|
|
|
|||||||
|
|||||||||
(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 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 ( 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
Recycling was determined by incubating cells for 20
min at 37 °C with
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
Figure 2:
Constitutive lysosomal targeting of
Figure 3:
Construction of
The specific
internalization rates of the different
Figure 4:
Specific internalization rates of
different
To confirm that internalization of the
different
Figure 5:
Approach to steady state binding of EGF
and anti-EGFR antibodies to cells expressing different
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
Figure 6:
Recycling of 125I-EGF associated with
different
Although there was a correlation between decreased recycling of EGF
and enhanced accumulation of ligand, absolute differences in recycling
between the 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
To demonstrate
that lower protein/mRNA ratios of some
Figure 7:
Turnover of wild type and
Figure 8:
Lack of tyrosine phosphorylation of
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 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 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.
The constitutive
turnover rate of the EGFR in B82 cells (25-28 h) is somewhat
slower than normal fibroblasts ( 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
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
Volume 270,
Number 9,
Issue of March 3, 1995 pp. 4325-4333
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) 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.
)or
insulin receptors. Their cellular distribution is highly dependent on
both ligand occupancy and activation of intrinsic receptor tyrosine
kinase activity(10, 11) .
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.
General
Mouse EGF was purified from submaxillary
glands (22) . EGF and monoclonal antihuman EGFR antibody 528
IgG (23) or Fab fragments of 13A9 IgG (24) were
iodinated with 
I (Amersham) using IODOBEADS (Pierce)
according to the manufacturer's recommendations. Free iodine was
separated from the radiolabeled ligands by dialysis or by passing the
mixture over a 0.8 
20 cm column of Sephadex G-10 equilibrated
with phosphate-buffered saline. The specific activity of I-labeled EGF was generally between 600 and 1,800
cpm/fmol, and I-labeled monoclonal antibodies was between
1,300 and 1,900 cpm/fmol. Fab fragments and intact anti-EGFR monoclonal
13A9 (24) were kind gifts from Marjorie Winkler of Genentech,
Inc. Monoclonal antibodies against human EGFR (528, 579, and
225(23) ) were purified from hybridomas obtained from the
American Type Culture Collection. Polyclonal rabbit antibody N-13
directed against a peptide corresponding to residues 1-13 in
human EGFR was a gift of Dr. Debora Cadena. Polyclonal rabbit
antibodies specific for phosphotyrosine were generated and
affinity-purified as described(25) . Polyclonal rabbit antibody
against the lysosomal marker lgp120 was a gift of Dr. Marilyn
Farquhar(26) . Secondary antibodies labeled with either Texas
Red or fluorescein were obtained from Cappel Laboratories.Construction and Expression of Mutant EGFR
c`688
EGFR cDNA was prepared by partial EcoRI digestion as described
previously(27) . c`688 f945-1022 and c`688
f945-1186 receptor cDNAs were generated by digestion of pX EGFR (28) with SmaI and HindIII and ligation of SmaI-HindIII fragments and EcoRI and HindIII fragments from pX EGFR or pX c`1022 EGFR(27) .
c`688 f945-991, c`688 f993-1022, and c`688 f1024-1186
EGFR cDNAs were prepared by excising the fusion segments as SalI to HindIII fragments from the appropriate mutant
EGFR (18) and ligating them to c`688 EGFR containing a
compatible linker oligonucleotide. Re-establishment of the SalI site places codons for V and D at the junction. All
constructions were verified by dideoxynucleotide sequencing. cDNAs were
transfected into B82L cells that lack endogenous EGFR by the calcium
phosphate procedure(29) . Stable clonal transfectants were
selected, and the EGFR gene was amplified by increasing concentrations
of methotrexate from 400 nM to 5 µM. At least two
independent transfections were used to select each cell line expressing
mutant EGFR. B82 cells were grown in Dulbecco's modified
Eagle's medium (Flow Laboratories) containing dialyzed 10% calf
serum (HyClone) and 5 µM methotrexate.Internalization and Recycling
Measurements
Internalization of both empty and occupied EGFR was
determined by first incubating the cells for 3 h at 0 °C with 0.5
µg/ml I-labeled 13A9 Fab fragment in either the
absence or presence of 0.5 µg/ml EGF. The cells were rinsed and
rapidly warmed to 37 °C by the addition of medium either without or
with EGF. Internalization of EGF alone was measured by changing to
medium containing the indicated concentrations of I-EGF
at 37 °C. The relative amounts of ligand associated with the
surface and interior of the cells were determined by acid-stripping at
0 °C using 50 mM glycine-HCl, 100 mM NaCl, 2
mg/ml polyvinylpyrrolidone, 2 M urea, pH 3.0(30) .
