A Leucine-based Determinant in the Epidermal Growth Factor
Receptor Juxtamembrane Domain Is Required for the Efficient Transport
of Ligand-Receptor Complexes to Lysosomes*
Song Jae
Kil
,
Michael
Hobert
§, and
Cathleen
Carlin
¶
From the
Department of Physiology and Biophysics,
School of Medicine, Case Western Reserve University and the ¶ Case
Western Reserve University Cancer Center,
Cleveland, Ohio 44106-4970
 |
ABSTRACT |
Ligand binding causes the epidermal growth factor
(EGF) receptor to undergo accelerated internalization with eventual
degradation in lysosomes. The goal of this study was to investigate the
molecular basis of endocytic sorting, focussing on post-internalization events. We have identified a sequence located between amino acid residues 675 and 697, encompassing a dileucine motif at residues 679 and 680, that enhances endosome-to-lysosome transport when conformational restraints in the EGF receptor carboxyl terminus are
removed by truncation. The same dileucine motif is also necessary for
efficient lysosomal transport of ligand-occupied full-length EGF
receptors. A L679A,L680A substitution diminished the degradation of
occupied full-length EGF receptors without affecting internalization but had a significant effect on recycling. Rapid recycling of mutant
receptors resulted in reduced intracellular retention of occupied EGF
receptors and delayed down-regulation of cell surface receptors. We
propose that the L679A,L680A substitution acts primarily to impair
transport of ligand-receptor complexes through an early endosomal
compartment, diverting occupied receptors to a recycling compartment at
the expense of incorporation into lysosome transport vesicles. We also
found that mutant receptors with truncations at the distal half of
tyrosine kinase domain (residues 809-957) were not efficiently
delivered to the cell surface but were destroyed in an endoplasmic
reticulum-associated degradative pathway.
 |
INTRODUCTION |
Epidermal growth factor
(EGF)1 and related
polypeptides elicit biological responses through binding to specific
cell surface receptors belonging to the ErbB family of protein-tyrosine
kinases (reviewed in Ref. 1). EGF regulates the intracellular
trafficking of ligand-EGF receptor (EGFR) complexes, causing
accelerated internalization from clathrin-coated pits, retention in
endosomes, and ultimately degradation in lysosomes (reviewed in Ref.
2). Ligand-regulated EGFR down-regulation therefore modulates cellular
responses to growth factors by controlling the duration of signaling
from the cell surface and by transporting activated EGFRs to
intracellular compartments where they continue to signal (reviewed in
Ref. 3). In addition, because EGFR is the only ErbB receptor that
undergoes ligand-induced down-regulation (4), signal transduction by different members of the ErbB family may be regulated by
compartmentalization in the endocytic pathway. The importance of EGFR
endocytic transport to normal proliferation is exemplified by the fact
that EGFRs that fail to internalize have been associated with cell
transformation (5) and tumorigenesis (6).
EGFR down-regulation is a complex process regulated by many factors,
including ligand occupancy, receptor aggregation, tyrosine kinase
activity, endosomal acidification, and intrinsic sorting signals (2).
It is also clear that the multiple transport steps in the endocytic
pathway are facilitated by different molecular interactions (7-10).
Internalization is regulated by endocytic codes located in the EGFR
carboxyl-terminal domain that are functionally interchangeable
with the internalization signal of the transferrin receptor (11).
Internalization also involves interactions between cytoplasmic
sequences in activated EGFRs and plasma membrane clathrin AP-2 adaptor
proteins (7, 11-15). Although a tyrosine-based signal encompassing
carboxyl-terminal domain residues 973-977 mediates AP-2 binding
in vitro, the physiological relevance of this interaction is
unclear, because EGFRs with an internal deletion of this region undergo
normal ligand-induced internalization (16). Interestingly, AP-2 also
interacts with two EGFR substrates: eps15, which is constitutively
associated with AP-2 in vivo (17), and SHC, which binds AP-2
in vitro (18). Another signaling intermediate required for
efficient EGFR internalization is Grb2, which binds to activated EGFRs
either directly or as part of a SHC-Grb2 complex (1). It has been
proposed that Grb2 provides a phosphoinositide-dependent link to dynamin (19, 20), a GTPase that regulates endocytosis (16, 21).
Taken together, these studies indicate that EGFR internalization from
clathrin-coated pits is facilitated by multiple interactions occurring simultaneously.
Following internalization, ligand-receptor complexes are diverted from
a recycling pathway to lysosomes (10). Endosome-to-lysosome sorting
presumably involves the signal-mediated transfer of ligand-receptor complexes to vesicles called endosomal carrier vesicles or
multivesicular bodies (ECV/MVBs), which transport material to late
endosomes (reviewed in Ref. 22). Although candidate sorting sequences have been identified in the carboxyl half of the cytoplasmic domain, a
consensus has not been reached regarding the structural and enzymatic
requirements for transporting EGFRs to lysosomes (9, 23, 24). One
candidate lysosomal sorting molecule, SNX-1, has been identified that
binds at the distal border of the tyrosine kinase domain (see Fig. 1
and Ref. 25).
The goal of this study was to further characterize the molecular basis
for selective transport of ligand-EGFR complexes to lysosomes. This was
accomplished by analyzing a series of EGFR proteins with progressive
truncations encompassing the entire cytoplasmic domain to test the
hypothesis that cryptic sorting sequences normally masked in unoccupied
full-length EGFRs would be exposed in truncated receptors. In contrast
to previous studies, we analyzed sequences in the juxtamembrane domain
as well as the carboxyl terminus for two reasons. First, the EGFR
cytoplasmic domain has numerous consensus leucine-based signals
implicated in lysosomal targeting of a number of membrane proteins
(26-29) located throughout the cytoplasmic domain. Second, the
juxtamembrane domain contains a sorting signal that regulates EGFR
basolateral delivery in polarized Madin-Darby canine kidney cells (30), suggesting that this region may have a broader role in vesicular transport. We have found that a sequence located between amino acid
residues 675 and 697 in the cytoplasmic juxtamembrane region enhances
endosome-to-lysosome transport of truncated EGFRs. Residues Leu679 and Leu680, which conform to a
leucine-based sorting signal, were shown to be a critical determinant
for efficient lysosomal transport of cytoplasmically truncated EGFRs.
This same motif was also required for the efficient transport of ligand
occupied full-length EGFR complexes to lysosomes.
 |
EXPERIMENTAL PROCEDURES |
Mutagenesis--
EGFR cytoplasmic truncation and substitution
mutants were made using polymerase chain reaction (PCR) to modify EGFR
coding sequences cloned in pCB6+, a eukaryotic expression
plasmid containing a human cytomegalovirus regulatory region,
transcription termination and polyadenylation signals from the human
growth hormone gene, an SV40 origin of replication and early region
promoter-enhancer, and a neomycin resistance gene (reviewed in Ref.
