Insulin resembles many peptide growth factors in that it can
elicit multiple biological responses in target cells. These cellular
responses include promotion of cellular growth, regulation of cell
differentiation programs, and regulation of cellular metabolism.
Despite the fact that individual growth factors may trigger distinct
biological responses on the part of the target cell, it is remarkable
that most of the receptors share in common an overlapping set of
downstream signaling pathways, e.g. activation of
phosphatidylinositol (PI) (
)3-kinase and activation of Ras
and the protein kinase cascade involving Raf and MAP kinase. In this
study, we have explored the consequences of the fact that two receptors
both compete to activate the same PI 3-kinase molecules. When PDGF
stimulates tyrosine phosphorylation of its receptor, this causes the
p85 regulatory subunit of PI 3-kinase to bind to phosphorylated
Tyr-Xaa-Xaa-Met motifs in the PDGF receptor(1, 2) .
Similarly, when insulin binds to its receptor, this leads to
phosphorylation of Tyr-Xaa-Xaa-Met motifs in the insulin receptor and
insulin receptor substrate-1 (IRS-1)(3) . Our data demonstrate
that the binding of phosphorylated insulin receptors and/or IRS-1 to
p85, competitively displaces p85 molecules from binding sites on
constitutively phosphorylated PDGF receptors. This phenomenon of
receptors competing to bind the same pool of downstream signaling
molecules provides a novel mechanism for cross-talk among receptors for
peptide growth factors. It is possible that this mechanism of
cross-talk may play an important role in the regulation of cell growth,
for example, the interactions among various peptide growth factors in
regulating the proliferation of malignant cells in vivo.
MATERIALS AND METHODS
Expression of Insulin Receptors by Transfection of cDNA
in Cultured Cells
NIH-3T3 cells were stably transfected with
pBPV (Pharmacia Biotech Inc.) expression vector and cDNA encoding
wild-type human insulin receptor (4) or
43 insulin
receptor cDNA, a truncated receptor lacking the 43 amino acid residues
at the COOH terminus of the
-subunit(5) , or Ile
mutant insulin receptor (a kinase-deficient mutant insulin
receptor in which Met
is replaced by Ile) (6) as
described previously(5) . Expression of insulin receptors was
assayed by measuring
I-insulin binding (7) and/or
immunoblotting(8) .
Immunoprecipitation
Confluent cells in 10-cm Petri
dishes, grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, were incubated in the presence
or the absence of 10
M insulin or in the
presence of 100 ng/ml of epidermal growth factor for 3 min at 37
°C. (In some experiments, serum was omitted from the tissue culture
medium.) When indicated in the figure legends, Na
VO
(100 µM) was added 15 min prior to insulin
stimulation. To test for the presence of an autocrine loop for PDGF,
the cells were incubated with 100 µg/ml of suramin 30 min prior to
the stimulation with insulin in some experiments(9) . The cells
were quickly washed once with ice-cold phosphate-buffered saline
followed by two washes with washing buffer (20 mM Tris-HCl, pH
7.5, 137 mM NaCl, 1 mM MgCl
, 1
mM CaCl
, 100 µM
Na
VO
). Thereafter, the cells were solubilized
in 500 µl of washing buffer containing Nonidet P-40 (1%), glycerol
(10%), phenylmethylsulfonyl fluoride (2 mM). After
normalization for protein concentration, about one-third of the cell
lysate was immunoprecipitated using either a polyclonal antibody
directed against the PDGF receptor at a dilution of 1:100(10) ;
a polyclonal antibody directed against the p85 regulatory subunit of PI
3-kinase (Upstate Biotechnology Inc., Lake Placid, NY; catalog number,
06-195) at a dilution of 1:250; B-10, an anti-insulin receptor antibody
directed against the
-subunit at a dilution of 1:100; or a
polyclonal anti-GAP (GTPase-activating protein) antibody (Upstate
Biotechnology Inc.; catalog number, 06-157) at a concentration of 6
µg/ml. The immune complexes were precipitated using either protein
A-agarose (Life Technologies, Inc.) in which nonspecific sites were
saturated by washing with a buffer containing Tris-HCl (10 mM,
pH 7.5) and albumin (10 mg/ml) or Ultra-Link Immobilized Protein A Plus
(Pierce). In some experiments, in which sequential immunoprecipitations
were performed, the supernatant was saved and immunoprecipitated again
with either anti-PDGF receptor antibody or anti-PI3 kinase antibody at
the same conditions as above. The immunoprecipitates were washed once
with phosphate-buffered saline containing Nonidet P-40 (0.1%) and
vanadate (100 µM) and twice with a buffer containing
Tris-HCl (10 mM, pH 7.5), NaCl (100 mM), EDTA (1
mM), and vanadate (100 µM).
