©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Endocytosis and Lysosomal Targeting of Epidermal Growth Factor Receptors Are Mediated by Distinct Sequences Independent of the Tyrosine Kinase Domain (*)

(Received for publication, August 23, 1994; and in revised form, November 29, 1994)

Lee K. Opresko (1) Chia-Ping Chang (2)(§) Birgit H. Will (1) Patrick M. Burke (1) Gordon N. Gill (2) H. Steven Wiley (1)

From the  (1)Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132 and the (2)Department of Medicine, University of California at San Diego, La Jolla, California 92093

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) leads to accelerated receptor degradation. Two models have been proposed to explain this. In the first model, induced internalization expands the intracellular pool of receptors, leading to enhanced lysosomal targeting. The second model proposes that activation of intrinsic receptor kinase activity induces inward vesiculation of endosomes, thus interrupting receptor recycling. To test these models, we created EGFR mutants that lack the conserved tyrosine kinase domain, but retain different parts of the distal carboxyl terminus regulatory region. Mutants lacking all distal regulatory sequences underwent slow internalization (0.02 min) and turnover (t 24 h), similar to unoccupied, holo-EGFR. Mutant receptors that lacked the kinase domain, but retained the entire distal regulatory domain, were constitutively internalized and targeted to lysosomes, even in the absence of EGF. The turnover of these receptors (t 11 h) was similar to that of occupied, kinase-active holo-EGFR (t 9.5 h). These results show that receptor tyrosine kinase activity is not required for the targeting of EGFR to lysosomes. Receptor mutants which expressed previously identified endocytic sequences underwent rapid internalization. Unexpectedly, enhanced turnover of EGFR mutants required additional sequences located between residues 945 and 991 in the holo-EGFR. Thus, internalization and lysosomal targeting of EGFR are separate processes mediated by distinct sequences. Our results indicate that induced internalization is necessary, but not sufficient, for enhanced EGFR degradation. Instead, down-regulation requires exposure of previously cryptic internalization and lysosomal targeting sequences. Occupied EGFR thus appear to be handled by the endocytic machinery in the same fashion as other constitutively internalized or lysosomally targeted receptors.


INTRODUCTION

The spatial distribution and trafficking of receptors is important for their function. The polarized distribution of the polymeric immunoglobulin receptor is required for the serosal-to-mucosal transport of dimeric IgA and pentameric IgM in various epithelial cells(1, 2) . The specific trafficking of the mannose 6-phosphate receptor between the Golgi apparatus and late endosomes is necessary for transport of many enzymes to lysosomes(3, 4) . Low density lipoprotein and transferrin receptors cycle between coated pits and endosomes to deliver nutrients to the cell interior (5, 6, 7) . Localization of these receptors to their correct intracellular destination occurs through the interaction of specific receptor targeting domains with components of the cellular trafficking machinery. Mutations in these targeting domains result in disruption of receptor distribution which can lead to disease(8) .

Most of the progress in identification and functional analysis of targeting domains has been achieved using receptors constitutively found in discrete cellular locations. Disruption of specific sequences in these receptors by site-directed mutagenesis leads to an altered cellular distribution of the mutated gene product(2, 8, 9) . A different situation is presented by signaling receptors, such as EGFR (^1)or insulin receptors. Their cellular distribution is highly dependent on both ligand occupancy and activation of intrinsic receptor tyrosine kinase activity(10, 11) .

Efficient EGFR internalization also requires the presence of specific sequences in the receptor carboxyl terminus. This region contains at least three endocytic domains that appear analogous to those found in constitutively internalized receptors. Endocytic motifs from transferrin and insulin receptors can be substituted for the endogenous EGFR sequences, but they work only if the resulting hybrid receptor displays intrinsic tyrosine kinase activity(12) . Lysosomal targeting of EGFR also requires ligand occupancy, but appears to depend on sequences distinct from those involved in occupancy-induced endocytosis (13) . This indicates that multiple domains are involved in regulating the EGFR trafficking pattern. The eventual consequence of ligand-induced internalization and lysosomal targeting is receptor down-regulation. This appears to play an important role in attenuating receptor signaling(14, 15) , but the relative importance of induced internalization versus lysosomal targeting to overall EGFR down-regulation is still unclear. It has been proposed that receptor degradation is primarily regulated by endocytosis, which controls the size of the intracellular receptor pool targeted for degradation(10) . Alternately, it has been suggested that interruption of receptor recycling is the primary mechanism that regulates receptor degradation(16) .

Although some of the structural requirements for EGFR down-regulation are known, it is unclear why tyrosine kinase activity is also necessary. Self-phosphorylation of the EGFR appears to result in a more open conformation, which has been proposed to expose trafficking domains(17) . Tyrosine phosphorylation must also work indirectly, however, because mutant EGFR that lack autophosphorylation sites but retain endocytic sequences can still undergo ligand-induced internalization(10, 12, 18) . It has been proposed that the indirect mechanism involves the formation of a complex between the receptor and a phosphorylated substrate. Formation of this complex would in turn expose endocytic domains in the EGFR(10, 12) . An alternate hypothesis has been advanced in which EGFR endocytosis is kinase-independent. Instead, ligand-induced kinase activity is proposed to prevent recycling of the receptor back to the cell surface by inducing inward vesiculation of multivesicular bodies(16, 19, 20) .