31). To create cDNAs encoding receptors with cytoplasmic
truncations, stop codon substitutions were made at codons for
Pro675, Ala698, Val810,
Pro886, Gln958, Tyr974,
Leu993, and Val1023. Forward primers (listed
below) were designed to anneal to sequences 5' to a novel restriction
enzyme site (in parentheses) in the EGFR coding sequence. Reverse
mutagenic primers (listed below) were designed to create a premature
stop codon (in bold) 400-500 nucleotides downstream of the forward
primer, as well as a restriction site (underlined and in parentheses)
compatible with the pCB6+ polylinker. PCR fragments were
gel-purified, digested at sites incorporated in the PCR products,
and ligated to pCB6+/EGFR digested with the same
restriction enzymes. cDNA-encoded proteins were named based on the
carboxyl-terminal amino acid residue in the EGFR coding region
(i.e. c'-674 has a P675STOP substitution). cDNAs with
stop codon substitutions for Arg652 and Arg724
have been described previously (32).
Forward Primers--
The forward primers used are: c'-674 and
c'-697, 5'-TGCGTCTCTTGCCGGAATGTCA-3' (BsmI); c'-809 and
c'-885, 5'-AGGCTGCTGCAGGAGAGGGA-3' (DraIII); c'-957,
5'-TCCGGGAACACAAAGACAAT-3' (ApaLI); and c'-973, c'-992, and
c'-1022, 5'-TCCGGGAACACAAAGACAAT-3' (BstEII).
Reverse Primers--
The reverse primers used are: c'-674,
5'-TCAATCGATCTAAGCTTCTCCACTGGGTGTAA-3'
(ClaI); c'-697,
5'-ATCATCGATTTAACCGGAGCCCAGCACTTTG-3' (ClaI); c'-809,
5'-CAGTCTAGATCACAAGCGACGGTCCTCCAAGT-3' (XbaI); c'-885,
5'-CGCAAGCTTCAGGTCATCAACTCCCAAAC-3' (HindIII); c'-957,
5'-AGTAAGCTTCAAATGACAAGGTAGCGCTGGGGGTCTC-3' (HindIII); c'-973,
5'-GTCAAGCTTCTAGAAGTTGGAGTCTGTAGGA-3' (HindIII); c'-992,
5'-ATTAAGCTTCTACTCGTCGGCATCCACCAC-3' (HindIII); and c'-1022,
5'-TGTAAGCTTCTAGGAATTGTTGCTGGTTGC-3' (HindIII).
The c'-697 cDNA containing a L679A,L680A substitution was
constructed using the "megaprimer" PCR method (33). Briefly, an initial PCR reaction was performed to convert codons for
Leu679 and Leu680 to alanine residues
(underlined) using a mutagenic primer
5'-CTCCCAACCAAGCTGCGAGGATCTTGAAGGAAACTGA-3' and a flanking
primer identical to the reverse primer listed above for c'-697. A
subsequent PCR reaction was carried out using the product of the first
reaction and a second flanking primer (same as the forward primer for
c'-674). The final PCR product was digested with BsmI and
ClaI and subcloned into pCB6+/EGFR. The
L679A,L680A substitution was introduced into the full-length molecule
by subcloning a 188-nucleotide Eco72I-EcoRI
fragment from pCB6+/697 containing the L679A,L680A substitution into an EGFR cDNA ligated to a pBK-CMV phagemid (Stratagene Cloning
Systems, La Jolla, CA).
PCR primers were designed using the DNASTAR software package (DNASTAR,
Inc., Madison, WI). PCR amplifications were carried out using a
RoboCycler 40 Temperature Cycler (Stratagene). Sequences of PCR
products were verified by dideoxy chain termination DNA sequencing
using a Sequenase II kit from U. S. Biochemical Corp.
Transient Transfections and Permanent Cell Lines--
COS-1
monkey cells expressing endogenous EGFRs were used for transient
transfections. Log-phase cells were seeded at a density of
approximately 5 × 103 cells/cm2 24 h
prior to transfection. Cells were rinsed twice with serum-free Dulbecco's modified Eagle's medium (DMEM) and then transfected with
10 µg of plasmid DNA using Lipofectin reagent (Life Technologies, Inc.) according to the manufacturer's instructions. The DNA-Lipofectin mixture was replaced with DMEM supplemented with 10% fetal bovine serum and 2 mM glutamine 5 h later. When multiple
dishes were transfected with the same plasmid, cells were pooled and
replated 24 h later to avoid plate-to-plate variability in
transfection efficiency.
NR6 cells lacking endogenous EGFRs (34, 35) were used to produce
permanent cell lines expressing human EGFRs. Cells were transfected
with human EGFR cDNAs using Lipofectin reagent as described
previously. Transfected cells were grown for 10-14 days in selection
medium containing G418 (0.8 mg/ml Geneticin; Life Technologies, Inc.).
Cells were then enriched for human EGFR expression by sterile sorting
on a flow cytometer (Cytofluorograph IIs; Ortho Instruments, Westwood,
MA) using the EGFR-specific monoclonal antibody (mAb) EGF-R1, which
detects an extracellular peptide core epitope in human EGFRs (36).
EGF-R1 also cross-reacts with endogenous EGFRs expressed in COS-1 cells.
Cell Labeling, Immunoprecipitation, and Western Blots--
Cells
were rinsed twice with methionine- and cysteine-free minimal essential
medium and then labeled with 50 µCi of Express Protein Labeling mix
(1175 Ci/mmol; New England Nuclear Research Products, Wilmington, DE)
per ml of methionine- and cysteine-free minimal essential medium
supplemented with 10% dialyzed fetal bovine serum and 0.2% BSA. In
some experiments, labeling medium was replaced with chase medium
consisting of serum-free DMEM supplemented with nonradioactive
methionine (0.75 mg/ml) and cysteine (1.2 mg/ml), and cells were
incubated for an additional period of time before harvesting. Cells
were lysed with 1% (w/v) Nonidet P-40 in 0.1 M Tris, pH
6.8, supplemented with 15% (w/v) glycerol, 2 mM EDTA, and
1 mM EGTA, 0.2 mM phenylmethylsulfonyl
fluoride, and 1 µM leupeptin. EGFRs were
immunoprecipitated with EGF-R1 adsorbed onto protein A-Sepharose CL-4B
beads (Sigma). Samples were solubilized with Laemmli buffer and
separated by SDS-polyacrylamide gel electrophoresis (PAGE) (37). Gels
were treated with En3Hance (New England Nuclear) for
fluorography. Labeled proteins were quantitated by phosphorstorage
autoradiography (Molecular Dynamics, Sunnyvale, CA). The percentage of
radioactivity remaining was plotted as a function of time on a semi-log
plot, and receptor half-lives (t1/2 values) were
calculated by linear regression analysis. For Western blotting,
proteins resolved by SDS-PAGE were transferred to nitrocellulose
according to standard procedure (38). EGFRs were detected by
immunoblotting with an affinity purified rabbit polyclonal antibody
specific to amino acids 1005-1016 in the EGFR cytoplasmic domain
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Phosphotyrosine-containing proteins were detected by immunoblotting
with an anti-phosphotyrosine-horseradish peroxidase conjugate
(Transduction Laboratories, Lexington, KY).
