 mM
Na2HPO4, pH 7.4) with or without CSF-1 or
insulin as indicated and incubated for 20 min at 37 °C in 1 ml of
the same buffer. The glucose uptake assay was initiated by replacement
of Krebs-Ringer's phosphate buffer in each well with Krebs-Ringer's
phosphate buffer containing 0.1 mM
2-deoxy-D[2,6-3H]glucose (1 µCi) for 10 min
at 37 °C. Termination of glucose uptake was performed by the rapid
removal of assay buffer followed by four rapid washes of each well with
1 ml of ice-cold buffer A containing 500 µM phloretin.
Cells were removed from each well with 0.4 ml of 0.1% SDS and counted
for radioactivity after the addition of 4 ml of Optifluor (Packard
Instrument Co).
Glycogen Synthesis--
The incorporation of glucose into
glycogen was measured as described previously (22). Fully
differentiated adipocytes in 2-cm wells were serum-starved for 3 h
in DMEM with 5.5 mM glucose and 2 mM glutamine.
Cells were washed in glycogen assay buffer (140 mM NaCl,
1.7 mM KCl, 0.9 mM CaCl2, 1.47 mM K2HPO4, 0.9 mM MgSO4, 25 mM Tris-HCl, pH 7.5, and 2 mg/ml
bovine serum albumin), stimulated with the indicated concentrations of
hormones for 20 min, and incubated with
D-[U-14C] glucose (1 µCi/ml, final specific
activity 0.26 µCi/µmol) for 30 min. Incubations were stopped by 3 rapid washes on ice with phosphate-buffered saline, and the cells were
solubilized in 1 ml of 0.1 M NaOH. Carrier glycogen (1 mg)
was added, and the samples were boiled for 30 min. Glycogen was
precipitated with 70% ethanol overnight at
20 °C. Each sample was
centrifuged at 2100 × g, washed once with 70%
ethanol, and resuspended in water. The amount of radioactivity
incorporated into glycogen was determined by scintillation counting.
Immunoprecipitation and Western Blot Analysis--
Quiescent
cells were stimulated with 10 nM CSF-1 for 10 min or left
untreated, washed once with ice-cold phosphate-buffered saline
containing 0.1 mM sodium orthovanadate, and lysed in buffer (50 mM HEPES, pH 7.8, 1% Triton X-100, 10 mM
NaF, 2 mM Na3VO4, 10 mM
sodium inorganic pyrophosphate, 5 mM EDTA, 10 µg/ml
aprotinin, 5 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride). Insoluble material was removed by
centrifugation at 14,000 × g for 10 min at 4 °C,
and the clarified lysates were incubated with appropriate antibodies.
Proteins precipitated with the indicated antibodies were resolved by
SDS-polyacrylamide gel electrophoresis on a 6% gel, electroblotted to
nitrocellulose membranes, and probed with monoclonal
anti-phosphotyrosine antibody RC20H conjugated with horseradish
peroxidase (1:1500), monoclonal antibody CT-1 (1:5000), or with
anti-IRS-1 (1:800), anti-IRS-2 (1:1000), or Ca-1 (1:2000) polyclonal
antibodies. Bound antibodies were detected with the appropriate
horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG
followed by ECL detection (Amersham Pharmacia Biotech) according to the
manufacturer's instructions.
