(Received for publication, August 23, 1994; and in revised form, November 29, 1994)
From the
Ligand-induced internalization of the epidermal growth factor
receptor (EGFR) leads to accelerated receptor degradation. Two models
have been proposed to explain this. In the first model, induced
internalization expands the intracellular pool of receptors, leading to
enhanced lysosomal targeting. The second model proposes that activation
of intrinsic receptor kinase activity induces inward vesiculation of
endosomes, thus interrupting receptor recycling. To test these models,
we created EGFR mutants that lack the conserved tyrosine kinase domain,
but retain different parts of the distal carboxyl terminus regulatory
region. Mutants lacking all distal regulatory sequences underwent slow
internalization (0.02 min) and turnover (t
24 h), similar to unoccupied,
holo-EGFR. Mutant receptors that lacked the kinase domain, but retained
the entire distal regulatory domain, were constitutively internalized
and targeted to lysosomes, even in the absence of EGF. The turnover of
these receptors (t
11 h) was similar to
that of occupied, kinase-active holo-EGFR (t
9.5 h). These results show that receptor tyrosine kinase
activity is not required for the targeting of EGFR to lysosomes.
Receptor mutants which expressed previously identified endocytic
sequences underwent rapid internalization. Unexpectedly, enhanced
turnover of EGFR mutants required additional sequences located between
residues 945 and 991 in the holo-EGFR. Thus, internalization and
lysosomal targeting of EGFR are separate processes mediated by distinct
sequences. Our results indicate that induced internalization is
necessary, but not sufficient, for enhanced EGFR degradation. Instead,
down-regulation requires exposure of previously cryptic internalization
and lysosomal targeting sequences. Occupied EGFR thus appear to be
handled by the endocytic machinery in the same fashion as other
constitutively internalized or lysosomally targeted receptors.
The spatial distribution and trafficking of receptors is important for their function. The polarized distribution of the polymeric immunoglobulin receptor is required for the serosal-to-mucosal transport of dimeric IgA and pentameric IgM in various epithelial cells(1, 2) . The specific trafficking of the mannose 6-phosphate receptor between the Golgi apparatus and late endosomes is necessary for transport of many enzymes to lysosomes(3, 4) . Low density lipoprotein and transferrin receptors cycle between coated pits and endosomes to deliver nutrients to the cell interior (5, 6, 7) . Localization of these receptors to their correct intracellular destination occurs through the interaction of specific receptor targeting domains with components of the cellular trafficking machinery. Mutations in these targeting domains result in disruption of receptor distribution which can lead to disease(8) .
Most of the progress in identification and
functional analysis of targeting domains has been achieved using
receptors constitutively found in discrete cellular locations.
Disruption of specific sequences in these receptors by site-directed
mutagenesis leads to an altered cellular distribution of the mutated
gene product(2, 8, 9) . A different situation
is presented by signaling receptors, such as EGFR ()or
insulin receptors. Their cellular distribution is highly dependent on
both ligand occupancy and activation of intrinsic receptor tyrosine
kinase activity(10, 11) .
Efficient EGFR internalization also requires the presence of specific sequences in the receptor carboxyl terminus. This region contains at least three endocytic domains that appear analogous to those found in constitutively internalized receptors. Endocytic motifs from transferrin and insulin receptors can be substituted for the endogenous EGFR sequences, but they work only if the resulting hybrid receptor displays intrinsic tyrosine kinase activity(12) . Lysosomal targeting of EGFR also requires ligand occupancy, but appears to depend on sequences distinct from those involved in occupancy-induced endocytosis (13) . This indicates that multiple domains are involved in regulating the EGFR trafficking pattern. The eventual consequence of ligand-induced internalization and lysosomal targeting is receptor down-regulation. This appears to play an important role in attenuating receptor signaling(14, 15) , but the relative importance of induced internalization versus lysosomal targeting to overall EGFR down-regulation is still unclear. It has been proposed that receptor degradation is primarily regulated by endocytosis, which controls the size of the intracellular receptor pool targeted for degradation(10) . Alternately, it has been suggested that interruption of receptor recycling is the primary mechanism that regulates receptor degradation(16) .
