©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Association of Epidermal Growth Factor Receptors with Coated Pit Adaptins via a Tyrosine Phosphorylation-regulated Mechanism (*)

(Received for publication, November 2, 1994; and in revised form, January 9, 1995)

Alexandre Nesterov Richard C. Kurten Gordon N. Gill (§)

From the Department of Medicine, University of California at San Diego, La Jolla, California 92093

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We investigated the mechanism by which ligand-activated epidermal growth factor receptors (EGFR) associate with coated pit adaptor protein (AP) complexes. In vivo association, assayed by coimmunoprecipitation of AP with mutant EGFR, required tyrosine kinase activity, intact autophosphorylation sites, and the regulatory carboxyl terminus of EGFR. The role of autophosphorylation of EGFR in interaction with AP was examined in vitro using a BIAcore instrument. Purified active EGFR, immobilized on the biosensor surface, was reversibly autophosphorylated or dephosphorylated by treatment with ATP or phosphatase. Autophosphorylation of EGFR significantly increased AP binding. Once formed, EGFRbulletAP complexes were resistant to disassembly by dephosphorylation of EGFR or competition with phosphotyrosine, indicating that phosphorylated tyrosine residues do not directly participate in AP binding. Induction of conformational changes in EGFR by treatment with urea increased AP binding up to 10-fold in the absence of EGFR autophosphorylation. A recombinant EGFR carboxyl terminus specifically bound the AP complex and each of the isolated alpha- and beta-subunits of AP2. We conclude that tyrosine autophosphorylation of EGFR exposes structural motif(s) in the carboxyl terminus of EGFR that interact specifically with AP2.


INTRODUCTION

Upon ligand binding, the diffusely distributed cell-surface receptors for epidermal growth factor (EGFR(^1)) and insulin (InsR) are activated and redistribute to clathrin-coated pits that are rapidly internalized into the cell(1) . Clearing receptors from the cell surface (down-regulation) attenuates signaling by making receptors unavailable for subsequent ligand binding and by degradation of internalized ligand (2, 3) . The process of ligand-induced internalization of EGFR and InsR requires intrinsic protein-tyrosine kinase activity and specific sequence motifs(4, 5, 6, 7, 8, 9) . Endocytic ``codes'' in EGFR and InsR are 4-6-amino acid sequences that contain obligatory tyrosine or phenylalanine residues (10, 11, 12) and are similar to those identified for endocytosis of many cell-surface receptors(13) . The requirement for ligand activation and intrinsic protein-tyrosine kinase activity of EGFR and InsR contrasts with the independence of these requirements for concentration of transferrin and low density lipoprotein receptors in coated pits(14, 15) . Because endocytic codes from EGFR can function in the context of the transferrin receptor (12) and because deletion mutants of EGFR that lack a kinase domain undergo high constitutive rates of internalization(16) , the tyrosine kinase activity of EGFR has been proposed to cause an autophosphorylation-dependent conformational change that exposes masked endocytic codes located in the regulatory carboxyl terminus(17) .

Sorkin and Carpenter (18) observed ligand- and temperature-dependent binding of EGFR to coated pit adaptors in vivo, suggesting that this was involved in receptor internalization. Previous in vitro studies identified interactions between adaptor complexes and the cytoplasmic endocytic code-containing domains of the low density lipoprotein receptor(19) , the mannose 6-phosphate receptor (20) , the asialoglycoprotein receptor(21) , and lysosomal acid phosphatase(22) . Adaptor proteins (AP), initially identified based upon their ability to promote the formation of coat structures from clathrin(23) , are a major component of coated pits. They consist of two 100-kDa polypeptides (adaptins) and two smaller polypeptides. Adaptor complex 1 (AP1) is restricted to the Golgi region, and AP2 is localized to plasma membrane coated pits; each has a distinct subunit composition(24) . The specific binding of liganddependent as well as constitutively internalized receptors to AP may thus be essential for endocytosis of these receptors.