125I-EGF Cross-linking--
Receptor grade mouse EGF
(Toyobo Biochemicals, Osaka, Japan) was labeled with 125I
(carrier-free, >350 mCi/ml; New England Nuclear) using chloramine-T. Cells were rinsed three times with ice-cold DMEM supplemented with
0.2% BSA and then incubated with approximately 10 nM
125I-EGF for 2 h at 4 °C. Cells were rinsed again
with the DMEM/BSA solution to remove unbound ligand and then incubated
with 2 mM disuccinimidyl suberate (Pierce) in a solution of
0.1 M HEPES, pH 7.4, supplemented with 0.12 M
NaCl, 0.05 M KCl, 8 mM glucose, and 1.2 mM MgSO4 for 15 min at room temperature. The
chemical cross-linker was quenched by a 5-min incubation with 0.05 M Tris, pH 7.4, at room temperature. Cell lysates were
prepared using 1% Nonidet P-40 exactly as described above, and total
cell protein was separated by SDS-PAGE.
Northern Blots--
Cells were trypsinized, lysed with TRIzol
reagent (Life Technologies, Inc.) for 5 min at room temperature, and
then extracted with chloroform:isoamyl alcohol (24:1 v/v). RNA was
precipitated with isopropanol, washed with 70% ethanol, and
resuspended in distilled H2O treated with 0.01%
diethylpyrocarbonate. 20 µg of total RNA was denatured, fractionated
by electrophoresis in a 1.4% agarose/formaldehyde gel, and transferred
to Biotrans (+) nylon membrane (ICN Pharmaceuticals, Inc., Costa Mesa,
CA) using standard techniques (39). An oligonucleotide complementary to nucleotides 2227-2247 in the human EGFR cDNA was labeled with [
-32P]dCTP (3000 Ci/mmol; Amersham Pharmacia Biotech)
using Klenow reagent and a Random Primed DNA Labeling kit from
Boehringer-Mannheim. Blots were prehybridized for 2 h at 65 °C
with a solution containing 1 mM EDTA, 250 mM
sodium phosphate, pH 7.2, 7% SDS, 1% BSA, and 50 µg/ml salmon sperm
DNA. Blots were hybridized with 1 × 106 cpm/ml of
32P-labeled oligonucleotide in the same solution for
18 h at 65 °C. Blots were washed three times with a solution of
20 mM sodium phosphate, pH 7.5, 1 mM EDTA, and
10% BSA at 65 °C, air-dried, and exposed to film for autoradiography.
Treatment with Brefeldin A--
Cells were preincubated in
medium supplemented with vehicle (1 µl of MeOH/ml) or brefeldin A
(BFA) (5 µg/ml) for 1 h, pulse-labeled with Express Protein
Labeling mix for 30 min, and then incubated in nonradioactive chase
medium for periods up to 2 h before lysis with Nonidet P-40.
Labeling and chase media were also supplemented with vehicle or BFA.
Digestion with Endoglycosidase H--
Pulse-labeled EGFRs
collected by immunoprecipitation were solubilized with 50 µl of a
solution of 1% SDS and 5%
-mercaptoethanol in 0.1 M
sodium citrate, pH 5.5. 20-µl aliquots were mixed with an equal
volume of distilled H2O and then incubated for 18 h at 37 °C with 1 milliunits endoglycosidase H (endo H; 40 units/mg enzyme; Boehringer-Mannheim). Equal aliquots were subjected to sham digestions.
Internalization Assays--
Cells were seeded at a density of
5 × 105 cells/well in six-well tissue culture plates
48 h before each assay and refed with DMEM supplemented with 25 mM HEPES and 0.2% BSA (D/H/B) 24 h later. Cells were
rinsed twice with cold D/H/B and then incubated with 250 ng/ml of
125I-labeled 528 mAb Fab or 1-100 ng/ml of
125I-labeled EGF diluted in the same medium for 2 h at
4 °C. The EGFR-specific 528 mAb Fab recognizes an extracellular
peptide epitope and is widely used, for example to track intracellular transport of unoccupied EGFRs (24). 528-Fabs (gift of Starla Glick,
Dept. of Pediatrics, Case Western Reserve University) were iodinated
exactly as described above. Cells were warmed to 37 °C for periods
up to 20 min and then rinsed rapidly with cold D/H/B to remove unbound
528-Fab or ligand. Cells were incubated with an acid-stripping solution
containing 50 mM glycine-HCl, pH 3.0, 100 mM
NaCl, 2 mg/ml polyvinylpyrrolidone, and 2 M urea for 6 min
on ice. The stripping efficiency was greater than 95%. This solution
was collected for
counting to estimate surface-bound radioactivity,
and cells were solubilized with 1 N NaOH to estimate cell-associated radioactivity. Internalization is represented as the
percentage of total radioactivity (counts released from the cell
surface by acid stripping plus counts remaining cell-associated after
acid-stripping) associated with the interior of the cell. Internalization rates were calculated by linear regression analysis.
Recycling Assays--
Cells seeded on six-well plates were
rinsed with cold D/H/B and incubated with 1 ng/ml 125I-EGF
at 4 °C for 1 h. Cells were then rinsed twice with cold D/H/B
and allowed to internalize receptors for 10 min at 37 °C. Cells were
rinsed with cold D/H/B, and 125I-EGF remaining on the cell
surface was removed by a 2.5-min mild acid wash (0.2 M
sodium acetate, 0.5 M NaCl, pH 4.5) as described elsewhere
(23). 125I-EGF-loaded cells were incubated with 100 ng/ml
of nonradioactive EGF at 4 °C for 1 h to saturate surface
receptors, and then switched to 37 °C for 0-40 min to allow for
receptor trafficking. At the end of each incubation period, cells were
placed on ice, and media were collected to determine the amount of
degraded and intact 125I-EGF. This was followed by a
2.5-min harsh acid wash (pH 2.8) to determine the amount of
surface-bound 125I-EGF. Cells were then solubilized with 1 N NaOH to determine the amount of intracellular
125I-EGF. To separate intact 125I-EGF from
degraded 125I-EGF products, trichloroacetic acid and
phosphotungstic acid were added to collected medium to the final
concentrations of 3 and 0.3%, respectively. This mixture was incubated
at 4 °C for 30 min and centrifuged to collect precipitates.
Precipitates, solubilized with 1 N NaOH, and supernatants
were used to calculate the amount of intact and degraded
125I-EGF, respectively. The amount of recycled
125I-EGF was calculated by summing the radioactivity
appearing on the cell surface and in the medium (intact) and was
expressed as fraction of the total radioactivity present in the cell
and media. The stripping efficiency of the pH 4.5 and 2.8 acid
solutions was greater than 90 and 95%, respectively. The efficiency of
precipitation with trichloroacetic acid and phosphotungstic acid was
greater than 95%.
