 |
INTRODUCTION |
Cell signaling events mediated by epidermal growth factor receptor
(EGFR)1 regulates survival,
proliferation, migration, and differentiation of many cell types. At
least five ligands are known to activate EGFR, including epidermal
growth factor (EGF) and transforming growth factor
(TGF
).
Progress has been made in the last two decades in elucidating
structure-function relationships for EGFR and other receptor tyrosine
kinases, particularly in how signal transduction is modulated by
self-phosphorylation of cytoplasmic tyrosine residues (1). This permits
access to the kinase domain of EGFR (2) and allows the receptor to bind
signaling proteins containing modular Src homology 2 (SH2) and
phosphotyrosine-binding domains (3, 4). Such interactions can affect
the activity of the bound protein through transmission of
conformational changes, enhancement of tyrosine phosphorylation,
and/or localization in proximity to membrane-associated target
molecules. One of the prominent signaling proteins activated by EGFR is
the
1 isoform of phospholipase C (PLC) (5). This enzyme, which has
two SH2 domains, catalyzes the hydrolysis of phosphatidylinositol
(4,5)-bisphosphate (PIP2), generating the second messengers
diacylglycerol and inositol triphosphate and liberating
PIP2-bound proteins (6). PLC-
1 activity is positively
modulated in vivo by association with EGFR and tyrosine
phosphorylation by the receptor kinase, providing a link to ligand
stimulation (7-10).
Another consequence of EGFR activation is clustering of ligand-receptor
complexes in clathrin-coated pits, which increases the rate of receptor
internalization (11). Following endocytosis, receptor-ligand complexes
and other components of the plasma membrane are delivered to early
endosomes, where molecules are sorted for recycling back to the cell
surface or degradation in lysosomes (12, 13). Since the degradative
route can yield down-regulation of total receptor mass and depletion of
ligand from the extracellular milieu, endocytic trafficking has been
recognized as an attenuation mechanism affecting long-term EGFR
function (14, 15). An unresolved question, however, is the contribution
to signaling of the steady-state EGFR pool residing in pre-degradative
internal compartments. It has been demonstrated that EGF remains
predominantly associated with EGFR in sorting endosomes, and that
internalized EGF-EGFR retain equal or greater tyrosine phosphorylation
stoichiometry as well as competency in binding and phosphorylating
signaling proteins (16-21). This suggests that meaningful signal
transduction might be extended after endocytosis of EGF (22, 23). In
contrast, the pH sensititivity of the TGF
-EGFR interaction and
differential trafficking of TGF
compared with EGF suggest that
TGF
dissociates from EGFR under the acidic conditions of endosomes
(24, 25). At the pH found at the surface, EGF and TGF
exhibit
indistinguishable affinities for EGFR in an equilibrium competition
assay (24). This disparity in ligand/receptor sorting could be
responsible for differences in the cell responses to EGF and TGF
. To
evaluate such a possibility, it is first necessary to know whether
internalized and surface complexes differ either qualitatively or
quantitatively in signaling.
We investigate here the effect of endocytosis and compartmentalization
of EGFR on the magnitude of signaling through the PLC pathway. Because
NR6 fibroblasts transfected with wild-type EGFR have been used
extensively in previous studies of both PLC-
1 activation (10,
26-28) and endocytic trafficking of the EGFR (14, 29-31), they were
chosen as our model system. We employed a ligand-based approach to
analyze the PLC pathway at three distinct points of regulation:
tyrosine phosphorylation of EGFR, tyrosine phosphorylation of PLC-
1,
and hydrolysis of PIP2. We found that internalized EGFR are
deficient in stimulating PLC function, and that the point of regulation
lies downstream of PLC-
1 tyrosine phosphorylation.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Quiescence Protocol--
NR6 mouse fibroblasts
transfected with wild-type human EGFR (NR6 WT) (14, 32) were cultured
in Corning tissue culture-treated dishes in a 5% CO2
environment. All cell culture reagents were obtained from Life
Technologies, Inc. The growth medium consisted of minimum essential
medium (MEM)
, 26 mM sodium bicarbonate with 7.5% fetal
bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM MEM nonessential
amino acids, and the antibiotics penicillin, streptomycin, and G418
(350 µg/ml). Cells were growth arrested at subconfluence using
restricted serum conditions without G418 (MEM-
, 26 mM
sodium bicarbonate with 1% dialyzed fetal bovine serum, 2 mM L-glutamine, 1 mM sodium
pyruvate, 0.1 mM MEM nonessential amino acids, and the
antibiotics penicillin/streptomycin) for 18-24 h prior to experiments.
Experiments were carried out in an air environment using MEM-
, 13 mM HEPES (pH 7.4 at 37 °C) with 0.5% dialyzed fetal
bovine serum, 2 mM L-glutamine, the antibiotics penicillin/streptomycin, and 1 mg/ml bovine serum albumin as the binding buffer.