One mechanism by which EGFR associates with cellular proteins involves binding of SH2 domains to sites of tyrosine autophosphorylation that are located in the regulatory carboxyl terminus(25, 26) . However, adaptor proteins lack SH2 domains, so the mechanisms for their association with EGFR are likely to be different. In this study, we demonstrate that autophosphorylation of EGFR is required for binding adaptor complexes via a mechanism that is distinct from binding to phosphotyrosine residues. Once formed, EGFRbulletAP complex stability is independent of tyrosine phosphorylation. Moreover, adaptor complexes and individual alpha- and beta-subunits of AP2 bind to the regulatory carboxyl terminus of EGFR expressed as a glutathione S-transferase fusion protein (GST-EGFR-CT). In vivo ligand- and temperature-dependent interaction of EGFR with AP2 requires the regulatory carboxyl terminus previously shown to be necessary for ligand-dependent endocytosis of EGFR(8, 12) . These results support the hypothesis that EGF- and autophosphorylation-dependent conformational changes in EGFR allow interaction of the carboxyl terminus with AP2. Such a mechanism could facilitate concentration of activated EGFR in coated pits.


EXPERIMENTAL PROCEDURES

Materials

Monoclonal antibody 13A9, specific to the extracellular domain of human EGFR(27) , was from Genentech Inc. Rabbit polyclonal antibody 4516-8, specific to residues 647-688 of human EGFR (28) , was from T. Hunter (Salk Institute, La Jolla, CA). Monoclonal antibody PY20, specific to phosphotyrosine(29) , was from Transduction Laboratories. Monoclonal antibody 12CA5 against the HA-1 epitope tag was from Berkeley Antibody Co. Monoclonal antibody 100/2, specific to alpha-adaptin(30) , and adaptor complexes isolated from bovine brain were provided by S. Schmid (Scripps Institute, La Jolla, CA)(31) . Monoclonal antibody AC1-M11, specific to alpha-adaptin(32) , and cDNA for mouse alphaC-adaptin were provided by M. Robinson (Cambridge University, Cambridge, United Kingdom). cDNA for rat beta-adaptin (33) was from T. Kirchhausen (Harvard Medical School, Boston, MA). Saccharomyces cerevisiae strain EGY48 (MATalpha, His3, Trp1, Ura3-52, Leu2:pLeu2LexAop6) and yeast expression vector pJG4-5 (34) were provided by R. Brent (Harvard Medical School). Peroxidase-conjugated sheep anti-mouse or donkey anti-rabbit immunoglobulin and the enhanced chemiluminescence (ECL) kit were from Amersham Corp. Protein G-Sepharose 4B and glutathione-agarose beads were from Sigma. Calf intestinal alkaline phosphatase was from New England Biolabs Inc. Zymolyase 100T was from ICN Biomedicals Inc.(Irvine, CA).

EGFR was immunopurified from A431 cells on a column containing immobilized monoclonal antibody 528, specific to the extracellular domain of EGFR(35) . Active EGFR was competitively eluted from the column with 0.1 mM EGF. To prepare denatured receptor, it was eluted with 6 M urea. Purity of proteins was verified by gel electrophoresis and Coomassie Blue staining to be >95%.

Construction and Expression of Mutant EGFR

Kinase-inactive (M) and carboxyl-terminally truncated (c`973) mutant EGFR were constructed as described previously(5, 8, 12, 36) . Mutant F5 EGFR contains Phe substitutions for all five known tyrosine autophosphorylation sites, i.e. Tyr-992, Tyr-1068, Tyr-1086, Tyr-1148, and Tyr-1173. This mutant was constructed using F3 EGFR as template(8) . Additional changes of Tyr to Phe at residues 992 and 1086 in F3 EGFR were constructed by oligonucleotide-directed mutagenesis(37) . Two oligonucleotides were used: residue 992, 5`-GTGGGATGAGGAATTCGTCGGCAT-3`; and residue 1086, 5`-GGCTGATTGTGAAAGCTTGGATTCTGCACAG-3`. All constructions were verified by dideoxynucleotide sequencing.

Clonal B82 cells expressing various EGFR constructs were prepared and maintained as described(12) . A431 cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% calf serum, 100 units/ml penicillin, 100 units/ml streptomycin, and 0.25 µg/ml amphotericin B. The medium was replaced with serum-free Dulbecco's modified Eagle's medium 18 h before experiments.