Nonspecific binding was measured using B82 cells lacking EGFR and was
less than 5% of total binding. Cell number was determined with a
Coulter counter.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.Quantification of EGFR Levels
Cells were incubated
at 37 °C for 24 h in 30 µCi/ml of Tran
S-label in
Dulbecco's modified Eagle's medium containing a 10% normal
concentration of methionine and cysteine. Cells were rinsed and removed
from the plates at 0 °C by scraping into 1.5 ml of D/H/B medium
containing a mixture of protease inhibitors (10 µg/ml concentration
each of chymostatin, pepstatin, leupeptin, aprotinin, and 4 mM iodoacetate(34, 35) ). The cells were gently
pelleted at 1000 g, rinsed once with
phosphate-buffered saline, and extracted for 10 min at 0 °C with
120 µl of 1% Triton X-100, 10% glycerol, 2 mM EDTA, 10
mM HEPES, pH 7.0, and a mixture of protease inhibitors (as
above but using a 10-fold greater concentration). Cell debris was
removed by centrifugation (10,000 g for 10 min), and
the rest of the cell extracts were snap-frozen in a methanol/dry ice
bath. After thawing, 2-µl aliquots were removed from each extract
sample and spotted onto Whatman 42 filter paper in a grid fashion 1.5
cm apart. The specific activity of the cellular protein was determined
by washing the filter paper with cold 7.5% trichloroacetic acid and
staining for protein by the method of Bramhall et
al.(36) . The protein-associated S on the
filter paper was first quantified by using the Bio-Rad G250 Molecular
Imager phosphorimager. Subsequently, the stained filter paper was cut
apart and the amount of protein in each stained spot was quantified by
eluting the dye with 66% methanol, 1% NH
OH and measuring
the absorbance at 610 nm. EGFR was then immunoprecipitated using 150
µg/ml of both monoclonal 528 IgG and rabbit anti-mouse secondary
antibody, and 20 µl of a 50% slurry of Protein A-Sepharose as
described previously(23, 37) . The final
immunoprecipitate was solubilized in 25 µl of 2% SDS, 20 mM dithiothreitol at 100 °C and run on a 5-15% gradient
polyacrylamide gel. The gel was dried and the amount of radioactivity
in the receptor bands was quantified using the G250 Molecular Imager.