Immunoblotting
After immunoprecipitation, the
complexes were boiled for 3 min in 40 µl of Laemmli sample buffer
containing dithiothreitol (80 mM). The samples were analyzed
by SDS-polyacrylamide gel electrophoresis. Proteins were transferred
from the gel to nitrocellulose sheets by electroblotting at 90 V for 1
h at 4 °C in a solution containing Tris (25 mM), glycine
(192 mM), and methanol (20%). The immunoblots were probed
either with 4G10 monoclonal antiphosphotyrosine antibody (Upstate
Biotechnology Inc.) at a concentration of 250 ng/ml or a monoclonal
antibody directed against the p85 subunit of PI 3-kinase at a
concentration of 1 µg/ml (Upstate Biotechnology Inc.; catalog
number, 05-217), a rabbit antibody directed against the human PDGF
receptor (1:500), or a polyclonal anti-GAP antibody (Upstate
Biotechnology Inc.) at a concentration of 2 µg/ml; proteins were
detected by enhanced chemiluminescence using either horseradish
peroxidase-labeled anti-mouse
-globulin or anti-rabbit
-globulin (Amersham Corp.) as described elsewhere(5) .
RESULTS
Insulin Inhibits Binding of PI 3-Kinase to
Phosphorylated PDGF Receptors
PI 3-kinase is an enzyme that
functions in the signaling pathways downstream from multiple cell
surface receptors. For example, the p85 regulatory subunit of PI
3-kinase binds to phosphotyrosine residues in the PDGF
receptor(2) , the insulin receptor (11, 12, 13) , and IRS-1(14) .
NIH-3T3 cells expressing recombinant human insulin receptors were
incubated in the presence or absence of insulin (10
M), and the extracts were immunoprecipitated with
anti-p85 antibody (Fig. 1, lanes 3 and 4). In cells
incubated in the absence of insulin, the major
phosphotyrosine-containing protein has M
190,000 (Fig. 1A, lane 3). Based upon the observation
that it is immunoprecipitated by antibody to the PDGF receptor, this M
190,000 phophoprotein is identified as the
PDGF receptor (Fig. 1A, lane 1). When cells
were incubated with insulin, this led to the disappearance of the
phosphorylated PDGF receptor from the immune complex immunoprecipitated
by anti-p85 antibody (Fig. 1A, lane 4).
However, when the extracts were immunoprecipitated by anti-PDGF
receptor antibody, we observed that insulin did not alter the
phosphotyrosine content of the PDGF receptor (Fig. 1A, lanes 1 and 2). Furthermore, as expected, insulin
stimulation led to the appearance of two additional
phosphotyrosine-containing proteins coimmunoprecipitated with p85. The M
95,000 band is the
-subunit of the
insulin receptor. Previously, using the criterion of
immunoprecipitation with antibody to IRS-1, we have demonstrated that
the M
180,000 band in these cells contains
phosphorylated IRS-1(5, 13) . When the extracts were
immunoprecipitated with anti-p85 antibody, insulin did not alter the
cellular content of p85 (Fig. 1B, lanes 3 and 4). In extracts of cells incubated in the absence of insulin,
antibody to the PDGF receptor co-immunoprecipitates p85 (Fig. 1B, lane 1), presumably due to the well
known ability of phosphotyrosine residues in the PDGF receptor to bind
to SH2 domains in the p85 subunit of PI 3-kinase(2) .
