(Received for publication, August 25, 1994; and in revised form, October 31, 1994)
From the
Plasma membrane clathrin-associated protein complexes (AP-2)
have been shown to co-immunoprecipitate with the epidermal growth
factor (EGF) receptor (Sorkin A., and Carpenter, G. (1993) Science 261, 612-615). Hence, we analyzed the stoichiometry of the
EGF receptor interaction with AP-2 using a new antibody that
efficiently immunoprecipitates native AP-2. EGF receptorAP-2
complexes were isolated from
S-labeled cells treated with
EGF by EGF receptor affinity chromatography followed by precipitation
with the antibody to AP-2. Quantitation of the relative molar
concentrations of the proteins found in the complex revealed that 1 mol
of AP-2 was associated with approximately 1.1 mol of EGF receptor. No
other proteins were present in significant molar concentrations
relative to AP-2, indicating that other proteins are not
stoichiometrically involved in the interaction of EGF receptors and
AP-2 in vivo. Co-immunoprecipitation experiments in cells
expressing a mutant EGF receptor demonstrated that the cytoplasmic
carboxyl-terminal 214 residues of the EGF receptor are essential for
interaction with AP-2.
The binding of epidermal growth factor (EGF) ()to its
receptor results in the rapid disappearance of receptors from the cell
surface(1) . Receptor down-regulation is due to the
EGF-accelerated endocytosis and degradation of EGF receptors (reviewed
in (2) and (3) ). It has been proposed that occupied
EGF receptors are internalized severalfold faster than unoccupied
receptors (4) and that the ligand-dependent acceleration of
receptor internalization is likely the rate-limiting step in receptor
down-regulation(2, 3, 4, 5) .
Morphological studies suggest that EGF increases receptor endocytosis by promoting receptor clustering into clathrin-coated pits on the plasma membrane which is followed by receptor internalization into clathrin-coated vesicles(6, 7, 8) . These observations together with similar analyses of other membrane receptors have lead to the view that plasma membrane-coated pits function as sorting organelles selectively recruiting receptors that contain internalization sequences or ``codes'' within their cytoplasmic domains (reviewed in Refs. 9 and 10).
A main structural
component of coated pits is the clathrin lattice anchored to the
cytoplasmic surface of the membrane by the associated protein complexes
or adaptors (APs) (11, 12, reviewed in Refs. 9 and 13-15). AP-2
is the most ubiquitous of the associated proteins found in coated
vesicles derived from the plasma membrane. It is a heterotetramer
containing two large subunits, and
2 (100-115 kDa), a
medium subunit µ2 (50 kDa), and a small subunit
2 (17 kDa)
(16, reviewed in Refs. 9, 15, 17). In addition, there are two isoforms
of the
-subunit,
A and
C, encoded by distinct but highly
homologous genes(19) .
The large 2 subunit plus the
medium and small subunits of AP-2 are very homologous to the
corresponding subunits (
1, µ1, and
1) of the Golgi
clathrin-associated protein complex,
AP-1(20, 21, 22, 23) . However, in
place of an
-subunit, AP-1 contains a
-subunit (
100 kDa)
that has a relatively low level of similarity to the
-subunit(24) . Studies of bovine brain-coated vesicles
suggest that for AP-2 each
-subunit is paired with a
2-subunit, whereas for AP-1,
-adaptin is complexed with a
1-subunit that migrates with slightly slow mobility on
SDS-polyacrylamide gel electrophoresis(19, 25) . The
subunit and isoform composition of APs are poorly described in other
cell types or cultured cell lines. The
subunits of AP-2 and AP-1
are known to bind clathrin(26) ; however, the function of other
subunits is still unclear.
Current data suggest that the interaction
of AP-2 with the intracellular domain of transmembrane receptors
mediates the selective recruitment of receptors into coated
pits(27, 28, 29, 30) . However, the
mechanism of receptorAP interaction and the universality of this
association with different classes of receptors are not yet understood.