PI 3' Kinase Assay--
Differentiated 3T3-L1 adipocytes in
35-mm culture dishes were serum-starved for 5 h in DMEM before
hormone stimulation. Cells were then stimulated with 10 nM
CSF-1 or 100 nM insulin for 2 min. The media was aspirated,
and the cell monolayers were washed once with ice-cold
phosphate-buffered saline containing 0.1 mM Na3VO4. The proteins were solubilized for 10 min in lysis buffer (40 mM Tris-HCl, pH 7.4, 2 mM Na3VO4, 20 mM NaF,
10 mM sodium inorganic pyrophosphate, 1 mM
EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin) containing 1% Triton X-100. Cell lysates containing equal amounts of protein were incubated with the
indicated antisera or antibodies for 2 h at 4 °C. Protein G-Sepharose (25 µl) was added to each immune complex, and incubation was continued for 2 h at 4 °C. The immunoprecipitates were
washed twice with each of the three following buffers: (a)
phosphate-buffered saline, pH 7.4, containing 1% Triton X-100,
(b) 100 mM Tris, pH 7.4, 0.5 M LiCl,
0.1 mM Na3VO4, and (c)
50 mM HEPES, pH 7.8, 100 mM NaCl, 1 mM EDTA, 0.1 mM Na3VO4.
The pellets were resuspended in 30 µl of 30 mM HEPES, 30 mM MgCl2, 0.4 mM EGTA. The kinase reactions were started by the addition of 15 µl of sonicated
substrate (0.6 mg/ml PI in 30 mM HEPES and 0.4 mM EGTA) and 10 µCi of [
-32P]ATP (6000 Ci/mmol)/tube. After mixing for 10 min at room temperature, the
reactions were stopped by the addition of 14 µl of 6 N
HCl and then extracted with 130 µl of methanol:chloroform (1:1). The samples were briefly separated by centrifugation, and the lower organic
phase (20 µl) was spotted on silica gel 6OA plates (Merck) that had
been pretreated with 1% potassium oxalate at 100 °C. The plates
were developed in chloroform/acetone/methanol/acetic acid/water
(35:15:13:12:8), dried, and detected by autoradiography. Phosphorylated
PI was quantified with a Molecular Dynamics densitometer.
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RESULTS |
Tyrosine Phosphorylation of the CSF1R/IR and CSF1R/IRA960
Chimeras--
Quiescent 3T3-L1 preadipocytes expressing the CSF1R/IR
and the CSF1R/IRA960 were incubated in the presence or absence of
CSF-1, and aliquots of cell lysates containing equal amounts of protein were immunoprecipitated with Ca-1 antibody directed against the cytoplasmic domain of the insulin receptor. Treatment of 3T3-L1 preadipocytes expressing the CSF1R/IR chimera with CSF-1 for 10 min
resulted in the tyrosine phosphorylation of the 170-kDa chimeric receptor protein (Fig. 1, 2nd
lane). The same treatment of 3T3-L1 cells expressing the
CSF1R/IRA960 chimera resulted in a slightly reduced extent of the
tyrosine phosphorylation of the mutant receptor (Fig. 1, 4th
lane) compared with the intact chimeric receptor, which is
consistent with prior studies (8). The intact and mutated chimera were
expressed equally, as determined by Western blot with Ca-1 antibody
(Fig. 1). The same effect of CSF-1 on phosphorylation of the CSF1R/IR
and CSF1R/IRA960 was observed in fully differentiated 3T3-L1 adipocytes
(data not shown).

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Fig. 1.
Phosphorylation of the CSF1R/IR and
CSF1R/IRA960 in 3T3-L1 cells. 3T3-L1 cells (2 × 105) expressing the CSF1R/IR or the CSF1R/IRA960 were left
untreated or treated with 10 nM CSF-1 for 10 min at
37 °C, lysed, and immunoprecipitated with antisera Ca-1 directed
against the cytoplasmic domain of the insulin receptor. Precipitated
proteins were resolved by SDS-polyacrylamide gel electrophoresis,
blotted to nitrocellulose, and probed with anti-phosphotyrosine
antibodies (left panel) or Ca-1 antisera (right
panel). M.W., molecular mass.
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CSF-1 Stimulates Glucose Uptake in 3T3-L1 Adipocytes Expressing the
CSF1R/IR--
Differentiation of 3T3-L1 preadipocytes into adipocytes
results in an increase in expression of the GLUT4 and in an increase in
insulin-stimulated glucose transport (23, 24). 3T3-L1 adipocytes or
3T3-L1 adipocytes expressing CSF1R/IR were treated for 20 min with a
maximally effective insulin concentration (100 nM) or with CSF-1 at the indicated concentrations before the addition of
2-[3H]deoxyglucose (Fig.
