(Received for publication, September 11, 1996, and in revised form, November 15, 1996)
From the Department of Pharmacology and the
§ Department of Microbiology and Immunology, Kimmel Cancer
Institute, Philadelphia, Pennsylvania 19107
Mutation of the autophosphorylation sites of
receptor protein-tyrosine kinases alters ligand dependent
internalization and down-regulation, indicating a critical role for
these sites in receptor processing. Currently, no differences in
receptor processing based on an individual autophosphorylation site
have been defined. By using a glutathione S-transferase
fusion protein containing the src homology 2 domains of
phospholipase C-1 to specifically recognize tyrosine 992 on the EGF receptor (Tyr(P)992), we have found differences
in this subpopulation of receptors. Following EGF stimulation, the
number of Tyr(P)992 receptors increased 2-fold over
receptors identified by an antibody that recognizes activated EGF
receptors (
-Act. EGFR) in A431 cells. Confocal fluorescence
microscopy showed that Tyr(P)992 receptors underwent
endocytosis at a slower rate and did not rapidly concentrate in
juxtanuclear bodies. Tyr(P)992 receptors were associated
with more SOS, Ras-GTPase activating protein, phosphatidylinositol
3-kinase, and SHPTP2/syp, but less Grb2, than receptors in
the general population, and these receptors were more heavily
phosphorylated than the general population of active receptors. These
findings suggest that autophosphorylation status is relevant to the
endocytosis, degradation, and effector molecule interaction of
individual EGF receptors. Further investigations based on
phosphorylation status should provide new insights into how receptor
protein-tyrosine kinase signaling is regulated.
Receptor protein-tyrosine kinases
(RPTKs)1 play an essential role in normal
cell growth and neoplasia (1). Following ligand activation, these
receptors oligomerize, activate, autophosphorylate, and rapidly
endocytose (1-6). Autophosphorylation sites play an important role in
signaling as well, since they serve as binding sites for proteins that
contain rc
omology-2 (SH2) domains (7), which in turn propagate the signals of the receptor. SH2
domains are found in many different proteins including enzymes, transcription factors, and adaptor proteins (8, 9). At least eight SH2
proteins bind to the epidermal growth factor (EGF) receptor following
activation. Each SH2 domain exhibits specificity based on amino acids
surrounding the anchor phosphotyrosine, so the phosphorylation status
of the receptor dictates subsequent interactions (10, 11).
Interestingly, site-directed mutagenesis on the EGF receptors'
autophosphorylation sites has shown that this receptor can be
processed differently when these sites are altered. Mutation of
Tyr1068, Tyr1148, or Tyr1173
slightly prolongs the half-life of this receptor (12, 13), and deletion
of all three results in a significant increase in half-life.
Several studies have now shown that receptor internalization plays a role in signaling. Upon activation, receptors are endocytosed in clathrin-coated pits, transferred to endosomes, and then multivesicular bodies, which ultimately fuse with lysosomes (5, 14, 15). Because the autophosphorylation sites and kinase domain of the receptor are oriented toward the cytoplasm during endocytosis (2, 16), the receptor can continue to signal until it is degraded in the lysosomal compartment (16-18). The EGF receptor kinase domain remains highly active during endocytosis (16), and certain substrates are phosphorylated following internalization. Futter et al. (19) have shown that annexin I is preferentially phosphorylated in the multivesicular body by the EGF receptor. A 55-kDa protein now identified as Shc has been shown to associate with the receptor in both membrane and endosomal fractions (20).
One implication of these findings is that receptor autophosphorylation sites could have a role in receptor internalization and degradation. Since the five autophosphorylation sites on the EGF receptor are not phosphorylated to the same extent (3, 21), there would be differential interaction of the EGF receptor with the SH2 domain containing effectors. Furthermore, since mutation of an individual autophosphorylation site can affect processing, another inference is that intact receptors may undergo differential processing based on which sites are phosphorylated, although this has never been directly visualized on intact receptors in situ.