Previously published data have shown that AP-2 co-immunoprecipitates
with EGF receptors from cells treated with EGF(31) . This
study, however, did not establish the molecular composition of EGF
receptor
AP-2 complexes. Here, we determine the stoichiometry of
components in this complex and assess whether AP-2 interacts directly
with the EGF receptor using a double affinity purification protocol to
analyze metabolically labeled EGF receptor
AP-2 complexes.
Immunoprecipitates were washed twice with TGH supplemented with 100
mM NaCl and then once with TGH. Typically, 7.5%
SDS-polyacrylamide gels were used to separate proteins. In indicated
experiments, 6 M urea was included in the separating gel as
described elsewhere (19) to separate - and
-subunits.
Transfer to nitrocellulose membrane and protein immunoblotting were
carried out as described(34) . Sheep antibodies to mouse IgG
(Cappel Inc.) or protein A (Zymed Inc.) conjugated with horseradish
peroxidase and enhanced chemiluminescence (Amersham or DuPont NEN) were
used to detect primary mouse or rabbit antibodies, respectively.
Stripping of the antibodies from the membrane was performed according
to the manufacturer's protocol (Amersham). Several films obtained
after various lengths of exposure times with the same blot were
analyzed to measure optical density within a linear range of
sensitivity. A Bio-Rad densitometer was used for quantitation.
The nonspecific precipitation
of AP-2 by nonimmune rabbit IgG used in the same quantity as antibody
986 to EGF receptor was negligible (0.1-0.2% of total AP-2)
compared to the specific association of AP-2 with the EGF receptor.
Nonspecific association of EGF receptors with control IgG was in some
experiments comparable with the specific co-immunoprecipitations with
anti-AP IgG and varied with the source of control IgG used. Therefore,
immunoprecipitation with Ab31 in the presence of C-ear-linker
peptide was used to control for nonspecific associations. To determine
specific co-precipitation of the receptor with AP-2, the amount of EGF
receptors in nonspecific immunoprecipitates was subtracted from that
amount recovered in the immunoprecipitates in the absence of
C-ear-linker.
To isolate AP-2
bound to receptors, 2 equal aliquots of the eluent from the EGF
affinity column were immunoprecipitated with Ab31 to C-subunit in
the absence or presence of recombinant
C-ear-linker protein (300
ng). Protein A-Sepharose immunoprecipitates were washed twice with TGH
containing 100 mM NaCl and twice in TGH without NaCl. The
immunoprecipitates were then separated on 7-11% gradient
SDS-gels. In some experiments, 3-15% gels were used to enable
detection of a larger molecular weight range of proteins. After the
gels were fixed and dried, radiolabeled proteins were analyzed with a
PhosphorImager or exposed to x-ray film. In some experiments, proteins
were transferred to PolyScreen transfer membrane. To detect
radiolabeled proteins, transfer membranes were exposed to the
PhosphorImager screens or x-ray films. Then the transfer membranes were
incubated with various antibodies to APs and EGF receptor, which were
detected by chemiluminescence.
S-Labeled bands were then
matched with the bands detected by blotting.
Ab32 was effective in immunoprecipitating a random
sample of all available APs. Comparative blotting of Ab32 precipitates
and cellular lysates with AC1-M11 indicates that in NIH 3T3 cells
approximately 60-70% of the solubilized AP-2 pool contains the
C isoform. Consistently, we estimate that the
C antibody
(Ab31) precipitates approximately 60-70% of the total solubilized
AP-2 pool in NIH 3T3 cells. In subsequent experiments, Ab31 was used to
minimize analysis of large subunits of AP-2 components, such as
1,
that are considered characteristic of AP-1.
Figure 1:
Time
course of EGF receptor association with AP-2. NIH 3T3 (WT) cells were
incubated with EGF (300 ng/ml) at 4 °C, and the temperature was
then shifted to 37 °C for the indicated times. Cell lysates for
each time point were divided into three equal aliquots. The first and
second aliquots were immunoprecipitated with Ab31 to AP-2 alone (lanes 1-5) or in the presence of an excess
C-ear-linker protein (lanes 6-10). The third
aliquot was immunoprecipitated with antibody 986 to the EGF receptor (lanes 11-15). EGF receptor and
-subunits of AP-2
were detected, respectively, by immunoblotting with antisera 2319 to
the EGF receptor and AC1-M11. The amount of EGF receptors
co-precipitated with AP-2 (closed circles) and AP-2
co-precipitated with EGF receptors (open circles) is expressed
in arbitrary units (a.u.), as described under
``Experimental Procedures.'' Results are representative of
three independent experiments.