Receptor Binding and Internalization Studies--
Mouse EGF
(Life Technologies, Inc.) or human TGF
(Peprotech) were iodinated
with 125I (NEN Life Science Products Inc.) using IODO-BEADS
(Pierce), according to the manufacturer's protocol. The specific
activities of labeled ligands were typically 150,000-200,000 cpm/ng
(
600 Ci/mmol). Quiescent cells in Corning 35-mm tissue culture
dishes were equilibrated in binding buffer for 15 min, on a warm plate that maintains cells at 37 °C, before challenge with
125I-labeled ligand. Surface-bound and internalized ligand
were discriminated essentially as described (11, 33). Briefly, free
ligand was removed by washing 6 times with ice-cold WHIPS buffer (20 mM HEPES, 130 mM NaCl, 5 mM KCl,
0.5 mM MgCl2, 1 mM
CaCl2, 1 mg/ml polyvinylpyrrolidone, pH 7.4). Surface-bound
ligand was then collected in ice-cold acid strip with urea (50 mM glycine-HCl, 100 mM NaCl, 1 mg/ml
polyvinylpyrrolidone, 2 M urea, pH 3.0) for 5-8 min, and
internalized ligand was released in 1 M NaOH overnight at
room temperature. Nonspecific binding (<2%) was assessed in the
presence of 2 µM unlabeled human EGF (Peprotech) and
subtracted from the total. Samples were quantified using a
-counter.
Removal of Surface-bound Ligand by Mild Acid Strip--
At
intermediate times during an experiment, surface-bound ligand was
removed without compromising cell viability, using brief (1-2 min)
treatments of ice-cold acid strip without urea (50 mM glycine-HCl, 100 mM NaCl, 1 mg/ml polyvinylpyrrolidone, pH
3.0) as indicated. By 1 min, this treatment is equally efficient in removing either EGF and TGF
(reproducibly 90-93%) from the surface of NR6 cells.
EGFR-Phosphotyrosine Sandwich ELISA--
High-binding ELISA
plates (Corning) were coated at room temperature overnight with 10 µg/ml anti-EGFR monoclonal antibody 225 in PBS, then incubated at
room temperature for 4-18 h in blocking buffer (10% horse serum,
0.05% Triton X-100 in PBS). After various treatments in binding buffer
as indicated, cells were washed once in ice-cold PBS supplemented with
1 mM sodium orthovanadate and 4 mM sodium
iodoacetate, scraped into ice-cold lysis buffer (50 mM
HEPES pH 7.0, 150 mM NaCl, 1% Triton X-100, 10% glycerol)
supplemented with 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM EGTA, 4 mM sodium iodoacetate, and 10 µg/ml each of aprotinin, leupeptin, chymostatin, and pepstatin, and transferred to an Eppendorf tube. After 20 min of incubation on ice, cellular debris was pelleted for 10 min at 16,000 × g, and the supernatant of each
sample was transferred to a new tube and kept on ice for analysis.
Total protein in each sample was assessed using a Micro BCA kit
(Pierce) according to the manufacturer's protocol. Each lysate was
diluted to various extents in blocking buffer supplemented with 1 mM sodium orthovanadate and incubated in anti-EGFR-coated
wells for 1 h at 37 °C. The wells were then rinsed four times
with wash buffer (10 mM Tris, pH 8.3, 300 mM
NaCl, 0.1% SDS, 0.05% Nonidet P-40) and incubated with 0.5 µg/ml
alkaline phosphatase-conjugated RC20 anti-phosphotyrosine antibody
(Transduction Laboratories) in blocking buffer for 1 h at
37 °C. After four additional washes, the wells were reacted with 1 mg/ml p-nitrophenyl phosphate (Sigma) in 10 mM
diethanolamine, 0.5 mM MgCl2, pH 9.5. The
reaction rate was monitored by measuring absorbance at 405 nm in a
15-min kinetic assay, using a Molecular Devices microplate reader. The
relative amount of EGFR-phosphotyrosine was determined from a binding
plot of reaction rate versus micrograms of total lysate
protein for each sample. Nonspecific control lanes in which the maximum
lysate load was incubated in wells without 225 antibody yielded similar activities to 225 wells incubated without lysate.
Immunoprecipitation and Western Blotting--
Cells were lysed
in 1% Triton X-100, and total cell protein was determined as detailed
above. Immunoprecipitations of equivalent total protein amounts were
performed at 4 °C for 90 min using 3-5 µg of primary antibody
precoupled to 10 µl of protein G-Sepharose beads per sample. The
beads were washed five times with ice-cold lysis buffer supplemented
with 1 mM sodium orthovanadate, and the residual liquid was
removed with a syringe. The beads in each tube were boiled for 5 min in
30 µl of sample buffer (62.5 mM Tris, pH 6.8, 2% SDS,
100 mM dithiothreitol, 10% glycerol, 0.005% bromphenol
blue), then clarified by centrifugation. Proteins were separated by
SDS-PAGE (34) on 7.5% acrylamide gels and transferred to
nitrocellulose membranes (35). Membranes were blotted for proteins as
indicated and visualized using horseradish peroxidase-conjugated secondary antibodies and SuperSignal Ultra detection reagent (Pierce). Bands were detected and quantified using a Bio-Rad chemiluminescence screen and Molecular Imager. When reprobing of a blot was desired, bound antibodies were first removed for 1 h at 55 °C in
stripping buffer (62.5 mM Tris, pH 6.8, 2% SDS, 100 mM
-mercaptoethanol).