We have found that an SH2 domain fusion protein can identify an EGF
receptor population that exhibits different rates of endocytosis, kinetics of activation, effector protein interaction, and level of
phosphorylation than the general pool of EGF receptors. Phospholipase C-1 contains two adjacent SH2 domains that bind to
Tyr(P)992 in the EGF receptor and maintain their
specificity for this single-site even when used at higher molar
concentrations (22). In this study, we produced a GST fusion protein of
the SH2 domains of PLC-
1 (PLC-SH2), and used this to
identify Tyr(P)992 EGF receptors during the process of
ligand activation and internalization in A431 cells, a human epidermoid
carcinoma cell line that overexpresses the EGF receptor.
The two SH2
domains of PLC-1 (nucleotides 1697-2468) (23) were
subcloned into EcoRI/BamHI sites of the vector
pGEX-5x-3 (Pharmacia Biotech Inc.). The GST fusion protein (PLC-SH2),
was produced and purified as described by the manufacturer. Protein was
dialyzed overnight against PBS, 5 mM dithiothreitol (24) at
4 °C and concentrated in a Centricon-10 apparatus (Amicon). The
fusion protein was quantitated by the Bio-Rad DC protein assay, supplemented with glycerol to 10%, and stored at
80 °C. For
immunofluorescence, PLC-SH2 was conjugated with Fluorolink Cy5 reactive
dye (Biological Detection Systems), at a molar ratio of 1:48 (fusion
protein:dye) and purified by centrifugation through a Centri-Sep column
(Princeton Separations) equilibrated in PBS. To produce a standard for
the stoichiometry experiments, a DNA fragment containing nucleotides 3217-3341 (amino acids 987-1028) of the EGF receptor was polymerase chain reaction-amplified from a mammalian expression vector
(pLTR2-CO12) containing the full-length EGF receptor cDNA and
cloned into EcoRI/BamHI sites of pGEX-5x-3. This
created a GST fusion protein containing the epitope for a monoclonal
antibody against the EGF receptor (
-EGFR, Transduction Laboratories)
and one autophosphorylation site from the EGF receptor
(Tyr992) and was called GST-EGFR-(987-1028). For use in
the stoichiometry experiments, this construct was phosphorylated
in vitro by the EGF receptor.
For lysate preparation, A431 cells were grown to 50-70% confluence in 100-mm tissue culture plates with Dulbecco's modified Eagle's medium, containing 100 units/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml kanamycin, and 10% fetal bovine serum. Cells were serum-starved for 16 h in OPTI-MEM (Life Technologies, Inc.), washed with PBS, and left untreated or given 20 ng/ml EGF (human; Life Technologies) in Dulbecco's modified Eagle's medium for 1, 5, 10, 15, 30, 60, or 120 min. The cells were then washed with PBS and lysed in cold TGH lysis buffer (50 mM HEPES, 100 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM NaVO4, 100 µg/ml phenylmethylsulfonyl fluoride, pH 7.4), sonicated two times for 10 s, and clarified. Protein concentrations were determined by the Bio-Rad DC protein assay.
For use in immunofluorescence studies, A431 cells were grown to 50-70% confluence on coverslips in six-well tissue culture plates. They were then washed two times with PBS, serum-starved for 16 h in OPTI-MEM (Life Technologies), and left untreated (5 min in Dulbecco's modified Eagle's medium) or given 100 ng/ml EGF for 1, 5, 10, 15, 30, or 60 min. The cells were then washed two times with ice-cold PBS and fixed in 2% formaldehyde in PBS. For the biotin-EGF experiments, in place of EGF the cells were incubated with 270 ng/ml of EGF conjugated to biotin (Boehringer Mannheim) for the same periods of time and processed as described.