The data presented in Fig. 1A (lanes 11-15) demonstrate the converse, i.e. that AP-2 co-immunoprecipitates with EGF receptors. Both A-
and
C-subunit isoforms (typically 2-4% of total solubilized
-subunit pool) were co-immunoprecipitated with EGF receptors. A
similar amount of AP-2 was associated with EGF receptors when cells
were exposed to EGF at 37 °C without preincubation at 4 °C
(data not shown). Co-immunoprecipitation of AP-2 with EGF receptors
also reached a maximum at 6-8 min following the shift to 37
°C (Fig. 1B, open circles).
These
results demonstrate that association of EGF receptors with AP-2 had
similar kinetics when monitored by either EGF receptor or AP-2
immunoprecipitation. In mouse cells, more C than
A isoform is
present. However, the
A/
C ratio in EGF receptor
co-immunoprecipitates was similar to the ratio of these isoforms in
cell lysates. This suggests that both types of AP-2, containing
A-
or
C-subunits, associate with the EGF receptor following the same
proportion relative to the cellular abundance of each isoform. Previous
data showed that in human fibroblasts and epidermoid carcinoma cells,
the ratio
A/
C was also similar in cell lysates and EGF
receptor immunoprecipitates, although
A was the predominant
isoform relative to
C in these cells(31) .
Figure 2:
Analysis of metabolically labeled AP-2
subunits. WT cells were metabolically labeled with
[S]methionine for 24 h, and, following cell
lysis in TGH, AP-2 was precipitated with Ab31 in the presence or
absence of
C-ear-linker. Immunoprecipitates were electrophoresed,
and radiolabeled proteins were detected using x-ray film (A).
The amount of radioactivity in the bands corresponding to AP-2 subunits (arrows) was quantitated with a PhosphorImager. The amount of
the protein specifically precipitated with Ab31 was calculated as a
difference between the radioactivity in the band in the Ab31
immunoprecipitate (lane
C-ear-) and the
radioactivity in the identical region of the gel of nonspecific
precipitations (lane
C-ear+). The 102-kDa
band contained both
C- and
2-subunits, as determined by
immunoblotting of labeled proteins. The molar concentrations of µ2
and
2 relative to
C/
2 adaptins (B) were
determined by normalizing the specific radioactivity of the bands to
the number of methionine residues in each protein. These numbers were
obtained from the sequences of rat µ2(21) , rat
2(20) , mouse
C(18) , and rat
2
subunits(23) . The molar concentration of
2 relative to
C was quantitated from separate experiments in which these
subunits were resolved on SDS-urea gels (data not shown). Data are
expressed as percent of the amount of the
C
subunit.
The molar ratio of individual AP-2
subunits in the immunoprecipitate was determined by measuring the
amount of radioactivity in bands corresponding to AP-2 subunits. Since
the sequences of known AP-2 subunits are almost identical among
mammalian species, we used the methionine content of the cloned rat
2, µ2, and
2 together with the mouse
C sequence (18, 20, 21, 22, 23) to
normalize the radioactivity in the each band to the abundance of
methionine residues. The molar ratio of
C- and
2-subunits was
estimated from SDS-urea gels. As summarized in Fig. 2B,
the four subunits of AP-2 were present in approximately equimolar
amounts.
Because the extent of protein labeling in vivo is
influenced by the rate of protein turnover, we also measured individual
degradation rates of the AP-2 subunits. Cells incubated with
[S]methionine were ``chased'' in a
medium containing unlabeled methionine, and AP-2 was immunoprecipitated
by Ab31 as described above. The amount of radioactivity in the bands
corresponding to the large (
C/
2), medium (µ2), and small
(
2) subunits was monitored. As seen in Fig. 3,
quantitation indicated a similar rate of degradation, t
30-36 h, for each of the AP-2 subunits. These results suggest
that all subunits are labeled to a similar extent during a 24-h
incubation with [
S]methionine, and that the
molar ratios of individual proteins in the AP-2 complex, as calculated
from the radioactivity in the gel bands (Fig. 2B) are
correct and do not need to be adjusted for different subunit turnover
rates. In contrast, the half-life of EGF receptors in the same
experiment was calculated at approximately 8-9 h, consistent with
previous measurements in similar cells(34) .