The relative amount of receptor radioactivity was corrected for both
specific activity and total amount of cellular protein, the
methionine/cysteine levels of the receptors as well as the fractional
labeling of the receptors using 15 h as the doubling time of the cells. Quantification of EGFR mRNA Levels
A ribonuclease
protection assay (Ambion RPA II kit) was used to quantify the levels of
both wild type and mutant EGFR in transfected cells. The EGFR probe
complements a 350-bp region encoding amino acids 613 to 730 in
holo-EGFR(38) . It was isolated as a NaeI/BamHI fragment and inserted into the BamHI/HindIII sites of BlueScript (Stratagene). The 
P-labeled antisense probe was transcribed from the T7
promoter essentially as described (39) . The 150-bp 
-actin
probe was generated from the plasmid obtained from Ambion, Inc., using
SP6 polymerase after linearizing with DdeI and using a 3-fold
lower specific activity of [P]UTP. All probes
were gel-purified before use. Total cellular RNA was isolated from
confluent 100-mm plates(40) . Mixtures of the EGFR and
-actin probes were hybridized overnight with 5 µg of total
cellular RNA followed by ribonuclease digestion using the
manufacturer's instructions. The protected RNA was precipitated,
denatured, and separated on a 5% polyacrylamide, 8 M urea gel
as described in the Ambion kit. The gels were dried, and the protected
probe was quantified using the Bio-Rad G250 Molecular Imager. The mRNA
from the mutant EGFR constructs protected approximately 225 bp of the
EGFR probe while the wild type EGFR mRNA protected the entire 350-bp
probe. For quantification, the amount of radioactivity protected was
corrected for the length of the probe and its specific activity. The
amount of EGFR mRNA was expressed as a ratio to the internal
-actin standard and normalized to the amount expressed in cells
transfected with c`688 EGFR. Two separate experiments using each cell
type were done in triplicate.Tyrosine Phosphorylation of Cellular
Proteins
Cells expressing different EGFR mutants were incubated
in the presence or absence of 200 ng/ml EGF at 37 °C. The cells
were rapidly rinsed at 0 °C, removed from the plates with a rubber
policeman, and extracted with the Triton X-100 solution described above
for immunoprecipitation, with the addition of 0.1 mM Na
VO(25) . After 5 min at 0
°C, cell debris was removed by centrifugation at 10,000 g for 3 min. Extracts were denatured at 100 °C by the
addition of an equal volume of 2% SDS and 12 mM dithiothreitol. Protein concentrations were determined by the
method of Bramhall(36) . Samples were separated on 5-10%
gradient gels, transferred to nitrocellulose in the presence of 0.1
mM NaVO, and blocked with 2.5% bovine
serum albumin, 0.1 mM NaVO, and 0.005%
Tween 20. Tyrosine phosphate was detected using 5 µg/ml
affinity-purified antiphosphotyrosine polyclonal
antibodies(25) . EGFR were detected in parallel blots using N13
polyclonal antibodies. These were visualized using 50 ng/ml I-Protein A for 30 min followed by exposure to either
film or phosphorimager plates.Fluorescence Microscopy
Cells were plated on
fibronectin-coated coverslips 48 h before the experiment. Cells treated
either without or with EGF were fixed for 15 min with freshly prepared
3% paraformaldehyde, 0.02% glutaraldehyde and then permeabilized for 15
min with 0.0125% saponin(41) . Free aldehyde groups were
quenched with 0.1% NaBH for 10 min. Cells were incubated
simultaneously with a mixture of anti-EGFR monoclonals 528, 579, 225,
and 13A9 (10 µg/ml each) and anti-lgp 120 (1:500) for 45 min
followed by staining with fluorescein isothiocyanate-labeled goat
anti-mouse and Texas Red-labeled goat anti-rabbit IgG antibodies
(1:100) for 45 min. The coverslips were then dehydrated through an
ethanol series and mounted in benzyl alcohol:benzyl benzoate (1:2) and
viewed with a DAGE Sit-11 camera and DSP-200 image processor attached
to a Zeiss microscope with a 100 objective. Images were averaged
over 32 frames and captured using a Data Translation QuickCapture
board. Images were scaled to 256 gray levels using Adobe Photoshop 2.0
on the Macintosh.Data Analysis
Values for surface-bound and
internalized ligand were corrected for nonspecific binding and for
spillover from the interior and surface of the cell,
respectively(33) . Specific internalization rates were
determined by plotting the integral of surface-associated ligand
against the amount internalized(30) . The slopes were then
determined by linear regression. Correlation coefficients of
internalization plots were generally >0.98. Nonlinear parameter
estimation was done with the program proFit (QuantumSoft) on the
Macintosh using the Levenberg-Marquardt algorithm.
Removal of the Tyrosine Kinase Domain Enhances
Constitutive Endocytosis of EGFR
To determine the contribution
of sequences within the conserved tyrosine kinase domain to EGFR
trafficking behavior, we deleted these residues between 689 and
944(42) . The resulting receptors are termed 
KD-EGFR.
