(Received for publication, December 23, 1994; and in revised form, April 20, 1995)
From the
We have demonstrated previously that dexamethasone treatment of HepG2 cells caused an enhancement of insulin's metabolic effects (Kosaki, A., and Webster, N. J.(1993) J. Biol. Chem. 268, 21990-21996). This correlated with increased expression of the mRNA encoding the B isoform of the insulin receptor (IR).
In the
present study, we have demonstrated that dexamethasone treatment
caused in addition an enhancement in insulin-stimulated immediate-early
gene expression (c-fos and egr-1). Dexamethasone
treatment caused an increase in in vivo IR autophosphorylation
and insulin receptor substrate-1 (IRS-1) phosphorylation both early
events in the insulin signaling pathway. Furthermore, the IRS-1
phosphorylation was distinctly left shifted, although the level of
IRS-1 protein was unchanged. Total cellular tyrosine phosphatase
activity was unaltered when assayed with P-labeled IR and
IRS-1. Studies in vitro on wheat-germ agglutinin-purified
receptors showed that the B isoform of the IR had increased kinase
activity both toward itself and the exogenous substrates
poly-glu
:tyr
and recombinant IRS-1 protein. In
addition, two-dimensional tryptic phosphopeptide maps indicated that
the B isoform has an additional phosphopeptide that is not seen for the
A isoform.
In conclusion, it appears that the B isoform of the IR signals more efficiently than the A isoform in HepG2 cells.
The insulin receptor (IR) ()protein is a
heterotetrameric protein composed of two
-subunits that confer the
ability to bind insulin and two
-subunits that contain the
membrane spanning and the tyrosine kinase
domains(1, 2, 3) . The
-subunit is
entirely extracellular and is linked by disulfide bounds to the
extracellular portion of the
-subunit. Following binding of
insulin to the
-subunits, the first observable event is
autophosphorylation on the cytoplasmic portion of the
-subunit
leading to an increase in the receptor's intrinsic tyrosine
kinase activity toward other
substrates(1, 4, 5) . The human IR is encoded
by a single gene that is located on chromosome 19 and composed of 22
exons(6) . The mature IR, however, exists as two isoforms,
designated A and B, which result from alternative splicing of the
primary transcript(6, 7, 8) . The A isoform
lacks exon 11, is expressed ubiquitously, and is the only isoform in
lymphocytes, brain, and spleen; the B isoform contains exon 11 and is
expressed predominantly in liver, muscle, adipocytes, and kidney (9, 10, 11, 12) . Exon 11 is
composed of 36 nucleotides that encode a 12-amino-acid segment
(residues 717-728) of the carboxyl terminus of the
-subunit
of IR. It has been reported that the affinity of the A isoform for
insulin is higher than that of the B isoform causing a left shift in
the insulin dose-response curves for insulin stimulation of
autophosphorylation, glycogen synthesis, and thymidine
uptake(12, 13, 14) . Two groups found that
the A isoform exhibits a higher insulin internalization and recycling
rate than the B isoform(14, 15) , whereas a third
found no difference in either the rate of internalization or the rate
of degradation(13) . However, all of these studies have been
performed in Chinese hamster ovary or Rat-1 fibroblast cell lines,
which are not good models for insulin-sensitive tissues. The fact that
the B isoform is expressed primarily in insulin-sensitive tissues
indicates that the B isoform of the IR must play an important role in
signaling in insulin-sensitive tissues.