Determination of Internal
EGFR-Phosphotyrosine--
Internalized EGFR were isolated by
labeling surface-accessible proteins for subsequent removal from cell
lysates (36). Briefly, cells were washed 3 times with ice-cold PBS, pH
8.0, after specific treatments, and surface proteins were biotinylated
at 4 °C with 5 mg of sulfo-NHS-LC-biotin (Pierce) per 10-cm plate.
Plates were washed once with PBS, once with PBS, 50 mM
glycine, and once again with PBS. Cells were lysed in 1% Triton X-100
as described above, and EGFR were immunoprecipitated using 225 antibody
precoupled to protein G-Sepharose. Proteins were eluted by boiling for
10 min in TNE buffer (50 mM Tris, pH 7.5, 140 mM NaCl, 5 mM EDTA) with 0.5% SDS. After
adding 1 volume of lysis buffer supplemented with 1 mM
sodium orthovanadate, biotinylated (surface) EGFR were removed
using immobilized streptavidin (Pierce). Supernatants were subjected to
SDS-PAGE and anti-phosphotyrosine immunoblotting.
PIP2 Hydrolysis Assay--
In vivo PLC
activity was determined essentially as described (10). Briefly, cells
were incubated with 5 µCi/ml
myo-[2-3H]inositol (American Radiolabeled
Chemicals) during the growth arrest protocol. Unincorporated
radioactivity was removed by two washes with PBS at 37 °C just
before the experiment. Following various treatments in binding buffer
as indicated, cells were washed once with ice-cold WHIPS buffer,
scraped into boiling dH2O, transferred to an Eppendorf
tube, and kept on ice. Samples were boiled for 5 min, and cellular
debris was pelleted for 5 min at 16,000 × g. The
concentration of cytosolic radioactivity in disintegrations/min/ml for
each supernatant was determined by liquid scintillation counting of
small aliquots, and equivalent volumes of samples were loaded onto
mini-columns packed with 0.5 ml of anion exchange resin (AG 1-X8,
formate, 100-200 mesh; Bio-Rad) each. After washing each column with
20 ml of dH2O and 20 ml of 5 mM sodium borate,
60 mM sodium formate, inositol phosphate fractions were
eluted with 200 mM ammonium formate, 100 mM
formic acid. The disintegrations/min of inositol phosphate that
accumulated during cell treatment was normalized to the total
disintegrations/min applied to the anion exchange column for each sample.
 |
RESULTS |
Differential Tyrosine Phosphorylation of Internalized EGFR by
EGF versus TGF
--
Given the central role of EGFR
autophosphorylation in initiating phospholipase C activity, we
determined whether the tyrosine phosphorylation stoichiometry of EGFR
(Tyr(P)/receptor) is altered upon internalization of EGF or
TGF
·EGFR complexes in NR6 WT cells. Based on the differential
binding affinities of these ligands at endosomal pH, we expected that
EGF would elicit a higher level of internal EGFR tyrosine
phosphorylation than TGF
. Saturating doses (20 nM) of
radioiodinated EGF or TGF
were used to follow the levels of
surface-bound and internalized ligand with time in NR6 WT cells (Fig.
1A). A decrease in surface
complexes to a level of about 60% of the total was observed within 30 min, with a parallel increase in internalized ligand, in agreement with
previously published results (14). The profiles of cell-associated EGF
and TGF
in both compartments were indistinguishable in these experiments. This indicated that the initial trafficking of EGFR in
these cells is similar following either EGF or TGF
treatment.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
Compartmentation and tyrosine phosphorylation
of EGF receptor. A, time course of 20 nM
125I-EGF (closed symbols) or
125I-TGF (open symbols) binding and
internalization. Surface-bound ( , ) and internalized ( , )
counts were determined in duplicate and normalized to the time-averaged
total cell-associated radioactivity ( , ) for NR6 WT cells, and
the experiment was repeated on three separate days (mean ± S.D.,
n = 3). B, time course of EGFR tyrosine
phosphorylation. NR6 WT cells were stimulated with 20 nM
EGF (closed symbols) or TGF (open symbols) in
a standard time course ( , ) or using a strip protocol ( , ),
as described in the text. The levels of Tyr(P)-EGFR in cell extracts
were determined using a sandwich ELISA as described under
"Experimental Procedures" and normalized to a common point (5 min
EGF stimulation); values are mean ± S.D., n = 3. C, representative analysis of Tyr(P)-EGFR time course ELISA
data from B; alkaline phosphatase reaction rate is plotted
versus total cellular protein for each well. In the interest
of presentation, only some of the samples processed on this day are
shown: unstimulated ( ), 5-min EGF ( , duplicates), 5-min
TGF ( ), 20-min TGF strip protocol ( ), and 25-min TGF
strip protocol ( ). The data were fit to a binding isotherm equation
with background: y(x) = A1 + A2x/(1 + A3x), with the relative level of
Tyr(P)-EGFR thus defined as A2
(r2 typically > 0.99).
|
|
To distinguish between surface-associated and intracellular activated
EGFR, we used a brief incubation with a mild acid wash. This treatment
rapidly removes surface-bound ligand (both EGF and TGF
are
dissociated equivalently). In addition, several studies have shown that
it does not compromise cell viability (37-39). The kinetics of EGFR
tyrosine phosphorylation were examined using two parallel stimulation
protocols: a standard time course of stimulation with EGF or TGF
(20 nM) at 37 °C, and a strip protocol (Fig. 1B).