2). After 10 min, the
2-[3H]deoxyglucose uptake was measured by scintillation
counting. Insulin stimulated an 8-10-fold increase in
2-[3H]deoxyglucose uptake in 3T3-L1 adipocytes (Fig. 2).
Tyrosine autophosphorylation of the CSF1R/IR is maximal at 10 nM and half-maximal at 1 nM CSF-1 (14). 3T3-L1
adipocytes expressing the CSF1R/IR were able to increase
2-[3H]deoxyglucose uptake approximately 7.5-fold in
response to 10 nM CSF-1 and to a slightly lesser extent
when 1 nM CSF-1 was used (Fig. 2). In contrast, 10 nM CSF-1 had no effect on glucose uptake in 3T3-L1 control
adipocytes (Fig. 2). These data suggest that physiological
concentrations CSF-1 are able to activate the CSF1R/IR and stimulate
glucose uptake in 3T3-L1 adipocytes.

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Fig. 2.
CSF-1-stimulated glucose uptake in 3T3-L1
adipocytes expressing the CSF1R/IR.
2-[3H]deoxyglucose uptake was measured in parental 3T3-L1
adipocytes and in adipocytes expressing the CSF1R/IR. Adipocytes in
35-mm dishes were treated with the indicated concentrations of insulin
or CSF-1, and glucose uptake was determined as described under
"Experimental Procedures." Results are representative of three
independent experiments.
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Effect of the CSF1R/IRA960 on Glucose Transport and Glycogen
Synthesis--
Tyr960 in the insulin receptor
juxtamembrane domain is essential for the ability of the receptor
kinase to phosphorylate IRS-1 and couple to downstream effectors (1,
25). The tyrosine in the CSF1R/IR corresponding to Tyr960
in the insulin receptor was changed to alanine, and the resulting CSF1R/IRA960 chimera was tested for its ability to stimulate glucose uptake upon stimulation with different doses of CSF-1. CSF-1 was equipotent to insulin in its ability to stimulate maximal glucose uptake in 3T3-L1 adipocytes expressing the CSF1R/IR (Fig.
3A). At submaximal
concentrations, CSF-1 appears more potent than insulin in its ability
to stimulate glucose uptake and glycogen synthesis (Fig. 3). This may
result from the fact that the expression of CSF1R/IR is slightly higher
than endogenous insulin receptors in the transfected 3T3-L1 adipocytes
(data not shown). In adipocytes expressing the CSF1R/IRA960, glucose
uptake in response to 100 nM CSF-1 was no greater than that
observed when the same cells were treated with 1 nM insulin
(Fig. 3A). However, glucose uptake stimulated by 1 nM and 10 nM CSF-1 was unaffected by the
Ala960 mutation (Fig. 3A).

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Fig. 3.
Glucose uptake and glycogen synthesis in
3T3-L1 adipocytes expressing the CSF1R/IR or CSF1R/IRA960. 3T3-L1
adipocytes expressing the CSF1R/IR or CSF1R/IRA960 were treated with
the indicated concentrations of insulin or CSF-1.
2-[3H]Deoxyglucose uptake (panel A) and
glycogen synthesis (panel B) were measured as described
under "Experimental Procedures." Results are normalized relative to
basal levels of activity. Basal glucose transport in CSF1R/IRA960 cells
averaged 166 ± 18% basal transport in cells expressing CSF1R/IR.
Basal glycogen synthesis in CSF1R/IRA960 cells averaged 134 ± 18% basal synthesis in cells expressing CSF1R/IR. Results are
presented as the mean ±S.D. of triplicate determinations and are
representative of three independent experiments for each
analysis.