The HepG2 cell is derived from a human hepatoblastoma and has been useful as a model for liver one of the major sites of insulin action (16, 17) . We have shown previously that dexamethasone (dex) causes an increase in sensitivity and responsiveness for insulin's metabolic effects (glucose incorporation into glycogen and 2-deoxyglucose transport) in these cells. This correlated with a switch in expression from the A (-exon 11) to the B (+exon 11) isoform of the IR similar to the ratio seen in adult liver(18) . The aim of the present study was to investigate whether similar enhancements would be seen for insulin's mitogenic effects and to determine whether the expression of the B isoform of the IR is responsible for the enhancement. We show that dex treatment does cause an enhancement in insulin-stimulated immediate-early gene expression (c-fos and egr-1). The B isoform couples more efficiently to the insulin receptor substrate 1 (IRS-1) in vivo than the A isoform. Furthermore, insulin-stimulated autophosphorylation and kinase activity in vitro are greater for the B isoform of the IR. Tryptic peptide mapping identified a novel phosphopeptide in the B isoform that may be involved in the enhanced kinase activity and/or signaling.
The c-fos primer pair consisted of oligonucleotides spanning nucleotides
140-169 (sense primer, 5`-GTTCTCGGGTTTCAACGCGGACTACGAGGC-3`) and
276-309 (antisense primer,
5`-GGCACTAGAGACGGACAGATCTGCGCAAAAGTCC-3`) which generate a fragment of
169 base pairs following amplification. The egr-1 primer pair
consisted of oligonucleotides spanning nucleotides 539-568 (sense
primer, 5`-GAGCCGAGCGAACAACCCTACGAGCACCTG-3`) and 763-791
(antisense primer, 5`-GCGCTGAGGATGAAGAGGTTGGAGGGTTGG-3`) which generate
a fragment of 252 base pairs following amplification. The L30 primer
pair consisted of oligonucleotides spanning nucleotides 74-98
(sense primer, 5`-GAAAGTACGTGCTGGGGTACAAACAGACTC-3`) and either
285-309 (antisense primer for c-fos measurement,
5`-ATCGGAATCACCTGGGTCAATGATAGCCAG-3`) or 224-254 (antisense
primer for egr-1 measurement,
5`-CCACACGCTGTGCCCAATTCAATGTTATTGC-3`) which generate a fragment of 238
and 181 base pairs, respectively. Five µl of the cDNA synthesis
reaction was used for polymerase chain reaction (PCR) amplification in a 50-µl final reaction volume (0.5
µM each oligonucleotide primer, 10 mM Tris-HCl,
pH 8.3, 50 mM KCl, 1.5 mM MgCl
, 0.1
mM dNTPs, 2 units of Taq DNA polymerase, and 1
µCi of [
-
P]dCTP). Twenty-five cycles of
amplification were performed using a Perkin-Elmer DNA thermal cycler
System 9600. Each cycle consisted of a 30-s denaturation at 94 °C,
a 30-s annealing at 55 °C, and a 60-s extension at 72 °C. The
number of cycles was optimized to ensure that the amplification lay
within the exponential phase. The products of the PCR amplification
were resolved by electrophoresis on 8% polyacrylamide gels. The gels
were dried and exposed to film at room temperature. The band densities
were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale,
CA). The counts of c-fos and egr-1 were normalized to
L30 as a internal standard.
For the
IRS-1 phosphorylation assay, equal amounts of WGA-purified receptor
were preincubated with increasing concentrations of insulin for 16 h at
4 °C, followed by the addition of 30 µCi of
[P]ATP in reaction buffer (25 mM HEPES,
pH 7.4, 50 µM ATP, 5 mM MnCl
, and 50
mM NaF) for 15 min at 23 °C. Recombinant IRS-1 protein
(0.3 µg) was added and the incubation continued for a further 90
min at 23 °C. The reaction was terminated by the addition of an
equal volume of 50 mM ATP, 4 mM sodium orthovanadate,
200 mM NaF, 20 mM sodium pyrophosphate, 10 mM EDTA, and 25 mM HEPES, pH 7.6. The IRS-1 proteins were
immunoprecipitated with an anti-IRS-1 antibody for 16 h at 4 °C
followed by precipitation with Pansorbin. Phosphorylated IRS-1 proteins
were visualized by SDS-PAGE and autoradiography and were quantified
using the PhosphorImager.