Because such a large deletion could affect the internalization of both
empty as well as ligand-occupied EGFR, we developed a method for
evaluating endocytosis which was independent of ligand binding. Fab
fragments of 
EGFR monoclonal Ab 13A9 were radiolabeled and bound
to cells in both the absence and presence of saturating concentrations
of EGF. This monoclonal Ab does not inhibit the binding of EGF to EGFR
nor does it affect receptor tyrosine kinase activity and thus can act
as a tag for both empty and occupied receptors(24) . After
warming the cells to 37 °C, the rate of antibody internalization
was determined. As shown in Fig. 1, antibody bound to wild type
EGFR was internalized much more rapidly when receptors were occupied.
Inactivation of receptor kinase activity by a point mutation in the ATP
binding site (Met
) virtually eliminated the increased
internalization rates induced by EGF binding. Deletion of
carboxyl-terminal sequences distal to residue 688 had no effect on
internalization of empty receptors, but eliminated the stimulatory
effect of ligand occupancy (Fig. 1, B and C).
In contrast, KD-EGFR were internalized rapidly in the absence of
EGF (Fig. 1D), but the addition of EGF still had a
small stimulatory effect, similar to that observed for Met receptors. Therefore, removal of the EGFR kinase domain appeared
to increase the constitutive rate of receptor endocytosis.
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.
Lysosomal Targeting of Receptors Lacking a Tyrosine
Kinase Domain
It has been proposed that ligand-induced
internalization should always lead to enhanced lysosomal targeting and
down-regulation of receptors(10, 43) . Because the
endocytic rate of KD-EGFR appeared to be higher than empty EGFR,
we also determined whether these receptors displayed enhanced targeting
to lysosomes. Cells were treated for 18 h either without or with 125
ng/ml EGF, fixed, permeabilized, and stained for EGFR as well as the
lysosomal marker lgp120(26) . We found that in the absence of
EGF, wild type EGFR were predominantly distributed at the cell surface (Fig. 2, top left). As expected, incubation with
saturating EGF concentrations induced a steady-state redistribution of
wild type EGFR to primarily lysosomes and endosomes. In contrast,
KD-EGFR were located in lysosomes and endosomes in both the
absence and presence of ligand (Fig. 2, middle panels).
Receptors that lack sequences carboxyl-terminal to the kinase domain
(c`688) remained localized to the cell surface, even in the presence of
EGF (Fig. 2, bottom panels). Thus, deletion of the
kinase domain not only resulted in the constitutive internalization of
EGFR, but also constitutive targeting to lysosomes.
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.
Multiple Domains Can Mediate Constitutive
Internalization
Previous studies indicated the existence of at
least three different endocytic motifs distributed within the carboxyl
terminus of the EGFR(12) . The ability of these sequences to
mediate internalization requires receptor kinase activity. Kinase
activation changes the receptor conformation, which could expose
previously cryptic endocytic domains(17) . Deletion of the
kinase domain could effect a similar change in receptor conformation.
If this is the case, then the previously identified endocytic domains
should function in the context of a kinase deletion mutant. To test
this hypothesis, we created EGFR mutants in which combinations of the
different endocytic domains were fused to c`688 EGFR (Fig. 3, top panel). Mouse B82 cells were stably transfected with
plasmids encoding these receptor constructs, and clones were selected
which expressed comparable levels of receptor mRNA (Fig. 3, bottom panel). These clonal lines were then used to evaluate
the trafficking behavior of the mutant EGFR.
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.
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.
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.
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.
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.
Altered Recycling and Turnover of Constitutively Active
Receptors
Although the results shown in Fig. 5confirm
that the endocytic behavior of KD-EGFR was constitutive, the rate
of accumulation of either antibody or EGF over a 2-h period did not
directly correlate with the specific internalization rate of the
receptor. For example, receptors containing only endocytic domain III
were internalized almost 3-fold faster than those containing domains I
+ II + III (Fig. 4). EGF bound to either of these
receptors, however, was accumulated to the same extent after 1 h.
Because intracellular accumulation of ligand depends on rates of
internalization, recycling, and degradation, this result suggested that
the KD-EGFR mutants differed in their ability to interact with
multiple components of the cellular trafficking machinery. Consistent
with this is the observation that the degradation rate of internalized
radiolabeled Fab fragments of Ab 13A9 bound to receptors containing
only endocytic domain III was 20% as fast as fragments bound to
receptors containing all three (1.9% versus 9.4% per h,
respectively; data not shown).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.