Interestingly, incubation of cells with insulin led to the
disappearance of p85 from the immune complex immunoprecipitated by
anti-PDGF receptor antibody (Fig. 1B, lanes 1 and 2). This suggests that insulin somehow led to a
displacement of phosphorylated PDGF receptors from binding sites in SH2
domains of PI 3-kinase. This effect of insulin was rapid, being
achieved within approximately 1 min (data not shown). In contrast, p85
was not co-immunoprecipitated with insulin receptors in extracts of
cells in basal state (Fig. 1B, lane 5).
However, incubation of the cells with insulin led to the appearance of
p85 in immune complexes immunoprecipitated by anti-insulin receptor
antibody (Fig. 1B, lane 6). This raises the
possibility that phosphorylated Tyr-Xaa-Xaa-Met motifs in insulin
receptors and IRS-1 bind to p85 and, as a result competitively inhibit
the binding of phosphorylated PDGF receptors.
Figure 1:
Insulin inhibits binding of PI 3-kinase
to phosphorylated PDGF receptors. Confluent monolayers (10 cm plates)
of stably transfected NIH-3T3 cells expressing the human insulin
receptor were incubated in the absence (lanes 1, 3,
and 5) or the presence (lanes 2, 4, and 6) of insulin (10-
M) for 3 min at 37
°C. The cells were lysed, and after solubilization cell extracts
were immunoprecipitated using either anti-PDGF receptor antibody (lanes 1 and 2), anti-p85 antibody (lanes 3 and 4), or anti-insulin receptor antibody (lanes 5 and 6) as described under ``Materials and
Methods.'' The immunoprecipitates were analyzed by
SDS-polyacrylamide (6.5%) gel electrophoresis and transferred to
nitrocellulose sheets by electroblotting. Blots were probed with either
antiphosphotyrosine antibody (panel A), anti-p85 antibody (panel B), or anti-PDGF receptor antibody (panel
C).
When extracts of cells
are immunoprecipitated with anti-PDGF receptor antibody, two bands are
detected by immunoblotting with the same antibody (Fig. 1C, lanes 1 and 2); the
intensities of these bands were not altered when the cells were
incubated with insulin. The upper band (M
190,000) co-migrates with the phosphorylated PDGF receptor (Fig. 1A, lanes 1-3). Although the
identity of the lower band is not known with certainty, it is possible
that it represents partially processed forms of the immature PDGF
receptor. In extracts of unstimulated cells, the phosphorylated PDGF
receptor was co-immunoprecipitated by anti-p85 antibody (Fig. 1C, lane 3, and Fig. 2A, lane 3). However, treatment of the cells with insulin led to
the disappearance of the PDGF receptor from anti-p85 immunoprecipitates (Fig. 1C, lane 4, and 2A, lane
4). These observations further support the conclusion that insulin
leads to displacement of phosphorylated PDGF receptors from binding
sites on p85. These experiments were extended by carrying out
sequential immunoprecipitation studies with two antibodies, anti-p85
and anti-PDGF receptor (Fig. 2, lanes 5-8). To
detect ``unbound'' PDGF receptors (i.e. unbound to
p85), supernatants of anti-p85 immunoprecipitates were subjected to
immunoprecipitation by anti-PDGF receptor antibody. Using this assay,
we demonstrated that insulin markedly increased the quantity of
``unbound'' phosphorylated PDGF receptors (Fig. 2A, lanes 5 and 6). We carried
out similar studies in which supernatants of anti-PDGF receptor
immunoprecipitates were subjected to immunoprecipitation by anti-p85
antibody. Prior to treatment of the cells with insulin, most of the p85
was bound to phosphorylated PDGF receptors (Fig. 2B, lanes 3 and 7). However, insulin treatment markedly
increased the quantity of p85 that was not bound to PDGF receptors (Fig. 2B, lane 8).