Western and Far Western Blot AnalysisA431 cell lysate (40 µg) was subjected to SDS-PAGE on 4-20% Tris/glycine gels,
transferred to nitrocellulose filters, and blocked for 1 h at room
temperature in TTBS (100 mM Tris, pH 7.5, 0.9% NaCl, 0.1%
Tween 20) with 5% nonfat dry milk. A monoclonal antibody against
phosphotyrosine (-Tyr(P); Upstate Biotechnology) was used at a
1:1000 dilution. Monoclonal antibodies against the EGF receptor
(
-EGFR), Grb2 (
-Grb2), SHPTP2/syp (
-SHPTP2), PI 3-kinase (
-PI3K), Ras-GAP (
-GAP), SOS (
-SOS), and
PLC-
1 (
-PLC
) (Transduction Laboratories) were used
at a 1:250 dilution. For blots in which PLC-SH2 was used in place of
primary antibody (far Western blots), all buffers were supplemented
with 5 mM dithiothreitol, the primary reagent was 5 µg/ml
PLC-SH2 in TTBS, 1% bovine serum albumin, the secondary reagent was a
monoclonal antibody against GST (
-GST; Santa Cruz Biotechnology) at
a 1:100 dilution, and the tertiary reagent was 125I-labeled
goat anti-mouse antibody at 5 × 105 cpm/ml. The
incubations and washes occurred at 4 °C.
For the quantitation of signals, the blots were exposed to a phosphor storage screen and analyzed on a PhosphorImager using ImageQuant software (Molecular Dynamics Inc.). Serial dilution experiments confirmed that signals from the PhosphorImager were linear over a 105 range (20-2 × 106 cpm), and this response was maintained over a 20-fold difference in exposure time on the phosphor storage screen (2-40 h).
ImmunofluorescenceFixed A431 cells were permeabilized for
10 min with 0.2% Triton X-100 in PBS; blocked in PBS, 5% nonfat dry
milk; and washed with PBS, 2% bovine serum albumin. The primary
reagents were -Act. EGFR, used at 1:500, and Cy5-conjugated PLC-SH2,
used at 10 µg/ml in PBS, 2% bovine serum albumin. To visualize
-Act. EGFR, FITC-conjugated goat anti-mouse antibody was used at a
1:20 dilution (Boehringer Mannheim) in PBS, 5% nonfat dry milk. The
cells treated with biotin-EGF were incubated with PLC-SH2, washed, and
then incubated with FITC conjugated to avidin (Boehringer Mannheim)
used at a 1:200 dilution. All incubations were for 30 min (except
permeabilization) and were performed in the dark at 4 °C. Both
primary reagents were incubated at the same time, and separate
experiments confirmed that there was no competition between these two
reagents for binding to the EGF receptor. Incubation with GST protein
alone (10 µg/ml), followed by
-GST at a 1:100 dilution and
FITC-conjugated goat anti-mouse antibody at a 1:20 dilution showed no
binding. The coverslips were mounted onto slides with SLOW-FADE
(Molecular Probes, Inc.) and sealed with nail polish. Microscopy was
performed using a Zeiss Axiovert 100 confocal microscope with a Bio-Rad MRC 600 krypton-argon mixed gas multiline scanning laser rated at 15 milliwatts. The cells were viewed at a magnification of × 63 using optical sections of 0.25-µm thickness. The excitation and
emission wavelengths were 488 and 520 nm for FITC and 647 and 667 nm
for Cy5, respectively.