For the strip protocol, cells were stimulated with EGF or TGF
(20 nM) for 15 min at 37 °C to allow internalization, treated with acid strip on ice for 1 min, and brought back to 37 °C
in the absence of ligand for 9 min. For EGF-treated cells, ligand was
then added back at 37 °C to determine whether receptor binding and
signaling capacities were intact following the acid wash. As shown in
Fig. 1B, EGF-treated NR6 WT cells displayed approximately 3 to 4 times higher EGFR-phosphotyrosine relative to TGF
-treated cells
following the surface strip, suggesting that the former ligand is more
effective in maintaining activation of EGFR in internal compartments.
Following readdition of EGF, Tyr(P)-EGFR returned to pre-strip levels,
showing that the treatment does not compromise signaling in these cells.
To determine the stoichiometry of EGFR tyrosine phosphorylation, the
levels of surface-bound and internalized 125I-ligand were
determined for the same time points and stimulation conditions shown in
Fig. 1B. The ratio of Tyr(P)-EGFR/total cell-associated ligand was then plotted versus the ratio of internalized
ligand/total cell-associated ligand. If EGFR maintains a constant
tyrosine phosphorylation stoichiometry, both with respect to time and
cellular location, such a plot will have zero slope. This was indeed
the case for EGF-treated NR6 WT cells (Fig.
2A). EGFR maintained a nearly
constant Tyr(P)-EGFR/cell-associated ligand before the strip, after the
strip, and following readdition of EGF. This type of plot can also be
used to determine whether EGFR is dephosphorylated following
endocytosis, since the ratio of Tyr(P)/ligand would change from the
surface to the internal value as the fraction of internalized ligand
increased. TGF
-treated cells displayed a decrease in Tyr(P)/ligand
as the internal ligand fraction increased, and the extrapolated
"surface" Tyr(P)/ligand value was very close to the mean
phosphorylation stoichiometry observed for EGF (Fig. 2B).
This suggests that in the case of cells treated with TGF
, a
significant fraction of internalized EGFR are dephosphorylated.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 2.
Analysis of EGFR tyrosine
phosphorylation stoichiometry. Surface-bound and internalized
125I-EGF were quantified for the same time points and
conditions used in Fig. 1B (n 3), and the
ratio of Tyr(P)-EGFR/total cell-associated ligand is plotted
versus the ratio of internal/total cell-associated ligand
(mean ± S.D. for both x and y axis values;
y value S.D. determined by propagation of error).
A, analysis of EGFR tyrosine phosphorylation stoichiometry
in response to EGF: , time course; , strip protocol; solid
line, mean of Tyr(P)-EGFR/ligand. B, analysis of EGFR
tyrosine phosphorylation stoichiometry in response to TGF : , time
course; , strip protocol; solid line, mean of
EGF-stimulated Tyr(P)-EGFR/ligand from A; dashed line,
theoretical line describing complete dephosphorylation of EGFR upon
internalization. C, tyrosine phosphorylation of internalized
EGFR. Cells were stimulated with 20 nM TGF
(T) or EGF (E) for 15 min at 37 °C. Where
indicated, acid washing was carried out for 2 min on ice, followed by 5 min equilibration in binding buffer at 37 °C. Surface biotinylation
and clearance with immobilized streptavidin was employed to isolate
internalized EGFR, as described under "Experimental Procedures,"
which was then subjected to SDS-PAGE and anti-phosphotyrosine
immunoblotting.
|
|
To examine the possibility that the receptor phosphorylation
stoichiometries elicited by EGF and TGF
simply reflect differential activation of surface complexes, cell surface proteins were
biotinylated and cleared from EGFR immunoprecipitates. The remaining
EGFR, presumably in intracellular compartments prior to cell lysis, were then subjected to anti-phosphotyrosine immunoblotting. As shown in
Fig. 2C, EGF elicited significantly higher Tyr(P)-EGFR than
TGF
in this assay, and phosphotyrosine levels were not altered by
acid washing. This demonstrates that EGF induces a greater extent of
internalized EGFR activation than TGF
, although tyrosine phosphorylation of internal EGFR in TGF
-treated cells is detectably higher than the unstimulated control. Taken together, our results indicate that tyrosine phosphorylation of internalized EGFR is strongly
correlated with ligand occupancy in endosomes.
The dose responses (0-20 nM) of EGF- and TGF
-stimulated
EGFR tyrosine phosphorylation were investigated as well, after 7.5 and
20 min of ligand challenge (Fig. 3). EGFR
exhibited half-maximal tyrosine phosphorylation at 1-2 nM
of either ligand, with TGF
values consistently and statistically
lower than EGF values for the same dose (Fig. 3). This is also
consistent with activation of surface EGFR to similar extents by the
two ligands and a greater degree of internalized EGFR activation by
EGF.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 3.