Figure 2:
Sequential immunoprecipitation. Cell
extracts of NIH-3T3 cells expressing the human insulin receptor were
immunoprecipitated using either anti-PDGF receptor antibody (lanes
1 and 2) or anti-p85 antibody (lanes 3 and 4) as in Fig. 1. The supernatant of the anti-p85
immunoprecipitation was subjected to a second immunoprecipitation with
anti-PDGF antibody (lanes 5 and 6), and the
supernatant of the anti-PDGF receptor immunoprecipitation was subjected
to a second immunoprecipitation with anti-p85 antibody (lanes 7 and 8). Finally, the immunoprecipitates were analyzed by
SDS-polyacrylamide (6.5%) gel electrophoresis followed by
immunoblotting either with antiphosphotyrosine (panel A) or
with anti-p85 antibody (panel B).
Requirement for Insulin Receptor Tyrosine Kinase
Activity
When insulin binds to the insulin receptor, this leads
to phosphorylation of multiple sites in the cytoplasmic domain of the
-subunit. One phosphorylation site (Tyr
), located
near the COOH terminus, is embedded in a Tyr-His-Thr-Met sequence that
is a binding site for the SH2 domains in the p85 subunit of PI
3-kinase(11, 12, 13) . In addition, the
insulin receptor phosphorylates IRS-1, a docking protein with multiple
Tyr-Xaa-Xaa-Met motifs that also provide binding sites for the SH2
domains of p85(14) . Therefore, it seems likely that
insulin-stimulated phosphorylation would provide phosphotyrosine
residues with the ability to competitively inhibit phosphorylated PDGF
receptors from binding to p85. Thus, we inquired whether receptor
tyrosine kinase activity is required for the ability of the insulin
receptor to mediate insulin-induced inhibition of PDGF receptor binding
to p85. To address this question, we studied NIH-3T3 cells expressing
Ile
mutant insulin receptors that are deficient in
receptor tyrosine kinase activity(6, 15) . As was
observed in cells expressing wild-type insulin receptors, p85 bound to
phosphorylated PDGF receptors as shown in co-immunoprecipitation
studies in extracts of cells expressing Ile
mutant
insulin receptors incubated in the absence of insulin (data not shown).
However, in contrast to what was observed with cells expressing
wild-type insulin receptors, there was no significant dissociation of
p85 from PDGF receptors when insulin was added to cells expressing
Ile
mutant insulin receptors. Similarly, in
untransfected NIH-3T3 cells that express relatively few insulin
receptors, insulin did not significantly affect the binding of p85 to
phosphorylated PDGF receptors (data not shown).Next, we inquired
whether phosphorylation of the Tyr-His-Thr-Met sequence at the COOH
terminus of the insulin receptor
-subunit is required to mediate
insulin's ability to dissociate phosphorylated PDGF receptors
from p85. To address this question, we studied cells overexpressing
43 mutant insulin receptors (Fig. 3, lanes
1-4). The
43 mutant is a truncated receptor that lacks
43 amino acid residues at the COOH terminus; these deleted amino acids
include the phosphorylation site at Tyr
. Previously, we
have demonstrated that deletion of this 43-amino acid sequence from the
carboxyl terminus of the insulin receptor abolishes binding of p85 to
the receptor(13) . This supports the conclusion that
Tyr
is the principal binding site for p85 on the
phosphorylated insulin receptor. Cells expressing
43 mutant
receptors were incubated in the presence or absence of insulin. As in
the experiments described above, extracts were immunoprecipitated with
antibodies directed against either p85 or the PDGF receptor. Incubation
of the cells with insulin led to a decrease in the quantity of p85
co-immunoprecipitated by anti-PDGF receptor antibody (Fig. 3B, lanes 1 and 3). Similarly,
insulin led to a decrease in the quantity of phosphorylated PDGF
receptor co-immunoprecipitated by anti-p85 antibody (Fig. 3A, lanes 2 and 4). These data
demonstrate that phosphorylation of Tyr
is not required
for the insulin receptor to mediate the effect of insulin to displace
phosphorylated PDGF receptors from binding sites on p85. Inasmuch as
43 mutant receptors retain the ability to phosphorylate
IRS-1(5, 16) , it is likely that binding of
phosphorylated IRS-1 to p85 is principally responsible for displacing
PDGF receptors.
