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(Received for publication, April 10, 1995; and in revised form, July 31, 1995) From the
pp120/HA4 is a hepatocyte membrane glycoprotein phosphorylated
by the insulin receptor tyrosine kinase. In this study, we have
investigated the role of pp120/HA4 in insulin action. Transfection of
antisense pp120/HA4 cDNA in H35 hepatoma cells resulted in inhibition
of pp120/HA4 expression and was associated with a 2-3-fold
decrease in the rate of insulin internalization. Furthermore, insulin
internalization in NIH 3T3 fibroblasts co-transfected with insulin
receptors and pp120/HA4 was increased 2-fold compared with cells
expressing insulin receptors alone. In contrast, no effect on
internalization was observed in cells overexpressing a naturally
occurring splice variant of pp120/HA4 that lacks the phosphorylation
sites in the intracellular domain. Insulin internalization was also
unaffected in cells expressing three site-directed mutants of pp120/HA4
in which the sites of phosphorylation by the insulin receptor kinase
had been removed (Y488F, Y488F/Y513F, and S503A). Our data suggest that
pp120/HA4 is part of a complex of proteins required for
receptor-mediated internalization of insulin. It is possible that this
function is regulated by insulin-induced phosphorylation of the
intracellular domain of pp120/HA4.
Insulin binding to its receptor triggers the rapid endocytosis
of the ligand-receptor complex(1, 2) . Internalization
of the insulin-insulin receptor complex constitutes the major mechanism
of insulin degradation and down-regulation of cell surface receptors (3, 4, 5) . The molecular events involved in
the internalization process, however, are yet to be well defined.
Specific sequences in the submembranous portion of the receptor are
required for internalization to occur. Furthermore, activation of the
receptor kinase is required for ligand-induced receptor
internalization(6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .
Following insulin-induced receptor autophosphorylation, several
intracellular substrates are phosphorylated, the best characterized of
which is insulin receptor substrate-1 (IRS-1)(16) . However,
the role of substrate phosphorylation in receptor internalization is
not established. In the present study, we have investigated the role
of pp120/HA4, a substrate of the insulin receptor kinase that is
predominantly expressed in liver, in the internalization process.
pp120/HA4 is a transmembrane glycoprotein that is phosphorylated by the
insulin receptor tyrosine kinase in intact cells (17, 18, 19, 20) and in cell-free
systems(21) . It is composed of a large extracellular domain
containing 16 sites of potential N-linked glycosylation and a
71-amino acid cytoplasmic domain(22) . Studies of site-directed
mutants indicate that the intracellular domain contains a site of
constitutive serine phosphorylation (Ser We report that insulin-induced receptor internalization is inhibited
2-3-fold in H35 hepatoma cells transfected with an antisense
pp120/HA4 cDNA. Inhibition of internalization paralleled the reduction
of expression of pp120/HA4. Moreover, expression of pp120/HA4 in NIH
3T3 cells transfected with insulin receptors increased the rate of
internalization of the insulin-insulin receptor complex. In contrast,
internalization rates were unaffected in NIH 3T3 cells expressing the
short isoform of pp120/HA4 or by expression of mutant pp120/HA4
molecules in which the potential phosphorylation sites of the
intracellular domain (Tyr
NIH 3T3 cells co-expressing human insulin
receptors and wild-type (WT) pp120/HA4 (full-length and truncated
isoforms) or phosphorylation-defective pp120/HA4 mutants (Y488F, Y513F,
Y488F/Y513F, and S503A) were described previously(20) .
Figure 1:
Expression of antisense pp120/HA4 RNA
in H35 hepatoma cells. Cell extracts were prepared from untransfected
H35 hepatoma cells (H35), H35 cells transfected with Neo
Figure 2:
Insulin internalization and degradation in
H35 cells. Insulin internalization (A) was assayed as
described under ``Experimental Procedures.'' Acid-resistant
radioactivity was measured in H35 cells (open circles), Neo
cells (solidcircles), and AS cells (opensquares) at the indicated times. Insulin degradation (B) was evaluated as trichloroacetic acid-soluble
radioactivity in the extracellular medium after 20 min at 37 °C.
These data represent mean ± S.D. of four experiments in
duplicates performed with at least two different clones of each cell
type.