Figure 3:
Degradation rate of AP-2 subunits and EGF
receptors. WT cells were metabolically labeled with
[S]methionine for 24 h and then were incubated
for the indicated times in the presence of unlabeled methionine as
described under ``Experimental Procedures.'' AP-2 and EGF
receptors were immunoprecipitated from TGH extracts with Ab31 and
antibody 986, respectively. The amount of radioactivity in bands
corresponding to the large AP-2 subunits
C/
2, 102 kDa (closed circles), medium subunit (µ2) (open
circles), and small subunit (
2) (closed triangles)
at each time point was quantitated as described in Fig. 2. The
amount of radiolabeled EGF receptor (open triangles) was
determined from the radioactivity in the major 175-kDa band from the
anti-EGF receptor precipitates as described previously(34) .
The amount of each protein is expressed as percent of the initial
amount of that protein recovered from the cells at time
0.
Finally, other unidentified radiolabeled proteins, for example a 65-kDa and a 250-kDa species, co-precipitated with AP-2 in a specific manner (see Fig. 2A). The molar concentration of these molecules, calculated from the average content of methionine residues in proteins (40) , was, however, less than 0.5 mol/mol of AP-2.
The results of this experiment (Fig. 4) show
that three bands corresponding to the mobilities C/
2, µ2,
and
2 subunits of AP-2, plus one band corresponding to the EGF
receptor were recovered in the specific AP-2 immunoprecipitates derived
from the EGF eluent. In addition, a faint
1 band was also
observed. As expected, the pattern and relative molar concentrations of
AP-2 subunits immunoprecipitated after EGF receptor purification was
similar to that obtained by direct immunoprecipitation from cell
lysates (compare Fig. 2A with Fig. 4). The
identity of
C- and
-subunits was confirmed by Western blot
analysis as described under ``Experimental Procedures'' (not
shown).
Figure 4:
Isolation
of EGF receptorAP-2 complexes. Metabolically prelabeled (24 h) WT
cells were incubated with EGF at 4 °C for 40 min, then shifted to
37 °C for 7 min, and subsequently lysed in TGH. EGF
receptor
AP-2 complexes were isolated by affinity chromatography
on EGF-Affi-Gel followed by immunoprecipitation with Ab31 as described
under ``Experimental Procedures.'' Immunoprecipitation with
Ab31 in the presence of the excess of
C-ear-linker protein was
used as a control for the nonspecific co-precipitation of labeled
proteins. The migration position of radiolabeled EGF receptors,
,
, µ2, and
2 AP-2 subunits are indicated by arrows. EGF receptors and large AP-2 subunits were matched
with the bands detected by blotting with the corresponding antibody, as
described under ``Experimental Procedures.'' The lane labeled Lysate represents 0.5% of the total cell lysate
used for the EGF receptor affinity purification with the same exposure
time.
The quantity of all bands in Fig. 4detectable by
PhosphorImager analysis was calculated as the difference between the
amount of radioactivity in identical regions of the gel corresponding
to the specific and nonspecific immunoprecipitates. The specific
radioactivity in the 180-kDa band, corresponding to phosphorylated
EGF receptor was approximately twice that present in the
C/
2
band. The amount of radioactivity present in these bands was then
normalized to the number of methionine residues in each protein and
also to the percentage of labeled protein. Since the t of
C/
2 is approximately 34 h (Fig. 2), approximately 40%
of solubilized pool of these subunits was labeled during the 24 h
incubation with [
S]methionine. In contrast, the t of EGF receptors was
8-9 h (Fig. 3),
indicating that in the same labeling period approximately 85% of the
total pool of EGF receptors was labeled with
[
S]methionine. The band intensities, therefore,
were also normalized to correct for the resultant differences in the
specific radioactivities of EGF receptors and AP-2 subunits. Using
these corrections, an average stoichiometry of 1.1 ± 0.2 mol of
EGF receptor per mol of AP-2 was estimated for five independent
experiments.