Ligand-induced EGFR Down-regulation--
To measure
down-regulation of cell surface EGFRs, cells were incubated with
nonradioactive EGF for periods up to 2 h. Cells were rinsed two
times with ice-cold D/H/B, and cell surface-associated EGF was removed
by incubating cells for 2.5 min on ice with the same acid-stripping
buffer (pH 4.5) described in the previous paragraph. Cells were rinsed
two times with D/H/B and then incubated with 100 ng/ml
125I-EGF for 1 h at 4 °C. Surface-bound
125I-EGF was removed by incubating cells with acid
stripping buffer (pH 2.8) for 2.5 min on ice, and radioactivity was
determined by
counting. To measure EGFRs down-regulation, cells
were pulse-labeled for 1 h, and then chased with serum-free DMEM
containing excess nonradioactive amino acid precursors for 3 h.
Cells were stimulated with 100 ng/ml nonradioactive EGF and harvested
for EGFR immunoprecipitation at various time points. Labeled EGFRs were
resolved by SDS-PAGE.
 |
RESULTS |
Expression and Stability of Human EGFRs with Cytoplasmic
Truncations--
To study the relative contribution of EGFR
cytoplasmic subdomains to protein stability, premature stop codons were
introduced at 10 different sites throughout this region to expose
cryptic sorting signals. Premature stop codons were always ligated
adjacent to transcription termination and polyadenylation signals in
the expression vector to rule out effects due to variability in 3' noncoding sequences. Three truncations were made in the carboxyl terminus (Fig. 1A). The most
distal carboxyl truncation (c'-1022) removes consensus tyrosine
NPXY-type signals located near the carboxyl-terminal end.
Carboxyl truncations to residues 992 and 973 remove one or both of the
known endocytic codes located in the CaIn domain, respectively (7, 10).
In addition to internalization, this region also regulates
ligand-mediated calcium responses (7). Truncation to residue 957 (c'-957) deletes sequences up to the distal border of the tyrosine
kinase core domain, exposing the binding site for the putative
lysosomal sorting molecule SNX-1 (25). Four truncations were made
within the kinase catalytic core domain (c'-697, c'-723, c'-809, and
c'-885), and another two truncations were created within the
juxtamembrane domain (c'-674 and c'-651).

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Fig. 1.
Steady-state expression of EGFRs with
cytoplasmic truncations in COS-1 cells. A, schematic
showing organization of transmembrane (TM) and cytoplasmic
domains for wild-type EGFR (WT) and corresponding domains
encoded by cDNAs with premature stop codons. Locations of a
dileucine motif (Leu679,Leu680) described in this study, the
SNX-1 binding site (25), the CaIn domain (7), and conserved
NPXY motifs (52) are also shown. B and
C, COS-1 cells transfected with cDNAs encoding each of
the proteins shown in A were assayed for EGFR expression
48 h post-transfection. B, cells were metabolically
labeled for 4 h, lysed with Nonidet P-40, and immunoprecipitated
with an EGFR mAb specific for an extracellular epitope common to monkey
and human EGFRs. C, intact cells were chemically
cross-linked with 125I-EGF. Immunoprecipitates
(B) or total cellular protein (C) were separated
on 7.5% SDS-PAGE gels. Locations of full-length 170-kDa and
cytoplasmically truncated EGFRs are indicated on the right.
Molecular mass standards: myosin, 200 kDa; -galactosidase, 116.3 kDa; phosphorylase B, 97.4 kDa. Jx, juxtamembrane.
|
|
To test the hypothesis that sorting signals that regulate EGFR
transport in the endocytic pathway to lysosomes were active in
truncated molecules, we first determined the steady-state expression of
receptor proteins depicted in Fig. 1A in transiently
transfected COS-1 cells. EGFRs were either immunoprecipitated from
metabolically labeled cells (Fig. 1B) or identified by
125I-EGF cell surface cross-linking (Fig. 1C).
Both of these methods also detect endogenous monkey EGFRs, which served
as an internal control for wild-type EGFR mobility on SDS-PAGE gels.
These analyses showed that cDNAs coding for proteins with
truncations in the carboxyl-terminal domain (c'-972, c'-992, and
c'-1022), in the proximal half of the kinase catalytic core (c'-697 and
c'-723), or in the juxtamembrane domain (c'-651 and c'-674) formed
stable products that were transported to the cell surface. In contrast, low levels of c'-957 receptors were seen after metabolic labeling, and
this receptor protein was not readily detectable following 125I-EGF cross-linking, suggesting these receptors are
probably degraded in the biosynthetic pathway. Products encoded by two
other EGFR cDNAs with truncations involving the distal half of the
kinase domain (c'-885 and c'-809) could not be detected by either
method, suggesting these molecules are either not produced or are
rapidly degraded.
To further characterize the protein stability of cytoplasmically
truncated EGFRs, transiently transfected COS-1 cells were metabolically
labeled for 3 h and then incubated in chase medium for either 3 or
15 h (Fig. 2). Consistent with
results in Fig. 1, c'-809, c'-885 and c'-957 receptors were not
detectable at either time point. EGFR proteins with truncations in the
carboxyl-terminal domain exhibited stability similar to full-length
EGFRs, as did c'-674 and c'-651 receptor proteins with truncations in
the juxtamembrane domain. However, stability of two proteins truncated
near the proximal border of the kinase domain, c'-697 and c'-723, was
markedly reduced compared with endogenous monkey EGFRs. Analysis of
EGFRs with cytoplasmic truncations therefore identified two classes of
receptors with reduced protein expression: those that failed to undergo
efficient transport to the cell surface (c'-809, c'-885, and c'-957)
and those that were transported to the cell surface but had reduced
stability compared with wild-type EGFRs under basal conditions (c'-697
and c'-723).

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Fig. 2.
Metabolic turnover of EGFRs with cytoplasmic
truncations. COS-1 cells transfected with EGFR cDNAs shown in
Fig. 1A were metabolically labeled for 3 h starting at
48 h post-transfection. Labeling medium was replaced with chase
medium, and cells were lysed with Nonidet P-40 either 3 or 15 h
later. EGFRs were immunoprecipitated with a receptor-specific mAb, and
immunoprecipitates were separated on 7.5% SDS-PAGE gels. Locations of
full-length 170-kDa and truncated EGFRs are indicated on the
left. WT, wild type; Jx,
juxtamembrane.
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|
Reduced Stability of EGFR Proteins with Cytoplasmic Truncations Is
Mediated by Two Distinct Post-translational Mechanisms--
Based on
results in Fig. 1, we hypothesized that EGFRs with truncations in the
distal kinase domain (c'-809, c'-885, and c-957) were degraded in the
biosynthetic pathway. To test that hypothesis, we first showed that
cDNAs encoding these truncated EGFRs produced stable mRNAs as
judged by Northern blot analysis (Fig.
3A). We then asked whether the
fungal metabolite BFA affected protein turnover, because the
endoplasmic reticulum (ER)-associated degradative pathway is
BFA-insensitive (40). Transfected COS-1 cells that had undergone a
mock-treatment or a preincubation with BFA were pulse-labeled for 45 min and then incubated in chase medium for periods up to 2 h (Fig.