For the immunoprecipitation study, the P-labeled IR was resuspended in 100 µl of 100 mM NMA, pH 8.3, following trypsin digestion. The sample was boiled
for 5 min and 0.5 mM phenylmethylsulfonyl fluoride added to
inactivate any remaining trypsin. The digested receptor was incubated
with an anti-COOH-terminal peptide (residues 1322-1341) antibody
for 16 h and then with Protein A-Sepharose for 2 h in 500 µl of NMA
at 4 °C. Sepharose-bound antibody was recovered by centrifugation
and washed twice with 1 ml of NMA. Phosphopeptides were eluted by
resuspending the complex in 500 µl of 1 M acetic acid
containing 10 µg of the cold carrier peptide, and mixing at 4
°C for 30 min. Acetic acid was removed by rotary evaporation, and
the phosphopeptide was washed extensively with water before analysis by
two-dimensional mapping as described above(24) .
Figure 1:
Insulin-stimulated c-fos and egr-1 gene expression. HepG2 cells were cultured with and
without 1 µM dexamethasone (Dex) in Ham's
F-12 mixture for 4 days and stimulated with increasing concentrations
of insulin for 45 min at 37 °C. The total cellular RNA was
extracted and subjected to RT-PCR to determine c-fos and egr-1 gene expression using the ribosomal protein L30 mRNA as
internal standard. Panel A, insulin stimulation of c-fos gene expression. B, insulin dose-response curves for
c-fos expression. Results are the mean ± S.E. of four
experiments and are normalized to L30 mRNA levels. Maximal insulin
stimulation is 1.6-fold higher in the presence of dexamethasone (p < 0.01, n = 4). The ED values were
9.8 and 5.6 nM for cells cultured without and with dex,
respectively, and are indicated by arrows (p <
0.05, n = 4). Panel C, Insulin stimulation of egr-1 gene expression. D, insulin dose-response
curves for egr-1 expression. Results are the mean ±
S.E. of four experiments and are normalized to L-30 mRNA levels.
Maximal insulin stimulation is 1.6-fold higher in the presence of
dexamethasone (p < 0.02, n = 4). The
ED
values were 5.0 and 2.1 nM for cells cultured
without and with dex, respectively, and are indicated by arrows (p < 0.02, n =
4).
Figure 2:
Insulin-stimulated tyrosine
phosphorylation in whole cells. HepG2 cells were cultured with and
without 1 µM dex (Dex) in Ham's F-12
mixture for 4 days and stimulated with increasing concentrations of
insulin for 1 min at 37 °C. Cells were solubilized and aliquots,
equalized for either insulin binding (panels A-C) or cell
number (panels D and E), were separated by SDS-PAGE,
and transferred to an Immobilon-P membrane. Tyrosine phosphorylated
proteins, IR, and IRS-1 were visualized by immunoblotting followed by
ECL detection. The band densities were quantified by densitometry. Panel A, anti-phosphotyrosine immunoblot. The -subunits
of the IR and IRS-1 are indicated by arrows. Panel B,
insulin dose-response curves for autophosphorylation of IR
-subunits. Results are the mean ± S.E. of eight
experiments. Maximal insulin stimulation is 1.5-fold higher in the
presence of dex (p < 0.01, n = 8). The
ED
values of the curves were 5.5 and 8.8 nM for
cells cultured without and with dex, respectively, and are indicated by
the arrows (p > 0.05, n = 8). Panel C, insulin dose-response curves for IRS-1
phosphorylation. Results are the mean ± S.E. of eight
experiments. Maximal insulin stimulation is 1.3-fold higher in the
presence of dex (p < 0.01, n = 8). The
ED
values of the curves were 5.4 and 2.6 nM for
cells cultured without and with dex, respectively, and are indicated by arrows (p < 0.04, n = 8). Panel D, effect of dex on IR protein levels. IRs were
visualized using a polyclonal anti-IR antibody. Dex treatment causes a
1.5-fold increase in IR level. Panel E, effect of dex on IRS-1
protein levels. IRS-1 was visualized using a polyclonal antibody to
IRS-1. Dex treatment has no effect on IRS-1
levels.