Coimmunoprecipitation and Western Blotting

Cells grown on 10-cm dishes were incubated for 1 h in ice-cold Dulbecco's modified Eagle's medium containing 20 mM HEPES, pH 7.4, without or with 200 ng/ml EGF. Cultures were then placed in a 37 °C water bath for 12 min and, at the end of the incubation, were placed back on ice. Dishes were washed four times with ice-cold phosphate-buffered saline, and cells were solubilized in 1 ml of immunoprecipitation (IP) buffer (50 mM HEPES, pH 7.4, 1% Triton X-100, 10% glycerol, 100 mM NaCl, 10 mM NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na(3)VO(4), 10 mM benzamidine, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) for 10 min at 4 °C. EGFR was immunoprecipitated with 10 µg of Ab 13A9 immobilized on protein G-Sepharose 4B for 1 h at 4 °C. Immunobeads were washed three times with IP buffer and boiled in SDS sample buffer. To dephosphorylate immobilized EGFR, immunobeads were washed twice with dephosphorylation buffer (10 mM Tris-HCl, pH 8.0, 10 mM MgCl(2), 50 mM NaCl, 0.1% Nonidet P-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride). Beads were then resuspended in 0.2 ml of dephosphorylation buffer, and 50 units of calf intestinal alkaline phosphatase was added for 15 min at 37 °C. Beads were then washed twice with IP buffer and processed for electrophoresis.

Proteins were resolved by electrophoresis, transferred to nitrocellulose filters, and probed with one of the following: rabbit polyclonal Ab 4516-8 or mouse monoclonal Ab PY20, 100/2, or AC1-M11. Both 100/2 and AC1-M11 recognized alpha-adaptins with approximately the same efficiency. Immunoreactive bands were detected using peroxidase-conjugated donkey anti-rabbit or sheep anti-mouse immunoglobulins and developed using enhanced chemiluminescence. To reprobe the same filter with a different antibody, it was then stripped for 30 min at 50 °C in 50 mM Tris-HCl, pH 7.5, in the presence of 2% SDS and 100 mM 2-mercaptoethanol.

Analysis of Protein Interactions Using the BIAcore Instrument

All experiments were performed using sensor chips CM5, research-grade (Pharmacia Biosensor AB) at a flow rate of 5 µl/min at 25 °C. The BIAcore running buffer used was 10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.005% P-20 (Pharmacia). To immobilize EGFR, the sensor chip was activated by injection of 70 µl of a 1:1 mixture of N-hydroxysuccinimide and N-ethyl-N`-(3-dimethylaminopropyl)carbodiimide hydrochloride (Pharmacia); 100 µl of Ab 13A9 at a concentration of 100 µg/ml in 10 mM sodium acetate, pH 5.6, was injected over the surface of the activated chip, and excess reactive groups were blocked with 1 M ethanolamine HCl, pH 8.5. Purified EGFR (50 µl) at 50-200 µg/ml was injected onto the covalently linked antibody. Enzymatically active EGFR was injected in 50 µl of preservation buffer (20 mM HEPES, pH 7.4, containing 1 mM EDTA, 6 mM 2-mercaptoethanol, 0.05% Triton X-100, 10% glycerol, and 130 mM NaCl); urea-denatured receptor was injected in 50 µl of BIAcore running buffer.

To autophosphorylate immobilized EGFR, 25 µl of 1 mM ATP in BIAcore running buffer containing 5 mM MnCl(2) was injected. To dephosphorylate EGFR, 25 µl of dephosphorylation buffer containing 250 units/ml calf intestinal alkaline phosphatase was injected. Efficiency of both phosphorylation and dephosphorylation of EGFR was estimated by injection of Ab PY20 in BIAcore running buffer at a concentration of 75 µg/ml. In some experiments, 10 mM Tyr(P) in BIAcore running buffer was used as a competitor. AP complexes were exchanged into BIAcore running buffer by gel filtration and were spun for 15 min in a microcentrifuge prior to use. AP complexes (35 µl) were injected at a concentration 0.3 mg/ml in BIAcore running buffer. Although the highly purified adaptor preparations used contained predominantly AP2(31) , the specific AP bound to EGFR on the biosensor surface was not identified.