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.
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.-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.
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.
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.
Constitutively Active Receptors Are Not Phosphorylated on
Tyrosine Residues
Under normal circumstances, the intrinsic
tyrosine kinase activity of EGFR is required for ligand-induced
internalization (10) . Although the constitutively active
KD-EGFR lack a kinase domain, it was still possible that these
receptors could be phosphorylated by other cellular tyrosine kinases.
To test for this possibility, extracts of cells expressing either wild
type or mutant EGFR were separated by gel electrophoresis. Receptors
and phosphotyrosine-containing proteins were then detected by Western
blot analysis using appropriate antibodies. As shown in Fig. 8A, no phosphotyrosine-containing proteins were
observed that corresponded to the positions of the mutant EGF
receptors. No change was observed upon receptor occupancy. Whereas the
addition of EGF to cells expressing holo-EGFR resulted in significant
receptor autophosphorylation, EGF had no effect on any KD-EGFR. In
addition, occupancy of KD-EGFR did not discernibly change the
phosphorylation state of other cell proteins. These data demonstrate
that the constitutive trafficking activities of the KD-EGFR do not
directly involve activated tyrosine kinases.
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.
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. 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.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.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.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.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.
)KD-EGFR, EGFR
lacking the tyrosine kinase domain (residues 689-944); k
, recycling rate constant; ,
fraction of recycled ligand; bp, base pair(s); Ab, antibody.)
We thank Marjorie Winkler for the gift of the 13A9
antibody. We also thank Douglas Lauffenburger and Rebecca Worthylake
for many helpful discussions regarding this manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Pennock and Z. Wang A Tale of Two Cbls: Interplay of c-Cbl and Cbl-b in Epidermal Growth Factor Receptor Downregulation Mol. Cell. Biol., May 1, 2008; 28(9): 3020 - 3037. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Li, R. Zhao, X. Wu, Y. Sun, M. Yao, J. Li, Y. Xu, and J. Gu Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics FASEB J, December 1, 2005; 19(14): 1978 - 1985. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Tsacoumango, S. J. Kil, L. Ma, F. D. Sonnichsen, and C. Carlin A novel dileucine lysosomal-sorting-signal mediates intracellular EGF-receptor retention independently of protein ubiquitylation J. Cell Sci., September 1, 2005; 118(17): 3959 - 3971. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Baldys and A. E. Aust Role of Iron in Inactivation of Epidermal Growth Factor Receptor after Asbestos Treatment of Human Lung and Pleural Target Cells Am. J. Respir. Cell Mol. Biol., May 1, 2005; 32(5): 436 - 442. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. S. Lazar, C. M. Cresson, D. A. Lauffenburger, and G. N. Gill The Na+/H+ Exchanger Regulatory Factor Stabilizes Epidermal Growth Factor Receptors at the Cell Surface Mol. Biol. Cell, December 1, 2004; 15(12): 5470 - 5480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Stoorvogel, S. Kerstens, I. Fritzsche, J. C. d. Hartigh, R. Oud, M. A. G. van der Heyden, J. Voortman, and P. M. P. van Bergen en Henegouwen Sorting of Ligand-activated Epidermal Growth Factor Receptor to Lysosomes Requires Its Actin-binding Domain J. Biol. Chem., March 19, 2004; 279(12): 11562 - 11569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-Y. He, J.