Although some of the structural requirements for EGFR down-regulation are known, it is unclear why tyrosine kinase activity is also necessary. Self-phosphorylation of the EGFR appears to result in a more open conformation, which has been proposed to expose trafficking domains(17) . Tyrosine phosphorylation must also work indirectly, however, because mutant EGFR that lack autophosphorylation sites but retain endocytic sequences can still undergo ligand-induced internalization(10, 12, 18) . It has been proposed that the indirect mechanism involves the formation of a complex between the receptor and a phosphorylated substrate. Formation of this complex would in turn expose endocytic domains in the EGFR(10, 12) . An alternate hypothesis has been advanced in which EGFR endocytosis is kinase-independent. Instead, ligand-induced kinase activity is proposed to prevent recycling of the receptor back to the cell surface by inducing inward vesiculation of multivesicular bodies(16, 19, 20) .
Unfortunately, because regulated trafficking of signaling receptors
depends on their state of activity, it is difficult to study this
process directly. Any mutation which alters the enzymatic activity of
receptors will also indirectly affect their intracellular trafficking.
For example, phosphorylation of the EGFR by protein kinase C at
Thr completely blocks ligand-induced internalization, but
this is due to inhibition of receptor kinase activation and not
interference with endocytosis per se(21) . In an
effort to circumvent the pleiotropic effects of kinase activity on
receptor trafficking behavior, we created mutant EGFR in which the
conserved tyrosine kinase domain was deleted. We show here that such
mutants undergo rapid internalization and lysosomal targeting, even in
the absence of ligand binding. The constitutive down-regulation of
these mutant receptors is absolutely dependent upon specific sequences
in the carboxyl terminus. These sequences can be segregated into a
class required for rapid internalization and a class required for
lysosomal targeting. Our results indicate that receptor down-regulation
is regulated at two distinct steps and that tyrosine kinase activity
works by stabilizing or amplifying receptor conformational changes
induced by ligand occupancy.
Recycling was determined by incubating cells for 20
min at 37 °C with I-EGF. The cells were rapidly
rinsed at 0 °C followed by removal of approximately 90%
surface-associated ligand with stripping buffer lacking urea. The cells
were rinsed two times with phosphate-buffered saline buffer and
returned to 37 °C by the addition of prewarmed medium containing 1
µg/ml unlabeled EGF to prevent rebinding of dissociated
ligand(31, 32) . At different times, the cells were
rinsed and the relative amount of label associated with either the
surface or inside of the cells was determined by acid
stripping(30) . As measured by release of radiolabeled
monoiodotyrosine (33) , no degradation of the internalized
antibody occurred during the incubation period.
Figure 1:
Relative internalization of wild type
and mutant EGF receptors in both occupied and empty states. Cells
expressing the indicated receptor types were incubated for 3 h at 0
°C with I-labeled Fab fragment of the anti-EGFR
monoclonal antibody 13A9 in either the absence (
) or presence
(
) of 0.5 µg/ml unlabeled EGF. Cells were then brought to 37
°C for the indicated times. Shown is the percent of initially bound
antibody which was internalized as determined by acid
stripping.
Figure 2:
Constitutive lysosomal targeting of
KD-EGFR. Cells expressing the receptors indicated in the
right-hand labels were treated for 18 h either without (Control) or with (+EGF) 125 ng/ml EGF. Cells
were then fixed, permeabilized, and stained for EGFR (left
panels) and the lysosomal protein lgp120 (right panels).
The two labels were simultaneously visualized. Arrows in the middle panels are for reference.
Figure 3:
Construction of KD-EGFR retaining
varying parts of the endocytic regulatory domains. A,
schematic of EGFR showing the location of the previously identified
endocytic domains. Numbers refer to amino acid residues in
human EGFR. Each of the indicated sequences was fused onto a c`688 EGFR
and expressed in B82 cells. B, relative levels of mRNA
encoding the different
KD-EGFR. Amino acids in the domain fused to
residue 688 are indicated above each lane. The ratios of the
KD-EGFR to
-actin bands as determined by direct counting is
indicated beneath each band.