Unfortunately, because regulated trafficking of signaling receptors depends on their state of activity, it is difficult to study this process directly. Any mutation which alters the enzymatic activity of receptors will also indirectly affect their intracellular trafficking. For example, phosphorylation of the EGFR by protein kinase C at Thr completely blocks ligand-induced internalization, but this is due to inhibition of receptor kinase activation and not interference with endocytosis per se(21) . In an effort to circumvent the pleiotropic effects of kinase activity on receptor trafficking behavior, we created mutant EGFR in which the conserved tyrosine kinase domain was deleted. We show here that such mutants undergo rapid internalization and lysosomal targeting, even in the absence of ligand binding. The constitutive down-regulation of these mutant receptors is absolutely dependent upon specific sequences in the carboxyl terminus. These sequences can be segregated into a class required for rapid internalization and a class required for lysosomal targeting. Our results indicate that receptor down-regulation is regulated at two distinct steps and that tyrosine kinase activity works by stabilizing or amplifying receptor conformational changes induced by ligand occupancy.


EXPERIMENTAL PROCEDURES

General

Mouse EGF was purified from submaxillary glands (22) . EGF and monoclonal antihuman EGFR antibody 528 IgG (23) or Fab fragments of 13A9 IgG (24) were iodinated with I (Amersham) using IODOBEADS (Pierce) according to the manufacturer's recommendations. Free iodine was separated from the radiolabeled ligands by dialysis or by passing the mixture over a 0.8 times 20 cm column of Sephadex G-10 equilibrated with phosphate-buffered saline. The specific activity of I-labeled EGF was generally between 600 and 1,800 cpm/fmol, and I-labeled monoclonal antibodies was between 1,300 and 1,900 cpm/fmol. Fab fragments and intact anti-EGFR monoclonal 13A9 (24) were kind gifts from Marjorie Winkler of Genentech, Inc. Monoclonal antibodies against human EGFR (528, 579, and 225(23) ) were purified from hybridomas obtained from the American Type Culture Collection. Polyclonal rabbit antibody N-13 directed against a peptide corresponding to residues 1-13 in human EGFR was a gift of Dr. Debora Cadena. Polyclonal rabbit antibodies specific for phosphotyrosine were generated and affinity-purified as described(25) . Polyclonal rabbit antibody against the lysosomal marker lgp120 was a gift of Dr. Marilyn Farquhar(26) . Secondary antibodies labeled with either Texas Red or fluorescein were obtained from Cappel Laboratories.

Construction and Expression of Mutant EGFR

c`688 EGFR cDNA was prepared by partial EcoRI digestion as described previously(27) . c`688 f945-1022 and c`688 f945-1186 receptor cDNAs were generated by digestion of pX EGFR (28) with SmaI and HindIII and ligation of SmaI-HindIII fragments and EcoRI and HindIII fragments from pX EGFR or pX c`1022 EGFR(27) . c`688 f945-991, c`688 f993-1022, and c`688 f1024-1186 EGFR cDNAs were prepared by excising the fusion segments as SalI to HindIII fragments from the appropriate mutant EGFR (18) and ligating them to c`688 EGFR containing a compatible linker oligonucleotide. Re-establishment of the SalI site places codons for V and D at the junction. All constructions were verified by dideoxynucleotide sequencing. cDNAs were transfected into B82L cells that lack endogenous EGFR by the calcium phosphate procedure(29) . Stable clonal transfectants were selected, and the EGFR gene was amplified by increasing concentrations of methotrexate from 400 nM to 5 µM. At least two independent transfections were used to select each cell line expressing mutant EGFR. B82 cells were grown in Dulbecco's modified Eagle's medium (Flow Laboratories) containing dialyzed 10% calf serum (HyClone) and 5 µM methotrexate.

Internalization and Recycling Measurements

Internalization of both empty and occupied EGFR was determined by first incubating the cells for 3 h at 0 °C with 0.5 µg/ml I-labeled 13A9 Fab fragment in either the absence or presence of 0.5 µg/ml EGF. The cells were rinsed and rapidly warmed to 37 °C by the addition of medium either without or with EGF. Internalization of EGF alone was measured by changing to medium containing the indicated concentrations of I-EGF at 37 °C. The relative amounts of ligand associated with the surface and interior of the cells were determined by acid-stripping at 0 °C using 50 mM glycine-HCl, 100 mM NaCl, 2 mg/ml polyvinylpyrrolidone, 2 M urea, pH 3.0(30) . Nonspecific binding was measured using B82 cells lacking EGFR and was less than 5% of total binding. Cell number was determined with a Coulter counter.

Recycling was determined by incubating cells for 20 min at 37 °C with I-EGF. The cells were rapidly rinsed at 0 °C followed by removal of approximately 90% surface-associated ligand with stripping buffer lacking urea. The cells were rinsed two times with phosphate-buffered saline buffer and returned to 37 °C by the addition of prewarmed medium containing 1 µg/ml unlabeled EGF to prevent rebinding of dissociated ligand(31, 32) . At different times, the cells were rinsed and the relative amount of label associated with either the surface or inside of the cells was determined by acid stripping(30) . As measured by release of radiolabeled monoiodotyrosine (33) , no degradation of the internalized antibody occurred during the incubation period.