-L. Huang, J. B. Gentry, and C. F. Chignell Epidermal Growth Factor Receptor Down-regulation Induced by UVA in Human Keratinocytes Does Not Require the Receptor Kinase Activity J. Biol. Chem., October 24, 2003; 278(43): 42457 - 42465. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. S. Hendriks, L. K. Opresko, H. S. Wiley, and D. Lauffenburger Coregulation of Epidermal Growth Factor Receptor/Human Epidermal Growth Factor Receptor 2 (HER2) Levels and Locations: Quantitative Analysis of HER2 Overexpression Effects Cancer Res., March 1, 2003; 63(5): 1130 - 1137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Cheng, H. Huang, Z.-T. Zhang, E. Shapiro, A. Pellicer, T.-T. Sun, and X.-R. Wu Overexpression of Epidermal Growth Factor Receptor in Urothelium Elicits Urothelial Hyperplasia and Promotes Bladder Tumor Growth Cancer Res., July 15, 2002; 62(14): 4157 - 4163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. Seto, H. J. Bellen, and T. E. Lloyd When cell biology meets development: endocytic regulation of signaling pathways Genes & Dev., June 1, 2002; 16(11): 1314 - 1336. [Full Text] [PDF]
|
|
|
|
|
|
Q. Lin, C. G. Lo, R. A. Cerione, and W. Yang The Cdc42 Target ACK2 Interacts with Sorting Nexin 9 (SH3PX1) to Regulate Epidermal Growth Factor Receptor Degradation J. Biol. Chem., March 15, 2002; 277(12): 10134 - 10138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. DeWitt, J. Y. Dong, H. S. Wiley, and D. A. Lauffenburger Quantitative analysis of the EGF receptor autocrine system reveals cryptic regulation of cell response by ligand capture J. Cell Sci., March 8, 2002; 114(12): 2301 - 2313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. Hamm-Alvarez Focus on "EGF receptor downregulation depends on a trafficking motif in the distal tyrosine kinase domain" Am J Physiol Cell Physiol, March 1, 2002; 282(3): C417 - C419. [Full Text] [PDF]
|
|
|
|
|
|
M. P. Oksvold, H. S. Huitfeldt, A. C. Ostvold, and E. Skarpen UV induces tyrosine kinase-independent internalisation and endosome arrest of the EGF receptor J. Cell Sci., February 15, 2002; 115(4): 793 - 803. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Y. Ting, K. H. Kain, R. L. Klemke, and R. Y. Tsien Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells PNAS, December 18, 2001; 98(26): 15003 - 15008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Butowt and C. S. von Bartheld Sorting of Internalized Neurotrophins into an Endocytic Transcytosis Pathway via the Golgi System: Ultrastructural Analysis in Retinal Ganglion Cells J. Neurosci., November 15, 2001; 21(22): 8915 - 8930. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Burke, K. Schooler, and H. S. Wiley Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis and Intracellular Trafficking Mol. Biol. Cell, June 1, 2001; 12(6): 1897 - 1910. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Kurten, A. Eddington, P Chowdhury, R. Smith, A. Davidson, and B. Shank Self-assembly and binding of a sorting nexin to sorting endosomes J. Cell Sci., January 5, 2001; 114(9): 1743 - 1756. [Abstract] [PDF]
|
|
|
|
|
|
N. L. Lill, P. Douillard, R. A. Awwad, S. Ota, M. L. Lupher Jr., S. Miyake, N. Meissner-Lula, V. W. Hsu, and H. Band The Evolutionarily Conserved N-terminal Region of Cbl Is Sufficient to Enhance Down-regulation of the Epidermal Growth Factor Receptor J. Biol. Chem., January 7, 2000; 275(1): 367 - 377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Beckman, L. L. Lin, M. E. Quinones, and G. D. Longmore Activation of the Erythropoietin Receptor Is Not Required for Internalization of Bound Erythropoietin Blood, October 15, 1999; 94(8): 2667 - 2675. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. W. Nordeng and O. Bakke Overexpression of Proteins Containing Tyrosine- or Leucine-based Sorting Signals Affects Transferrin Receptor Trafficking J. Biol. Chem., July 23, 1999; 274(30): 21139 - 21148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Luton and K. E. Mostov Transduction of Basolateral-to-Apical Signals across Epithelial Cells: Ligand-stimulated Transcytosis of the Polymeric Immunoglobulin Receptor Requires Two Signals Mol. Biol. Cell, May 1, 1999; 10(5): 1409 - 1427. [Abstract] [Full Text]