Following receptor-mediated internalization, insulin molecules are
released from the receptor by the acidic pH of the endocytic vesicles,
and insulin is subjected to intracellular degradation. Insulin
degradation was measured as trichloroacetic acid-soluble radioactivity
in clones expressing antisense pp120/HA4 RNA (Fig. 2B).
Consistent with the decreased internalization rate, insulin degradation
was also decreased 2-fold in AS cells (15% of bound insulin, following
20 min incubation at 37 °C) compared with both untransfected and
Neo-transfected cells (35 and 42% of bound insulin, respectively).
After 30 min at 37 °C, 78% of bound insulin was degraded in H35,
82% in Neo cells, and 28% in AS cells, indicating that insulin
degradation occurred at a slower rate in cells expressing antisense
pp120/HA4 RNA. Similar results were also obtained when insulin
degradation was measured in cell lysates (data not shown). Insulin-induced receptor down-regulation was analyzed in AS and
control cells following incubation with 100 nM insulin for 24
h at 37 °C. At the end of the incubation period, the receptor
number on the surface of control cells (H35 and Neo) was decreased by
15-20%. In contrast, AS cells did not down-regulate the number of
cell surface receptors in response to insulin (data not shown).
Figure 3:
Insulin receptor internalization in NIH
3T3 cells expressing pp120/HA4. Insulin internalization in NIH 3T3
cells expressing hIR alone (opencircles),
co-expressing hIR and WTpp120 (opensquares), or
co-expressing hIR and
Figure 4:
PDGF
receptor internalization in NIH 3T3 cells expressing pp120/HA4. Binding
of
Insulin receptor internalization was also studied using biotin
labeling of cell surface proteins followed by immunoprecipitation with
anti-insulin receptor antibody and immunodetection with
[
Figure 5:
Internalization of biotin-labeled insulin
receptors. After labeling of cell surface protein with biotin,
monolayers were incubated at 37 °C for 20 min in the presence (fullbars) or absence (hatchedbars) of 100 nM insulin. After incubation with
Pronase, cells were lysed and immunoprecipitated with anti-insulin
receptor antibodies (Ab50). These data were obtained by PhosphorImager
quantitation of blots from three separate
experiments.
Thus, expression of full-length
pp120/HA4, but not of the truncated isoform, was associated with
increased internalization of insulin-receptor complexes in NIH 3T3
cells.
In NIH
3T3 cells co-transfected with hIR and phosphorylation-defective mutants
of pp120/HA4 (Y488F, Y488F/Y513F, or S503A), insulin internalization
rates were similar to those observed in cells expressing hIR alone and
cells co-expressing hIR and
Figure 6:
Insulin internalization and degradation in
cells expressing pp120/HA4 phosphorylation-defective mutants. Insulin
internalization (upperpanel) and degradation (lowerpanel) were measured as in the legend to Fig. 2. Insulin internalization rates (upperpanel) were determined according to Lund et al.(27) . Insulin degradation (lowerpanel)
represents the fraction of trichloroacetic acid-soluble radioactivity
detected in the extracellular medium after 20 min at 37 °C. All
results are expressed as the mean ± S.D. of three triplicate
experiments with at least two different clones of each cell
type.
Receptor down-regulation was evaluated in clones
transfected with hIR and different forms of pp120/HA4. In hIR cells,
insulin binding decreased by 37% after 24 h of insulin treatment. In
contrast, at the same time point, the number of surface receptors in
WTpp120 cells decreased by 50% (Fig. 7). In cells transfected
with the truncated isoform of pp120/HA4 (
Figure 7:
Insulin-induced receptor down-regulation
in NIH 3T3 transfected cells. Down-regulation was measured in hIR (opencircles), WTpp120 (opensquares),
pp120/HA4 is a transmembrane glycoprotein that is
phosphorylated on tyrosine residues by the insulin receptor
kinase(17, 18, 19, 20, 21) .