One possible explanation for the increase in sensitivity and maximal phosphorylation of IRS-1 seen in cells treated with dex is that the level of IRS-1 protein has increased. Consequently, we measured IR and IRS-1 levels by immunoblotting. Cell lysates from equal numbers of the cells were immunoblotted with a polyclonal anti-IR antibody (Fig. 2D). The amount of IR was 1.5-fold higher in cells treated with dex similar to the increment in insulin binding that we reported previously(18) . Immunoblotting with an anti-IRS-1 antibody indicated that dex treatment did not alter the amount of IRS-1 protein (Fig. 2E). It should be noted that the increase in IR protein is not the origin of the increased in vivo autophosphorylation as extracts were normalized for insulin binding prior to antiphosphotyrosine immunoblotting (Fig. 2A). However, IRS-1 levels are unaffected by dex treatment so less IRS-1 is loaded in the antiphosphotyrosine immunoblot for cells treated with dex. When adjusted for equal cell number and therefore equal IRS-1 levels, the maximal insulin-stimulated phosphorylation of IRS-1 is 2.2-fold higher in cells treated with dex rather than the 1.3-fold shown in Fig. 2A (data not shown).
Figure 3:
In vitro autophosphorylation of
IR isoforms. HepG2 cells were cultured with and without 1 µM dex (Dex) in Ham's F-12 mixture for 4 days. IRs
were purified by WGA affinity chromatography. Equal numbers of IRs,
normalized by insulin binding, were subjected to in vitro autophosphorylation in the presence of
[-
P]ATP. The receptors were
immunoprecipitated with an anti-IR antibody for 16 h at 4 °C
followed by precipitation with Pansorbin. Phosphorylated
-subunits
were visualized by SDS-PAGE and autoradiography and quantified on a
PhosphorImager. Panel A, insulin stimulation of IR
autophosphorylation. PanelB, insulin dose-response
curves for in vitro autophosphorylation. Results are the mean
± S.E. of four experiments. Maximal insulin-stimulated
autophosphorylation is 1.7-fold higher in the presence of dex (p < 0.01, n = 4). The ED
values are
indicated by arrows (ED
: 9.8 nM for
-Dex; 10.2 nM for +Dex). Panel C,
partially purified IRs normalized for insulin binding were separated by
SDS-PAGE, transferred to an Immobilon-P membrane and subjected to
immunoblotting using an anti-IR antibody to verify equal receptor
number.
Figure 4:
In vitro kinase activity of IR
isoforms. HepG2 cells were cultured with and without 1 µM dex (Dex) in Ham's F-12 mixture for 4 days.
WGA-purified IRs normalized for insulin binding were subjected to an in vitro kinase assay using poly-glu:tyr
or recombinant IRS-1 protein as a substrate. Panel A,
insulin dose-response curves for phosphorylation of
poly-glu
:tyr
plotted as phosphorylation above
basal versus insulin concentration. Samples were normalized
for insulin binding. Results are the mean ± S.E. of four
experiments. Maximal insulin-stimulated phosphorylation is 1.6-fold
higher in the presence of dex (p < 0.01, n = 4). The ED
values are 21.7 and 19.7 nM for -Dex and +Dex, respectively, and are indicated by
the arrows. Panel B, insulin-stimulated
phosphorylation of recombinant IRS-I. Equal numbers of IRs were used to
phosphorylate recombinant IRS-1 in vitro. Phosphorylated IRS-1
proteins were immunoprecipitated then visualized by SDS-PAGE and
autoradiography. A representative autoradiogram is shown. Maximal
insulin-stimulated phosphorylation is 1.5-fold higher in the presence
of dex. Panel C, insulin-stimulated of IR autophosphorylation.