The EGF receptor was the only co-precipitating protein
found in specific AP-2 immunoprecipitates in a significant amount, i.e. more than 0.2 mol/mol of AP-2. Control experiments using
3-15% gels did not reveal the presence of additional bands with
relative mobilities of greater than 10 kDa or less than 500 kDa.
Thus, we suggest that no other proteins are necessary in stoichiometric
amounts to maintain the equimolar interaction of AP-2 and EGF receptor.
It is possible that other proteins were not detected because of their
low content of methionine and/or low rate of biosynthesis. However, the
molar concentration of such proteins would have to be smaller than that
of the
2 subunit which has a long half-life (
36 h) and which
contains only 4 methionine residues.
The data in Fig. 5show that in the absence of EGF AP-2 does not interact with WT or Dc214 receptors. When cells were incubated with EGF at 4 °C and then allowed to internalize EGF at 37 °C, AP-2 co-immunoprecipitated with WT receptor, but not with the Dc214 receptor mutant.
Figure 5:
Interaction of AP-2 with wild-type and
Dc214 EGF receptors. NIH 3T3 cells expressing wild-type (WT) or Dc214
truncated EGF receptors were subjected as indicated to
K-depletion, incubated with EGF (300 ng/ml) at 4
°C in buffer B, and placed at 37 °C for 15 min as described
under ``Experimental Procedures.'' Control cells, i.e. no K
depletion, were incubated at 4 °C in
buffer C in the presence or absence of EGF as indicated. EGF-treated
cells were further incubated at 37 °C for 9 min. After incubations,
cells were solubilized in TGH, and EGF receptors were
immunoprecipitated with EGF receptor antibody. Aliquots of lysates
corresponding to 5% of the amount used for immunoprecipitation were
electrophoresed to compare with immunoprecipitates. The
-subunits
of AP-2 were detected in receptor immunoprecipitates (panel B)
and lysates (panel C) by immunoblotting with AC1-M11. EGF
receptor was probed with monoclonal antibody LA22 (panel
A).
In cell cultures, I-EGF is
poorly internalized by Dc214 receptors(5, 34) ,
indicating that this mutant EGF receptor may not enter coated pits. To
test whether the failure of the Dc214 mutant to associate with AP-2 was
due to its inability to enter coated pits, we used K
depletion conditions to compare AP-2 association with wild-type
and Dc214 receptors in the absence of coated pits(31) . In WT
cells, K
depletion resulted in a significant increase
in AP-2 co-immunoprecipitated with the EGF receptor, similar to results
obtained with human cells(31) . The data in Fig. 5A demonstrate, however, no significant increase of AP-2 association
with the Dc214 mutant receptor under the same conditions. Therefore,
residues 973-1186 in the cytoplasmic domain of the EGF receptor are
critical for receptor interaction with AP-2. These results also
indicate that the low internalization efficiency of the Dc214 receptor
may be attributable to its inability to associate with AP-2.
AP-2 is implicated in functions essential for the dynamic cycle of clathrin-coated pits and vesicles. One of these functions is the selective recruitment of membrane proteins into coated pits by the recognition of internalization ``codes'' located within the cytoplasmic domains of receptors (reviewed in Refs. 9, 10, and 15). Initial evidence for this model was based on the in vitro binding of purified AP-2 to the intracellular domains of transmembrane proteins known to be capable of efficient clustering in coated pits, such as low density lipoprotein, mannose 6-phosphate, asialoglycoprotein receptors, and lysosomal acid phosphatase(27, 28, 29, 30) . These experiments showed that although the in vitro interaction was apparently specific and required an internalization motif, the affinity and stoichiometry of the interaction was very low (29, 30) . More recent experiments have shown that EGF receptors associate in vivo with AP-2 following EGF addition to intact human cells at 37 °C(31) . Although the association was stable, the experimental design did not allow a determination of the stoichiometry interaction in vivo, nor did it allow a test of whether there was direct interaction between receptors and AP-2.