The specific
internalization rates of the different KD-EGFR mutants were
determined and compared to the internalization of the holo-EGFR at both
low and high occupancies. As shown in Fig. 4, all fusion
receptors displayed higher specific internalization rates than the
c`688 receptor. The relative activity of the endocytic domains was III
> I > II, which is the same in the context of kinase-active
receptor mutants(12) . Internalization rates for each of the
KD-EGFR mutants were similar at both low and high ligand
concentrations, indicating that internalization is constitutive (data
not shown). There was no evidence of synergism between the different
endocytic domains, similar to the situation observed in the analogous
kinase-active receptor mutants(12) . Because internalization
rates of the
KD-EGFR mutants are constitutive, they should be the
same as fully occupied holo-EGFR, assuming the rate-limiting step of
the two receptor types is the same. As shown in Fig. 4, the
specific internalization rate of fully occupied holo-EGFR (0.084
min
) is similar to those displayed by the
KD-EGFR mutants, with the exception of the c`688 f1022-1186
receptor which is internalized much faster. These data show that all
EGFR endocytic domains are functional when the kinase domain is
deleted. However, the higher internalization rate of the c`688
f1022-1186 receptor indicates that the rate-limiting steps for
internalization of the kinase-active and
KD-EGFR mutants are not
necessarily the same.
Figure 4:
Specific internalization rates of
different KD-EGFR mutants. The specific internalization rate of
EGF was determined using cells expressing the indicated
KD-EGFR
using a concentration of 1 ng/ml EGF. Internalization of wild type
receptors was measured at either 1 or 110 ng/ml as indicated in the
figure. The number of independent experiments is shown in parentheses. Error bars indicate
S.D.
To confirm that internalization of the
different KD-EGFR mutants was independent of their state of
occupancy, cells expressing the receptors were incubated with either
Ilabeled EGF or
I-labeled 528
EGFR
IgG. As shown in Fig. 5, the steady-state distribution of
labeled receptor between the cell surface and inside was similar in the
case of either the activating ligand or nonactivating monoclonal
antibody. The accumulation of labeled antibody was more pronounced than
EGF, possibly because of differences between receptor dissociation or
lysosomal degradation.
Figure 5:
Approach to steady state binding of EGF
and anti-EGFR antibodies to cells expressing different KD-EGFR.
Cells expressing the
KD-EGFR mutants c`688
(
-
), c`688 f945-1186
(
-
), c`688 f1022-1186
(
-
), c`688 f945-991
(
-
), c`688 f945-1022
(
-
), or c`688 f993-1022 (x-x)
were incubated with either 10 ng/ml
I-EGF (top
panel) or 200 ng/ml
I-labeled 528 monoclonal Ab (bottom panel). At the indicated times, the ratio of label
associated with the inside to surface of the cells was determined by
acid stripping.
We have previously shown that the
carboxyl terminus of EGFR contains sequences which appear involved in
endosomal retention and lysosomal targeting(13, 32) .
To determine whether the ability of some of the KD-EGFR mutants to
efficiently accumulate ligand was due to the presence of these
retention sequences, rates of recycling were directly compared. Cells
were incubated for 20 min with
I-EGF. Surface-associated
ligand was removed with a mild acid treatment, and the cells were then
chased with excess unlabeled ligand. As shown in Fig. 6, EGF
bound to
KD-EGFR containing only endocytic domains II or III
recycled with a k
of 0.18-0.19
min
and to an extent of 90% (
= 0.9).
EGF was recycled both more slowly and to a lesser extent when bound to
the
KD-EGFR mutant that contained the entire regulatory
cytoplasmic domain (k
= 0.14
min
and
= 0.7). The
KD-EGFR
containing only endocytic domain I also displayed slower recycling
rates and greater endosomal retention (k
=
0.15 min
and
= 0.8), although not to
the extent of the receptor containing all three endocytic domains.
These results suggest that endosomal retention of EGFR is facilitated
by sequences located in the region between residues 945 and 991.
Figure 6:
Recycling of 125I-EGF associated with
different KD-EGFR. Cells expressing c`688 f945-1186 (I
+ II+ III) (
-
), c`688
f1022-1186 (III) (
-
), c`688
f993-1022 (II) (x-x), or c`688 f945-991 (I)
(
-
) receptors were incubated for 20 min at 37
°C with
I-EGF. Surface-associated ligand was removed
at 0 °C followed by a chase at 37 °C. The amount of
I-EGF remaining inside the cells at the indicated times
was determined and expressed as a percent of the intracellular ligand
prior to the chase. The curves through the data points were
calculated by nonlinear regression.