3B). BFA treatment had no effect on turnover of c'-809
receptors (Fig. 3B) or c'-885 receptors (not shown) and
little effect on turnover of c'-957 receptor (Fig. 3B). The molecular weight of endogenous EGFRs was reduced in BFA-treated cells,
consistent with BFA-induced blockade of Golgi-mediated carbohydrate
processing. These data suggest that although a fraction of c'-957
receptors appear to be degraded in a post-Golgi compartment, receptors
with truncations in the distal half of the kinase domain are mostly
disposed of by a BFA-insensitive, ER-associated degradative pathway
(41).

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Fig. 3.
Reduced stability of EGFR proteins with
cytoplasmic truncations is mediated by two distinct mechanisms.
A, Northern blot analysis of human EGFR mRNA expression.
20 µg of total RNA from cells transfected with cDNAs encoding
truncated EGFRs listed in the figure were fractionated in 1.4%
agarose/formaldehyde gels and transferred to nitrocellulose. Blots were
incubated with a 32P-labeled oligonucleotide probe
complementary to nucleotides 2227-2247 in the human EGFR cDNA.
Sizes of transcripts are indicated beneath each lane. B and
C, effect of BFA on degradation of EGFRs with cytoplasmic
truncations. COS-1 cells transfected with cDNAs encoding c'-957 or
c'-809 EGFRs (B), or c'-973 or c'-697 EGFRs (C),
received a mock treatment (1 µl of MeOH/ml) or were preincubated with
BFA (5 µg/ml) for 1 h, pulse-labeled for 30 min, and then
incubated in chase medium for times indicated before lysis with Nonidet
P-40. Labeling and chase media were also supplemented with vehicle or
BFA. Cell lysates were immunoprecipitated with a receptor-specific mAb,
and immunoprecipitates were separated on 7.5% SDS-PAGE gels.
WT, wild type; kb, kilobases.
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|
Similar to truncated receptors such as c'-973 (Fig. 3C),
which exhibits normal stability, c'-697 (Fig. 3C) and c'-723
(not shown) receptors were not subject to BFA-insensitive,
ER-associated degradation. In addition, c'-697 and c'-723 receptors
were transported through the Golgi complex with approximately the same
kinetics as wild-type EGFRs. This was shown using endo H digestion to
distinguish mature EGFRs containing a mixture of complex and high
mannose-type N-linked oligosaccharides from EGFR precursors
containing only high mannose N-linked oligosaccharides (36,
42). Because endo H only cleaves high mannose oligosaccharides (43),
endo H resistance correlates with transit through Golgi compartments
where complex oligosaccharides are formed (44). Cells were
pulse-labeled for 45 min and then harvested at 30-min intervals during
a nonradioactive chase for immunoprecipitation with an EGFR mAb and
endo H digestion. c'-697 receptors, truncated EGFRs exhibiting normal
stability (i.e. c'-651 or c'-674), and endogenous monkey
EGFRs had all acquired endo H resistance by 90 min of chase (Fig.
4). Similar results were obtained for
c'-723 receptors (not shown). Taken together, these data suggest that
c'-697 and c'-723 receptors exit the ER and undergo biosynthetic
transport with normal kinetics. Once at the plasma membrane, however,
these receptors exhibit reduced half-lives compared with wild-type
EGFRs.

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Fig. 4.
Acquisition of endo H resistance by EGFRs
with cytoplasmic truncations involving juxtamembrane or proximal kinase
domains. COS-1 cells transfected with cDNAs encoding EGFR
proteins listed in the figure were pulse-labeled for 20 min starting at
48 h post-transfection. Labeling medium was replaced with chase
medium for times indicated. Cells were lysed with Nonidet P-40 and
immunoprecipitated with a receptor-specific mAb. Half of each
immunoprecipitate was subjected to a mock digestion
( EndoH), and the remainder was incubated for 18 h at
37 °C with 1 milliunit of endo H (+EndoH).
Immunoprecipitates were separated on 7.5% SDS-PAGE gels. P,
deglycosylated precursor protein; M, mature protein.
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Internalization of c'-697 Receptors--
Because c'-674 receptors
do not have a shortened half-life, we hypothesized that EGFR residues
675-697 encompass a cryptic signal that regulates sorting in the
endocytic pathway. To determine whether this region contains a cryptic
internalization signal, we made permanent NR6 cell lines expressing
c'-697 or c'-674 receptors. Similar to results obtained with COS-1
cells, c'-697 receptors exhibited reduced stability in NR6 cells
compared with receptors truncated to residue 674 (not shown). The
uptake of a nonactivating 125I-labeled EGFR mAb Fab was
similar in cells expressing either c'-697 or c'-674 receptors (Fig.
5). These data suggest that enhanced turnover of c'-697 receptors is not due to accelerated internalization. We therefore hypothesized that truncation to residue 697 unmasks a
potential endosomal sorting signal able to divert basally internalized c'-697 receptors from a recycling pathway to lysosomes.

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Fig. 5.
Internalization of 125I-labeled
EGFR-specific Fab into cell lines expressing truncated EGFRs. Cell
lines encoding c'-674 (open circles) or c'-697 (closed
circles) EGFRs were incubated with 250 ng/ml
125I-labeled EGFR-specific Fab for 2 h at 4 °C.
Cells were then warmed to 37 °C for periods up to 15 min and rinsed
extensively to remove unbound Fab. Surface-bound radioactivity was
removed by acid stripping, and cells were solubilized with 1 N NaOH to determine internalized radioactivity.
Internalization is represented as the percentage of radioactivity
associated with the interior of the cell compared with the amount of
surface-bound radioactivity without a 37 °C warm-up. The values are
the means ± S.D. (n = 3). Some standard error
bars are obscured by symbols.
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Role for Leu679-Leu680 in c'-697 Receptor
Degradation--
To understand the molecular basis of the enhanced
turnover of c'-697 and c'-723 receptors, we examined the amino acid
sequence of EGFR residues 675-697 to identify potential consensus
sorting signals (Fig. 6). Residues
Leu679 and Leu680 were of particular interest
because leucine-based motifs are known to regulate lysosomal
trafficking of several membrane proteins (reviewed in Ref. 45).
Furthermore, Leu679-Leu680 and adjacent amino
acids (residues 679-683) are identical to sequences at the carboxyl
terminus of an adenovirus early region 3 membrane protein called
E3-13.7 (46). Because E3-13.7 re-routes recycling EGFRs to ECV/MVB
lysosomal transport vesicles, we hypothesize that E3-13.7 has co-opted
an intrinsic sorting signal normally used during lysosomal transport of
ligand-EGFR complexes (47). We therefore tested the potential role of
the Leu679-Leu680 motif in the reduced
stability phenotype of c'-697 receptors, by changing this dileucine to
a dialanine using PCR-based site-directed mutagenesis. Receptor
half-lives were determined by harvesting cells beginning 3 h after
the end of a pulse label to allow time for transport of labeled protein
to the plasma membrane. The EGFR immunoprecipitates were then
quantitated by phosphorstorage autoradiography. All EGFRs examined
exhibited first-order exponential decay kinetics (Fig.