In the present study we have shown that inhibition of the expression of
pp120/HA4 is associated with decreased endocytosis of insulin. We have
also shown, using transfected cells, that overexpression of pp120/HA4
is associated with increased insulin endocytosis. Thus, manipulation of
pp120/HA4 expression correlates with changes in insulin
receptor-mediated internalization and degradation of insulin. Moreover,
this effect of pp120/HA4 appears to require phosphorylation by the
insulin receptor or other kinases, inasmuch as expression of
phosphorylation-defective mutants of pp120/HA4 (20) is not
associated with changes in internalization of the insulin-insulin
receptor complex. The effect is specific for insulin endocytosis,
because internalization of PDGF, a ligand that is also internalized via
a receptor tyrosine kinase, is not affected by expression of pp120/HA4,
nor is internalization of the unrelated molecule transferrin. Our data
do not indicate whether there is a causal relation between expression
of pp120/HA4 and endocytosis of insulin. It is, however, interesting to
note that pp120/HA4 is predominantly expressed on the plasma membrane
of the hepatocyte, a major site of insulin clearance from the
circulation. The mechanism by which pp120/HA4 might affect insulin
receptor internalization is not clear. Our data suggest that
phosphorylation of pp120/HA4 by the insulin receptor kinase may play a
role in this process. The lack of a dominant negative effect by
phosphorylation-defective mutants of pp120/HA4 on insulin
internalization is consistent with the notion that there might exist
pp120-dependent and pp120-independent internalization pathways. The
tissue distribution of pp120/HA4, which is predominantly found in
liver, is also consistent with the idea that pp120 may play a specific
role in hepatic insulin clearance. pp120/HA4 may be part of a complex
of proteins contributing to the interaction of insulin receptors with
clathrin-coated pits(30, 31, 32) . In support
of the latter hypothesis, it is of interest to note that the amino acid
sequence of pp120/HA4 shares homology with tyrosine-containing
sequences thought to be important recognition elements for AP-2
adaptors binding(33, 34) . The sequences are found at
positions 488-491 (Tyr-Ser-Val-Leu) and 513-516
(Tyr-Ser-Val-Val) of pp120/HA4. Insulin's ability to stimulate
thymidine incorporation is increased in H35 cells transfected with
antisense pp120/HA4 cDNA and decreased in NIH 3T3 cells overexpressing
the full-length isoform of pp120/HA4. This finding is in agreement with
a recent report showing that increased endocytosis of a splice variant
of the IGF-1 receptor is associated with a decreased response to the
mitogenic action of IGF-1 (35) . It remains to be seen whether
this effect is due to down-regulation, ligand degradation, or
sequestration of substrates. The process of ligand-induced
endocytosis of insulin and growth factor receptors can be summarized in
the following steps: 1) binding of the ligand; 2) receptor
autophosphorylation and kinase activation; 3) surface redistribution of
receptors from microvilli to the nonvillous surface of the cell; and 4)
interaction with clathrin and various components of the coated pits,
such as adaptins (AP-2) and perhaps other
proteins(11, 30) . Carpentier and co-workers (11) have recently shown that insulin-induced activation of
receptor autophosphorylation releases a constraint maintaining the
receptor on microvilli. Whether a phosphorylated intermediate such as
pp120/HA4 is involved at this level cannot be determined from our
experiments. Further, it is not clear whether the insulin receptor
binds directly to adaptor proteins or clathrin. On the other hand, it
has also been demonstrated by co-immunoprecipitation experiments that
epidermal growth factor receptor associates with AP-2 complex upon
epidermal growth factor stimulation of cells(31, 32) . In conclusion, our data are compatible with the hypothesis that
pp120/HA4 is involved in insulin internalization and degradation.
Further studies are needed to understand the mechanism by which this
effect is elicited. Differential expression of the two isoforms of
pp120/HA4 in liver may be important to modulate insulin clearance and
to regulate receptor number at the cell surface.
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24073-24077
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
POTENTIAL ROLE OF pp120/HA4, A SUBSTRATE OF THE INSULIN RECEPTOR
KINASE (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) as well as one
tyrosine residue that is phosphorylated in response to insulin
(Tyr
)(20, 23) . Alternative splicing of
pp120/HA4 mRNA generates two isoforms, one of which lacks 61 amino
acids at the C terminus of the cytoplasmic domain, including all the
potential phosphorylation sites(24) . The function of pp120/HA4
is still unknown. It has been proposed to serve as a
Ca
/Mg
-dependent ecto-ATPase (22) and a bile acid transporter(25, 26) .
and Ser
) had been
replaced by site-directed mutagenesis with nonphosphorylatable amino
acids.