For the PLC-SH2
precipitations, 500-4000 µg of fresh A431 cell lysate, untreated or
treated with 20 ng/ml EGF, was incubated for 4 h at 4 °C with
10-200 µg of PLC-SH2 or 10 µg of GST protein alone prebound to
50-300 µl of glutathione-Sepharose (Pharmacia) in TGH buffer
supplemented with dithiothreitol at 5 mM for 1 h. at
4 °C. The pellets were washed three times with TGH and resuspended in the same buffer. For the co-precipitation experiments, the pellets
were washed three times with HNTG buffer (20 mM HEPES, pH
7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol) and 5 mM dithiothreitol at 4 °C with a 5-min rocking period
between washes at 4 °C. For antibody immunoprecipitations, 150-400
µg of A431 cell lysate was incubated with 10-50 µg of a sheep
polyclonal antibody against the EGF receptor (Life Technologies), 10 µg of a monoclonal antibody against phosphotyrosine (Transduction
Laboratories), 5 µg of -Act. EGFR, or 10 µg of a monoclonal
antibody against phospholipase C-
1 prebound to 25-75
µl of protein G + A-agarose (Oncogene Science) and 10-20 µl of
formalin-fixed Staph A cells (Life Technologies) with the same buffers
and times as above. For the PLC-SH2 characterization and stoichiometry
experiments, equal amounts of pellets were run on SDS-PAGE. In the
co-precipitation experiments, the amount of pellet sample run on
SDS-PAGE was adjusted to achieve equal
-EGFR signals between the
pellets (see figure legends).
To determine phosphorylation
stoichiometry of the EGF receptor, we generated the
GST-EGFR-(987-1028) reagent, which could be used to quantitate both
the number of phosphates and the number of receptors via Western blot
analysis. This protein was phosphorylated using an in vitro
phosphorylation reaction with the EGF receptor. A431 cells treated with
100 ng/ml EGF for 5 min were lysed in HNTG, and the supernatant was
immunoprecipitated with a monoclonal antibody against the EGF receptor
(Promega). GST-EGFR-(987-1028) was added to the pellet at a final
concentration of 91 µM in kinase reaction buffer (10 µM MnCl2 and 50 µM ATP in HNTG
buffer), and the reaction proceeded for 15 min at 4 °C with rocking.
After the addition of 1 ml of HNTG, the reaction was centrifuged, and the supernatant was removed and immunoprecipitated overnight with a
monoclonal antibody against phosphotyrosine to ensure that only phosphorylated GST-EGFR-(987-1028) was used in the experiment. Control
experiments showed that GST alone was not phosphorylated in this
system. To determine the concentration of the phosphorylated GST-EGFR-(987-1028), 10 µl of the immunoprecipitate and serial dilutions of a GST fusion protein of known concentration were subjected
to Western blot analysis with -GST and analyzed on a
PhosphorImager. The signals from the serially diluted standard were
graphed, and a curve was determined by linear regression. The signal
from the EGFR-(987-1028) reagent was then plotted on the standard
curve to determine the concentration.
Duplicate SDS-PAGE gels were prepared containing known amounts of
GST-EGFR-(987-1028) and the -Act. EGFR and PLC-SH2 precipitations from 500 µg of the lysate of A431 cells that were serum-starved and
either untreated or treated with 20 ng/ml EGF for 1, 30, and 60 min.
The gels were transferred to nitrocellulose, and the filters were
blotted with
-EGFR and
-Tyr(P). PhosphorImager analysis was
performed to identify the amount of receptor and phosphotyrosine in
each precipitation using the signals from EGFR-(987-1028) to determine
a signal/molecule ratio. The experiment was performed three times, and
Fig. 6 represents the average of those three experiments.
During studies on the activation kinetics of the EGF
receptor, far Western blot analysis with the SH2 domains of
PLC-1 revealed an interesting pattern (Fig.
1A). Quantitation of the receptor signal and
normalization to the 1-min time point showed that the maximum signal
occurred at 60 min, with a 2-fold increase over time (Fig.
1B). In contrast, it has been shown that maximal receptor autophosphorylation occurs within 1-5 min, and this level remains relatively constant for up to 1 h (2, 25). Therefore, it was
possible that we had identified a subset of receptors with activation
and phosphorylation kinetics different from the general pool of active
receptors. One potential explanation for our observation may be simply
due to differences in the reagents used. The previous studies used
anti-phosphotyrosine antibodies to identify active EGF receptors and
hence reflect the amount of phosphotyrosine at each time point, not the
amount of active receptor. PLC-SH2 recognizes only a single
autophosphorylation site on the EGF receptor (tyrosine 992) and thus
directly represents a portion of active receptor. To determine if our
observation was indeed due simply to valence differences of the
reagents, we compared PLC-SH2 blots with an antibody that recognizes
activated EGF receptors (Transduction Laboratories; Ref. 25), which
we call
-Act. EGFR.