Expression of the Carboxyl Terminus of EGFR

Residues 975-1186 of EGFR was expressed in Escherichia coli as a fusion with glutathione S-transferase. For isolation of proteins, 100 ml of overnight culture was inoculated into 1 liter of 2 times yeast tryptone medium and induced with isopropylthiogalactopyranoside for 5 h at 37 °C. Cells were harvested by centrifugation and lysed in buffer containing 0.2% lauroyl sarcosine. Glutathione-agarose was used to purify proteins from the lysates. Glutathione-agarose beads (1 ml) were incubated with lysates for 1 h at 4 °C and then washed three times with 50 ml of phosphate-buffered saline. The beads were resuspended in phosphate-buffered saline containing 50% glycerol and stored at -20 °C.

Expression of Isolated alpha- and beta-Adaptin Subunits in Yeast

Mouse alphaC-adaptin (residues 2-938) and rat beta-adaptin (residues 2-951) were expressed as fusion proteins with a 12-kDa leader sequence containing the HA-1 epitope tag (YPYDVPDYA) using the expression vector pJG4-5(34) . The 5`-fragment of alphaC-adaptin cDNA was amplified from pBluescript SK using Pfu polymerase and two primers (5`-AGTCATCTAGAGAATTCGTATCCAAAGGGGACGGGATGCGAG-3` and 5`-CATGGTGGATGATCAGGCTGATGGC-3`) to create an XbaI site and an additional EcoRI site at the 5`-end of the cDNA and to remove the first methionine. The XbaI/BclI fragment of the original cDNA was then replaced with the corresponding product of the polymerase chain reaction. The EcoRI fragment containing the whole coding region was then cloned into the EcoRI site of pJG4-5. The same two-step strategy was applied to clone beta-adaptin cDNA into the same expression vector. Modified cDNA for beta-adaptin in pBluescript SK with NdeI and EcoRI sites flanking the coding region (38) was used as template for the polymerase chain reaction. The oligonucleotides 5`-AGTCATCATATGGAATTCACTGAGTCCAAGTACTTCACAACC-3` and 5`-AGTCATCCGAGGCGTCACTCGCTCACAGAT-3` were used to amplify the 5`-fragment of the cDNA, to remove the first methionine, and to create an additional EcoRI site between the NdeI site and the beginning of the coding region. The NdeI/NsiI fragment of cDNA was replaced with the corresponding fragment of the amplified 5`-region, and the whole coding region of the cDNA was excised with EcoRI and cloned into the EcoRI site of pJG4-5. All constructions were verified by dideoxynucleotide sequencing.

Both expression constructs were transformed into S. cerevisiae strain EGY48 and selected on Trp medium. Expression of fusion proteins was induced by growth in 2% galactose. Cells were then spun down, washed with water, and resuspended in 50 ml of 100 mM Tris/SO(4), pH 9.4, containing 20 mM dithiothreitol. After incubation for 10 min at room temperature, cells were pelleted by centrifugation and resuspended in 5 ml of sorbitol buffer (1 MD-sorbitol, 20 mM Tris-HCl, pH 7.5, containing 6.7 g/liter yeast nitrogen base). 1 mg (100 lytic units) of zymolyase was then added, and cells were incubated for 30 min at 30 °C with continuous shaking. Spheroplasts were gently washed to remove zymolyase by two centrifugations using 50 ml of ice-cold sorbitol buffer containing 10 mM benzamidine and 1 mM EDTA. Spheroplasts were solubilized in 1 ml of IP buffer for 20 min at 4 °C, and insoluble material was pelleted by centrifugation.


RESULTS

Role of Autophosphorylation in the Formation of EGFRbulletAP Complexes in Vivo

The observation that tyrosine kinase activity (6, 8) and autophosphorylation (39, 40, 41) are important factors in ligand-induced EGFR endocytosis raised the possibility that autophosphorylation may also serve as a signal for EGFR association with coated pit adaptors. Therefore, we first analyzed the ability of two mutant EGFR with impaired autophosphorylation to form complexes with adaptors in coimmunoprecipitation experiments. One mutant EGFR (M) contained a lysine-to-methionine change in the ATP-binding site of the catalytic domain, making it functionally and enzymatically inactive (5) . Another receptor (F5) lacking all known autophosphorylation sites was shown to retain signaling activity, but its autophosphorylation ability was dramatically reduced(43) . EGFR were immunoprecipitated from mouse B82 cells treated without or with EGF, and bound material was analyzed by Western blotting (Fig. 1A). Consecutive reprobing of the same filter with antibody to EGFR, antibody to phosphotyrosine, and antibody to alpha-adaptin, which distinguishes plasma membrane adaptor AP2 from the AP1 adaptor complex of the Golgi apparatus, revealed that inhibition of EGFR autophosphorylation dramatically reduced its ability to bind the AP2 adaptor complex in an EGF-dependent fashion. AP2 was detected in anti-EGFR Ab immunoprecipitates from EGF-treated M and F5 EGFR expressor cells, but the amount was much less than that associated with wild-type EGFR. To confirm that the observed EGFR/AP2 association took place in vivo and not after solubilization of cells, we confirmed the temperature dependence of complex formation reported by Sorkin and Carpenter(18) . Exposure of cells to EGF at low temperature did not result in increased EGFR/AP2 association, presumably due to the increased viscosity of the lipid bilayer preventing receptors from reaching coated pits, but the ability of EGFR to autophosphorylate was not impaired. As shown in Fig. 1B, EGFR formed complexes with AP2 only after the cells exposed to EGF at 4 °C were placed at physiological temperature. Association was detected at 5 min and was maximum after 10-15 min at 37 °C.


Figure 1: Coimmunoprecipitation of AP2 with EGFR in B82 cells. A, parental B82 cells (Par) and B82 cells expressing wild-type EGFR (WT), enzymatically inactive EGFR (M), and EGFR lacking five autophosphorylation sites (F5) were treated without or with 200 ng/ml EGF, and EGFR were immunoprecipitated using anti-EGFR Ab 13A9. The immunoprecipitated material was electrophoresed and transferred to nitrocellulose, and the filter was cut in half. The top half was probed with anti-Tyr(P) Ab PY20 and then reprobed with anti-EGFR Ab 4516-8, while the bottom half was probed with anti-alpha-adaptin Ab AC1-M11. B, B82 cells expressing wild-type EGFR were incubated with 200 ng/ml EGF at 4 °C for 1 h and then placed at 37 °C for 0, 5, 10, 15, and 20 min. EGFR were immunoprecipitated, and immmunoprecipitated material was then analyzed by electrophoresis and Western blotting. The same filter was consecutively probed with anti-EGFR Ab 4516-8 and Ab 100/2 to alpha-adaptin.



Role of Autophosphorylation in the Formation of EGFRbulletAP Complexes in Vitro

To directly investigate the role of EGFR autophosphorylation in ligand-dependent association with coated pit adaptors, a biosensor approach was used that allows study of interactions between molecules in real time. In this procedure, one of the molecules is immobilized on the surface of a sensor chip while a second is injected over the surface at a continuous flow rate. If the soluble molecule binds to the immobilized one, the refractive index of the medium at the surface of the sensor is changed in direct proportion to the bound mass. This change is detected using the phenomenon of surface plasmon resonance in a BIAcore instrument and is expressed as resonance units(44, 45, 46) .

We first established that we could immobilize EGFR on the sensor surface, that tyrosine kinase activity was retained, and that EGFR could be reversibly autophosphorylated and dephosphorylated. Active EGFR was immunoisolated from A431 cells using Ab 528, which competes with EGF for receptor binding(47) . EGFR was eluted from the 528 IgG column with excess EGF and injected over the biosensor surface containing immobilized Ab 13A9, which binds to the ectodomain of EGFR but does not compete with EGF(27) . Due to the high affinity of interaction, dissociation of EGFR from Ab 13A9 was negligible (Fig. 2). At the end of the EGFR injection, a base line was established, and monoclonal antiphosphotyrosine antibody PY20 was injected before and after injection of ATP/Mn. As shown in Fig. 2, Ab PY20 bound the biosensor only after injection of ATP, demonstrating the ability of captured EGFR to autophosphorylate. Injection of Tyr(P) as a competitor completely eluted bound Ab PY20, confirming the specificity of the interaction. Calf intestinal alkaline phosphatase was then injected to dephosphorylate immobilized EGFR. Phosphatase treatment abolished the ability of subsequently injected Ab PY20 to bind to EGFR. Failure of Ab PY20 to bind to EGFR was due to receptor dephosphorylation and not to proteolysis or other reasons because a second injection of ATP/Mn restored binding of Ab PY20.


Figure 2: Phosphorylation and dephosphorylation of EGFR immobilized on the biosensor surface. Active EGFR was captured by Ab 13A9 covalently linked to the carboxymethylated dextran layer on the surface of the biosensor chip. Ab PY20 (Anti-P-Tyr), ATP/Mn (ATP), Tyr(P) (P-Tyr), and calf intestinal alkaline phosphatase (CIP) were injected over the surface of the biosensor as indicated. Arrows point to the beginning and the end of the injections. Plotted is the relative response in resonance units (RU) versus time. Note that the off-scale response observed when EGFR was injected was due to the presence of glycerol in the injection buffer, but a stable base line was then established.



The binding of the adaptor complex isolated from bovine brain to unphosphorylated and autophosphorylated EGFR was compared (Fig. 3). When AP was injected over the surface of the biosensor after immobilization of Ab 13A9 but prior to injection of EGFR, no significant binding was detected. EGFR was then captured on the biosensor, and AP was injected before and after ATP. The amount of AP bound to EGFR was significantly increased following injection of ATP/Mn, which allowed autophosphorylation of immobilized EGFR. The gradually decreasing base line reproducibly observed during injection of AP presumably reflects dissociation of a substance weakly associated with AP. Because Mn is reported to cause aggregation of the intracellular domain of EGFR, implying a conformational change(48) , Mn was also tested for effects on AP binding to EGFR. Mn (5 mM) injection without ATP resulted in either no or a small positive effect on binding of subsequently injected AP to immobilized EGFR (data not shown), in contrast to the reproducibly increased AP binding following injection of ATP/Mn.


Figure 3: Binding of adaptor complexes to EGFR immobilized on the biosensor surface. Ab 13A9 was coupled to the biosensor chip, and consecutive injections of bovine brain AP complexes were performed before EGFR was captured, after active but nonphosphorylated EGFR was captured, and after EGFR was autophosphorylated by injection of ATP/Mn. RU, resonance units.



EGFRbulletAP Complexes Are Stable to Receptor Dephosphorylation

Although significantly fewer adaptor complexes associate with unphosphorylated EGFR than with phosphorylated EGFR, the overall pattern of binding and dissociation appeared similar, suggesting that phosphorylated tyrosine residues of EGFR molecules do not directly participate in forming the EGFRbulletAP complex. To test this possibility more rigorously, we attempted to dissociate the complex formed between phosphorylated EGFR and AP either by competition with Tyr(P) or by dephosphorylation with phosphatase. For this purpose, the receptor was phosphorylated by injection of ATP/Mn, and after binding of AP, Tyr(P) and calf intestinal alkaline phosphatase were consecutively injected (Fig. 4A). Neither of these treatments resulted in any detectable dissociation of the AP complex. Dephosphorylation of EGFR was almost complete as judged by failure of Ab PY20 to recognize EGFR after phosphatase treatment. Rephosphorylation of immobilized EGFR restored the ability of Ab PY20 to bind, and this binding was competed by phosphotyrosine (Fig. 4A). One can argue, however, that phosphotyrosine residues responsible for adaptor association might be masked by bound adaptor and therefore be unaccessible to either phosphatase or antiphosphotyrosine antibody. To investigate this possibility, EGFR was immunoprecipitated from EGF-stimulated A431 cells, and immunobeads were then treated with calf intestinal alkaline phosphatase. Western blotting with Ab PY20 demonstrated that no detectable Tyr(P) remained in the EGFR molecule after phosphatase treatment (Fig. 4B). However, reprobing of the same filter with antibody to alpha-adaptin revealed that the amount of coimmunoprecipitated AP2 remained unchanged. These results indicate that once formed, the EGFRbulletAP2 complex remains stable even though tyrosine residues are dephosphorylated.