Antibodies
pp120/HA4 antibody (
-295) is a
rabbit antipeptide polyclonal antiserum directed against the sequence
VLLLAHNLPQEFQV (amino acids 51-64) of the extracellular domain of
rat liver pp120(32) . Anti-insulin receptor antibody 50 was
described in previous publications(20) .Cell Culture
H35 hepatoma cells were grown in RPMI
medium (Biofluids Inc., Rockville, MD) supplemented with 10% fetal calf
serum. NIH 3T3 cells were maintained in Dulbecco's modified
Eagle's medium (Biofluids Inc., Rockville, MD) containing 10%
fetal calf serum (UBI, Lake Placid, NY) and 2 mM glutamine
(Biofluids, Inc). Transfected cells were routinely maintained at 37
°C in 5% CO
.Construction and Transfection of Antisense pp120/HA4 cDNA
cDNA corresponding to nucleotides -8 to 1574 of pp120/HA4 (20, 22) was ligated in a reverse orientation into a
bovine papilloma virus-based expression vector (pBPV, Pharmacia Biotech
Inc.) at the XhoI/NotI sites. Transfections of H35
cells at 70% confluence were carried out in 100-mm plates with 20, 50,
or 100 µg of the expression vector (pBPV/ASpp120) and 1, 2.5, and 5
µg, respectively, of a plasmid containing the neomycin resistance
gene (pRSVNeo) in the presence of 90 µl of Lipofectamine (Life
Technologies, Inc.) according to the manufacturer's instructions.
Single G418-resistant clones were isolated and analyzed for pp120/HA4
expression by immunoblotting with -295 antibody as described
previously(20) .Insulin Binding, Internalization, and
Degradation
Confluent monolayers of H35 hepatoma and NIH 3T3
cells were washed three times with phosphate-buffered saline and
incubated at 4 °C for 4 h in binding buffer (100 mM HEPES,
120 mM NaCl, 1.2 mM MgSO
, 1 mM EDTA, 15 mM CH
COONa, 10 mM glucose,
1% bovine serum albumin, pH 7.4) containing 30 pM [I]insulin. Unbound ligand was removed by
washing with ice-cold binding buffer. Cells were either lysed with 1 N NaOH (to measure total insulin binding) or further incubated
with the same buffer at 37 °C for the indicated times (to measure
insulin internalization). At each time point, cells were washed with
ice-cold phosphate-buffered saline at pH 3.0 and incubated in the same
buffer twice for 5 min to remove insulin that was still bound at the
cell surface. Following an additional wash with ice-cold
phosphate-buffered saline, pH 7.4, cells were solubilized in 1 N NaOH, and internalization rates were calculated as described by
Lund et al.(27) . Insulin degradation was determined
in the incubation medium or in cell lysates (to measure intracellular
degradation products) by precipitation with 10% trichloroacetic acid.
PDGF
Untransfected
NIH 3T3 cells or WTpp120 cells were incubated with 
InternalizationI-PDGF
(Amersham Corp.) as described by Mori(28) . Thereafter,
internalization of labeled ligand was measured using the same acid wash
procedure as described above.
Insulin Receptor Down-regulation
Confluent
monolayers were incubated at 37 °C in serum-free culture media
(RPMI for H35 cells and Dulbecco's modified Eagle's medium
for NIH 3T3 cells), containing 1% bovine serum albumin and 10 mM HEPES, with or without 100 nM insulin. Surface-bound
insulin was removed by the acid wash technique described above, and
insulin binding was determined by further incubating cells with
[I]insulin for 4 h at 4 °C, as described
above.
Insulin Receptor Internalization
Cell monolayers
were incubated with biotin (0.5 mg/ml) as described(29) .