Quantification of EGFR Levels

Cells were incubated at 37 °C for 24 h in 30 µCi/ml of TranS-label in Dulbecco's modified Eagle's medium containing a 10% normal concentration of methionine and cysteine. Cells were rinsed and removed from the plates at 0 °C by scraping into 1.5 ml of D/H/B medium containing a mixture of protease inhibitors (10 µg/ml concentration each of chymostatin, pepstatin, leupeptin, aprotinin, and 4 mM iodoacetate(34, 35) ). The cells were gently pelleted at 1000 times g, rinsed once with phosphate-buffered saline, and extracted for 10 min at 0 °C with 120 µl of 1% Triton X-100, 10% glycerol, 2 mM EDTA, 10 mM HEPES, pH 7.0, and a mixture of protease inhibitors (as above but using a 10-fold greater concentration). Cell debris was removed by centrifugation (10,000 times g for 10 min), and the rest of the cell extracts were snap-frozen in a methanol/dry ice bath. After thawing, 2-µl aliquots were removed from each extract sample and spotted onto Whatman 42 filter paper in a grid fashion 1.5 cm apart. The specific activity of the cellular protein was determined by washing the filter paper with cold 7.5% trichloroacetic acid and staining for protein by the method of Bramhall et al.(36) . The protein-associated S on the filter paper was first quantified by using the Bio-Rad G250 Molecular Imager phosphorimager. Subsequently, the stained filter paper was cut apart and the amount of protein in each stained spot was quantified by eluting the dye with 66% methanol, 1% NH(4)OH and measuring the absorbance at 610 nm. EGFR was then immunoprecipitated using 150 µg/ml of both monoclonal 528 IgG and rabbit anti-mouse secondary antibody, and 20 µl of a 50% slurry of Protein A-Sepharose as described previously(23, 37) . The final immunoprecipitate was solubilized in 25 µl of 2% SDS, 20 mM dithiothreitol at 100 °C and run on a 5-15% gradient polyacrylamide gel. The gel was dried and the amount of radioactivity in the receptor bands was quantified using the G250 Molecular Imager. The relative amount of receptor radioactivity was corrected for both specific activity and total amount of cellular protein, the methionine/cysteine levels of the receptors as well as the fractional labeling of the receptors using 15 h as the doubling time of the cells.

Quantification of EGFR mRNA Levels

A ribonuclease protection assay (Ambion RPA II kit) was used to quantify the levels of both wild type and mutant EGFR in transfected cells. The EGFR probe complements a 350-bp region encoding amino acids 613 to 730 in holo-EGFR(38) . It was isolated as a NaeI/BamHI fragment and inserted into the BamHI/HindIII sites of BlueScript (Stratagene). The P-labeled antisense probe was transcribed from the T7 promoter essentially as described (39) . The 150-bp beta-actin probe was generated from the plasmid obtained from Ambion, Inc., using SP6 polymerase after linearizing with DdeI and using a 3-fold lower specific activity of [P]UTP. All probes were gel-purified before use. Total cellular RNA was isolated from confluent 100-mm plates(40) . Mixtures of the EGFR and beta-actin probes were hybridized overnight with 5 µg of total cellular RNA followed by ribonuclease digestion using the manufacturer's instructions. The protected RNA was precipitated, denatured, and separated on a 5% polyacrylamide, 8 M urea gel as described in the Ambion kit. The gels were dried, and the protected probe was quantified using the Bio-Rad G250 Molecular Imager. The mRNA from the mutant EGFR constructs protected approximately 225 bp of the EGFR probe while the wild type EGFR mRNA protected the entire 350-bp probe. For quantification, the amount of radioactivity protected was corrected for the length of the probe and its specific activity. The amount of EGFR mRNA was expressed as a ratio to the internal beta-actin standard and normalized to the amount expressed in cells transfected with c`688 EGFR. Two separate experiments using each cell type were done in triplicate.

Tyrosine Phosphorylation of Cellular Proteins

Cells expressing different EGFR mutants were incubated in the presence or absence of 200 ng/ml EGF at 37 °C. The cells were rapidly rinsed at 0 °C, removed from the plates with a rubber policeman, and extracted with the Triton X-100 solution described above for immunoprecipitation, with the addition of 0.1 mM Na(3)VO(4)(25) . After 5 min at 0 °C, cell debris was removed by centrifugation at 10,000 times g for 3 min. Extracts were denatured at 100 °C by the addition of an equal volume of 2% SDS and 12 mM dithiothreitol. Protein concentrations were determined by the method of Bramhall(36) . Samples were separated on 5-10% gradient gels, transferred to nitrocellulose in the presence of 0.1 mM Na(3)VO(4), and blocked with 2.5% bovine serum albumin, 0.1 mM Na(3)VO(4), and 0.005% Tween 20. Tyrosine phosphate was detected using 5 µg/ml affinity-purified antiphosphotyrosine polyclonal antibodies(25) . EGFR were detected in parallel blots using N13 polyclonal antibodies. These were visualized using 50 ng/ml I-Protein A for 30 min followed by exposure to either film or phosphorimager plates.