EGFR is a monoclonal antibody that recognizes the
ligand-activated and phosphorylated form of the EGF receptor but does
not recognize other tyrosine-phosphorylated proteins. It recognizes an
EGF receptor mutant that lacks Tyr1068,
Tyr1148, and Tyr1173 and is not inhibited by
0.1 M phosphoserine, phosphothreonine, or phosphotyrosine;
thus, it is an antibody that recognizes an active conformation rather
than phosphotyrosine (25). This antibody can recognize the EGF receptor
in nondenaturing and denaturing conditions, and cleavage studies have
shown that the epitope for this antibody is on the carboxyl terminus of
the receptor distal to amino acid 1052 (25). Competition experiments
using a 100-fold molar excess of PLC-SH2 failed to affect binding of
this antibody to EGF receptors in Western blots and
immunocytochemistry, which confirmed that PLC-SH2 and -Act. EGFR
recognize distinct epitopes.2
EGFR revealed an increase of 1.4-fold from 1 to 5 min, and, similar to studies using anti-phosphotyrosine antibodies, the signal remained essentially the same until 60 min and then decreased by 120 min (Fig. 1B), suggesting that the differences observed with PLC-SH2 were not merely due to differences in reagents used.
Specificity of PLC-SH2Having observed these initial
differences, we wished to confirm the specificity of this reagent.
Lysates from A431 cells untreated or stimulated with EGF for 5 min were
used for immunoprecipitation with an antibody to the EGF receptor (Fig.
2A). Pellets were subjected to Western blot
analysis with an antibody against the EGF receptor (-EGFR) or
PLC-SH2, which confirmed that PLC-SH2 recognized the EGF receptor and
required EGF-induced activation for its recognition. Next, we
determined if the PLC-SH2 reagent recognized other
tyrosine-phosphorylated proteins. Total cell lysate was examined by
Western blot analysis with
-Tyr(P) and PLC-SH2. While
-Tyr(P)
recognized numerous bands in addition to the EGF receptor in stimulated
cells, the PLC-SH2 reagent recognized only the EGF receptor (Fig.
2B). To verify that PLC-SH2 recognized phosphorylated EGF
receptors in their native form, and only Tyr(P)992
receptors, a greater than 100-fold molar excess of PLC-SH2 was used to
precipitate the EGF receptor from EGF-stimulated A431 cells (Fig.
2C). Western blot analysis of the supernatant and pellet
with
-EGFR demonstrated that only 15-20% of the phosphorylated receptors were precipitated under conditions in which all of the PLC-SH2 was precipitated. This is in agreement with the fraction of EGF
receptor that has been reported to be phosphorylated on this site (9,
10). An
-Tyr(P) immunoprecipitation blotted with
-EGFR and
PLC-SH2 showed that the decrease in mobility of the EGF receptor band
in the PLC-SH2 precipitation pellet lanes was due to phosphorylation of
the receptor. Immunoprecipitation of lysates from EGF-stimulated A431
cells with a monoclonal antibody against PLC-
1 showed
that, under conditions in which all of the native PLC-
1
is precipitated, only a very small fraction of the total EGF receptor
pool was co-precipitated (Fig. 2D).
Differential Trafficking of the Tyr(P)992 Subset
After ligand binding, most EGF receptors are internalized
in clathrin-coated pits within 1-10 min and then transit through the
endosomal system (2, 4-6, 12, 15, 17, 26, 27). By 5-15 min, the
majority of internalized receptors are concentrated in the juxtanuclear
area in multivesicular bodies, where they enter into lysosomes and are
degraded. The temporal differences identified in the first experiment
prompted us to investigate any differences in the endocytosis of EGF
receptors. PLC-SH2 and -Act. EGFR were used to label EGF receptors
in A431 cells during the process of activation and internalization.