Following incubation at 37 °C for 20 min in the absence or presence
of 100 nM insulin, cells were incubated with Pronase (2.5
mg/ml) for 1 h at 4 °C and solubilized in a buffer containing 50
mM HEPES, pH 7.6, 150 mM NaCl, 1% Triton X-100, 1
mM phenylmethylsulfonyl fluoride and a mixture of protease
inhibitors. Cell lysates were immunoprecipitated with anti-insulin
receptor antiserum (Ab50) and analyzed by Western blotting as
described(29) . PhosphorImager analysis was used to quantitate
insulin receptors.Thymidine Incorporation
Subconfluent 12-well
plates were incubated in serum-free medium for 24 h. Thereafter,
insulin was added at the indicated concentrations for an additional 16
h. Subsequently, the incubation medium was replaced with medium
supplemented with [H]thymidine (500 nCi/ml), and
cells were incubated for 1 h at 37 °C. After being washed twice
with phosphate-buffered saline pH 7.4, twice with 5% trichloroacetic
acid, and twice with ethanol, cells were solubilized in 300 µl of 1 N NaOH for 1 h at 37 °C. An equal volume of 1 N HCl was added to the solubilized extracts, and the radioactivity
was measured by liquid scintillation counting.
Insulin Internalization and Degradation in H35 Cells
Expressing Antisense pp120/HA4 RNA
H35 hepatoma cells were
transfected with antisense pp120/HA4 cDNA and analyzed by
immunoblotting with anti-pp120/HA4 polyclonal antiserum (Fig. 1, upperpanel). In six indepedent clones (AS1-AS6), the
steady state levels of pp120/HA4 were decreased 4-6-fold compared
with untransfected H35 cells and with mock-transfected cells (Fig. 1, neo1, neo2). Insulin binding and
receptor autophosphorylation were similar in all clones studied and in
control cells (data not shown). To measure internalization, cells were
allowed to bind [I]insulin at 4 °C and then
were transferred to 37 °C for various lengths of time. In control
cells (untransfected H35 and Neo-transfected cells), 50% of bound
[
I]insulin was internalized after 1 min at 37
°C (Fig. 2A). In contrast, cells expressing
antisense pp120/HA4 RNA (AS) internalized only 35% of bound
insulin after 1 min at 37 °C (Fig. 2A).
vector, and clones transfected with antisense pp120/HA4 cDNA
(AS1-AS6) and analyzed by immunoblotting with -295 (anti-pp120/HA4
polyclonal antibody; upperpanel). Quantitation was
done by phosphorImager analysis of the radioactive blot (lowerpanel). This figure shows one of two different
representative experiments.
Insulin Receptor Internalization in NIH 3T3 Cells
Transfected with pp120/HA4
Next, we studied insulin receptor
internalization as a measure of insulin endocytosis in NIH 3T3 cells
co-expressing insulin receptors (hIR) and either full-length (WTpp120)
or the truncated form (
448) of pp120/HA4 that is expressed as a
splice variant in liver. In cells expressing hIR, 37% of bound hormone
was internalized after 10 min at 37 °C (Fig. 3). In cells
co-transfected with hIR and full-length pp120/HA4 (WTpp120), incubation
at 37 °C resulted in internalization of 70% of bound insulin after
10 min. In contrast, in cells co-transfected with truncated pp120/HA4
(448) and hIR, insulin internalization was comparable with that of
cells expressing hIR alone. This effect appeared to be specific for the
insulin receptor in that ligand-dependent internalization of the PDGF
receptor, another member of the receptor tyrosine kinase family, was
unaffected by transfection of pp120/HA4. In untransfected NIH 3T3
cells, 70% of bound I-PDGF was internalized after 20 min
at 37 °C (Fig. 4). In WTpp120 cells, internalization of PDGF
proceeded at a slightly faster rate and peaked at 15 min but was
otherwise similar in extent to that observed in untransfected cells.
Likewise, we failed to detect differences between hIR cells and WTpp120
cells when we measured internalization of transferrin by the
transferrin receptor, a constitutively recycled, non-tyrosine kinase
receptor (data not shown). Thus, it appears that the effect of
pp120/HA4 to increase internalization is specific for insulin.
448 (solidcircles) was
measured as described in the legend to Fig. 2. The data
represent mean ± S.D. from three different experiments performed
in triplicate on at least two different clones of each cell
type.