Fluorescence Microscopy

Cells were plated on fibronectin-coated coverslips 48 h before the experiment. Cells treated either without or with EGF were fixed for 15 min with freshly prepared 3% paraformaldehyde, 0.02% glutaraldehyde and then permeabilized for 15 min with 0.0125% saponin(41) . Free aldehyde groups were quenched with 0.1% NaBH(4) for 10 min. Cells were incubated simultaneously with a mixture of anti-EGFR monoclonals 528, 579, 225, and 13A9 (10 µg/ml each) and anti-lgp 120 (1:500) for 45 min followed by staining with fluorescein isothiocyanate-labeled goat anti-mouse and Texas Red-labeled goat anti-rabbit IgG antibodies (1:100) for 45 min. The coverslips were then dehydrated through an ethanol series and mounted in benzyl alcohol:benzyl benzoate (1:2) and viewed with a DAGE Sit-11 camera and DSP-200 image processor attached to a Zeiss microscope with a 100times objective. Images were averaged over 32 frames and captured using a Data Translation QuickCapture board. Images were scaled to 256 gray levels using Adobe Photoshop 2.0 on the Macintosh.

Data Analysis

Values for surface-bound and internalized ligand were corrected for nonspecific binding and for spillover from the interior and surface of the cell, respectively(33) . Specific internalization rates were determined by plotting the integral of surface-associated ligand against the amount internalized(30) . The slopes were then determined by linear regression. Correlation coefficients of internalization plots were generally >0.98. Nonlinear parameter estimation was done with the program proFit (QuantumSoft) on the Macintosh using the Levenberg-Marquardt algorithm.


RESULTS

Removal of the Tyrosine Kinase Domain Enhances Constitutive Endocytosis of EGFR

To determine the contribution of sequences within the conserved tyrosine kinase domain to EGFR trafficking behavior, we deleted these residues between 689 and 944(42) . The resulting receptors are termed DeltaKD-EGFR. Because such a large deletion could affect the internalization of both empty as well as ligand-occupied EGFR, we developed a method for evaluating endocytosis which was independent of ligand binding. Fab fragments of alphaEGFR monoclonal Ab 13A9 were radiolabeled and bound to cells in both the absence and presence of saturating concentrations of EGF. This monoclonal Ab does not inhibit the binding of EGF to EGFR nor does it affect receptor tyrosine kinase activity and thus can act as a tag for both empty and occupied receptors(24) . After warming the cells to 37 °C, the rate of antibody internalization was determined. As shown in Fig. 1, antibody bound to wild type EGFR was internalized much more rapidly when receptors were occupied. Inactivation of receptor kinase activity by a point mutation in the ATP binding site (Met) virtually eliminated the increased internalization rates induced by EGF binding. Deletion of carboxyl-terminal sequences distal to residue 688 had no effect on internalization of empty receptors, but eliminated the stimulatory effect of ligand occupancy (Fig. 1, B and C). In contrast, DeltaKD-EGFR were internalized rapidly in the absence of EGF (Fig. 1D), but the addition of EGF still had a small stimulatory effect, similar to that observed for Met receptors. Therefore, removal of the EGFR kinase domain appeared to increase the constitutive rate of receptor endocytosis.


Figure 1: Relative internalization of wild type and mutant EGF receptors in both occupied and empty states. Cells expressing the indicated receptor types were incubated for 3 h at 0 °C with I-labeled Fab fragment of the anti-EGFR monoclonal antibody 13A9 in either the absence (circle) or presence (bullet) of 0.5 µg/ml unlabeled EGF. Cells were then brought to 37 °C for the indicated times. Shown is the percent of initially bound antibody which was internalized as determined by acid stripping.



Lysosomal Targeting of Receptors Lacking a Tyrosine Kinase Domain

It has been proposed that ligand-induced internalization should always lead to enhanced lysosomal targeting and down-regulation of receptors(10, 43) . Because the endocytic rate of DeltaKD-EGFR appeared to be higher than empty EGFR, we also determined whether these receptors displayed enhanced targeting to lysosomes. Cells were treated for 18 h either without or with 125 ng/ml EGF, fixed, permeabilized, and stained for EGFR as well as the lysosomal marker lgp120(26) . We found that in the absence of EGF, wild type EGFR were predominantly distributed at the cell surface (Fig. 2, top left). As expected, incubation with saturating EGF concentrations induced a steady-state redistribution of wild type EGFR to primarily lysosomes and endosomes. In contrast, DeltaKD-EGFR were located in lysosomes and endosomes in both the absence and presence of ligand (Fig. 2, middle panels). Receptors that lack sequences carboxyl-terminal to the kinase domain (c`688) remained localized to the cell surface, even in the presence of EGF (Fig. 2, bottom panels). Thus, deletion of the kinase domain not only resulted in the constitutive internalization of EGFR, but also constitutive targeting to lysosomes.


Figure 2: Constitutive lysosomal targeting of DeltaKD-EGFR. Cells expressing the receptors indicated in the right-hand labels were treated for 18 h either without (Control) or with (+EGF) 125 ng/ml EGF. Cells were then fixed, permeabilized, and stained for EGFR (left panels) and the lysosomal protein lgp120 (right panels). The two labels were simultaneously visualized. Arrows in the middle panels are for reference.



Multiple Domains Can Mediate Constitutive Internalization

Previous studies indicated the existence of at least three different endocytic motifs distributed within the carboxyl terminus of the EGFR(12) . The ability of these sequences to mediate internalization requires receptor kinase activity. Kinase activation changes the receptor conformation, which could expose previously cryptic endocytic domains(17) . Deletion of the kinase domain could effect a similar change in receptor conformation. If this is the case, then the previously identified endocytic domains should function in the context of a kinase deletion mutant. To test this hypothesis, we created EGFR mutants in which combinations of the different endocytic domains were fused to c`688 EGFR (Fig. 3, top panel). Mouse B82 cells were stably transfected with plasmids encoding these receptor constructs, and clones were selected which expressed comparable levels of receptor mRNA (Fig. 3, bottom panel). These clonal lines were then used to evaluate the trafficking behavior of the mutant EGFR.