The data in this report confirm, in NIH 3T3
cells expressing human EGF receptors, the EGF- and
temperature-dependent interaction of EGF receptors with AP-2 in mouse
cells (Fig. 2) similar to that previously observed in human
cells(31) . The amount of AP-2 co-immunoprecipitated with EGF
receptors in NIH 3T3 cells (Fig. 1) was significantly smaller,
however, than that detected in A-431 cells. Moreover,
immunoprecipitation of AP-2 using an C-specific antibody showed
that a relatively small pool of EGF receptors, equivalent to
approximately 5-10
10
receptors per cell, is
associated with AP-2 at any one time under these experimental
conditions. The small fraction of receptors and AP-2 detected in these
complexes is likely due to the transient nature of receptor
AP-2
association during internalization through coated pits. Also, it is
possible that the rate of receptor transition through the early stages
of endocytosis is dependent on the particular receptor and cell type.
For example, the maximal rate constant for EGF internalization is
approximately 50% higher in mouse fibroblasts (5, 34) than in A-431 cells (42) or human
fibroblasts(42, 43) . This may partially explain the
quantitative differences in the extent of association between the EGF
receptor and AP-2 in NIH 3T3 and A-431 cells.
Interaction of EGF
receptor with proteins that have src homology 2 (SH2) domains
is mediated by phosphotyrosine-containing motifs in the
carboxyl-terminal domain of the receptor(44) . Some
SH2-containing proteins, termed adaptors, are known to mediate
association with other proteins; for example, GRB-2 mediates
association of the ras guanine nucleotide exchanger (Sos) with
the EGF receptor(45) . Since AP-2 subunits do not have SH2
domains, it seemed plausible that AP-2 interaction with the activated
EGF receptor might be mediated by an SH2-containing adaptor protein.
However, in preparations of metabolically labeled EGF
receptorAP-2 complexes, we did not detect other radiolabeled
proteins in significant amounts. This indicates that other proteins do
not mediate the stoichiometric association of EGF receptor and AP-2
which is consistent with a direct interaction of EGF receptor and AP-2.
Our data do not rule out the involvement of additional regulatory
proteins which may have a catalytic function in complex formation.
The novel two-step isolation of receptorAP-2 complexes
indicates the relative stability of these complexes and demonstrates
that an average of one AP-2 tetramer is associated with one EGF
receptor monomer. Although EGF receptors (46, 47) and
AP-2 (48) are both capable of aggregation, we suggest that
predominantly monomeric forms of each comprise the complex in TGH
lysates for the following reasons. First, the stability of receptor
dimers in Triton X-100 is low due to reduced EGF binding
affinity(47, 49) . Glycerol gradient centrifugation of
TGH lysates reveals that more than 95% EGF receptors are monomers. (
)Second, AP-2 tends to aggregate at a much higher
concentration than the concentration of AP-2 in either the TGH lysates
or the EGF affinity column eluent(48, 50) . Lastly, in
these experiments, large aggregates were removed by high speed
centrifugation. It is possible, though, that EGF receptor dimerization
is important for the formation of EGF receptor
AP-2 complexes in vivo.
Finally, the working model developed here and in
previous studies (31, 34) suggests the direct
interaction of AP-2 with the activated EGF receptors at an early step
in endocytosis. EGF-induced receptor tyrosine kinase activity has been
shown to be necessary for rapid internalization of EGF
receptors(5, 34, 43) . Activation of the
receptor kinase leads to autophosphorylation and conformational changes
in the intracellular domain of the receptor which may make receptor
internalization motifs accessible to AP-2(34) . This is
consistent with preliminary results showing that the tyrosine kinase
inhibitor genistein prevents EGF receptor interaction with AP-2. Although several sequences within the carboxyl terminus of the
EGF receptor can serve as internalization codes(41) , the
identification of the exact binding site(s) for AP-2 in the native EGF
receptor remains to be completed. Studies of the influence of point
mutations within the putative EGF receptor internalization motifs on
the receptor association with AP-2 are in progress.