Following immunoprecipitation of the IRS-1 proteins, the supernatants
were subjected to SDS-PAGE and autoradiography to visualize the IR
autophosphorylation.
Figure 5:
Two-dimensional tryptic phosphopeptide
maps of IR isoforms. WGA purified IRs from cells cultured without (panel A) and with (panel B) dex were incubated with
100 nM insulin for 16 h at 4 °C, followed by the addition
of 30 µCi of [P]ATP in reaction buffer for
30 min at 4 °C. The reaction was terminated by the addition of
excess cold ATP with phosphatase inhibitors. The IRs were
immunoprecipitated with an anti-IR antibody for 16 h at 4 °C
followed by precipitation with Pansorbin and subjected to SDS-PAGE.
Phosphorylated
-subunits were eluted from gel and digested with
TPCK-treated trypsin.
P-Labeled tryptic phosphopeptides
were separated on thin layer cellulose plates by electrophoresis at pH
1.9 followed by ascending chromatography. A representative
autoradiogram is shown.
The position of phosphopeptide E suggested that it may be derived
from the carboxyl terminus of the -subunit. We attempted to
confirm this assignment by immunoprecipitation with an antibody that
had been raised against the COOH-terminal peptide (residues
1322-1341) that contains the two known auto-phosphorylation sites
(tyrosines 1328 and 1334) (Fig. 6). Peptide F was precipitated
along with peptide G and a fraction of peptide D (Fig. 6B). However, none of fragment E was precipitated
so it is unlikely the E is derived from the COOH terminus. Peptide D
contains the doubly phosphorylated tryptic peptide (amino acids
1156-1168) having an approximate charge of +1.5 at pH 1.9.
The marker dye dinitrophenyl lysine has a charge of +1.7 at pH
1.9. The doubly phosphorylated COOH-terminal peptide (amino acids
1327-1341) is predicted to have an approximate charge of
+1.5 whereas the singly phosphorylated form has a charge of
+2.5. Thus these two peptides should migrate either side of the
dinitrophenyl lysine marker. Only a fraction of D is immunoprecipitated
by the antibody, so D most likely represents a mixture of the doubly
phosphorylated COOH terminus peptide (amino acids 1327-1341) and
the doubly phosphorylated peptide from the triple tyrosine region
(amino acids 1156-1168). The singly phosphorylated peptide
derived from the COOH-terminal peptide is thus G by analogy with
peptides A and B and C and D(27) . Phosphopeptide F is likely
to be a doubly phosphorylated tryptic peptide that contains a single
additional lysine or arginine residue due to incomplete cleavage (amino
acids 1326-1341 or 1327-1342). This would cause the peptide
to have an additional +1 charge. The identity of peptide E is
unknown.
Figure 6:
Immunoprecipitation of tryptic fragments
of IR. IRs were labeled and digested as in Fig. 5. The
tryptic-phosphopeptides were incubated with an anti-COOH-terminal
peptide antibody for 16 h and then with Protein A-Sepharose for 2 h in
500 µl of 100 mM NMA at 4 °C. Sepharose-bound antibody
was recovered by centrifugation and washed twice with 1 ml of NMA.
Phosphopeptide was eluted by resuspending the complex in 500 µl of
1 M acetic acid containing 10 µg of the cold carrier
peptide at 4 °C for 30 min. Acetic acid was removed by rotary
evaporation and the phosphopeptide was washed extensively with water.
Then P-labeled tryptic phosphopeptides were separated on
thin layer cellulose plates by electrophoresis at pH 1.9 followed by
ascending chromatography. A representative autoradiogram is shown. Panel A, total phosphopeptides. Panel B,
immunoprecipitated phosphopeptides.