Figure 3: Construction of DeltaKD-EGFR retaining varying parts of the endocytic regulatory domains. A, schematic of EGFR showing the location of the previously identified endocytic domains. Numbers refer to amino acid residues in human EGFR. Each of the indicated sequences was fused onto a c`688 EGFR and expressed in B82 cells. B, relative levels of mRNA encoding the different DeltaKD-EGFR. Amino acids in the domain fused to residue 688 are indicated above each lane. The ratios of the DeltaKD-EGFR to beta-actin bands as determined by direct counting is indicated beneath each band.



The specific internalization rates of the different DeltaKD-EGFR mutants were determined and compared to the internalization of the holo-EGFR at both low and high occupancies. As shown in Fig. 4, all fusion receptors displayed higher specific internalization rates than the c`688 receptor. The relative activity of the endocytic domains was III > I > II, which is the same in the context of kinase-active receptor mutants(12) . Internalization rates for each of the DeltaKD-EGFR mutants were similar at both low and high ligand concentrations, indicating that internalization is constitutive (data not shown). There was no evidence of synergism between the different endocytic domains, similar to the situation observed in the analogous kinase-active receptor mutants(12) . Because internalization rates of the DeltaKD-EGFR mutants are constitutive, they should be the same as fully occupied holo-EGFR, assuming the rate-limiting step of the two receptor types is the same. As shown in Fig. 4, the specific internalization rate of fully occupied holo-EGFR (0.084 min) is similar to those displayed by the DeltaKD-EGFR mutants, with the exception of the c`688 f1022-1186 receptor which is internalized much faster. These data show that all EGFR endocytic domains are functional when the kinase domain is deleted. However, the higher internalization rate of the c`688 f1022-1186 receptor indicates that the rate-limiting steps for internalization of the kinase-active and DeltaKD-EGFR mutants are not necessarily the same.


Figure 4: Specific internalization rates of different DeltaKD-EGFR mutants. The specific internalization rate of EGF was determined using cells expressing the indicated DeltaKD-EGFR using a concentration of 1 ng/ml EGF. Internalization of wild type receptors was measured at either 1 or 110 ng/ml as indicated in the figure. The number of independent experiments is shown in parentheses. Error bars indicate S.D.



To confirm that internalization of the different DeltaKD-EGFR mutants was independent of their state of occupancy, cells expressing the receptors were incubated with either Ilabeled EGF or I-labeled 528 alphaEGFR IgG. As shown in Fig. 5, the steady-state distribution of labeled receptor between the cell surface and inside was similar in the case of either the activating ligand or nonactivating monoclonal antibody. The accumulation of labeled antibody was more pronounced than EGF, possibly because of differences between receptor dissociation or lysosomal degradation.


Figure 5: Approach to steady state binding of EGF and anti-EGFR antibodies to cells expressing different DeltaKD-EGFR. Cells expressing the DeltaKD-EGFR mutants c`688 (down triangle-down triangle), c`688 f945-1186 (box-box), c`688 f1022-1186 (circle-circle), c`688 f945-991 (-), c`688 f945-1022 (Delta-Delta), or c`688 f993-1022 (x-x) were incubated with either 10 ng/ml I-EGF (top panel) or 200 ng/ml I-labeled 528 monoclonal Ab (bottom panel). At the indicated times, the ratio of label associated with the inside to surface of the cells was determined by acid stripping.



Altered Recycling and Turnover of Constitutively Active Receptors

Although the results shown in Fig. 5confirm that the endocytic behavior of DeltaKD-EGFR was constitutive, the rate of accumulation of either antibody or EGF over a 2-h period did not directly correlate with the specific internalization rate of the receptor. For example, receptors containing only endocytic domain III were internalized almost 3-fold faster than those containing domains I + II + III (Fig. 4). EGF bound to either of these receptors, however, was accumulated to the same extent after 1 h. Because intracellular accumulation of ligand depends on rates of internalization, recycling, and degradation, this result suggested that the DeltaKD-EGFR mutants differed in their ability to interact with multiple components of the cellular trafficking machinery. Consistent with this is the observation that the degradation rate of internalized radiolabeled Fab fragments of Ab 13A9 bound to receptors containing only endocytic domain III was 20% as fast as fragments bound to receptors containing all three (1.9% versus 9.4% per h, respectively; data not shown).

We have previously shown that the carboxyl terminus of EGFR contains sequences which appear involved in endosomal retention and lysosomal targeting(13, 32) . To determine whether the ability of some of the DeltaKD-EGFR mutants to efficiently accumulate ligand was due to the presence of these retention sequences, rates of recycling were directly compared. Cells were incubated for 20 min with I-EGF. Surface-associated ligand was removed with a mild acid treatment, and the cells were then chased with excess unlabeled ligand. As shown in Fig. 6, EGF bound to DeltaKD-EGFR containing only endocytic domains II or III recycled with a k(x) of 0.18-0.19 min and to an extent of 90% ( = 0.9). EGF was recycled both more slowly and to a lesser extent when bound to the DeltaKD-EGFR mutant that contained the entire regulatory cytoplasmic domain (k(x) = 0.14 min and = 0.7). The DeltaKD-EGFR containing only endocytic domain I also displayed slower recycling rates and greater endosomal retention (k(x) = 0.15 min and = 0.8), although not to the extent of the receptor containing all three endocytic domains. These results suggest that endosomal retention of EGFR is facilitated by sequences located in the region between residues 945 and 991.