Dose response of EGF receptor tyrosine
phosphorylation. NR6 WT cells were stimulated with the indicated
doses of TGF ( ) or EGF ( ) at 37 °C for times of 7.5 min
(A) or 20 min (B). The levels of Tyr(P)-EGFR in
cell extracts were determined by sandwich ELISA and expressed relative
to the maximum value obtained on the same day. Values are mean ± S.E., n 3; *, Student's t test,
p < 0.05; **, Student's t test,
p < 0.01 between EGF and TGF at a particular ligand
concentration.
|
|
EGF and TGF
Are Equipotent in Stimulating PLC-mediated
PIP2 Hydrolysis--
Having established that EGF yields
higher levels of tyrosine phosphorylation of internalized EGFR than
TGF
, we next investigated whether these naturally occurring ligands
could stimulate the PLC pathway to different extents. To this end, we
employed a functional assay that assesses the hydrolysis of
PIP2 in intact cells. In vitro reactions using
immunoisolated PLC-
1 can be misleading, since the concentrations of
PIP2 and other membrane-associated signaling molecules in
various compartments might be different. Following the liberation of
soluble inositol triphosphate from PIP2, inositol
phosphatases rapidly metabolize this intermediate to free inositol.
Cell exposure to Li+ inhibits the breakdown of inositol
phosphate (IP), potentiating its accumulation in the cytosol. Previous
studies using NR6 WT and other NR6 transfectants in conjunction with
the specific PLC inhibitor U73122 demonstrated that this assay is
indeed a direct readout of PIP2 hydrolysis (10).
PLC dose-response experiments were performed by incubating NR6 cells
with 20 mM LiCl for 15 min, followed by stimulation in the
continued presence of LiCl. Control experiments indicated that IP
accumulation is roughly linear with time for at least 30 min of 20 nM EGF stimulation, that lithium is required for observable
IP accumulation, that the basal level of IP in the absence of
stimulation does not increase detectably with time, and that lithium
treatment does not affect EGFR internalization (data not shown). The
dose responses of EGF- and TGF
-stimulated PIP2
hydrolysis were examined for stimulation times of 15 and 30 min (Fig.
4). These time scales allow for
sufficient internalization of ligand to occur (Fig. 1A), and
for stimulated IP accumulation to achieve adequate signal/noise ratios.
As shown in Fig. 4, EGF did not gain any noticeable advantage over
TGF
with respect to stimulation of the PLC pathway over the course
of 30 min, despite higher levels of total cellular EGF-mediated EGFR
activation at all doses (Fig. 3). This might be expected if the
activation of PLC were saturable, i.e. if PLC-
1 or
PIP2 were stoichiometrically limiting at submaximal
Tyr(P)-EGFR (40). However, both ligand-induced PIP2
hydrolysis and EGFR phosphotyrosine were half-maximal at similar EGF
and TGF
concentrations (1-2 nM). Therefore, these results indirectly suggest that activated EGFR in internal compartments are deficient in stimulating PLC function.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4.
Dose response of EGFR-mediated
PIP2 hydrolysis. NR6 WT cells were stimulated with the
indicated doses of TGF ( ) or EGF ( ) at 37 °C in the
presence of 20 mM LiCl for times of 15 min (A)
or 30 min (B). The accumulated levels of inositol phosphate
resulting from hydrolysis of cellular PIP2 were measured as
described under "Experimental Procedures." The unstimulated
background was subtracted from stimulated values, and these are
expressed relative to the maximum value obtained on the same day
(mean ± S.E., n 3). EGF and TGF values were
assessed in parallel, so the normalized levels of PIP2
hydrolysis represent a direct comparison in which saturating
concentrations (20 nM) of EGF and TGF resulted in
equivalent inositol phosphate production.
|
|
PIP2 Hydrolysis Is Not Stimulated by Activated EGFR in
the Endocytic Pathway--
Although it seemed possible that active
EGFR do not have access to PLC-
1 and/or PIP2 in
intracellular trafficking compartments, our dose-response results did
not address this point directly. The internal pool of EGFR does not
constitute a large fraction of the total cellular EGFR in the NR6 cell
line, obscuring its potential contribution to PLC activation. Thus,
mild acid washing was again employed to test the relationship between
cell surface and intracellular receptor pools with regard to signaling.
NR6 WT cells were pretreated for 15 min with 20 nM EGF or
TGF
at 37 °C, in the absence of lithium, to saturate and permit internalization of surface EGFR. This was followed by incubation with
ice-cold acid wash for 2 min to remove surface-bound ligand. Cells were
then returned to 37 °C in the presence of 20 mM LiCl and
various concentrations of TGF
(0-20 nM), regardless of
whether the cells were pretreated with EGF or TGF
. This "surface
titration" protocol allowed us to vary the level of surface-activated
EGFR, relative to a constant level of internal-activated EGFR that
depends on whether the cells were pretreated with EGF or TGF
. This
differs from the standard dose-response experiment, in which internal receptor activation is coupled to the level of surface receptor activation. PIP2 hydrolysis was assayed 15 min following
LiCl addition, and Tyr(P)-EGFR was assayed in a separate experiment 7.5 min following LiCl addition as an intermediate time point. Control
experiments demonstrated that accumulation of inositol phosphate was
evident within 5 min of lithium addition (data not shown). The control
protocol was 15 min pretreatment with no ligand at 37 °C, acid wash
treatment, then a return to 37 °C with 20 mM LiCl and no ligand.