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To determine whether the mutation of Tyr960 inhibited only
the ability of the chimera to stimulate maximal glucose uptake, we examined glycogen synthesis in 3T3-L1 adipocytes expressing the CSF1R/IR and CSF1R/IRA960. In cells expressing the CSF1R/IR, glycogen synthesis stimulated by CSF-1 resembled the dose-dependent
increases in synthesis observed with insulin (Fig. 3B). As
seen with glucose uptake, however, CSF-1 was unable to stimulate
maximal glycogen synthesis in cells expressing the CSF1R/IRA960 above
the level of synthesis observed with 1 nM insulin (Fig.
3B).
CSF-1-stimulated Tyrosine Phosphorylation of IRS-1 and IRS-2 in
3T3-L1 Adipocytes Expressing the CSF1R/IR and CSF1R/IRA960--
The
ability of the Ala960 mutation to blunt but not abolish
CSF-1-stimulated glucose uptake and glycogen synthesis in adipocytes expressing the CSF1R/IRA960 indicated that the juxtamembrane mutation did not completely uncouple the CSF1R/IR from downstream effectors that
regulate these biological activities. IRS-1 is readily detectable in
adipocytes (26) permitting examination of the ability of the CSF1R/IR
and CSF1R/IRA960 to tyrosine-phosphorylate this endogenous substrate of
the insulin receptor kinase. Cells were left untreated or treated with
CSF-1 and lysed, and IRS-1 was immunoprecipitated and analyzed by
Western blotting with anti-phosphotyrosine antibodies. CSF-1 stimulated
tyrosine phosphorylation of IRS-1 in adipocytes expressing the intact
CSF1R/IR (Fig. 4). Consistent with
previous studies (27), substitution of Tyr960 with
alanine-blocked CSF-1 stimulated tyrosine phosphorylation of IRS-1 in
adipocytes expressing the CSF1R/IRA960 (Fig. 4). Stripping and
reprobing of the blot verified that equivalent amounts of IRS-1 were
present in each immunoprecipitate from each cell line (Fig. 4,
middle panel). In contrast, CSF-1-treated 3T3-L1 adipocytes expressing the CSF1R/IRA960 were comparable with 3T3-L1 adipocytes expressing the CSF1R/IR in their ability to phosphorylate IRS-2 (Fig.
4). Stripping and reprobing of the blot also revealed comparable levels
of expression of IRS-2 in each immunoprecipitate from each cell line
(Fig. 4).

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Fig. 4.
Phosphorylation of IRS-1 and IRS-2 in 3T3-L1
adipocytes expressing the CSF1R/IR and CSF1R/IRA960. A,
3T3-L1 adipocytes expressing the CSF1R/IR and CSF1R/IRA960 were treated
with or without 10 nM CSF-1 for 10 min, lysed, and
immunoprecipitated with antibodies to IRS-1 (left four
lanes) or IRS-2 (right four lanes). Immunoprecipitated
proteins were resolved by SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose, and probed with antibodies to
phosphotyrosine. B, blot shown in panel A was
stripped and reprobed with polyclonal antibodies against IRS-1.
C, blot in panel B was stripped and reprobed with
antibodies against IRS-2. M.W., molecular mass.
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Interaction of IRS-2 with the CSF1R/IR and the CSF1R/IRA960--
A
second tyrosine-phosphorylated protein co-precipitated with IRS-2 from
CSF-1-treated cells (Fig. 4). To determine the identity of this
protein, the CSF1R/IR or the CSF1R/IRA960 were left untreated or
treated with 10 nM CSF-1, and lysates were
immunoprecipitated with antibodies to IRS-2 or to the carboxyl terminus
of the insulin receptor cytoplasmic domain (CT-1). IRS-2 was
phosphorylated in CSF-1-treated cells expressing the CSF1R/IR or the
CSF1R/IRA960. However, the second tyrosine-phosphorylated protein
co-precipitated with IRS-2 in cells expressing the CSF1R/IR but did not
co-precipitate with IRS-2 in cells expressing the CSF1R/IRA960 (Fig.