|
|
|
|
|
|
R. Worthylake, L. K. Opresko, and H. S. Wiley ErbB-2 Amplification Inhibits Down-regulation and Induces Constitutive Activation of Both ErbB-2 and Epidermal Growth Factor Receptors J. Biol. Chem., March 26, 1999; 274(13): 8865 - 8874. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mukherjee, T. T. Soe, and F. R. Maxfield Endocytic Sorting of Lipid Analogues Differing Solely in the Chemistry of Their Hydrophobic Tails J. Cell Biol., March 22, 1999; 144(6): 1271 - 1284. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Bost, R. McKay, M. Bost, O. Potapova, N. M. Dean, and D. Mercola The Jun Kinase 2 Isoform Is Preferentially Required for Epidermal Growth Factor-Induced Transformation of Human A549 Lung Carcinoma Cells Mol. Cell. Biol., March 1, 1999; 19(3): 1938 - 1949. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Kil, M. Hobert, and C. Carlin A Leucine-based Determinant in the Epidermal Growth Factor Receptor Juxtamembrane Domain Is Required for the Efficient Transport of Ligand-Receptor Complexes to Lysosomes J. Biol. Chem., January 29, 1999; 274(5): 3141 - 3150. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Schaefer, R. W. Akita, and M. X. Sliwkowski A Discrete Three-amino Acid Segment (LVI) at the C-terminal End of Kinase-impaired ErbB3 Is Required for Transactivation of ErbB2 J. Biol. Chem., January 8, 1999; 274(2): 859 - 866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Lauffenburger, G. T. Oehrtman, L. Walker, and H. S. Wiley Real-time quantitative measurement of autocrine ligand binding indicates that autocrine loops are spatially localized PNAS, December 22, 1998; 95(26): 15368 - 15373. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Levkowitz, H. Waterman, E. Zamir, Z. Kam, S. Oved, W. Y. Langdon, L. Beguinot, B. Geiger, and Y. Yarden c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor Genes & Dev., December 1, 1998; 12(23): 3663 - 3674. [Abstract] [Full Text]
|
|
|
|
|
|
A. Subtil, A. Rocca, and A. Dautry-Varsat Molecular Characterization of the Signal Responsible for the Targeting of the Interleukin 2 Receptor beta Chain toward Intracellular Degradation J. Biol. Chem., November 6, 1998; 273(45): 29424 - 29429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Blagoveshchenskaya, E. W. Hewitt, and D. F. Cutler A Balance of Opposing Signals within the Cytoplasmic Tail Controls the Lysosomal Targeting of P-selectin J. Biol. Chem., October 23, 1998; 273(43): 27896 - 27903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. N. Ghosh, W. G. Mallet, T. T. Soe, T. E. McGraw, and F. R. Maxfield An Endocytosed TGN38 Chimeric Protein Is Delivered to the TGN after Trafficking through the Endocytic Recycling Compartment in CHO Cells J. Cell Biol., August 24, 1998; 142(4): 923 - 936. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kang, L. Liang, C. D. Parker, and J. F. Collawn Structural Requirements for Major Histocompatibility Complex Class II Invariant Chain Endocytosis and Lysosomal Targeting J. Biol. Chem., August 7, 1998; 273(32): 20644 - 20652. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Straley, B. L. Daugherty, S. E. Aeder, A. L. Hockenson, K. Kim, and S. A. Green An Atypical Sorting Determinant in the Cytoplasmic Domain of P-Selectin Mediates Endosomal Sorting Mol. Biol. Cell, July 1, 1998; 9(7): 1683 - 1694. [Abstract] [Full Text]
|
|
|
|
|
|