Both reagents strongly stained cell surface membrane ruffles 1 min
after the addition of EGF. However, the cell membrane did not always
uniformly label with the PLC-SH2 reagent in comparison with -Act.
EGFR (Fig. 3, 1 min.). Punctate staining
within the cytoplasm corresponding to early endosomes was seen only
with
-Act. EGFR, a difference that persisted after 5 min of EGF
exposure. At 10 min, PLC-SH2 did show punctate staining in most cells.
A major difference was that juxtanuclear structures could be detected with
-Act. EGFR, but despite the size and extent of these bodies there was no staining by PLC-SH2. Fig. 4A
shows double labeled micrographs of one cell displaying this
phenomenon. The top micrograph shows the signal from the
-Act. EGFR antibody, and the arrows indicate the presence
of large, juxtanuclear structures stained with this reagent. The
bottom micrograph shows the signal from PLC-SH2. While
punctate staining corresponding to early endosomes can readily be seen,
there is no staining of the juxtanuclear structures. After 15 min, more
extensive punctate staining was seen with PLC-SH2 as well as labeling
of the juxtanuclear structures. However, while these structures were
uniformly labeled by
-Act. EGFR, only distinct portions were labeled
by PLC-SH2, suggesting that vesicles containing PLC-SH2-labeled
receptors had fused separately to this putative multivesicular body
(Fig. 3, 15 min., inset). Greater co-localization
within these bodies was noted at 30 min, although it was possible in a
few cells to identify juxtanuclear structures that were stained by
PLC-SH2 but not by the antibody against the activated EGF receptor.
This observation may reflect the fact that at this time point a greater
proportion of endocytosed receptors are stained by PLC-SH2. After 60 min, no differences in the localization of the two reagents could be
found. To confirm these findings, experiments were also performed using
EGF conjugated to biotin as the reagent for detecting activated
receptors, which produced a staining pattern similar to that of
-Act. EGFR.2 Additionally, double labeling occasionally
revealed the presence of cells that stained very weakly or not at all
with the SH2 domain reagent yet were avidly stained with
-Act. EGFR.
Fig. 4B shows micrographs of a cell that exhibits this
staining pattern. The top micrograph shows staining with
-Act. EGFR, and both plasma membrane and juxtanuclear structure
staining can readily be seen. The bottom micrograph shows
staining of the same cell with PLC-SH2 at the same laser intensity.
Despite the fact that adjacent cells show staining, the cell identified
by the arrow is devoid of any staining by this reagent.
Differential Association of SH2 Proteins with the Tyr(P)992 Subset
We wondered if any differences
existed in the binding of SH2 proteins to the general population of EGF
receptors versus the Tyr(P)992 subset. Lysates
from A431 cells treated with EGF for 30 min were precipitated with
either an antibody against the EGF receptor or PLC-SH2, and the
supernatants (S) and pellets (P) were used for
Western blot analysis with antibodies to various EGF
receptor-associating SH2 proteins (Fig. 5). Preliminary
experiments were performed so that equal amounts of EGF receptor were
loaded in the pellet lanes. This showed that EGF receptors precipitated
with PLC-SH2 showed an enhanced association with Ras-GAP, PI 3-kinase,
and SHPTP2/syp. In contrast, the -EGFR-precipitated
receptors exhibited a 1.5-fold greater ability to co-precipitate Grb2
than the PLC-SH2 precipitated receptors. Interestingly, although SOS
indirectly associates with the EGF receptor through Grb2, these
Tyr(P)992 receptors were still capable of co-precipitating
a greater amount of SOS. These results show that differences can exist
in the association of SH2 proteins with the Tyr(P)992
population versus the general population of EGF receptors.