Figure 6: Recycling of 125I-EGF associated with different DeltaKD-EGFR. Cells expressing c`688 f945-1186 (I + II+ III) (box-box), c`688 f1022-1186 (III) (circle-circle), c`688 f993-1022 (II) (x-x), or c`688 f945-991 (I) (-) receptors were incubated for 20 min at 37 °C with I-EGF. Surface-associated ligand was removed at 0 °C followed by a chase at 37 °C. The amount of I-EGF remaining inside the cells at the indicated times was determined and expressed as a percent of the intracellular ligand prior to the chase. The curves through the data points were calculated by nonlinear regression.



Although there was a correlation between decreased recycling of EGF and enhanced accumulation of ligand, absolute differences in recycling between the DeltaKD-EGFR mutants were relatively small. However, steady-state ligand accumulation required incubation times of greater than 1 h (see Fig. 5). The t of EGF recycling was 5 to 10 min, meaning that multiple rounds of internalization and recycling would occur, amplifying small differences between receptors. Because the small differences in absolute recycling rates made it difficult to use this parameter to reliably evaluate receptor behavior, we developed a more robust assay based on specific protein/mRNA ratios.

For any given protein, the ratio of mRNA to protein mass is directly proportional to turnover rates(44) . Because lysosomal targeting of receptors is required for their degradation, increases in the rate of lysosomal targeting between two receptor mutants will decrease their relative protein/mRNA ratios. To determine the levels of EGFR mRNA in transfected cells, a ribonuclease protection assay was used with beta-actin as an internal standard. Receptor protein mass was determined by quantitative immunoprecipitation of S-labeled cell extracts. As shown in Table 1, the constitutive protein/mRNA ratio was similar between unoccupied holo- and c`688 EGFR. DeltaKD-EGFR mutants containing only endocytic domains II or III (between residues 993 and 1186) also displayed protein/mRNA ratios similar to unoccupied wild type receptors. However, all DeltaKD-EGFR containing endocytic domain I displayed significantly lower protein/mRNA ratios, indicating a higher constitutive turnover rate. The protein/mRNA ratio of the DeltaKD-EGFR containing the entire carboxyl-terminal regulatory region was only about 1/3 of that displayed by unoccupied holo-EGFR. Significantly, the addition of a saturating amount of EGF to cells expressing holo-EGFR decreased their steady state receptor mass to 25-35% of the initial value (data not shown). These data indicate that turnover of DeltaKD-EGFR that contain the entire regulatory carboxyl terminus is similar to occupied, kinase-active holoreceptors.



To demonstrate that lower protein/mRNA ratios of some DeltaKD-EGFR mutants were due to accelerated turnover, this parameter was measured directly. Cells expressing either wild type EGFR or DeltaKD-EGFR displaying either low or normal protein/mRNA ratios were labeled with S-amino acids and then chased in either the absence or presence of saturating concentrations of EGF. The turnover of the receptors was then determined by immunoprecipitation. As shown in Fig. 7, in the absence of EGF, the turnover of wild type receptors was similar to the DeltaKD-EGFR mutant containing only endocytic domain III (25-28 h). In contrast, the turnover of the full-length DeltaKD-EGFR containing the entire carboxyl terminus (I + II + III) was significantly faster (11 h). In the presence of EGF, turnover of wild type holoreceptors was accelerated to 10 h, whereas turnover of the DeltaKD-EGFR containing the entire c` terminus or the III domain alone was unaffected. These data establish that the rate of degradation of DeltaKD-EGFR (c`688 f945-1186) is the same as fully occupied wild type EGFR. In addition, this enhanced degradation requires specific sequences within the carboxyl region of the receptor.


Figure 7: Turnover of wild type and DeltaKD-EGFR mutants in the absence and presence of EGF. Cells expressing wild type (bullet-bullet), c`688 f945-1186 (box-box), or c`688 f1022-1186 (circle-circle) receptors were metabolically labeled with S-amino acids and then chased in unlabeled medium in the absence (upper panel) or the presence (lower panel) of 1 µg/ml EGF. The amount of label remaining in the receptors was determined at the indicated times by immunoprecipitation followed by quantitation using a Molecular Imager. The results are the average of four separate experiments ± S.E.



Constitutively Active Receptors Are Not Phosphorylated on Tyrosine Residues

Under normal circumstances, the intrinsic tyrosine kinase activity of EGFR is required for ligand-induced internalization (10) . Although the constitutively active DeltaKD-EGFR lack a kinase domain, it was still possible that these receptors could be phosphorylated by other cellular tyrosine kinases. To test for this possibility, extracts of cells expressing either wild type or mutant EGFR were separated by gel electrophoresis. Receptors and phosphotyrosine-containing proteins were then detected by Western blot analysis using appropriate antibodies. As shown in Fig. 8A, no phosphotyrosine-containing proteins were observed that corresponded to the positions of the mutant EGF receptors. No change was observed upon receptor occupancy. Whereas the addition of EGF to cells expressing holo-EGFR resulted in significant receptor autophosphorylation, EGF had no effect on any DeltaKD-EGFR. In addition, occupancy of DeltaKD-EGFR did not discernibly change the phosphorylation state of other cell proteins. These data demonstrate that the constitutive trafficking activities of the DeltaKD-EGFR do not directly involve activated tyrosine kinases.