Although there was a correlation between decreased recycling of EGF
and enhanced accumulation of ligand, absolute differences in recycling
between the KD-EGFR mutants were relatively small. However,
steady-state ligand accumulation required incubation times of greater
than 1 h (see Fig. 5). The t
of EGF
recycling was 5 to 10 min, meaning that multiple rounds of
internalization and recycling would occur, amplifying small differences
between receptors. Because the small differences in absolute recycling
rates made it difficult to use this parameter to reliably evaluate
receptor behavior, we developed a more robust assay based on specific
protein/mRNA ratios.
For any given protein, the ratio of mRNA to
protein mass is directly proportional to turnover rates(44) .
Because lysosomal targeting of receptors is required for their
degradation, increases in the rate of lysosomal targeting between two
receptor mutants will decrease their relative protein/mRNA ratios. To
determine the levels of EGFR mRNA in transfected cells, a ribonuclease
protection assay was used with -actin as an internal standard.
Receptor protein mass was determined by quantitative
immunoprecipitation of
S-labeled cell extracts. As shown
in Table 1, the constitutive protein/mRNA ratio was similar
between unoccupied holo- and c`688 EGFR.
KD-EGFR mutants
containing only endocytic domains II or III (between residues 993 and
1186) also displayed protein/mRNA ratios similar to unoccupied wild
type receptors. However, all
KD-EGFR containing endocytic domain I
displayed significantly lower protein/mRNA ratios, indicating a higher
constitutive turnover rate. The protein/mRNA ratio of the
KD-EGFR
containing the entire carboxyl-terminal regulatory region was only
about 1/3 of that displayed by unoccupied holo-EGFR. Significantly, the
addition of a saturating amount of EGF to cells expressing holo-EGFR
decreased their steady state receptor mass to 25-35% of the
initial value (data not shown). These data indicate that turnover of
KD-EGFR that contain the entire regulatory carboxyl terminus is
similar to occupied, kinase-active holoreceptors.
To demonstrate
that lower protein/mRNA ratios of some KD-EGFR mutants were due to
accelerated turnover, this parameter was measured directly. Cells
expressing either wild type EGFR or
KD-EGFR displaying either low
or normal protein/mRNA ratios were labeled with
S-amino
acids and then chased in either the absence or presence of saturating
concentrations of EGF. The turnover of the receptors was then
determined by immunoprecipitation. As shown in Fig. 7, in the
absence of EGF, the turnover of wild type receptors was similar to the
KD-EGFR mutant containing only endocytic domain III (25-28
h). In contrast, the turnover of the full-length
KD-EGFR
containing the entire carboxyl terminus (I + II + III) was
significantly faster (11 h). In the presence of EGF, turnover of wild
type holoreceptors was accelerated to 10 h, whereas turnover of the
KD-EGFR containing the entire c` terminus or the III domain alone
was unaffected. These data establish that the rate of degradation of
KD-EGFR (c`688 f945-1186) is the same as fully occupied wild
type EGFR. In addition, this enhanced degradation requires specific
sequences within the carboxyl region of the receptor.
Figure 7:
Turnover of wild type and KD-EGFR
mutants in the absence and presence of EGF. Cells expressing wild type
(
-
), c`688 f945-1186
(
-
), or c`688 f1022-1186
(
-
) receptors were metabolically labeled with
S-amino acids and then chased in unlabeled medium in the
absence (upper panel) or the presence (lower panel)
of 1 µg/ml EGF. The amount of label remaining in the receptors was
determined at the indicated times by immunoprecipitation followed by
quantitation using a Molecular Imager. The results are the average of
four separate experiments ± S.E.
Figure 8:
Lack of tyrosine phosphorylation of
KD-EGFR mutants. A, Western blot analysis of extracts of
cells expressing the
KD-EGFR mutants c`688 f945-1022 (A), c`688 f945-991 (B), c`688 f993-1022 (C), c`688 f1022-1186 (D), or c`688
f945-1186 (E). Equal amounts of extract were run on
5-10% gradient SDS gels and transferred to nitrocellulose. The
membranes were then probed with polyclonal antibodies against either
the EGFR (top panel) or phosphotyrosine (bottom
panel) followed by
I-labeled Protein A. The images
were obtained directly from a Molecular Imager. B, Western
blot analysis of tyrosine-phosphorylated proteins in cells expressing
the
KD-EGFR mutants that were either treated without(-) or
with (+) 200 ng/ml EGF for 5 min at 37 °C.