EGF-pretreated cells yielded statistically higher levels of total EGFR
phosphotyrosine than TGF
-pretreated cells for each chase TGF
concentration (Fig. 5A),
consistent with the continued EGF-stimulated phosphorylation of
internalized EGFR. Despite this difference, PIP2 hydrolysis
activities were equivalent when EGF- and TGF
-pretreated cells were
stimulated with ligand in the chase (0.5-20 nM TGF
;
Fig. 5B). For the 20 nM chase concentration, using EGF instead of TGF
in the chase did not effect the level of
PIP2 hydrolysis observed in this assay (data not shown).
Furthermore, in the absence of ligand in the chase, TGF
-pretreated
cells stimulated minimal PIP2 hydrolysis, even though
tyrosine phosphorylation of some internalized EGFR was detected under
these conditions (see also Figs. 1C and 2C).
These results are consistent with stimulation of PIP2
hydrolysis by surface-localized EGFR only.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 5.
Surface titration experiment. NR6 WT
cells were pretreated with 20 nM TGF ( ) or EGF ( )
at 37 °C then incubated with ice-cold acid wash for 2 min to remove
surface-bound ligand. Cells were then brought back to 37 °C with 20 mM LiCl and the indicated doses of TGF in the chase.
Cells were assayed for EGFR phosphotyrosine after 7.5 min
(A) or stimulated PIP2 hydrolysis after 15 min
(B). Values are mean ± S.E., n 3;
*, Student's t test, p < 0.05; **,
Student's t test, p < 0.01; +, Student's
t test, p > 0.95 between EGF- and
TGF -pretreated cells for the same chase conditions. C,
inhibition of recycled receptor-ligand complex signaling by an
anti-EGFR antibody. NR6 WT cells were treated, and PIP2
hydrolysis was determined, as in B following pretreatment
with 20 nM TGF (T) or EGF (E).
Where indicated, 10 µg/ml anti-EGFR monoclonal antibody 225 was
included in the chase medium. Values are expressed as ligand-stimulated
inositol phosphate accumulation normalized to the basal level
(mean ± S.E., n 2).
|
|
In apparent disagreement with this conclusion, however, EGF-pretreated
cells exhibited statistically higher PIP2 hydrolysis in the
absence of ligand in the chase (Fig. 5B), in apparent
disagreement with this conclusion. One possible explanation for this
disparate result is that some internalized EGF, but not TGF
, is
recycled back to the surface complexed with EGFR (30), an effect that would be masked by exogenous ligand added to the medium. To determine whether recycled EGF/EGFR could account for the enhanced
PIP2 hydrolysis, a receptor-blocking antibody (10 µg/ml
225 anti-EGFR) was included in the medium after acid wash treatment
(Fig. 5C). This antibody causes accelerated dissociation of
surface-bound EGF (data not shown) and therefore should reduce the
level of surface signaling from recycled EGFR. Indeed, the presence of the antagonistic anti-EGFR antibody in the chase was able to inhibit PIP2 hydrolysis in EGF-pretreated cells by 60%, but it had
no effect on PIP2 hydrolysis in TGF
-pretreated cells
(Fig. 5C). Taken together, these results indicate that
active EGFR in internal compartments do not participate in PLC signaling.
To examine this hypothesis further, stimulated PIP2
hydrolysis was plotted versus EGFR phosphotyrosine for both
standard dose-response and surface titration experiments. Since the
extent of EGFR tyrosine phosphorylation constitutes a readout and
modulator of EGFR kinase activity, in addition to the defined role of
EGFR phosphotyrosine in docking PLC-
1 and other signaling proteins,
it represents the input for cell signaling at the receptor level. If
signaling downstream of EGFR autophosphorylation is not affected by
internalization of the receptor, then the relationship between receptor
phosphorylation and PIP2 hydrolysis would be identical for
both EGF and TGF
. As shown in Fig. 6,
this is clearly not the case for the PLC pathway in NR6 cells. For the
surface titration experiments, the curves for TGF
- and
EGF-pretreated cells are parallel and shifted to the right by the
constant level of internal Tyr(P)-EGFR, indicating that these receptors
are not contributing to PIP2 hydrolysis. In the case of the
standard ligand treatment protocol, the curves of PIP2
hydrolysis versus Tyr(P)-EGFR for both TGF
and EGF
overlap until intracellular EGF concentrations become high enough to
start occupying receptors. At this point the curves diverge, with EGF values shifted to the right of TGF
values since EGF is more
effective than TGF
in stimulating internal Tyr(P)-EGFR (Fig. 6).