5). Reprobing of the blots with CT-1
antibody revealed that the CSF1R/IR, but not the CSF1R/IRA960,
coprecipitated with phosphorylated IRS-2 protein.

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Fig. 5.
Interaction of IRS-2 with the CSF1R/IR and
the CSF1R/IRA960. 3T3-L1 adipocytes expressing the CSF1R/IR or the
CSF1R/IRA960 were treated with or without 10 nM CSF-1 and
lysed. Identical samples were immunoprecipitated with antibodies
against IRS-2 or against the carboxyl terminus of the insulin receptor
cytoplasmic domain (CT-1). Immunoprecipitated proteins were resolved by
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,
and detected on blots with antibodies against phosphotyrosine, followed
by stripping and reprobing the blots with antibody CT-1 or with
anti-IRS-2 antibody. M.W., molecular mass.
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CSF-1-stimulated Activation of PI 3' Kinase in 3T3-L1 Adipocytes
Expressing the CSF1R/IR or the CSF1R/IRA960--
PI 3' kinase is
activated by the insulin receptor via association with
tyrosine-phosphorylated intermediate proteins including IRS-1
(28). To further investigate the role of the juxtamembrane Tyr960 in downstream signaling by the insulin receptor, we
examined the ability of the CSF1R/IRA960 to activate PI 3' kinase
associated with IRS-1 and IRS-2 or with tyrosine-phosphorylated
proteins. 3T3-L1 adipocytes expressing the CSF1R/IR or the CSF1R/IRA960 were serum-deprived for 5 h and then treated with 10 nM CSF-1 or with 100 nM insulin for 2 min. The
cells were lysed and immunoprecipitated with antibodies to IRS-1,
IRS-2, or phosphotyrosine. In adipocytes expressing the CSF1R/IR,
insulin and CSF-1 stimulated PI 3' kinase activity in
anti-phosphotyrosine immunoprecipitates as well as in anti-IRS-1 or
anti-IRS-2 immunoprecipitates (Fig. 6).
In contrast, CSF-1 was unable to stimulate PI 3' kinase in anti-IRS-1
immunoprecipitates from adipocytes expressing the CSF1R/IRA960 (Fig.
6). CSF-1 stimulated PI 3' kinase activity in anti-IRS-2 and
anti-phosphotyrosine immunoprecipitates from adipocytes expressing the
CSF1R/IRA960 (Fig. 6), consistent with its ability to stimulate the
phosphorylation of IRS-2.

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Fig. 6.
Activation of PI 3' kinase associated with
IRS proteins or tyrosine-phosphorylated proteins in 3T3-L1
adipocytes. 3T3-L1 adipocytes expressing the CSF1R/IR or the
CSF1R/IRA960 were left untreated or treated with 10 nM
CSF-1 or 100 nM insulin and lysed. Cell lysates were
immunoprecipitated with antibodies to IRS-1, IRS-2, or phosphotyrosine.
Immunoprecipitates were analyzed for the presence of PI 3' kinase
activity as described under "Experimental Procedures." Quantitative
analysis of PI 3' kinase activity associated with immunoprecipitates of
IRS-1, IRS-2, and tyrosine-phosphorylated proteins was performed using
storage phosphor technology. The data are the mean ±S.D. of four
independent experiments.
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DISCUSSION |
These data demonstrate that the juxtamembrane phosphorylation site
Tyr960 within the insulin receptor cytoplasmic domain is an
essential determinant for the tyrosine phosphorylation of IRS-1 but not for the phosphorylation of IRS-2 by the insulin receptor kinase (Fig.
