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 KilDagger , Michael HobertDagger §, and Cathleen CarlinDagger parallel

From the Dagger  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
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Abstract
Introduction
References

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
Top
Abstract
Introduction
References

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 [alpha -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% beta -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 gamma  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 gamma  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; beta -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.

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.

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.

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.

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.

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.

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 gamma  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.

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.

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.


    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 gamma  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 alpha -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.

parallel 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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Abstract
Introduction
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