Finally, for high ligand concentrations (and therefore high
Tyr(P)-EGFR), the standard dose-response and surface titration curves
for the same ligand converge, confirming that the nature of signaling is not affected by differences in the two experimental designs. These
results are entirely consistent with the hypothesis that internalized
EGFR, even when biochemically active, are far less effective than
surface receptors in stimulating PLC-mediated PIP2 hydrolysis.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 6.
Plot of PIP2 hydrolysis
versus Tyr(P)-EGFR for dose response and surface
titration experiments. Standard dose-response data for TGF
( ) and EGF ( )-stimulated cells are taken from Figs. 3
(x axis values) and 4 (y axis values), with
values at each of the two time points averaged (7.5 and 20 min for
Tyr(P)-EGFR, 15 and 30 min for accumulated PIP2
hydrolysis). Surface titration data for TGF ( ) and EGF
( )-pretreated cells are taken from Fig. 5, A
(x axis values) and B (y axis values);
the unstimulated control ( ) is also shown.
|
|
Compartmentalization of PLC Activity Is Not Due to Differences in
PLC-
1 Tyrosine Phosphorylation--
Our dose-response and surface
titration experiments indicated that PLC activity is inhibited
following EGFR endocytosis, and that loss of signaling occurs between
EGFR activation and PIP2 hydrolysis. To determine if this
was due to an inability of the EGFR to induce tyrosine phosphorylation
of PLC-
1, we used the same surface titration protocol used above
(EGF or TGF
pretreatment, surface strip, and TGF
chase). The same
conditions used to measure EGFR phosphorylation in Fig. 5A
were used to examine PLC-
1 phosphorylation.
Tyrosine-phosphorylated PLC-
1 was immunoprecipitated using either
anti-phosphotyrosine or anti-PLC-
1 antibodies. After electrophoresis and membrane transfer, the blots were probed for the presence of
PLC-
1 or pY. Shown in Fig.
7A is a typical experiment in
which PLC-
1 was visualized following immunoprecipitation with
anti-Tyr(P) antibodies. Qualitatively, PLC-
1 tyrosine
phosphorylation mirrored EGFR tyrosine phosphorylation in NR6 WT cells,
in that EGF-pretreated cells always exhibited higher Tyr(P)-PLC-
1
than TGF
-pretreated cells for the same chase stimulation.
Essentially identical results were obtained when PLC-
1 was
immunoprecipitated and then visualized with anti-Tyr(P) antibodies.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
Analysis of PLC- 1
tyrosine phosphorylation. NR6 WT extracts were prepared as for
Fig. 5A, using the surface titration protocol with TGF
(T) or EGF (E) pretreatments.
Tyrosine-phosphorylated PLC- 1 was immunoprecipitated from equal
levels of total cellular protein using PY20 anti-phosphotyrosine
antibody (Transduction Laboratories) and detected by immunoblotting
with an anti-PLC- 1 mixed monoclonal (Upstate Biotechnology)
(n = 2; representative data shown, A). As a
check, Tyr(P)-PLC- 1 was also detected once by anti-PLC- 1
immunoprecipitation/anti-phosphotyrosine immunoblotting with similar
results; this blot was reprobed with anti-PLC- 1 to confirm roughly
equal total levels of PLC- 1 (not shown). B, plot of
Tyr(P)-PLC- 1 versus Tyr(P)-EGFR. For each of the three
Tyr(P)-PLC- 1 experiments, the data was expressed relative to the
maximum band intensity, and the mean for each condition is plotted
versus Tyr(P)-EGFR from Fig. 5A to compare
TGF - ( ) and EGF ( )-pretreated cells. The unstimulated point
( ) is also shown. The dotted line is the least squares
linear fit of all the data points (R2 > 0.99).
|
|
We quantified the results of these experiments using a Bio-Rad
Molecular Imager. Control experiments verified that there was a linear
relationship between micrograms of total protein from the same lysate
subjected to immunoprecipitation and the detected band intensity (data
not shown). To ascertain quantitatively whether tyrosine
phosphorylation of PLC-
1 is affected by EGFR endocytosis, Tyr(P)-PLC-
1 (averaged over three experiments) was analyzed as a
function of Tyr(P)-EGFR for each condition (Fig. 7B). The
relationship between tyrosine phosphorylation of the EGFR and PLC-
1
was the same in the case of both EGF- and TGF
-pretreated cells,
showing that tyrosine phosphorylation of PLC-
1 is not affected by
the localization of active receptors. As EGFR-mediated PIP2
hydrolysis is dependent on a surface localization, this suggests a
signaling restriction downstream of PLC-
1 phosphorylation.