Figure 7:
Two-dimensional tryptic peptide maps of
IRS-1. Equal numbers of IRs from cells cultured without (panel
A) and with (panel B) dex were preincubated with 100
nM insulin for 16 h at 4 °C, followed by the addition of
30 µCi of [P]ATP in reaction buffer for 20
min at 23 °C. Recombinant IRS-1 protein (0.3 µg) was added and
the incubation continued for a further 90 min at 23 °C. The
reaction was terminated by the addition of excess cold ATP with
phosphatase inhibitors. The IRS-1 proteins were immunoprecipitated with
anti-IRS-1 antibody for 16 h at 4 °C followed by precipitation with
Pansorbin. Phosphorylated IRS-1 proteins were separated by SDS-PAGE,
eluted from gel, and digested with TPCK-treated trypsin.
P-Labeled tryptic phosphopeptides were separated on thin
layer cellulose plates by electrophoresis at pH 1.9 followed by
ascending chromatography. A representative autoradiogram is
shown.
Figure 8:
Effect of dexamethasone on phosphatase
activity in HepG2 Cells. Cells cultured with and without dex (Dex) were homogenized in lysis buffer (see
``Experimental Procedures''). After centrifugation, the
Triton X-100-soluble total fraction was incubated with in vitroP-labeled IR and IRS-1 for indicated period at 30
°C. The reaction was terminated by boiling, and samples were
separated by SDS-PAGE to determine dephosphorylation of IR and IRS-1. A
representative autoradiogram is shown.
We have published previously that dexamethasone treatment of HepG2 hepatoma cells causes a switch in insulin receptor isoform expression from A to B. This alteration is accompanied by an increase in responsiveness and insulin sensitivity for two of insulin's metabolic effects, namely glucose incorporation into glycogen and 2-deoxyglucose transport. Changes in isoform expression and insulin response are also observed during the differentiation of 3T3-L1 adipocytes. In the present study, we demonstrate that similar enhancements are observed for two of insulin's mitogenic effects, namely induction of the c-fos and egr-1 genes. This suggested to us that it was expression of the B isoform of the IR upon dexamethasone treatment that caused the enhanced signaling as the metabolic and mitogenic pathways are thought to diverge at a very early point, perhaps at the insulin receptor itself. Consequently, we examined two of the earliest events in the signaling pathway, IR autophosphorylation and IRS-1 phosphorylation in vivo. Maximal insulin-stimulated phosphorylation of both proteins was elevated in cells treated with dexamethasone. Furthermore, the sensitivity for IRS-1 phosphorylation, but not for IR autophosphorylation, was increased. This increase in sensitivity for IRS-1 phosphorylation is likely to be the origin for the observed enhancement in signaling as it has been demonstrated that IRS-1 is involved in both insulin-stimulated DNA synthesis in Rat-1 fibroblasts and Glut-4 translocation in 3T3-L1 cells(28, 29) .
Tyrosine phosphorylation is
regulated in vivo by a balance of kinase and phosphatase
activities. Alteration in either activity could cause the observed
increase in phosphorylation. Therefore, we undertook experiments on
receptors purified by wheat-germ agglutinin affinity chromatography. In vitro, the B isoform of the IR autophosphorylates to a
greater extent than the A isoform and has increased kinase activity
toward the synthetic substrate poly-glu:tyr
and
recombinant IRS-1 protein. Conversely, we were not able to detect any
alterations in total cellular tyrosine phosphatase activity when
assayed with in vitro
P-labeled IR and IRS-1.
Thus it appears that the enhanced phosphorylation seen in vivo is due to differences in the intrinsic kinase activities of the IR
isoforms. There is still a discrepancy between the in vitro and in vivo results, however, as there is no difference
in insulin sensitivity for IRS-1 phosphorylation in vitro.
Although IRS-1 is a direct substrate for the IR kinase in
vitro, there may be additional factors in vivo that
enhance the interaction between the two molecules causing the
left-shift in the insulin dose-response curve. Alternatively, the
receptor may require the intact plasma membrane for correct function.