Figure 8: Lack of tyrosine phosphorylation of DeltaKD-EGFR mutants. A, Western blot analysis of extracts of cells expressing the DeltaKD-EGFR mutants c`688 f945-1022 (A), c`688 f945-991 (B), c`688 f993-1022 (C), c`688 f1022-1186 (D), or c`688 f945-1186 (E). Equal amounts of extract were run on 5-10% gradient SDS gels and transferred to nitrocellulose. The membranes were then probed with polyclonal antibodies against either the EGFR (top panel) or phosphotyrosine (bottom panel) followed by I-labeled Protein A. The images were obtained directly from a Molecular Imager. B, Western blot analysis of tyrosine-phosphorylated proteins in cells expressing the DeltaKD-EGFR mutants that were either treated without(-) or with (+) 200 ng/ml EGF for 5 min at 37 °C. Tyrosine-phosphorylated proteins were visualized as described in panel A. Shown is an autoradiograph of the gel.




DISCUSSION

Receptors involved in signal transduction typically display regulated cellular trafficking. Mutations which inactivate the signaling capacity of these receptors usually abrogate occupancy-induced changes in receptor internalization or recycling(11, 45, 46, 47) . The reasons for this have not been clear. In the case of EGFR or insulin receptors, point mutations which inactivate tyrosine kinase activity eliminate ligand-induced endocytosis(27, 48) . Mutants in which tyrosine phosphorylation sites are replaced with phenylalanine residues also lack the ability to undergo ligand-induced internalization(49, 50) . One effect of self-phosphorylation, however, is a conformational change of the receptor(17) . SH2 domain-containing proteins subsequently bind to the occupied, activated receptors, perhaps stabilizing or enhancing conformational changes(51) . Thus, it has been unclear whether tyrosine phosphorylation per se is required for ligand-induced endocytosis or whether consequent conformational changes in the receptor are responsible.

The results of the current study suggest that an important component of ligand-induced internalization is a conformational change in the receptor. Removal of the conserved tyrosine kinase domain of EGFR results in the constitutive internalization and lysosomal targeting of the receptor. This indicates that sequences mediating endocytosis and lysosomal targeting normally exist in a cryptic state and are exposed upon receptor activation. It is possible that the altered trafficking of the mutant receptors we studied was mediated by sequences that do not normally serve that function in the holoreceptor, but this appears unlikely for several reasons. First, the specific internalization rate of the DeltaKD-EGFR containing the entire regulatory carboxyl terminus was the same as that of the fully occupied holo-EGFR (Fig. 3). Second, the cellular distribution, protein/mRNA ratios, and receptor turnover rates of DeltaKD-EGFR and occupied holo-EGFR were the same. Finally, endocytic and lysosomal targeting sequences mapped to the same receptor regions in both DeltaKD-EGFR and holoreceptors. It seems most likely that the similar behavior of occupied holo- and DeltaKD-EGFR in these three independent parameters is due to their similar functional states.

It has been suggested previously that receptor kinase activity is not required for ligand-induced internalization of the EGFR(16, 19) . By using a very sensitive assay for internalization of both empty and occupied receptors, we found that ligand occupancy of kinase-inactive Met EGFR does have a small stimulatory effect on endocytosis (Fig. 1). This positive effect of receptor occupancy was also seen for DeltaKD-EGFR that retained endocytic sequences. It thus appears that ligand occupancy per se promotes a receptor conformation that is more favorable for its interaction with the endocytic apparatus. Nevertheless, kinase-active receptors displayed a 5-10-fold greater stimulation of endocytosis. We have previously shown that kinase-dependent endocytosis is both specific and saturable and have postulated that the responsible rate-limiting ``internalization component'' is a substrate for the receptor kinase activity(30, 52, 53) . Interestingly, endocytosis of DeltaKD-EGFR does not appear to be saturable, indicating that these mutations bypass the normal rate-limiting step for EGFR endocytosis. If this is the case, then receptor kinase activity could work by phosphorylating the internalization component, which then could bind to the EGFR and induce a conformation change sufficient to expose the endocytic codes. The previously described high affinity saturable endocytosis would therefore be due to the high affinity binding of the internalization component to the EGFR. The primary role of receptor kinase activity would thus be to amplify the conformational change induced by receptor occupancy to expose sequences necessary for regulated receptor trafficking.

A significant finding in this study was that rapid internalization was not sufficient for accelerated turnover of EGFR. Even though c`688 f1022-1186 EGFR displayed internalization rates almost 10-fold higher than either c`688 or unoccupied holo-EGFR, the turnover rate of these receptors was the same. Thus, the flux of receptors through the endocytic pathway is not normally rate-limiting to receptor turnover and suggests that ligand-induced endocytosis per se is not sufficient to lead to receptor down-regulation. Sequences between 945 and 991 appear to be necessary to direct lysosomal targeting. Because antibodies bound to receptors containing these sequences were degraded at an accelerated rate, this targeting must occur from the endocytic pathway. DeltaKD-EGFR containing the entire regulatory carboxyl terminus were targeted more efficiently to lysosomes than those containing only sequences between 945 and 991 (region I). This could indicate the presence of additional lysosomal targeting sequences outside of region I. Alternatively, the conformation of the DeltaKD-EGFR containing the entire regulatory carboxyl terminus could be more conducive to interacting with the postendocytic trafficking machinery. Nevertheless, receptors lacking region I displayed the same turnover rates and mRNA/protein ratios as empty holoreceptors. This indicates that region I is required for accelerated turnover of EGFR.