The fact that greater amounts of SH2 proteins and SOS associate with the Tyr(P)992 population suggests that this subset may
play a significant role in signaling.
Phosphotyrosine Stoichiometry of the Tyr(P)992 EGFR Subset
Our previous results suggested that there might be a
difference in the stoichiometry of tyrosine phosphorylation of
Tyr(P)992 receptors. To examine this directly, lysates
from A431 cells that were serum-starved and either untreated or treated
with EGF for 1, 30, and 60 min were precipitated with either -Act.
EGFR or PLC-SH2, and the pellets were subjected to Western blot
analysis. These blots also contained a GST fusion protein encoding the
epitopes for
-EGFR and a single tyrosine phosphorylation site
(GST-EGFR-(987-1028)). Thus, following a kinase reaction and
anti-phosphotyrosine purification (see "Experimental Procedures"),
this protein would provide a known standard for both tyrosine
phosphorylation and EGF receptor content. This analysis showed that the
number of phosphotyrosines per receptor for both
-Act. EGFR- and
PLC-SH2-precipitated receptors remained constant over the period of EGF
treatment (Fig. 6). For both the
-Act. EGFR and
PLC-SH2 precipitations, there was a low, basal level of phosphorylation
in the unstimulated lysates. Following EGF treatment, the ratio for
-Act. EGFR was approximately 1 phosphotyrosine/molecule from 1 to 60 min of EGF treatment. Interestingly, we found that the
Tyr(P)992 subset was more heavily phosphorylated, with
approximately 2 phosphates/molecule from 1 to 60 min of EGF treatment,
demonstrating yet another difference in this population of receptors.
These results suggest that the basis for the enhanced SH2 protein
association with this population of receptors was a higher degree of
phosphorylation.
We interpret these data as demonstrating that
Tyr(P)992 receptors form a specific subset of active EGF
receptors in A431 cells. In comparison with the total pool of active
EGF receptors, Tyr(P)992 receptors exhibited numerous
differences. The pool of Tyr(P)992 receptors increased
substantially post-EGF treatment, possessed a greater level of
phosphorylation and enhanced interaction with several SH2 proteins, and
exhibited an overall slower rate of endocytosis. The properties of this
subclass of receptors as compared with the general population of active
receptors are summarized in Fig. 7.
One potential concern in this study was that endogenous
PLC-1 could be affecting the activity of PLC-SH2
in vivo. This was highly unlikely for a number of reasons.
Studies on SH2 domain affinities have shown that along with a very high
rate of association, there is also a high rate of dissociation, which
causes a rapid exchange of proteins on the receptor (28). Hence, each
receptor can interact with a succession of SH2 proteins, which makes it feasible for exogenous SH2 domains to bind receptors as the endogenous SH2 proteins turn over. Second, and as a possible consequence of this
rapid turnover, there is a low fraction of proteins bound to the
receptor at any time (Ref. 29, Fig. 2D), despite the high
EGF receptor expression level in A431 cells. To support this, staining
with
-PLC
1 antibody confirmed that there was no
formation of vesicles or juxtanuclear bodies with time as assayed by
confocal microscopy.2 Finally, there is a 20-fold molar
excess of receptor capable of binding to PLC-
1 per cell
(based on ~2 × 105 Tyr(P)992 receptors
and ~104 copies of PLC-
1 (30)). Taken
together, these facts indicate that PLC-SH2 detects the general
population of Tyr(P)992 receptors as opposed to receptors
that cannot bind PLC
1.