Tyrosine-phosphorylated proteins were visualized as described in panel A. Shown is an autoradiograph of the
gel.
Receptors involved in signal transduction typically display regulated cellular trafficking. Mutations which inactivate the signaling capacity of these receptors usually abrogate occupancy-induced changes in receptor internalization or recycling(11, 45, 46, 47) . The reasons for this have not been clear. In the case of EGFR or insulin receptors, point mutations which inactivate tyrosine kinase activity eliminate ligand-induced endocytosis(27, 48) . Mutants in which tyrosine phosphorylation sites are replaced with phenylalanine residues also lack the ability to undergo ligand-induced internalization(49, 50) . One effect of self-phosphorylation, however, is a conformational change of the receptor(17) . SH2 domain-containing proteins subsequently bind to the occupied, activated receptors, perhaps stabilizing or enhancing conformational changes(51) . Thus, it has been unclear whether tyrosine phosphorylation per se is required for ligand-induced endocytosis or whether consequent conformational changes in the receptor are responsible.
The results of the current study suggest
that an important component of ligand-induced internalization is a
conformational change in the receptor. Removal of the conserved
tyrosine kinase domain of EGFR results in the constitutive
internalization and lysosomal targeting of the receptor. This indicates
that sequences mediating endocytosis and lysosomal targeting normally
exist in a cryptic state and are exposed upon receptor activation. It
is possible that the altered trafficking of the mutant receptors we
studied was mediated by sequences that do not normally serve that
function in the holoreceptor, but this appears unlikely for several
reasons. First, the specific internalization rate of the KD-EGFR
containing the entire regulatory carboxyl terminus was the same as that
of the fully occupied holo-EGFR (Fig. 3). Second, the cellular
distribution, protein/mRNA ratios, and receptor turnover rates of
KD-EGFR and occupied holo-EGFR were the same. Finally, endocytic
and lysosomal targeting sequences mapped to the same receptor regions
in both
KD-EGFR and holoreceptors. It seems most likely that the
similar behavior of occupied holo- and
KD-EGFR in these three
independent parameters is due to their similar functional states.
It
has been suggested previously that receptor kinase activity is not
required for ligand-induced internalization of the
EGFR(16, 19) . By using a very sensitive assay for
internalization of both empty and occupied receptors, we found that
ligand occupancy of kinase-inactive Met EGFR does have a
small stimulatory effect on endocytosis (Fig. 1). This positive
effect of receptor occupancy was also seen for
KD-EGFR that
retained endocytic sequences. It thus appears that ligand occupancy per se promotes a receptor conformation that is more favorable
for its interaction with the endocytic apparatus. Nevertheless,
kinase-active receptors displayed a 5-10-fold greater stimulation
of endocytosis. We have previously shown that kinase-dependent
endocytosis is both specific and saturable and have postulated that the
responsible rate-limiting ``internalization component'' is a
substrate for the receptor kinase
activity(30, 52, 53) . Interestingly,
endocytosis of
KD-EGFR does not appear to be saturable, indicating
that these mutations bypass the normal rate-limiting step for EGFR
endocytosis. If this is the case, then receptor kinase activity could
work by phosphorylating the internalization component, which then could
bind to the EGFR and induce a conformation change sufficient to expose
the endocytic codes. The previously described high affinity saturable
endocytosis would therefore be due to the high affinity binding of the
internalization component to the EGFR. The primary role of receptor
kinase activity would thus be to amplify the conformational change
induced by receptor occupancy to expose sequences necessary for
regulated receptor trafficking.
A significant finding in this study
was that rapid internalization was not sufficient for accelerated
turnover of EGFR. Even though c`688 f1022-1186 EGFR displayed
internalization rates almost 10-fold higher than either c`688 or
unoccupied holo-EGFR, the turnover rate of these receptors was the
same. Thus, the flux of receptors through the endocytic pathway is not
normally rate-limiting to receptor turnover and suggests that
ligand-induced endocytosis per se is not sufficient to lead to
receptor down-regulation. Sequences between 945 and 991 appear to be
necessary to direct lysosomal targeting. Because antibodies bound to
receptors containing these sequences were degraded at an accelerated
rate, this targeting must occur from the endocytic pathway.