The two receptor isoforms differ by 12 amino acids in the carboxyl
terminus of the extracellular -subunit. The intracellular kinase
domains of the two isoforms are identical. How is it possible that the
two isoforms have different kinase activities? Kellerer and co-workers (26) found that if the receptors are activated by trypsin
cleavage rather than insulin stimulation then no differences in
activity are observed. So how is insulin able to activate one kinase
more than the other? The B isoform autophosphorylates to a greater
extent than the A isoform both in vivo and in vitro.
There are two possible explanations for this finding. It has been shown
that following insulin stimulation only 43% of insulin receptors
isolated from human adipocytes are phosphorylated on
tyrosine(30) . This has lead to the proposal that there are
distinct pools of kinase competent and incompetent receptors. In
non-insulin-dependent diabetes mellitis, fewer than 15% of IRs are
phosphorylated on tyrosine suggesting that the proportion of
incompetent receptors is greater leading to a kinase
defect(30) . However, these findings can be interpreted
differently as the phosphorylation reaction is inherently reversible.
The observed autophosphorylation is a balance between the forward
phosphorylation and the reverse dephosphorylation reactions. So an
increase in the forward reaction due to an increase in V
would shift the equilibrium toward a more
fully phosphorylated state.
An alternative explanation could be that
the B isoform may be utilizing autophosphorylation sites that are
unavailable in the A isoform due to conformational restraints imposed
by the extracellular -subunits. This latter alternative can be
tested by tryptic phosphopeptide mapping. Kellerer et al.(26) used HPLC analysis of the tryptic peptides but were
not able to observe any differences. However, direct comparison of HPLC
and thin layer peptide mapping of insulin receptor phosphopeptides has
shown that HPLC lacks the sensitivity and resolution of two-dimensional
thin layer mapping(27) . Therefore, we utilized two-dimensional
tryptic phosphopeptide mapping (Fig. 5). The intensity of all
phosphopeptides was higher in the B isoform suggesting that the
equilibrium has been shifted to a more highly phosphorylated state
consistent with the increase in V
. More
interestingly, an additional phosphopeptide E was detected in the B
isoform that is not seen in the A isoform. The exact identity of
peptide E remains to be determined, but two possibilities are the
peptides spanning amino acids 1086-1092 and 1102-1127,
which include tyrosines 1087 and 1122, respectively. Both these
peptides are predicted to have a charge of +1.5 at pH 1.9 if
phosphorylated on tyrosine. Interestingly, both peptides are derived
from domains that are conserved among tyrosine kinases; however neither
are known autophosphorylation sites(31) .
In conclusion, dexamethasone causes an increase in responsiveness and sensitivity for insulin-stimulated immediate early gene expression in HepG2 cells. The results presented here indicate that it is the expression of the B isoform of the IR upon dex treatment that is responsible for this enhancement. The B isoform has a greater insulin-stimulated kinase activity, phosphorylates IRS-1 more efficiently in vitro, and couples to IRS-1 more efficiently in vivo. Autophosphorylated tyrosine residues on tyrosine kinases are the docking sites for signaling proteins containing SH2 domains, and the identification of a new phosphopeptide in the B isoform raises the possibility that this isoform could interact with signaling molecules that are not available to the A isoform(32, 33, 34) . It will be interesting to identify this new autophosphorylation site and determine whether the B isoform couples to novel SH2-containing proteins. It should be stated, however, that we cannot rule out the possibility that dexamethasone could be having other effects on the cell that could contribute to the enhanced signaling. For example, it has been shown that dex treatment of IM-9 and Fao cells causes changes in the carbohydrate composition of the IR, but it is not known whether this effects signaling(35) . Alternatively, dex may effect membrane fluidity which could alter the function of the IR. Although these are possibilities, we believe that the major determinant for enhanced signaling is the switch in expression of IR isoforms from A to B. A method of modulating the alternative splicing in vivo that does not require hormonal treatment will be required to rule out these alternatives definitively.