7). The half-life of the unoccupied
endogenous COS-1 receptor was calculated to be approximately 25 h,
consistent with values reported in other studies (48). In contrast,
half-lives for c'-723 and c'-697 receptors were reduced to 9 and
12 h, respectively. The half-life for c'-697 receptors with a
L679A,L680A substitution (c'-697/L679A,L680A), however, was
indistinguishable from the half-life for endogenous receptors (Fig. 7).
These results support the hypothesis that
Leu679-Leu680 is part of a cryptic lysosomal
sorting signal exposed in c'-697 and c'-723 receptors.

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Fig. 6.
Amino acid sequence and predicted structure
of EGFR residues 675-697. A, organization of EGFR
cytoplasmic domain and amino acid sequence of a putative signal
encompassing Leu679-Leu680. Also shown are two
other putative lysosomal sorting signals: residues 945 and 957 encompassing the binding site for SNX-1 in the kinase catalytic core
domain (25) and residues 1022-1123 (Ly) in the
carboxyl-terminal domain (23). Amino-terminal post-translational
phosphorylation sites (mitogen-activated protein kinase sites
Thr669 and Ser671) and acidic residues
(Glu673) are characteristic of other known consensus
leucine-based motifs. An amphipathic helix predicted for this region is
highlighted by cross-hatched bar. B, amphipathic
helical representation of residues 675-697, with charged residues
(+/ ) aligned on one side of the helix and hydrophobic residues
(in italics) on the other side. The L679A,L680A substitution
is also indicated.
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Fig. 7.
Metabolic half-lives of cytoplasmically
truncated EGFRs. Cells were metabolically labeled for 3 h
starting at 48 h post-transfection, changed to chase medium, and
lysed with Nonidet P-40 at indicated times. Cell lysates were
immunoprecipitated with a receptor-specific mAb, and immunoprecipitates
were separated on 7.5% SDS-PAGE gels. Radiolabeled endogenous EGFRs
and EGFRs with cytoplasmic truncations (top) were
quantitated by phosphorstorage autoradiography (bottom).
Data are presented as the percentage of radioactive EGFRs at the 3-h
chase time point.
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Role of Leu679-Leu680 in Ligand-induced
EGFR Down-regulation--
To test the hypothesis that a
Leu679-Leu680-based sorting signal is utilized
during ligand-induced transport, we expressed the L679A,L680A substitution in the context of a full-length EGFR (EGFR/L679A,L680A). As shown in Fig. 8A, permanent
NR6 cell lines transfected with plasmids encoding either wild-type EGFR
or EGFR/L679A,L680A expressed 170-kDa proteins that were reactive with
EGFR-specific antibodies directed against both extracellular and
cytoplasmic domains. Under normal circumstances, efficient
ligand-induced EGFR down-regulation requires intrinsic tyrosine kinase
activity (2). Although Leu679-Leu680 lies
outside of the kinase catalytic domain, it was nevertheless possible
that the L679A,L680A substitution would have an adverse effect on
ligand-induced kinase activity. To test that possibility, the NR6 cell
lines were assayed for ligand-induced receptor autophosphorylation by
Western blotting using a phosphotyrosine-specific antibody. As shown in
Fig. 8B, EGF stimulation induced tyrosine phosphorylation of
both proteins to a similar extent and also caused a molecular weight
shift reflecting the increase in tyrosine phosphorylation. These data
indicate that the L679A,L680A substitution does not interfere with
ligand-induced EGFR activation.

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Fig. 8.
Expression of full-length wild-type EGFR or
EGFR/L679A,L680A in NR6 cells. Permanent NR6 cells expressing
wild-type EGFR or EGFR/L679A,L680A were incubated with or without 100 ng/ml EGF for 15 min and lysed with Nonidet P-40. A, cell
lysates were immunoprecipitated (IP) with EGF-R1.
Immunoprecipitates were transferred to nitrocellulose and immunoblotted
(IB) using a second EGFR-specific antibody directed against
a carboxyl-terminal epitope. B, cell lysates were
immunoprecipitated with a biotin-conjugated phosphotyrosine
(PY) antibody. Immunoprecipitates were resolved by SDS-PAGE,
transferred to nitrocellulose, and immunoblotted with an horseradish
peroxidase-conjugated phosphotyrosine antibody.
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|
Two approaches were used to determine the effect of L679A,L680A
substitution on ligand-induced EGFR down-regulation. First, NR6 cells
expressing either wild-type EGFR or EGFR/L679A,L680A were assayed for
steady-state 125I-EGF binding following a preincubation
with unlabeled EGF for periods up to 2 h. Down-regulation of
wild-type EGFRs reached a plateau of 60% in cells that had been
preincubated with 1 ng/ml ligand for 1 h and remained at the same
level with longer preincubations (Fig.
9). In contrast, EGFR/L679A,L680A
receptors were down-regulated by less than 20% after 1 h of
preincubation with unlabeled ligand and continued to undergo
down-regulation at later time points (Fig. 9). The second approach for
analyzing ligand-induced down-regulation was to measure EGFR
half-lives. Cells that had been incubated in chase medium for 3 h
after a 45-min pulse-label were stimulated with ligand for periods up
to 2 h, and cell lysates were immunoprecipitated using an
EGFR-specific mAb (Fig. 10). As shown
in Fig. 10, the pool of radiolabeled receptors was relatively constant
in cells expressing EGFR/L679A,680A during a 2-h incubation with EGF,
compared with cells expressing wild-type EGFRs where the majority of
receptors were degraded. Interestingly, we consistently observed an
increased amount of radioactivity associated with EGFR/L679A,680A at
the 60-min time point, suggesting that these molecules may have
enhanced solubility in Nonidet P-40. These data therefore indicate that the mutant EGFRs are degraded less efficiently than wild-type EGFRs
following ligand occupation.

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Fig. 9.
Ligand-induced down-regulation of cell
surface 125I-EGF binding capacity. NR6 cells that had
been stimulated with unlabeled EGF (1 ng/ml) at 37 °C for the
periods of time indicated were incubated with a mild acid-stripping
solution to remove surface bound ligand. Cells were then incubated with
125I-EGF (100 ng/ml) for 1 h at 4 °C and subjected
to harsh stripping to determine surface-associated radioactivity by counting. Data are represented as the percentage of surface
125I-EGF binding without a 37 °C warm-up. The values are
the means ± S.D. (n = 6). Some standard error
bars are obscured by symbols. WT, wild type.
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Fig. 10.
Ligand-induced EGFR down-regulation.
NR6 cell lines were metabolically labeled for 45 min, changed to chase
medium for 4 h, and then stimulated with EGF (100 ng/ml) for
periods up to 2 h. Cell lysates were immunoprecipitated with a
receptor-specific mAb, and immunoprecipitates were separated on 7.5%
SDS-PAGE gels.