A. W. Gagnon, L. Kallal, and J. L. Benovic Role of Clathrin-mediated Endocytosis in Agonist-induced Down-regulation of the beta 2-Adrenergic Receptor J. Biol. Chem., March 20, 1998; 273(12): 6976 - 6981. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Blagoveshchenskaya, J. P. Norcott, and D. F. Cutler Lysosomal Targeting of P-selectin Is Mediated by a Novel Sequence within Its Cytoplasmic Tail J. Biol. Chem., January 30, 1998; 273(5): 2729 - 2737. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Worthylake and H. S. Wiley Structural Aspects of the Epidermal Growth Factor Receptor Required for Transmodulation of erbB-2/neu J. Biol. Chem., March 28, 1997; 272(13): 8594 - 8601. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-J. S. Huang, M. Nagane, C. K. Klingbeil, H. Lin, R. Nishikawa, X.-D. Ji, C.-M. Huang, G. N. Gill, H. S. Wiley, and W. K. Cavenee The Enhanced Tumorigenic Activity of a Mutant Epidermal Growth Factor Receptor Common in Human Cancers Is Mediated by Threshold Levels of Constitutive Tyrosine Phosphorylation and Unattenuated Signaling J. Biol. Chem., January 31, 1997; 272(5): 2927 - 2935. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Bohm, L. M. Khitin, S. P. Smeekens, E. F. Grady, D. G. Payan, and N. W. Bunnett Identification of Potential Tyrosine-containing Endocytic Motifs in the Carboxyl-tail and Seventh Transmembrane Domain of the Neurokinin 1Receptor J. Biol. Chem., January 24, 1997; 272(4): 2363 - 2372. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Kornilova, T. Sorkina, L. Beguinot, and A. Sorkin Lysosomal Targeting of Epidermal Growth Factor Receptors via a Kinase-dependent Pathway Is Mediated by the Receptor Carboxyl-terminal Residues 1022-1123 J. Biol. Chem., November 29, 1996; 271(48): 30340 - 30346. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sorkin, M. Mazzotti, T. Sorkina, L. Scotto, and L. Beguinot Epidermal Growth Factor Receptor Interaction with Clathrin Adaptors Is Mediated by the Tyr974-containing Internalization Motif J. Biol. Chem., June 7, 1996; 271(23): 13377 - 13384. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Zwart, C. B. Brewer, J. Lazarovits, Y. I. Henis, and M. G. Roth Degradation of Mutant Influenza Virus Hemagglutinins Is Influenced by Cytoplasmic Sequences Independent of Internalization Signals J. Biol. Chem., January 12, 1996; 271(2): 907 - 917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. G. Thomas, K. M. Baker, T. J. Motel, and T. J. Thekkumkara Angiotensin II Receptor Endocytosis Involves Two Distinct Regions of the Cytoplasmic Tail J. Biol. Chem., September 22, 1995; 270(38): 22153 - 22159. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Nesterov, R. C. Kurten, and G. N. Gill Association of Epidermal Growth Factor Receptors with Coated Pit Adaptins via a Tyrosine Phosphorylation-regulated Mechanism J. Biol. Chem., March 17, 1995; 270(11): 6320 - 6327. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-F. Gaulin, A. Fiset, S. Fortier, and R. L. Faure Characterization of Cdk2-Cyclin E Complexes in Plasma Membrane and Endosomes of Liver Parenchyma. INSULIN-DEPENDENT REGULATION J. Biol. Chem., May 26, 2000; 275(22): 16658 - 16665. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-S. Chin, M. C. Raynor, X. Wei, H.-Q. Chen, and L. Li Hrs Interacts with Sorting Nexin 1 and Regulates Degradation of Epidermal Growth Factor Receptor J. Biol. Chem., March 2, 2001; 276(10): 7069 - 7078. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Cherqui, V. Kalatzis, G. Trugnan, and C. Antignac The Targeting of Cystinosin to the Lysosomal Membrane Requires a Tyrosine-based Signal and a Novel Sorting Motif J. Biol. Chem., April 13, 2001; 276(16): 13314 - 13321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.E. Futter, L.M. Collinson, J.M. Backer, and C.R. Hopkins Human VPS34 is required for internal vesicle formation within multivesicular endosomes J. Cell Biol., December 24, 2001; 155(7): 1251 - 1264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Jones, S. K. Foreman, B. B. Shank, and R. C. Kurten EGF receptor downregulation depends on a trafficking motif in the distal tyrosine kinase domain Am J Physiol Cell Physiol, March 1, 2002; 282(3): C420 - C433. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |