(Received for publication, September 15, 1995; and in revised form, November 30, 1995)
From the
Four transmembrane tyrosine kinases constitute the ErbB receptor
family: the epidermal growth factor (EGF) receptor, ErbB-2, ErbB-3, and
ErbB-4. We have measured the endocytic capacities of all four members
of the EGF receptor family, including ErbB-3 and ErbB-4, which have not
been described previously. EGF-responsive chimeric receptors containing
the EGF receptor extracellular domain and different ErbB cytoplasmic
domains (EGFR/ErbB) have been employed. The capacity of these growth
factor-receptor complexes to mediate I-EGF
internalization, receptor down-regulation, receptor degradation, and
receptor co-immunoprecipitation with AP-2 was assayed. In contrast to
the EGF receptor, all EGFR/ErbB receptors show impaired ligand-induced
rapid internalization, down-regulation, degradation, and AP-2
association. Also, we have analyzed the heregulin-responsive wild-type
ErbB-4 receptor, which does not mediate the rapid internalization of
I-heregulin, demonstrates no heregulin-regulated receptor
degradation, and fails to form association complexes with AP-2. Despite
the substantial differences in ligand-induced receptor trafficking
between the EGF and ErbB-4 receptors, EGF and heregulin have equivalent
capacities to stimulate DNA synthesis in quiescent cells. These results
show that the ligand-dependent down-regulation mechanism of the EGF
receptor, surprisingly, is not a property of any other known ErbB
receptor family member. Since endocytosis is thought to be an
attenuation mechanism for growth factor-receptor complexes, these data
imply that substantial differences in attenuation mechanisms exist
within one family of structurally related receptors.
The epidermal growth factor (EGF) ()receptor is a
transmembrane glycoprotein possessing a cytoplasmic tyrosine kinase
active site, which is activated by specific growth factor binding to an
external ligand-binding domain(1, 2) . Three
additional transmembrane molecules have considerable sequence homology
to the EGF receptor. These four transmembrane tyrosine kinases
constitute the ErbB receptor family: ErbB-1 or the EGF receptor, ErbB-2 (3) , ErbB-3(4) , and ErbB-4(5) .
Ligands that specifically bind to and activate the EGF receptor include EGF and several EGF-like gene products(6) . Activation of the EGF receptor by ligand binding includes receptor dimerization, activation of intrinsic receptor tyrosine kinase activity, autophosphorylation of the receptor carboxyl terminus, and tyrosine phosphorylation of and/or association with intracellular signaling molecules(1, 2, 7) . Ligands that specifically bind to the EGF receptor do not directly interact with ErbB-2, ErbB-3, or ErbB-4. While a specific ligand for ErbB-2, a putative receptor, has not been identified, the heregulin family of growth factors, which do not interact with the EGF receptor or ErbB-2, bind with low affinity to ErbB-3 (8, 9) and with high affinity to ErbB-4(9) . Although heregulins do not directly interact with ErbB-2 by itself, heterodimers of ErbB-2 and ErbB-3 are reported to constitute a second high affinity binding site for heregulin(10) .
Binding of EGF to the EGF receptor at 37
°C rapidly induces the clustering of ligand-receptor complexes in
coated pits, internalization of the complexes, and ultimately lysosomal
degradation of both EGF and its receptor(11) . The endocytic
pathway, therefore, may function as a mechanism for the gradual
attenuation of plasma membrane signaling complexes(12) . While
a molecular mechanism for the rapid internalization of growth
factor-receptor complexes has not been established, recent evidence
suggests that direct interaction of a plasma membrane
clathrin-associated protein complex, termed AP-2 for adaptor protein,
with the carboxyl terminus of the EGF receptor may facilitate receptor
internalization through coated pits(13) . AP-2 (14) is
a heterotetramer of two large subunits, and
(100-115
kDa), a medium subunit, µ2 (50 kDa), and a small subunit,
2
(17 kDa). The nature of AP-2 interaction with the EGF receptor carboxyl
terminus is not clear, but the interaction is stoichiometric, direct,
and requires receptor kinase activity(15, 16) .
However, SH2 domains and phosphotyrosine residues seem not to be direct
mediators of this association, as AP-2 subunits do not contain SH2 or
SH3 domains.
Using a chimeric receptor composed of the extracellular
EGF receptor binding domain and the cytoplasmic domain of the ErbB-2
molecule, one study (17) showed that EGFR/ErbB-2 receptors
internalize I-EGF severalfold more slowly than the EGF
receptor. This study also indicated that the impaired internalization
capacity of this receptor was due to sequences in the ErbB-2
carboxyl-terminal domain. A more recent study (18) showed that
the wild-type ErbB-2 receptor failed to associate with AP-2 and was not
internalized. Surprisingly, however, the oncogenic form of ErbB-2, neu, did form a constitutive complex with AP-2 and was
internalized.
In this study, we have measured the internalization and AP-2 association capacities of all four members of the EGF receptor family, including ErbB-3 and ErbB-4, which have not been described previously. We have used both chimeric receptors, containing an EGF receptor extracellular domain with each of the ErbB cytoplasmic domains, as well as the wild-type ErbB-4 receptor that responds to heregulin.
As determined by I-EGF binding assays at 5 °C (in binding medium of
DMEM supplemented with 20 mM Hepes, pH 7.4, and 0.1% bovine
serum albumin), the average number of receptors/cell was approximately
0.7-2.0
10
for the wild-type EGF receptor,
2-6
10
for EGFR/ErbB-2, 0.6-1.5
10
for EGFR/ErbB-3, and 0.8-4.5
10
for EGFR/ErbB-4. Similar assays indicated the presence of
approximately 1.2
10
I-heregulin
binding sites/cell in cells expressing the wild-type ErbB-4 receptor.
In all cell lines, human EGFR/ErbB molecules or wild-type ErbB
molecules are over-expressed by more than 100-fold compared with the
endogenous levels of mouse ErbB or EGF receptors.
The degradation of
metabolically labeled receptors in the absence or presence of EGF or
heregulin was measured as described
previously(17, 30) . Cells were radiolabeled by
incubation with S-labeled amino acids overnight at 37
°C and subsequently washed with DMEM to remove unincorporated
radiolabeled amino acids. The monolayers were then incubated at 37
°C without or with EGF (300 ng/ml) or heregulin (100 ng/ml) in
binding medium for 0, 2, and 6 h before solubilization in TGH buffer
(1% Triton X-100, 10% glycerol, 50 mM Hepes, pH 7.2) plus 100
mM NaCl and protease and phosphatase inhibitors (1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride,
10 µg/ml aprotinin, 10 µg/ml leupeptin, and 544 µM iodoacetamide). Receptors were immunoprecipitated with polyclonal
antibodies as described below and isolated on SDS gels. The amount of
radioactivity in bands corresponding to each receptor was quantitated
using a PhosphorImager (Molecular Dynamics).
For adaptin binding experiments, GST fusion proteins bound to glutathione-agarose beads were washed 3 times in cold TGH/NaCl buffer. Lysates from NIH-3T3 murine fibroblasts, prepared in TGH/NaCl buffer as described above for the immunoprecipitation protocol, were incubated for 1.5 h at 4 °C with the GST fusion proteins. The ratio of protein lysate to GST fusion protein was approximately 1000:1. After the incubation, the beads were washed 5 times with TGH/NaCl and processed for immunoblotting of AP-2 as described above.
Figure 1:
EGF-dependent activation
of chimeric receptors. Cells expressing EGF receptors or EGFR/ErbB
receptors were incubated overnight in medium containing 0.5% calf serum
and then incubated for 1 h at 4 °C without or with EGF (100 ng/ml). Panel A, after two washes with cold
Ca,Mg
-free PBS, cells were lysed,
and an aliquot (1 mg) of each lysate was then used to assay receptor
autophosphorylation by receptor immunoprecipitation and
anti-phosphotyrosine Western blotting, as described under
``Experimental Procedures.'' Panel B, a second
aliquot (100 µg) of each lysate was directly electrophoresed and
Western blotted with anti-phosphotyrosine to detect tyrosine
phosphorylated cellular proteins.
Figure 2:
I-EGF internalization by
chimeric receptors. Cells expressing EGF receptors or EGFR/ErbB
receptors were incubated for 1-6 min at 37 °C with
I-EGF (
2 ng/ml). At the indicated times, the
monolayers were washed with cold DMEM to remove unbound
I-EGF and then acid-washed to remove surface bound
I-EGF, as described under ``Experimental
Procedures.'' Radioactivity present in the acid washes was
quantitated as surface-associated
I-EGF. The remaining
cell-associated, or internalized, radioactivity was quantitated
following solubilization of the cells. To compare the internalization
capacity of different cell lines, data at each time point are expressed
as the ratio of internalized to surface
radioactivity.
Recent studies (13, 15, 16) have shown
that the EGF receptor associates with the plasma membrane coated-pit
adaptor complex, AP-2, which may facilitate internalization of occupied
receptors. This association is increased when cells are incubated with
EGF at 37 °C, but is not enhanced following ligand binding at 4
°C(13) . We have, therefore, determined whether the
impaired EGF internalization by ErbB-2, ErbB-3, and ErbB-4 chimeric
receptors is paralleled by deficient association with AP-2. Cells
expressing the EGF receptor or the indicated chimeric receptors were
incubated with EGF at 4 °C or 37 °C, and their association with
AP-2 was measured. As a control, association of each receptor with the
tyrosine kinase substrate Shc also was assayed. Unlike AP-2, Shc
contains specific phosphotyrosine recognition sequences, both SH2 (34) and PTB domains(35, 36, 37) ,
and its association with activated receptors is
temperature-independent(38) . The cells were also incubated in
a K-free media, which impairs coated-pit formation and
endocytosis and, thereby, enhances the accumulation of AP-2-receptor
complexes(13) . The data presented in Fig. 3show that
while there was a low basal level (i.e. 4 °C) of
-adaptin present in all receptor immunoprecipitates, enhanced
adaptin-receptor association at 37 °C was only detected with the
EGF receptor. Western blots show that the level of receptors in all
immunoprecipitates was not dramatically different and that all
receptors showed substantial levels of co-immunoprecipitated Shc.
Interestingly, the p46 and p66 isoforms of Shc varied with different
ErbB family members, but the p52 isoform associated with all ErbB
chimeric molecules.
Figure 3:
Analysis of -adaptin association with
chimeric receptors. Cells expressing EGF or EGFR/ErbB receptors were
placed in potassium-depleted media (13) and then incubated at 4
°C for 45 min with EGF (100 ng/ml). To induce maximal AP-2
association, the cultures were then shifted to 37 °C and incubated
for 10 min, while control cultures were maintained at 4 °C. Cell
lysis, receptor immunoprecipitation, and Western blotting for
-adaptin, Shc, and receptors were performed as described under
``Experimental Procedures.''
The net result of ligand-induced receptor internalization
and degradation is a decrease in the number of functional receptors on
the cell surface, a phenomenon termed down-regulation. Down-regulation,
which may have a role in regulating the level of growth factor
signaling, is also influenced by other factors, such as the relative
rates of receptor synthesis and receptor recycling. Therefore, the
capacity of EGF to down-regulate various chimeric ErbB family members
was measured. Cells were incubated with unlabeled EGF for varying
periods of time and subsequently washed to remove unbound ligand and
surface-associated EGF. Then the capacity of the cells to bind I-EGF was assayed at 4 °C. As shown in Fig. 4,
EGF receptors were down-regulated by approximately 65%, while
down-regulation of the EGFR/ErbB-2 and EGFR/ErbB-4 receptors was
negligible, about 15%. Under these conditions, the EGFR/ErbB-3 receptor
exhibited an intermediate level of down-regulation, approximately 40%.
The mechanism of the apparent EGFR/ErbB-3 down-regulation is unclear,
but it may reflect its short half-life compared with other ErbB family
members.
Figure 4:
EGF-induced down-regulation of chimeric
receptors. Cells expressing EGF or EGF/ErbB receptors were incubated
without or with EGF (300 ng/ml) for the indicated times at 37 °C.
Thereafter, the monolayers were rinsed with cold DMEM, and
surface-bound EGF was removed by cold acid washes followed by two
rinses with DMEM. The number of the EGF binding sites on the cell
surface was then determined by incubating the cells with I-EGF (100 ng/ml) at 4 °C for at least 1 h. Data are
expressed as the percentage of
I-EGF binding capacity
relative to cells not exposed to unlabeled
EGF.
To measure the capacity of ErbB-4 to mediate the internalization of I-heregulin, an experimental protocol analogous to that
shown in Fig. 2for
I-EGF internalization was
followed. Control experiments showed that when
I-heregulin was incubated at 4 °C with ErbB-4
overexpressing cells, approximately 95% of the surface-bound ligand was
subsequently removed by washing with an acid (pH 4.5) buffer. The
results of the internalization assays for
I-heregulin
and, for comparison,
I-EGF are shown in Fig. 5.
Clearly, the data indicate that the ErbB-4 receptor is markedly
deficient, compared with the EGF receptor, in its capacity to mediate
ligand internalization. We have also measured
I-heregulin
internalization by endogenous receptors in the MDA-MB-453 and SK-BR-3
mammary carcinoma cell lines, which express high levels of ErbB-2 and
ErbB-3 and relatively low levels of ErbB-4(40, 46) .
The results (data not shown) demonstrate low levels of
I-heregulin internalization, comparable with that
observed in Fig. 5with ErbB-4 transfected cells.
Figure 5:
I-Heregulin internalization
by wild-type ErbB-4. Cells expressing ErbB-4 or EGF receptors were
incubated for 1-10 min at 37 °C with approximately 2 ng/ml of
either
I-heregulin or
I-EGF, respectively.
Internalized and surface-bound radioactivity for each time point were
assayed and plotted as described in Fig. 2.
The results
in Fig. 5would suggest that heregulin is not likely to alter
the metabolic half-life of ErbB-4. However, it is possible for a ligand
to induce receptor degradation without internalization, e.g. by protease activation at the cell surface. Therefore, we have
measured ErbB-4 receptor metabolic half-life in the absence and
presence of heregulin. Following a protocol analogous to that in Table 1, cells transfected with native ErbB-4 were incubated with S-labeled amino acids, washed, and incubated in
nonradioactive media in the absence or presence of heregulin (100
ng/ml). After immunoisolation, the ErbB-4 half-life was calculated. The
results indicate that the half-life of ErbB-4 is 5.5 and 5.0 h,
respectively, in the absence and presence of heregulin (data not
shown). Therefore, the high affinity native ErbB-4 receptor for
heregulin does not demonstrate significantly enhanced metabolic
turnover in the presence of heregulin. In this experiment, the
immunoprecipitation of ErbB-4 employed an antibody that recognizes the
cytoplasmic domain of ErbB-4. Autoradiographs of the radiolabeled
ErbB-4 immunoprecipitates did not reveal lower molecular weight
degradation products, suggesting that proteolytic cleavage of a small
fragment of the ErbB-4 molecule is also not induced by heregulin
binding.
Finally, we examined the capacity of heregulin to induce the autophosphorylation of ErbB-4 and to stimulate the association of AP-2 with the ErbB-4 receptor (Fig. 6). These data show that while EGF did induce association of AP-2 with the EGF receptor at 37 °C, heregulin failed to significantly stimulate ErbB-4 interaction with AP-2 at 37 °C (Fig. 6A). Since AP-2 association in vivo requires receptor autophosphorylation (15, 16) , the capacity of heregulin to induce ErbB-4 autophosphorylation was examined. The data in Fig. 6B demonstrate that heregulin did significantly stimulate ErbB-4 autophosphorylation. Similar levels of heregulin-induced ErbB-4 autophosphorylation were induced at 37 (10 min) or 4 °C (1 h). We also tested AP-2 association with ErbB-4 compared with the EGF receptor in an in vitro system, employing GST fusion proteins containing either the carboxyl terminus of the EGF receptor or the carboxyl terminus of ErbB-4 (Fig. 7). Consistent with the results of Nesterov et al.(16) , we obtained a significant level of AP-2 association with the EGF receptor-GST fusion protein. However, association of AP-2 with the ErbB-4-GST fusion protein was not detectable.
Figure 6:
Heregulin activation of ErbB-4 and AP-2
association. Panel A, cells expressing ErbB-4 or EGF receptors
were placed in potassium-depleted medium (13) and then
incubated at 4 °C for 45 min with heregulin (100 ng/ml) or EGF (100
ng/ml), respectively. To induce maximal AP-2 association, the cultures
were then incubated at 37 °C for 10 min, while control cultures
were maintained at 4 °C. Cell lysis, receptor immunoprecipitation,
and Western blotting for -adaptin were performed as described
under ``Experimental Procedures.'' Panel B, cells
expressing ErbB-4 or EGF receptors were incubated overnight in DMEM
plus 0.5% calf serum and then incubated at 4 °C for 1 h with
heregulin (100 ng/ml) or EGF (100 ng/ml), respectively. Cell lysis,
receptor immunoprecipitation, and Western blotting with phosphotyrosine
antibody were performed as described under ``Experimental
Procedures.''
Figure 7:
Interaction of AP-2 with EGF or ErbB-4
receptor carboxyl termini GST fusion proteins. GST fusion proteins
containing the carboxyl terminus of either the EGF receptor (residues
944-1186) or ErbB-4 (residues 990-1264) were obtained from bacterial
expression and purified on glutathione-agarose beads. A volume of beads
containing 5-10 µg of each GST fusion protein was washed 3
times with cold TGH/NaCl buffer and incubated with NIH-3T3 cell lysate
(5-10 mg of total protein) for 1.5 h at 4 °C. After the
incubation, the beads were washed and processed for immunoblotting with
an -adaptin antibody, as described under ``Experimental
Procedures.''
The receptor system described above offers the opportunity to ask whether a receptor that does not undergo ligand-induced internalization and down-regulation may be more mitogenic than a similar receptor, which is rapidly down-regulated, particularly at low growth factor concentrations. Therefore, we have used NIH-3T3 cells expressing EGF receptors or ErbB-4 receptors to assay the mitogenicity of increasing concentrations of EGF or heregulin (Fig. 8). The data indicate that for this comparison there is no substantial difference in the capacity of activated EGF and ErbB-4 receptors to elicit increased DNA synthesis in quiescent cells.
Figure 8:
Comparison of EGF and heregulin-induced
DNA synthesis. Cells expressing either wild-type ErbB-4 or EGF
receptors were grown in six-well culture dishes (Corning) until
confluent and then incubated for 48 h in DMEM plus 0.5% calf serum.
Subsequently, the cells were incubated with indicated concentrations of
heregulin or EGF in binding medium for 24 h. 2 h before the end of the
incubation, [H]thymidine was added to the medium
(1 µCi/ml). The medium was aspirated, and the monolayers were
washed 3 times with cold 10% trichloroacetic acid, and the
acid-insoluble fraction remaining on the dishes was resuspended in 0.5 N NaOH. A 50-µl aliquot was assayed for protein using
bicinchoninic acid (Pierce), and the remainder of the sample was used
to measure the amount of acid-insoluble
[
H]thymidine by scintillation counting. The
radioactivity incorporated per mg of cellular protein (cpm/mg) was
calculated for each ligand concentration and expressed as the -fold
increase compared with untreated cells.
The phenomenon of receptor-mediated internalization of growth
factors was first described for EGF and its receptor(20) , and
the basic observation has since been established for a wide variety of
growth factors and their receptors(11) . Hence, it was
unexpected that within the EGF receptor family, only the EGF receptor
would mediate rapid ligand internalization and growth factor-induced
receptor down-regulation. The data of Sorkin et al. (17) did show that the chimeric EGFR/ErbB-2 is deficient in
receptor-mediated endocytosis and that the carboxyl terminus of the
receptor determines the endocytic capacity. As the chimeric ErbB-2
receptor failed to mediate rapid ligand internalization in either
NIH-3T3 or the human mammary adenocarcinoma cell line MDA-MD-134, it
seems unlikely that cell background defines internalization capacity.
However, no ligand for ErbB-2 has been identified and, therefore, the
conclusions are based on the behavior of chimeric EGFR/ErbB-2
molecules. No data have been reported for the ErbB-3 and ErbB-4
molecules. The data in this manuscript show that chimeric EGFR/ErbB-3
and EGFR/ErbB-4 molecules fail to exhibit receptor-mediated endocytosis
of EGF and that the wild-type ErbB-4 receptor fails to mediate
heregulin internalization or heregulin-dependent receptor trafficking,
such as enhanced metabolic degradation. Therefore, the analysis of
ErbB-4 has utilized a chimeric molecule, which responds to EGF, as well
as the wild-type receptor. The results with both molecules are
consistent; the activated ErbB-4 cytoplasmic domain fails to mediate
the rapid ligand-dependent receptor trafficking demonstrated by the EGF
receptor. Using the data in Fig. 2and Fig. 5, we
calculate that the I-EGF internalization rate constant, k
, is approximately 0.13 for the wild-type EGF
receptor, approximately 0.04 for the chimeric ErbB-2, ErbB-3, ErbB-4
receptors, and about 0.07 for the native ErbB-4-mediated
internalization of
I-heregulin. These low internalization
rate constants for ErbB-2, -3, -4 are equivalent to our published
internalization rate of the kinase-negative EGF receptor (39) and imply that only a constitutive basal level of receptor
endocytosis is operative for the ErbB-2, -3, and -4 molecules.
The
analysis of ErbB-3 relies on data from chimeric molecules, as does the
previously published analysis (17) of ErbB-2. Heterodimeric
complexes of ErbB-3 and ErbB-2 are reported to form a high affinity
receptor for heregulin (10) and to be the basis of co-operative
signaling and neoplastic transformation by these two ErbB family
members(10, 40) . This raises the possibility that a
heterodimer of ErbB-2 and ErbB-3 might mediate rapid heregulin
internalization. However, mammary tumor lines that express high levels
of ErbB-2 and ErbB-3, relative to ErbB-4, also demonstrate a low rate
of I-heregulin internalization. In the NIH-3T3 cells
employed in this study, each receptor has been over-expressed in a cell
line in which the endogenous levels of EGF receptor, ErbB-2, ErbB-3,
and ErbB-4 are relatively low. The overexpression of each transfected
receptor is approximately 100-fold relative to the level of endogenous
ErbB molecules. Therefore, it seems unlikely that significant levels of
heterodimerization between ErbB family complicate our analyses of
ligand binding.
While defined internalization codes have been
identified in certain non-growth factor receptors, such as the low
density lipoprotein, mannose 6-phosphate, and transferrin
receptors(41) , internalization codes have not been clearly
identified in any growth factor receptor. Using deletion analysis,
Chang et al.(42) came to the conclusion that the
ligand internalization capacity of the EGF receptor is dependent on
multiple or functionally redundant internalization codes. Primarily
identified were sequences QQGFF and
FYRAL
within the cytoplasmic domain of the EGF receptor. Neither of these
putative internalization codes, however, is preserved in the
cytoplasmic domains of ErbB-3 and ErbB-4. The cytoplasmic domain of
ErbB-2 does contain FYRSL and QQGFF sequences at approximately the same
relative position as the proposed internalization codes in the EGF
receptor. However, the only studies reporting ErbB-2 internalization
are experiments in which antibodies to the ErbB-2 extracellular domain
have been used to stimulate the internalization
process(18, 44, 45) . It is unclear and
perhaps unlikely that antibody-induced internalization is analogous to
the rapid internalization process promoted by growth factors. Efficient
EGF receptor internalization and down-regulation are also thought to
involve the phosphorylation of Ser
-Ser
in
the receptor carboxyl terminus(43) . While Ser
is retained in ErbB-2 and -4, Ser
is not retained
in ErbB-2, -3, or -4.
The mechanisms of receptor-mediated internalization of growth factors are not understood, but recent data suggest that the clathrin-coated pit adaptor protein AP-2 might mediate activated EGF receptor association with coated pits and hence internalization(13, 15, 16) . If the association of AP-2 and the EGF receptor is functionally related to the internalization process, then a testable prediction would be that receptors that do not undergo rapid internalization should not form complexes with AP-2. The data in this manuscript show that this correlation holds for all three ErbB molecules that are internalization impaired.