We have found two distinct properties of the Tyr(P)992 EGF
receptors during signaling in A431 cells. One property is a slower rate
of endocytosis. Since the autophosphorylation sites of the EGF receptor
are oriented toward the cytoplasm during endocytosis (2) and the
receptor remains active during this time (16), it is capable of
transmitting signals until it is degraded. The rate of internalization
will affect signaling by affecting the duration of interaction with
effector molecules. While Tyr(P)992 EGF receptors
constitute only 15-20% of all active receptors, a substantial amount
of the enzyme can be activated due to the slower rate of
internalization. This would explain other experiments, which showed
that despite the majority of EGF receptor being internalized within 30 min, the activation of PLC-1 increased linearly up to 60 min following EGF stimulation (31), which was paralleled by an increase
in tyrosine phosphorylation of PLC-
1 (32) and the
association of phosphorylated PLC-
1 with the
cytoskeleton (33).
A second characteristic of the Tyr(P)992 receptor subpopulation in A431 cells is a higher stoichiometry of phosphorylation and greater binding of SOS, Ras-GAP, PI 3-kinase, and SHPTP2/syp. This would suggest that, despite being a small percentage of the active receptor pool, this subset of receptors may have a greater role in linking the EGF receptor to a diversity of downstream signaling pathways. Of particular interest was the observation that greater amounts of SOS were associated with Tyr(P)992 receptors, although this population bound relatively less Grb2. SOS is a guanine nucleotide exchange factor for Ras, and the formation of a complex with the EGF receptor and Grb2 stimulates Ras activation (34-36). We speculate that Ras activation is preferentially associated with these receptors. Recently, it has been shown that the EGF receptor and other signal transduction proteins can be segregated into specific intracellular compartments, and our results could provide further clues into how these proteins are sorted. Di Guglielmo et al. (37) found greater amounts of SHC, Grb2, and SOS with endosomal as opposed to plasma membrane EGF receptors. They also concluded that receptor internalization can serve as a mechanism to prolong signaling. It would be of interest to determine if Tyr(P)992 receptors are the subset that associates with SOS in endosomes.
Evidence that SH2 domain-containing proteins affect receptor processing
is beginning to surface. The high affinity binding sites for PI
3-kinase on the platelet-derived growth factor receptor are necessary
in the early steps of endocytic trafficking (38), and PI 3-kinase
catalytic activity is required to divert the receptor to a degradative
pathway (39). Furthermore, it has been shown that disruption of Grb2
binding to the EGF receptor prevents the endocytosis of that receptor
(40). The results presented here suggest that some protein that binds
to the Tyr(P)992 receptor subclass, perhaps
PLC-1 itself or PI 3-kinase, may be involved in
distinguishing the intracellular routing of the EGF receptor.
Collectively, the findings presented here and those of Joly et
al. (38, 39), Sorkin et al. (13), Helin et
al. (12), and Wang and Moran (40) show that autophosphorylation sites are essential for proper internalization and, moreover, that an
individual site may determine specific routing of the receptor. It is
possible that RPTKs undergo continuous
phosphorylation/dephosphorylation, and this also contributes to the
pattern of internalization. However, the fact that the number of
Tyr(P)992 receptors remains constant over time (Fig. 6),
suggests that this phenomenon does not play a large role in A431 cells.
Regardless, this phenomenon would not alter our conclusions about the
role of autophosphorylation sites in the differential trafficking of EGF receptors. It is important to keep in mind that these
characteristics have been identified in A431 cells that overexpress the
EGF receptor and thus may not be extrapolative to cells with normal
levels of receptors. Present evidence indicates, however, that some of these findings are also true in other cells.
Using other SH2 domains with the methods described might provide further insights into receptor processing and signaling. For example, another SH2 domain may define the population of receptors that rapidly undergoes endocytosis and degradation. One implication would be that the signals from the SH2 proteins that bind this autophosphorylation site would be rapidly induced and terminated. Employing SH2 domains from proteins involved in activating Ras, such as Grb2 or SHC, may reveal new aspects of how oncogenic signals are transmitted from the EGF receptor. Undoubtedly, there are a wide variety of other RPTKs and physiologic situations that could be studied using SH2 domains as phosphotyrosine site-specific reagents.
We acknowledge P. Hahn and J. Keen for assistance with confocal microscopy and Hansjuerg Alder for help with the PhosphorImager.