KD-EGFR containing the entire regulatory carboxyl terminus were
targeted more efficiently to lysosomes than those containing only
sequences between 945 and 991 (region I). This could indicate the
presence of additional lysosomal targeting sequences outside of region
I. Alternatively, the conformation of the
KD-EGFR containing the
entire regulatory carboxyl terminus could be more conducive to
interacting with the postendocytic trafficking machinery. Nevertheless,
receptors lacking region I displayed the same turnover rates and
mRNA/protein ratios as empty holoreceptors. This indicates that region
I is required for accelerated turnover of EGFR.
The constitutive
turnover rate of the EGFR in B82 cells (25-28 h) is somewhat
slower than normal fibroblasts (10 h(54) ), but is similar
to the range displayed by A431, NA, and Ca9-22 cells (15-23
h(55) ) and is somewhat faster than the rate observed in UCVA-1
cells (
37 h (55) ). The factors regulating the absolute
rate of EGFR turnover in a given cell type are not understood, but
could involve the net flux of membrane through the endocytic pathway as
well as specific components that target receptors to lysosomes. We do
know that the relatively slow turnover of the EGFR we observed was cell
type-dependent because human EGFR expressed in CHO cells displayed a
half-life of less than 10 h (data not shown). Despite the slow absolute
rates of EGFR degradation observed in B82 cells, the mechanisms
responsible for ligand-dependent lysosomal targeting appear to be
intact. For example, EGF treatment reduces total steady state EGFR mass
in B82 cells by 65-72% as compared to 76-83% in normal
human fibroblasts and 60% in normal human mammary epithelial cells. (
)Furthermore, removal of all cytoplasmic sequences in the
EGFR distal to c`647 results in an increase in receptor half-life to
approximately 60 h (data not shown). This indicates that B82 cells
retain components which regulate both the constitutive as well as
ligand-induced turnover of the EGFR.
Previously, we demonstrated
that kinase-active EGFR truncated to residue 973 displayed specific and
saturable lysosomal targeting(32) . This indicates that
sequences between 945 and 973 mediate occupancy-induced lysosomal
targeting of EGFR. Receptors truncated to residue 958 have also been
shown to be transferred to lysosomes(13) . Because the minimal
sequence required for accelerated degradation in constitutively active
KD-EGFR was between residues 945 and 991, the active sequences are
most likely located between residues 945 and 958. The sequence YLVI,
found at residues 954-958 in EGFR, has been proposed to be a
lysosomal targeting sequence for lysosomal-associated membrane protein
1(56) . It resembles the tyrosine-containing and di-leucine
motifs previously identified as potential mediators of lysosomal
targeting(57, 58, 59) , but involvement of
this sequence in postendocytic trafficking of EGFR will require more
direct analysis.
It has been proposed that tyrosine kinase activity prevents receptor recycling and directs lysosomal targeting of ligand-activated internalized EGFR(16, 19, 20, 60) . Our data, and results from other investigators, do not support this hypothesis(10, 32, 49, 61, 62) . Instead, it appears more likely that the occupancy-induced exposure of specific lysosomal targeting domains is the mechanism leading to endosomal retention and enhanced lysosomal targeting of EGFR. It has been postulated that occupancy-induced ubiquitination of platelet-derived growth factor receptor is involved in enhanced degradation following ligand binding(63) . We have examined immunoprecipitated EGFR for ubiquitination in either the empty or occupied state by Western blot analysis, but have found no evidence for this covalent modification (data not shown). Because, the degradation rate of the platelet-derived growth factor receptor is almost 10-fold greater than that of the EGFR(47, 63) , ubiquitination may be involved in an alternate pathway of very rapid turnover of some receptor species.
It appears that occupancy-induced receptor
down-regulation is a two-step process of internalization followed by
lysosomal targeting. It has been suggested that common sequence motifs
are responsible for both internalization and lysosomal targeting of
some proteins, such as CD3 (57) . Other proteins, like
P-selectin, have distinct endocytic and lysosomal targeting
domains(64) . The segregation of the regulatory
carboxyl-terminal domain of KD-EGFR into regions required for
internalization and lysosomal targeting indicates that distinct
sequences mediate these two processes as well. Because
KD-EGFR
mutants appear to contain all of the information necessary to specify
normal postendocytic compartmentation, occupied EGFR appear to be
handled by the endocytic machinery in the same fashion as other
constitutively internalized or lysosomally targeted receptors.