4). Phosphorylation of IRS-2 by the CSF1R/IRA960 is functional as it
stimulates the activation of PI 3' kinase bound to IRS-2 (Fig. 6). The
Ala960 mutation blocks the ability of the chimeric CSF1R/IR
to maximally stimulate glucose uptake and glycogen synthesis but does
not impair stimulation of glucose transport by submaximal amounts of
CSF-1 (Fig. 3). If IRS-2 is capable of mediating insulin-stimulated GLUT4 translocation (29), then these data suggest that interaction of
IRS-2 with the insulin receptor juxtamembrane domain may not be a
required event to induce glucose uptake. The ability of the CSF1R/IRA960 to phosphorylate IRS-2 and to stimulate glucose transport may explain why ectopic expression of IRS-1 domains that interact with
the insulin receptor juxtamembrane domain were able to block insulin-stimulated mitogenic effects mediated by IRS-1 but did not
inhibit insulin-stimulated GLUT 4 translocation or glucose transport
(30, 31). The data presented here demonstrate that competitive
inhibition of interaction with the juxtamembrane domain is not required
for tyrosine phosphorylation of IRS-2.
These data also suggest an important but not exclusive role for
Tyr960 and IRS-1 in signaling by the insulin receptor
kinase (14, 15, 30, 31). The conclusion that other mechanisms, in
addition to the phosphorylation of IRS-1, specifically contribute to
insulin-stimulated glucose uptake is consistent with several forms of
evidence. Microinjection of antibodies and peptides or the exogenous
expression of IRS-1 domains disrupt protein interaction with the
insulin receptor juxtamembrane region (30, 31). IRS-1
/
mice show mild resistance to insulin and impaired glucose tolerance (32, 33). Adipocytes isolated from IRS-1
/
mice are
impaired but not completely resistant to insulin-stimulated glucose
uptake (33). IRS-2
/
mice also demonstrate peripheral
insulin resistance, but the severity of the phenotype is compounded by
a coinciding lack of pancreatic
-cell compensation (34).
The Ala960 mutation does appear to affect the interaction
of the insulin receptor cytoplasmic domain with IRS-2. Western blotting revealed that the CSF1R/IR co-precipitated with IRS-2, but the CSF1R/IRA960 was not detected in anti-IRS-2 immunoprecipitates. These
data indicate that the juxtamembrane domain is a point of interaction between the insulin receptor cytoplasmic domain and IRS-2
as demonstrated previously (35, 36). The observation that IRS-2 is
still tyrosine-phosphorylated upon activation of the CSF1R/IRA960 may
emphasize the importance of additional domains of interaction between
the insulin receptor cytoplasmic domain and IRS-2. Recent observations
that IRS-1 and IRS-2 are distributed differently in 3T3-L1 cells and
that they translocate from intracellular membrane compartments to
cytosol after insulin stimulation (37) also raises the possibility of
differential interactions between the insulin receptor cytoplasmic
domain and these intracellular substrates.
Two-hybrid analyses (35, 36) have demonstrated that IRS-2 interacts
with phosphotyrosines in the insulin receptor kinase activation loop
through a region termed the kinase regulatory loop binding domain (38).
Furthermore, it has been suggested that phosphorylation of
Tyr624 and Tyr628 within the kinase regulatory
loop binding domain of IRS-2 may inhibit interaction of this domain
with the insulin receptor kinase (38). If these data are correct, then
the phosphorylation-induced decrease in affinity between the insulin
receptor and IRS-2 might explain the inability to co-precipitate IRS-2
and the CSF1R/IRA960 (Fig. 5).
The insulin receptor has multiple intracellular substrates (39-45)
that may serve as distinct signaling intermediates for the pleiotropic
effects of insulin. The ability of the insulin receptor kinase to
mediate diverse signals through distinct intracellular substrates may
be determined not only by the phosphorylation state of the substrate
but by the nature of the signaling complex formed upon interaction
between the receptor and each substrate. The data presented here
demonstrate the utility of the chimeric CSF1R/IR for probing the
molecular details of the interactions between the insulin receptor
kinase and its various intracellular effectors.