I-PDGF to 75% confluent monolayers was carried out as
indicated in (28) . Thereafter, PDGF internalization in NIH 3T3
cells (opensquares) or cells expressing WTpp120 (filledsquares) was measured by the acid wash
technique as described. Experiments were performed in
triplicate.
I]streptavidin. Cell surface proteins were
biotinylated at 4 °C and then transferred to a 37 °C incubator
for 15 min in the absence or presence of 100 nM insulin (Fig. 5). Thereafter, monolayers were treated with Pronase to
remove residual cell surface proteins, lysed with Triton X-100,
immunoprecipitated with anti-insulin receptor antibody (Ab50) and
probed with [
I]streptavidin. In the absence of
Pronase treatment, two major bands were detected at approximately 130
and 95 kDa, corresponding to
- and 
-subunits of the insulin
receptor(29) . Pronase treatment resulted in a 90-95%
decrease of cell surface receptors (Fig. 5, solid
bars). Preincubation with insulin rendered a larger fraction of
insulin receptors expressed in NIH 3T3 cells resistant to digestion
with Pronase as a result of having undergone internalization (Fig. 5, hatchedbars). Consistent with
insulin internalization data, the amount of internalized receptors was
2-fold higher in WTpp120 cells (33 ± 5% of the total
biotinylated receptors were internalized) than in hIR (16 ± 4%)
and 448 cells (12 ± 3%).
Insulin Receptor Internalization in Cells Transfected
with Phosphorylation-defective Mutants of pp120/HA4
We have
recently shown that mutation of Tyr (Y488F) impaired
phosphorylation of pp120/HA4 by the insulin receptor in vitro and in intact cells(20) . Similarly, mutation of both
Tyr
and Tyr
(Y488F/Y513F) also impaired
insulin-induced phosphorylation. In contrast, mutation of Tyr
did not affect the ability of pp120/HA4 to undergo
insulin-stimulated phosphorylation. Finally, substitution of
Ser
with alanine (S503A) impaired basal as well as
insulin-induced phosphorylation of pp120/HA4(20) .
448 (Fig. 6A). In
contrast, insulin internalization rates in cells transfected with Y513F
mutant were similar to those observed in cells expressing WTpp120 (Fig. 6A). Thus, we observed a correlation between
increased rates of insulin endocytosis and expression of
phosphorylation-competent forms of pp120/HA4.
Insulin Degradation and Insulin-induced Receptor
Down-regulation in NIH 3T3 Transfected Cells
As shown in Fig. 6B, insulin degradation products were detected in
the medium of hIR cells after 20 min at 37 °C (7 ± 2% of
bound insulin). Over the same time period, insulin degradation in
WTpp120 cells was 2-fold higher (15 ± 3%), whereas in 448
cells insulin degradation was similar to that measured in hIR cells (6
± 3%). Further incubation at 37 °C resulted in increased
degradation of insulin in all clones, so that at 60 min 52% of insulin
was degraded in hIR cells, 59% in WTpp120 cells, and 50% in 448
cells (data not shown). In cells expressing phosphorylation-defective
mutants of pp120/HA4 (Y488F, Y488F/Y513F, and S503A) degradation of
insulin was similar to hIR and 448 cells (Fig. 6B). In contrast, Y513F cells internalized and
degraded insulin at the same rate as cells transfected with WTpp120 (Fig. 6B). Thus, in all clones studied we observed a
good correlation between insulin endocytosis and intracellular
degradation.448), insulin receptor
down-regulation was similar to hIR cells (34%). Insulin-induced
receptor down-regulation of Y488F, Y488F/Y513F, and S503A cells was
comparable with that observed in 448 cells. In cells expressing
Y513F, receptor down-regulation displayed a pattern similar to that of
WTpp120 cells, with 47% of receptors being removed from the cell
surface by 24 h (Fig. 7).
448 (opentriangles),
Y488F (solidsquares), Y513F (solidcircles), Y488F/Y513F (solidtriangles), and S503A (opendiamond)
cells as described under ``Experimental Procedures.'' Data
are means ± S.D. of three triplicate experiments with at least
two clones of each cell type.