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The Role of L679A,L680A on Internalization of Full-length
EGFRs--
We next asked whether the inability of ligand to
down-regulate EGFR/L679A,680A as efficiently as wild-type EGFR was
associated with differences in internalization. To measure basal
internalization, cells were incubated with 250 ng/ml
125I-Fab for 2 h at 4 °C and then brought to
37 °C for periods up to 15 min. Internalization is represented as
the percentage of total radioactivity (cell surface plus cell interior)
associated with the interior of the cell as a function of time. As
shown in Fig. 11A, the ligand-independent internalization of
EGFR/L679A,L680A was indistinguishable from that of wild-type EGFRs.
When ligand-induced internalization was monitored by measuring
125I-EGF uptake, both cell lines exhibited a similar
internalization rate using two different concentrations of ligand (1 or
100 ng/ml), as shown in Table I. However,
cells expressing the mutant EGFR did not accumulate intracellular
ligand to the same extent as cells with wild-type EGFRs (Fig.
11B). The steady-state
amount of ligand internalized by cells expressing the wild-type EGFR was approximately 50%, compared with approximately 30% for cells expressing EGFR/L679A,L680A. These data suggest that the L679A,L680A substitution does not interfere with ligand-dependent
internalization but that the mutant receptors have a shorter endosomal
retention time than wild-type EGFRs.
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Table I
Intracellular transport kinetics for wild-type and mutant human EGFRs
expressed in NR6 cells
Basal and nonsaturating ligand-induced (1 ng/ml) internalization rates
(KIn) were calculated by linear regression analysis
of data in Fig. 11, panels A and B, respectively.
Data for deriving ligand-induced KIn under
saturating conditions (100 ng/ml) are not shown. Ligand-induced
recycling rates (KRe) were calculated by linear
regression analysis of data from Fig. 12. Ligand-induced EGFR
half-lives (t1/2) were calculated by linear
regression analysis of values derived from PhosphorImager
quantitation of data in Fig. 10.
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Fig. 11.
Internalization of EGFR-specific
125I-Fab or 125I-EGF by NR6 cell lines
expressing human EGFRs. NR6 cells were preincubated for 2 h
at 4 °C with either EGFR-specific 125I-Fabs (250 ng/ml)
(A) or 125I-labeled EGF (1 ng/ml) (B)
and then switched to 37 °C for times indicated in the figure. After
removing surface-bound Fab or ligand by acid-stripping, cells were
solubilized with 1 N NaOH to determine internalized
radioactivity. Internalization is represented as the percentage of
total radioactivity associated with the interior of the cell. The
values are the means ± S.D. (A, n = 6;
B, n = 6). Some standard error bars are
obscured by symbols. WT, wild type.
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The Role of L679A,L680A on Recycling of Full-length
EGFRs--
Because ligand-occupied mutant receptors had a longer
half-life than wild-type EGFRs, we hypothesized that the reduced
endosomal retention exhibited by EGFR/L679A,L680A was due to increased
recycling. This hypothesis was tested by measuring 125I-EGF
recycling kinetics in NR6 cells expressing either wild-type EGFR or
EGFR/L679A,L680A. Recycling was initiated by incubating cells that had
been preloaded with 125I-EGF (1 ng/ml) with an excess of
nonradioactive EGF for up to 40 min at 37 °C. As shown in Fig.
12, the L679A,L680A substitution was
associated with increased ligand recycling compared with wild-type EGFR. Recycling rates were 4.52% min
1 for occupied
mutant receptors, versus 2.72% min
1 for
occupied wild-type receptors (Table I). In addition to having a faster
recycling rate, the amount of recycled 125I-EGF in cells
expressing EGFR/L679A,L680A reached a maximum of approximately 55%
after 20 min of incubation. In contrast, only 40% of
125I-EGF-wild-type EGFR complexes had recycled in the same
time interval. Together with data in the previous sections, this
suggests that the L679A,L680A substitution inhibits the degradation of
occupied full-length EGFRs by diverting ligand-receptor complexes to a recycling pathway without affecting internalization.

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Fig. 12.
125I-EGF recycling kinetics in
NR6 cell lines expressing full-length human EGFRs. NR6 cells were
preincubated for 2 h at 4 °C with 125I-labeled EGF
(1 ng/ml) and then switched to 37 °C for 5 min to allow for
internalization. After removing surface-bound ligand by mild
acid-stripping, 125I-EGF-loaded cells were incubated with a
100-fold excess of unlabeled EGF for 1 h at 4 °C and then
incubated at 37 °C for the indicated periods of time. At the end of
each incubation, media were collected to determine the amount of intact
and degraded 125I-EGF as described under "Experimental
Procedures." Surface bound 125I-EGF was removed by a
harsh acid strip, and cells were solubilized with 1 N NaOH
to determine cell-associated radioactivity. The sum of intact
125I-EGF in the media and surface-bound
125I-EGF released by a harsh acid strip was expressed as
the percentage of total radioactivity in the media and the cells at
each time point. The values of are the means ± S.D.
(n = 6). Some standard error bars are obscured by
symbols. WT, wild type.
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|
 |
DISCUSSION |
In this study, we have characterized the molecular basis for
selective transport of ligand-EGFR complexes to lysosomes. By examining
cytoplasmically truncated EGFRs exhibiting reduced protein stability,
we identified two categories of receptor proteins with enhanced
degradation phenotypes: those exhibiting enhanced degradation after
reaching the cell surface and those undergoing rapid degradation shortly after biosynthesis. In addition, we demonstrated that a
dileucine motif in the EGFR juxtamembrane domain is required for
efficient lysosomal transport of truncated EGFRs as well as ligand
occupied full-length EGFRs.
The mutant receptors c'-697 and c'-723, which terminate in the proximal
half of the kinase catalytic core domain, both exhibited enhanced
endosome-to-lysosome transport after delivery to the cell surface.
Because c'-674 receptors did not have a shortened half-life, we
hypothesized that EGFR residues 675-697 encompass a cryptic signal
that regulates sorting in the endocytic pathway. Examination of the
amino acid sequence between residues 675 and 697 revealed a potential
leucine-based sorting signal at amino acids,
Leu679-Leu680. To test the relative importance
of Leu679-Leu680, these residues were changed
to alanines in the context of a c'-697 truncation. The L679A,L680A
substitution abolished the enhanced degradation phenotype, suggesting
that Leu679-Leu680 acts to divert recycling
molecules to lysosomes when conformational restraints in the
carboxyl-terminal regulatory domain are removed by truncation.
To test the hypothesis that Leu679-Leu680 is
part of a signal that regulates lysosomal sorting of ligand-receptor
complexes, the L679A,L680A substitution was also studied in the context
of a full-length receptor. Identical to wild-type EGFR,
EGFR/L679A,L680A mediated slow uptake of a nonactivating EGFR mAb Fab
fragment consistent with constitutive membrane turnover (8). Ligand binding induced intrinsic tyrosine kinase activity and led to accelerated internalization of mutant and wild-type EGFRs to similar extents. These results indicate that
Leu679-Leu680 is not required for
internalization of full-length EGFRs, in agreement with findings from
other investigators who have shown that the EGFR juxtamembrane domain
is not important for ligand-induced receptor uptake (7, 11).