 |
DISCUSSION |
While receptor down-regulation and ligand depletion via the
endocytic pathway are known to negatively modulate signal transduction mediated by EGFR and other receptor tyrosine kinases (14, 15), there is
no a priori reason to suspect that internalized receptors in
sorting endosomes cannot participate in signaling. Because its kinase
and substrate binding activities continue to face the cytosol in early
endosomes, internalized EGFR have the potential to signal as long as
they remain ligated (20). Indeed, internalized EGFR in rat liver are
competent in both binding and phosphorylating the adaptor protein Shc,
which helps localize the exchange factor Sos for potential interactions
with the Ras GTPase (21). In this cellular context, surface complexes
are rapidly desensitized, while internal complexes somehow escape this
regulation, implying that compartmentalized feedback mechanisms exist
(16, 18). Another possibility is that endocytosis might affect
signaling specificity. Cells overexpressing a dominant-negative mutant
of the dynamin GTPase are defective in both inducible endocytosis of
EGFR and EGF-responsive tyrosine phosphorylation of the cytoplasmic signaling protein phosphatidylinositol 3-kinase. This suggested that
receptor internalization is required for full manifestation of some
signals but not others (15). However, it has been reported recently
that the mutant cells are also defective in high affinity EGFR/ligand
binding, suggesting that dynamin might modulate aspects of signal
transduction at the surface (41). The concept of compartmentalized "separation" of signaling pathways has also been implicated in the
activation of sphingomyelinases mediated by tumor necrosis factor (23,
42). Thus, it is possible that signaling from the endosomal compartment
versus the surface plays an important role in dictating the
outcome of receptor tyrosine kinase stimulation.
It is now appreciated that many intracellular reactions, especially
those at or just beyond the receptor level, are regulated by
subcellular localization (43, 44). Interestingly, post-receptor targets
such as PIP2 and Ras are membrane-associated, implying that
they are readily compartmentalized. Since previous studies on EGFR
signaling in internal compartments have not probed beyond tyrosine
phosphorylation of cytoplasmic proteins, we examined activation of the
PLC pathway by the EGFR to the level of PIP2 hydrolysis. We
used a physiologically relevant ligand-based approach, rather than
comparing results from variant cell lines, based on previous studies
suggesting that TGF
dissociates from EGFR in endosomes to a much
greater extent than EGF (24, 25). Our study is the first to show that
the magnitude of signal transduction through a specific pathway, at the
level of proximal target modification, is affected by EGFR internalization.
We found that internalized EGF·EGFR complexes retain a maximal
tyrosine phosphorylation stoichiometry, whereas EGFR internalized in
response to TGF
binding are dephosphorylated to a significant extent. However, the two ligands are equipotent in stimulating PIP2 hydrolysis, the functional outcome of PLC-
1
activation. By manipulating the relative levels of surface and internal
receptor activation independently, we showed that active EGFR in
internal compartments stimulate little if any hydrolysis of
PIP2. This deficiency was not due to a location-specific
difference in PLC-
1 tyrosine phosphorylation, since this event
correlates with receptor phosphorylation in either compartment.
Therefore, we concluded that the spatial requirements for
PIP2 hydrolysis are defined at a step subsequent to PLC
phosphorylation by EGFR.
The simplest interpretation of our data is that PLC-
1 associated
with EGFR in pre-degradative trafficking organelles (endocytic vesicles, early endosomes, and recycling endosomes) do not have access
to PIP2. Studies in numerous cell types have indicated that
active maintenance of PIP2 levels is required for
meaningful PLC signaling. This function is carried out by
phosphatidylinositol transfer protein, which directs transport of
phosphatidylinositol between cellular membranes (45). Therefore,
exchange among lipid pools by membrane-phase sorting must be much
slower than enzymatic turnover by PLC and other enzymes (46),
indicating that the concentrations of PIP2 and other lipids
present in low amounts are not likely to be homogeneous among distinct
cellular membranes. Interestingly, phosphatidylinositol transfer
protein forms a signaling complex with EGFR, phosphatidylinositol
4-kinase, and PLC-
1 in response to EGF, suggesting that
PIP2 supply and hydrolysis are coupled. When
phosphatidylinositol transfer protein and PLC-
1 are depleted from
the cytosol of A-431 cells, both proteins must be added exogenously to
reconstitute EGFR-mediated PLC activity (47). The requirement of
cofactors for maintenance of PLC function could restrict
PIP2 hydrolysis to the plasma membrane, especially if one
or more of these proteins are preferentially recruited by surface EGFR.
Furthermore, PIP2 concentrated in caveolae microdomains may
comprise the EGFR-responsive substrate pool (48), which is probably
segregrated from the bulk membrane delivered to endosomes via
clathrin-coated pits.
Our results also have implications regarding the functional difference
between EGF and TGF
as naturally occurring agonists for the same
receptor. Physiologically, EGF and TGF
may have evolved as ligands
that vary in their ability to mediate long-term receptor/ligand
processing (24, 30, 49) but not in their compartmentalization of signal
transduction. Although EGFR-transfected NR6 fibroblasts do not maintain
a high percentage of internalized receptors at steady state, other
cells display a rapid redistribution of receptors to internal pools
(50). In these cases, internalization could act as a potent shut-off
mechanism in response to chronic stimulation of EGFR. While the above
situation likely applies to regulation of PLC-
1 signaling, it is
unclear whether other EGFR-mediated pathways are activated or even
augmented in endosomes as has been suggested. If internalized receptors
can signal through other intermediates, an important question is
whether endocytosis is a requirement for signaling or if internal
receptors simply continue activities initiated at the cell surface. In
either case, compartmentalization of membrane-associated molecules
would provide an additional level of signaling control, by affecting
the spatiotemporal selectivity of enzymes that coordinate different
cell functions.