Figure 3:
COOH terminus of the insulin receptor is
not required for insulin-induced inhibition of binding of PDGF receptor
to p85. Confluent monolayers of stably transfected NIH-3T3 cells
expressing
43 human insulin receptor, a truncated receptor lacking
the 43 amino acid residues from the COOH terminus of the
-subunit (lanes 1-4) were incubated in the absence (lanes 1 and 2) or the presence (lanes 3 and 4)
of insulin. In the same experiment, NIH-3T3 cells expressing the
wild-type human insulin receptor were incubated with epidermal growth
factor (100 ng/ml) (lanes 5 and 6). After
solubilization, the cell extracts were immunoprecipitated using either
anti-PDGF receptor antibody (lanes 1, 3, and 5) or anti-p85 antibody (lanes 2, 4, and 6). Immunoprecipitates were analyzed by SDS-polyacrylamide
(6.5%) gel electrophoresis followed by immunoblotting either with
antiphosphotyrosine (panel A) or with anti-p85 antibody (panel B).
Specificity of Insulin's Effect upon the Binding of
p85 to Phosphorylated PDGF Receptors
We inquired whether this
effect of insulin to cause displacement of PDGF receptors from p85 was
specific. To address this question, we carried out experiments in which
we studied the interaction between phosphorylated insulin receptors and
GAP. When cells were incubated with insulin, we observed only a slight
decrease in the quantity of phosphorylated PDGF receptor that was
co-immunoprecipitated by anti-GAP antibody (Fig. 4, lanes 5 and 6); this slight decrease was not reproducible in
other experiments. As a control, we demonstrated that, even in extracts
from cells incubated in the presence of insulin, PDGF receptors were
co-immunoprecipitated by anti-GAP antibodies (Fig. 4, lanes
7 and 8). In contrast, incubation with insulin led to a
near total displacement of phosphorylated PDGF receptors from anti-p85
immunoprecipitates (Fig. 4, lanes 3 and 4).
Furthermore, we demonstrated that epidermal growth factor did not mimic
the action of insulin to dissociate p85 from PDGF receptors in NIH-3T3
cells over-expressing insulin receptors (Fig. 3, lanes 5 and 6).
Figure 4:
Insulin stimulation slightly decreases
PDGF receptor binding to GAP. Cell extracts of NIH-3T3 cells expressing
the human insulin receptor were immunoprecipitated using either
anti-PDGF receptor antibody (lanes 1 and 2), anti-p85
antibody (lanes 3 and 4), or anti-GAP antibody (lanes
5-8) as described under ``Materials and Methods.''
Immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis followed by immunoblotting either with
antiphosphotyrosine (lanes 1-6) or with anti-GAP
antibody (lanes 7 and 8).
Phosphorylation of PDGF Receptors in the Basal
State
Under our experimental conditions, we observed easily
detectable phosphorylation of PDGF receptors in the basal state. While
phosphorylation appeared to be slightly higher when NIH-3T3 cells were
incubated in the presence of serum (Fig. 5, lanes
1-4), phosphorylated PDGF receptors were also detected in
cells cultivated in the absence of serum (Fig. 5, lanes
9-12). This suggests that basal phosphorylation of PDGF
receptors did not absolutely require the presence of a factor present
in fetal bovine serum. Furthermore, addition of suramin to the culture
medium did not abolish basal phosphorylation of PDGF receptors (Fig. 5, lanes 5-8). This suggests that
phosphorylation was not due to autocrine stimulation by endogenously
secreted PDGF(9) .