Insulin-stimulated Thymidine Incorporation
We
measured thymidine incorporation in cells transfected with sense and
antisense cDNA encoding pp120/HA4 as an index of the mitogenic effect
of insulin (Table 1). Maximal thymidine incorporation in all
clones was observed between 10 and 10
M insulin. However, cells expressing antisense pp120/HA4
RNA showed a leftward shift in the dose-response to insulin (ED
= (1.5 ± 0.3)
10M), compared with control H35 (ED
=
(4.9 ± 0.2)
10M) and Neo
cells (ED
= (5.1 ± 0.3)
10M). Conversely, cells transfected with
WTpp120 showed a rightward shift of the dose-response curve for insulin
(ED
= (3.0 ± 0.4)
10M) compared with hIR cells (ED
=
(0.7 ± 0.1)
10M) and
448 cells (ED = (0.6 ± 0.2)
10M). Therefore, cells overexpressing hIR
alone are more sensitive to the mitogenic effects of insulin than cells
expressing WTpp120/HA4. Cells co-transfected with hIR and Y513F showed
a dose-response curve similar to WTpp120 cells (ED
= (2.4 ± 0.2)
10M), whereas cells co-transfected with hIR and one of the
three phosphorylation-defective mutants displayed ED
= (0.8 ± 0.4)
10M (Y488F), (0.7 ± 0.2)
10M (Y488F/Y513F), and (0.8 ± 0.1)
10M (S503A).
)
We thank Dr. Simeon Taylor for support and advice
during the course of this work and Drs. Mohammed Taouis, Paris Roach,
and Jean-Louis Carpentier for helpful discussions and technical
comments. We also thank Dr. Derek LeRoith and Prof. Nicola Perrotti
(University of Catanzaro, Italy) for critical reading of the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 S. Sadekova, N. Lamarche-Vane, X. Li, and N. Beauchemin The CEACAM1-L Glycoprotein Associates with the Actin Cytoskeleton and Localizes to Cell-Cell Contact through Activation of Rho-like GTPases Mol. Biol. Cell, January 1, 2000; 11(1): 65 - 77. [Abstract] [Full Text]
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M. Huber, L. Izzi, P. Grondin, C. Houde, T. Kunath, A. Veillette, and N. Beauchemin The Carboxyl-terminal Region of Biliary Glycoprotein Controls Its Tyrosine Phosphorylation and Association with Protein-tyrosine Phosphatases SHP-1 and SHP-2 in Epithelial Cells J. Biol. Chem., January 1, 1999; 274(1): 335 - 344. [Abstract] [Full Text] [PDF]
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C. V. Choice, M. J. Howard, M. N. Poy, M. H. Hankin, and S. M. Najjar Insulin Stimulates pp120 Endocytosis in Cells Co-expressing Insulin Receptors J. Biol. Chem., August 28, 1998; 273(35): 22194 - 22200. [Abstract] [Full Text] [PDF]
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S. M. Najjar, C. V. Choice, P. Soni, C. M. Whitman, and M. N. Poy Effect of pp120 on Receptor-mediated Insulin Endocytosis Is Regulated by the Juxtamembrane Domain of the Insulin Receptor J. Biol. Chem., May 22, 1998; 273(21): 12923 - 12928. [Abstract] [Full Text] [PDF]
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S. L. Calzi, C. V. Choice, and S. M. Najjar Differential effect of pp120 on insulin endocytosis by two variant insulin receptor isoforms Am J Physiol Endocrinol Metab, October 1, 1997; 273(4): E801 - E808. [Abstract] [Full Text] [PDF]
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S. M. Najjar, Y. R. Boisclair, Z. T. Nabih, N. Philippe, Y. Imai, Y. Suzuki, D.-S. Suh, and G. T. Ooi Cloning and Characterization of a Functional Promoter of the Rat pp120 Gene, Encoding a Substrate of the Insulin Receptor Tyrosine Kinase J. Biol. Chem., April 12, 1996; 271(15): 8809 - 8817. [Abstract] [Full Text] [PDF]
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M. N. Poy, R. J. Ruch, M. A. Fernstrom, Y. Okabayashi, and S. M. Najjar Shc and CEACAM1 Interact to Regulate the Mitogenic Action of Insulin J. Biol. Chem., January 4, 2002; 277(2): 1076 - 1084. [Abstract] [Full Text] [PDF]
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