Although wild-type and mutant receptors facilitated the rapid
accumulation of intracellular ligand, steady-state levels of accumulated ligand were substantially lower in cells expressing the
mutant EGFR than in cells expressing wild-type EGFR. The reduced ability of cells expressing mutant receptors to accumulate
intracellular ligand was also evident in down-regulation assays
monitoring the disappearance of cell surface ligand binding sites.
Additionally, mutant EGFRs were not as efficiently transported to
lysosomes as wild-type EGFRs, as shown by differences in the
biosynthetic half-lives of ligand-occupied EGFRs. EGFR/L679A,L680A
receptors did, however, exhibit faster recycling kinetics than
wild-type EGFRs. These data suggest that reduced ligand accumulation
and down-regulation exhibited by EGFR/L679A,L680A receptors is due to
enhanced recycling of internalized receptors from endosomes.
A closer examination of the amino acid sequence surrounding
Leu679-Leu680 reveals that this region conforms
to other known leucine-based signals by several criteria. For example,
the targeting activity of leucine-based motifs is often influenced by
an acidic residue four or five residues amino-terminal to the leucine
pair (49). In addition, leucine-based signals may be regulated by
post-translational modification, because they often have nearby
phosphorylation sites (45). The putative signal encompassing
Leu679-Leu680 has both an appropriately
distanced acidic residue (Glu673) as well as nearby
mitogen-activated protein kinase phosphorylation sites
(Thr669 and Ser671) (Fig. 6). Although
leucine-based signals implicated in lysosomal transport were originally
identified in membrane proteins with relatively short (20-30 amino
acids) cytoplasmic tails, critical leucine motifs also have been found
in juxtamembrane regions of other membrane proteins with more extensive
cytoplasmic domains, such as the T-cell receptor CD3
subunit (29)
and the insulin receptor (50).
The altered transport of the EGFR/L679A,L680A is probably due to the
fact these molecules do not fully interact with sorting factor(s)
required for efficient transport to lysosomes, because lysosomal
transport is thought to be signal-mediated (reviewed in Ref. 22).
Although we do not know whether a signal encompassing Leu679-Leu680 facilitates protein-protein
interactions, computer-based modeling suggests that this region can
form an amphipathic
-helix. As shown in Fig. 6B, nonpolar
residues Asn676, Ala678, Leu679,
Leu680, Ile682, and Leu683 are
aligned on one side of a theoretical amphipathic helix and charged
residues Arg681, Lys684, and Glu685
on the opposite side. The hydrophobic face of this structure could
directly specify protein-protein interactions, or alternatively it
could confer stability to a signal located on the hydrophilic face by
interacting with surrounding molecules. In either case, leucine
side-chains are critical because activity of this signal is abolished
by substitution with less hydrophobic alanine residues.
Because endosome-to-lysosome transport is a multi-step process,
it is reasonable to hypothesize that each step is regulated by distinct
structural elements. In addition to the signal described in this study,
two other intrinsic EGFR lysosomal targeting signals have been reported
by other investigators (Fig. 6). One of these signals, located between
residues 945 and 957 at the distal border of the kinase catalytic core
(9), encompasses the binding site for SNX-1 (25). Another lysosomal
sorting signal has been localized to residues 1022-1123 at the
carboxyl terminus (23). It has also been proposed that sequestration of
EGFRs into internal vesicles of ECV/MVBs requires phosphorylation of
the EGFR substrate annexin I (51). Differences in their requirements
for kinase activity suggest that some signals act relatively early in
the endocytic pathway before kinase activity is extinguished, whereas
others act relatively late in the pathway after kinase inactivation
and/or ligand dissociation. Although these diverse sequences may be
redundant signals that regulate the same transport step, we consider it more likely that multiple lysosomal sorting signals act sequentially at
different steps in the endosome-to-lysosome pathway. Although the
relation of the Leu679-Leu680 signal to other
lysosomal sorting signals is currently unknown, the fact that steps
regulated by this signal occur within minutes of adding ligand
indicates that it acts relatively early in the endocytic pathway. In
addition, an adenovirus E3 protein that down-regulates EGFR by
diverting constitutively recycling EGFRs to ECV/MVBs contains an
identical motif at its carboxyl terminus (47). If the adenovirus
protein mimics a normal trafficking event, the dileucine motif in EGFR
probably acts either before or during incorporation into forming
ECV/MVBs.
Receptor proteins undergoing rapid degradation shortly after
biosynthesis had truncations involving the distal half of the kinase
domain. The rapid turnover of these receptors was not restricted to a
particular cell type, because similar results were obtained when these
proteins were expressed in other cell lines including NR6, Chinese
hamster ovary, and Madin-Darby canine kidney cells (data not shown).
These receptors exhibited half-lives in the order c'-957 > c'-809 > c'-885 (data not shown). Degradation of c'-885 and
c'-809 receptors, and to a lesser extent c'-957 receptors, was mediated
by an BFA-insensitive pathway (Fig. 3). One interpretation for these
results is that these molecules are disposed of by an ER quality
control apparatus that recognizes aberrant proteins (41). In contrast
to c'-885 and c'-809 receptors, a portion of c'-957 receptors appeared
to be degraded in a post-ER compartment. If the SNX-1 binding site is
constitutively active in the c'-957 receptors, these data suggest that
SNX-1 may mediate transport of c'-957 receptors that escape the ER
degradative pathway from the trans-Golgi network to lysosomes.
In summary, our results indicate that the efficient transport of
ligand-receptor complexes to lysosomes depends on an intrinsic signal
encompassing Leu679-Leu680 located at EGFR
juxtamembrane domain. The Leu679-Leu680 signal
exerts its effect by facilitating movement of occupied EGFRs through an
early endocytic compartment. Along with other studies that have
identified a basolateral sorting signal in this same region (30), we
suggest that the EGFR juxtamembrane domain may have a broad role in
vesicular transport.
 |
ACKNOWLEDGEMENTS |
We thank Ed Greenfield, Martin Snider, and
Leslie Friend for helpful comments during the course of this work, Jim
Crish for help with Northern blot analysis, and Starla Glick for EGFR
mAb 528 Fab fragments.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants CA49540 and DK45669 (to C. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported in part by National Institutes of Health Training Grant
T32-HL07717.
To whom correspondence should be addressed: Dept. of
Physiology and Biophysics, School of Medicine, 10900 Euclid Ave.,
Cleveland, OH, 44106-4970. Tel.: 216-368-8939; Fax: 216-368-5586;
E-mail: cxc39{at}po.cwru.edu.
The abbreviations used are:
EGF, epidermal
growth factor; EGFR, EGF receptor; ECV/MVB, endosomal carrier
vesicle/multivesicular body; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; BFA, brefeldin A; endo H, endoglycosidase H; ER, endoplasmic reticulum; PCR, polymerase chain
reaction; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin.
 |
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