Figure 5:
Phosphorylation of PDGF receptors in the
basal state. Confluent monolayers of NIH-3T3 cells expressing the human
insulin receptor were incubated in the absence (lanes 1, 3, 5, 7, 9, and 11) or the
presence of insulin (lanes 2, 4, 6, 8, 10, 12, 13, and 14).
Under our standard experimental conditions, cells were incubated in
media containing fetal bovine serum; however, serum was omitted in the
experiments shown in lanes 9-12. In some samples suramin
(100 µg/ml) (lanes 5-8) or vanadate (100
µM) (lanes 13 and 14) was added to the
cells, either 30 min (Suramin) or 15 min (Vanadate)
prior to stimulation by insulin. After solubilization, the cell
extracts were immunoprecipitated using either anti-PDGF receptor
antibody (lanes 1, 2, 5, 6, 9, 10, and 13) or anti-p85 antibody (lanes 3, 4, 7, 8, 11, 12, and 14). Immunoprecipitates were analyzed by
SDS-polyacrylamide gel electrophoresis followed by immunoblotting with
antiphosphotyrosine.
Nevertheless, PDGF receptors were not
maximally phosphorylated in the basal state. For example, if vanadate
is added to the culture medium, this led to a marked increase in the
phosphotyrosine content of PDGF receptors (Fig. 5, lane
13). Under these conditions, insulin retained its ability to
effect a total displacement of phosphorylated PDGF receptors from p85 (Fig. 5, lane 14). Moreover, the presence of vanadate
plus insulin led to a marked increase in the phosphorylation of a band
with slightly higher mobility than the PDGF receptor, presumably
corresponding to IRS-1 (Fig. 5, lane 14).
DISCUSSION
The receptors for insulin and PDGF share a common signaling
pathway involving PI 3-kinase. The p85 regulatory subunit of PI
3-kinase binds to phosphotyrosine residues in various phosphoproteins
including the PDGF receptor, the insulin receptor, and
IRS-1(2, 12, 14, 17) . Using NIH-3T3
cells overexpressing the human insulin receptor, we demonstrate in the
present study that insulin decreases the binding of the p85 the
regulatory subunit of PI3-kinase to constitutively phosphorylated PDGF
receptors.
Cross-talk between Receptors for PDGF and Insulin via PI
3-Kinase
In our experimental system, there is detectable
phosphorylation of tyrosine residues in the PDGF receptor, even in the
basal state. Furthermore, these constitutively phosphorylated PDGF
receptors bind to p85. Incubation of the cells with insulin stimulates
phosphorylation of the insulin receptor and IRS-1. Possibly as a
consequence of the binding of p85 to phosphotyrosine residues in IRS-1,
PDGF receptors are no longer found in association with p85. Insulin
receptor tyrosine kinase activity is required for the ability of
insulin to promote the dissociation of p85 from PDGF receptors (data
not shown). However, the Tyr-Xaa-Xaa-Met motif
(Tyr
-His-Thr-Met) in the COOH terminus of the insulin
receptor
-subunit is not required (Fig. 3). This latter
observation is consistent with the hypothesis that it is IRS-1 rather
than the insulin receptor that directly competes with PDGF receptors
for binding of p85. Moreover, vanadate treatment does not prevent the
release of PDGF receptor from p85 binding; this observation suggests
that the dissociation of p85 is not due to dephosphorylation of PDGF
receptors by insulin-activated phosphotyrosine phosphatases.
Specificity of Insulin Action
The ability of the
p85 subunit of PI 3-kinase to bind to multiple
phosphotyrosine-containing proteins is not unique among SH2-domain
containing intracellular proteins. In fact, many such proteins function
downstream in the signaling pathways initiated by multiple receptor
tyrosine
kinases(18, 19, 20, 21, 22) .