The constitutive turnover rate of the EGFR in B82 cells (25-28 h) is somewhat slower than normal fibroblasts (10 h(54) ), but is similar to the range displayed by A431, NA, and Ca9-22 cells (15-23 h(55) ) and is somewhat faster than the rate observed in UCVA-1 cells (37 h (55) ). The factors regulating the absolute rate of EGFR turnover in a given cell type are not understood, but could involve the net flux of membrane through the endocytic pathway as well as specific components that target receptors to lysosomes. We do know that the relatively slow turnover of the EGFR we observed was cell type-dependent because human EGFR expressed in CHO cells displayed a half-life of less than 10 h (data not shown). Despite the slow absolute rates of EGFR degradation observed in B82 cells, the mechanisms responsible for ligand-dependent lysosomal targeting appear to be intact. For example, EGF treatment reduces total steady state EGFR mass in B82 cells by 65-72% as compared to 76-83% in normal human fibroblasts and 60% in normal human mammary epithelial cells. (^2)Furthermore, removal of all cytoplasmic sequences in the EGFR distal to c`647 results in an increase in receptor half-life to approximately 60 h (data not shown). This indicates that B82 cells retain components which regulate both the constitutive as well as ligand-induced turnover of the EGFR.

Previously, we demonstrated that kinase-active EGFR truncated to residue 973 displayed specific and saturable lysosomal targeting(32) . This indicates that sequences between 945 and 973 mediate occupancy-induced lysosomal targeting of EGFR. Receptors truncated to residue 958 have also been shown to be transferred to lysosomes(13) . Because the minimal sequence required for accelerated degradation in constitutively active DeltaKD-EGFR was between residues 945 and 991, the active sequences are most likely located between residues 945 and 958. The sequence YLVI, found at residues 954-958 in EGFR, has been proposed to be a lysosomal targeting sequence for lysosomal-associated membrane protein 1(56) . It resembles the tyrosine-containing and di-leucine motifs previously identified as potential mediators of lysosomal targeting(57, 58, 59) , but involvement of this sequence in postendocytic trafficking of EGFR will require more direct analysis.

It has been proposed that tyrosine kinase activity prevents receptor recycling and directs lysosomal targeting of ligand-activated internalized EGFR(16, 19, 20, 60) . Our data, and results from other investigators, do not support this hypothesis(10, 32, 49, 61, 62) . Instead, it appears more likely that the occupancy-induced exposure of specific lysosomal targeting domains is the mechanism leading to endosomal retention and enhanced lysosomal targeting of EGFR. It has been postulated that occupancy-induced ubiquitination of platelet-derived growth factor receptor is involved in enhanced degradation following ligand binding(63) . We have examined immunoprecipitated EGFR for ubiquitination in either the empty or occupied state by Western blot analysis, but have found no evidence for this covalent modification (data not shown). Because, the degradation rate of the platelet-derived growth factor receptor is almost 10-fold greater than that of the EGFR(47, 63) , ubiquitination may be involved in an alternate pathway of very rapid turnover of some receptor species.

It appears that occupancy-induced receptor down-regulation is a two-step process of internalization followed by lysosomal targeting. It has been suggested that common sequence motifs are responsible for both internalization and lysosomal targeting of some proteins, such as CD3 (57) . Other proteins, like P-selectin, have distinct endocytic and lysosomal targeting domains(64) . The segregation of the regulatory carboxyl-terminal domain of DeltaKD-EGFR into regions required for internalization and lysosomal targeting indicates that distinct sequences mediate these two processes as well. Because DeltaKD-EGFR mutants appear to contain all of the information necessary to specify normal postendocytic compartmentation, occupied EGFR appear to be handled by the endocytic machinery in the same fashion as other constitutively internalized or lysosomally targeted receptors.


FOOTNOTES

*
These studies were supported by National Institutes of Health Grants PO1HD28528 (to H. S. W.) and PO1CA 58689 (to G. N. G.) and by National Science Foundation Grant BCS91-11940 (to H. S. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Bristol-Myers Squibb Research Institute, 5 Research Pkwy., Wallingford, CT 06492.

(^1)
The abbreviations used are: EGFR, epidermal growth factor receptor; mutant EGFR are indicated by the carboxyl-terminal amino acid residue (c`) and by the amino acids of the segment fused in-frame to the carboxyl terminus (f), i.e. c`688 f945-1186; EGFR corresponds to EGFR truncated at residue 688 followed in-frame by residues 945-1186; DeltaKD-EGFR, EGFR lacking the tyrosine kinase domain (residues 689-944); k, recycling rate constant; , fraction of recycled ligand; bp, base pair(s); Ab, antibody.

(^2)
B. H. Will, unpublished observations.


ACKNOWLEDGEMENTS

We thank Marjorie Winkler for the gift of the 13A9 antibody. We also thank Douglas Lauffenburger and Rebecca Worthylake for many helpful discussions regarding this manuscript.


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