For example, GAP binds to phosphotyrosine residues in the receptors for
PDGF and epidermal growth factor (among other receptors)(20) ,
but not to either the insulin receptor or IRS-1. Assuming our
hypothesis is correct, one would predict that insulin-induced tyrosine
phosphorylation would not lead to competitive displacement of GAP from
binding sites on the PDGF receptor. Indeed, as predicted, we did not
detect binding of GAP to either IRS-1 or the insulin receptor;
moreover, insulin did not significantly alter the binding of GAP to the
PDGF receptor.
Constitutive Phosphorylation of PDGF
Receptors
Unlike many other published reports, there was a
detectable level of tyrosine phosphorylation in PDGF receptors in our
NIH-3T3 cells. Nevertheless, we confirmed that PDGF (both PDGF-A and
PDGF-B) increased the phosphotyrosine content of PDGF receptors.
However, the increment in phosphorylation due to PDGF was approximately
2-fold, considerably less than the magnitude of effect that is usually
reported(10, 23) . We wondered whether the basal
phosphorylation might be the result of an autocrine loop due to
endogenous production of PDGF. However, the observation that suramin
did not decrease receptor phosphorylation made this explanation
unlikely. Based upon the report that the E5 protein of bovine papilloma
virus increases the phosphorylation of the PDGF
receptor(24, 25) , we also considered the possibility
that the E5 protein might contribute to the increased phosphorylation
of the PDGF receptor. However, we did not detect the presence of the E5
protein in cells transfected with the pBPV expression vector. Moreover,
the basal level of phosphorylation was detectable even in
nontransfected NIH-3T3 cells. Thus, we have not identified the cause of
the increased basal phosphorylation of the PDGF receptors observed in
these NIH-3T3 cells.
Significance of Receptor Cross-talk
Assuming that
the effect of insulin is due to competition between IRS-1 and PDGF
receptors for binding to p85, one would predict that the effect of
insulin would be favored by the presence of large concentrations of
phosphorylated IRS-1. Therefore, because overexpression of recombinant
insulin receptors increases the cellular content of phosphorylated
IRS-1 molecules(26) , this is predicted to maximize the ability
of insulin to promote the dissociation of p85 from PDGF receptors.
Nevertheless, it seems likely that this phenomenon occurs to some
extent in target cells that express physiological numbers of insulin
receptors. What, then, is the physiological significance of this type
of receptor cross-talk in which multiple phosphotyrosine-containing
proteins compete for the binding of p85? While the answer to this
question is not clear, several possibilities deserve consideration.
First, multiple proteins containing phosphorylated Tyr-Xaa-Xaa-Met
motifs share in common the ability to bind to p85, thereby activating
PI 3-kinase. Nevertheless, it is possible that some phosphoproteins may
exert large effects and other phosphoproteins may exert small effects
upon PI 3-kinase activity. Second, different phosphoproteins may be
located in different subcellular compartments. For example, the PDGF
receptor is located at the plasma membrane while IRS-1 is a cytosolic
protein. If 3-phosphorylated inositol derivatives interact with
multiple proteins located in distinct subcellular compartments, the
subcellular localization of PI 3-kinase may determine distinct
signaling functions for the same messenger molecule. Third, PI 3-kinase
may not function in isolation but as part of signaling complex. Thus,
when PI 3-kinase binds to the PDGF receptor, it is located in close
proximity to one set of signaling molecules; when PI 3-kinase binds to
IRS-1, it is located in proximity to a different set of signaling
molecules. It is possible that this sort of combinatorial specificity
may determine which biological responses are initiated in the target
cell.In conclusion, many intracellular signaling molecules such as
PI 3-kinase function in pathways downstream from receptors for multiple
growth factors and peptide hormones. These overlapping signaling
pathways create the possibility of many different types of interactions
among the various pathways. In principle, these interactions may be
additive, synergistic, or antagonistic. In this investigation, we have
demonstrated the possibility of competitive interactions whereby
insulin-stimulated tyrosine phosphorylation causes the p85 subunit of
PI 3-kinase to dissociate from the PDGF receptor and rather to
associate with IRS-1 and/or the insulin receptor. Future studies may
provide important insights into the physiological significance of this
type of receptor cross-talk.