Figure 4: Effect of EGFR dephosphorylation and competition with Tyr(P) on preformed EGFRbulletAP2 complexes. A, a complex between phosphorylated EGFR and AP was formed on the biosensor chip. Tyr(P) (P-Tyr) and calf intestinal alkaline phosphatase (CIP) were then consecutively injected. The activity of the phosphatase was confirmed by injection of Ab PY20 (Anti-P-Tyr), and the activity of EGFR was demonstrated by injection of ATP/Mn (ATP) followed by injection of Ab PY20. The specificity of Ab PY20 binding is demonstrated by competitive elution with Tyr(P). Note that the base-line resonance unit (RU) was set to that of the Ab 13A9bulletEGFR complex. B, the EGFRbulletAP2 complex was coimmunoprecipitated from A431 cells treated without or with EGF using anti-EGFR Ab 13A9. Immunobeads were treated with calf intestinal alkaline phosphatase where indicated, and bound material was then analyzed by electrophoresis and Western blotting. The same filter was consecutively probed with anti-EGFR Ab 4516-8, Ab PY20, and Ab 100/2 to alpha-adaptin.



Denaturation of EGFR Enhances Binding of Adaptors

Because autophosphorylation was required, but the phosphorylated tyrosine residues of EGFR did not appear to participate directly in association with AP2, a model in which tyrosine phosphorylation-induced conformational changes in EGFR facilitated exposure of motifs responsible for AP2 binding was considered. Observations that autophosphorylation induces an increase in the Stokes radius of the intracellular domain of EGFR (17) are consistent with this model. To test this hypothesis, changes in receptor conformation were introduced by treatment with denaturing agent. Immunopurified EGFR was denatured with 6 M urea and immobilized on the biosensor surface, and binding of AP was tested. As shown in Fig. 5A, denatured EGFR did not possess any kinase activity, and tyrosine phosphorylation was not detected by Ab PY20 binding. However, the AP complex was bound efficiently. The AP/EGFR molar ratio measured in several experiments revealed that denaturation increased the ability of EGFR to bind AP up to 10-fold (Fig. 5B).


Figure 5: Binding of bovine brain adaptors to denatured EGFR. A, EGFR was denatured by 6 M urea, transferred to BIAcore running buffer by gel filtration on Sephadex G-25, and captured by Ab 13A9 coupled to the biosensor surface, followed by injection of AP. ATP/Mn (ATP) and Ab PY20 (Anti-P-Tyr) were injected prior to injection AP to confirm lack of enzymatic activity and consequent tyrosine phosphorylation of the denatured receptor. RU, resonance units. B, efficiency of AP binding to native unphosphorylated, native phosphorylated, or urea-denatured receptors was determined. The stoichiometry of stable complexes formed 5 min post-injection of AP was calculated from the ratio of the relative responses corrected for the difference in molecular mass of both species. Similar results were obtained in two independent sets of experiments.



Regulatory Carboxyl Terminus of EGFR Is Required for Adaptor Binding

These results support the hypothesis that autophosphorylation-dependent exposure of EGFR sequence motifs is the basis for ligand-induced binding of AP2 to EGFR, but do not distinguish whether these sequences reside in the tyrosine kinase core or in the regulatory carboxyl terminus. Because it was previously shown that truncation of EGFR to residue 973, which deletes the carboxyl-terminal 214 amino acids, abolished EGF-induced internalization(8) , EGFR truncated to residue 973 were analyzed for their ability to coimmunoprecipitate AP2. Deletion of the carboxyl terminus of EGFR completely abolished association of EGFR with the AP2 complex (Fig. 6A).


Figure 6: Binding of AP2 to the EGFR carboxyl terminus. A, wild-type EGFR (wt) and EGFR lacking the entire carboxyl terminus (c`973) were expressed in B82 cells. The cells were treated without or with 200 ng/ml EGF, and EGFR were immunoprecipitated with Ab 13A9. The immunoprecipitated material was electrophoresed and transferred to nitrocellulose, and the filter was cut in half. The top half was probed with anti-EGFR Ab 4516-8, while the bottom half was probed with anti-alpha-adaptin Ab 100/2. B, glutathione-agarose beads (10 µl) containing 30 µg of glutathione S-transferase (GST) or 10 µg of glutathione S-transferase fused to residues 975-1186 of human EGFR (GST-EGFR-CT) were first incubated in IP buffer with 10 mg/ml bovine serum albumin. Beads were pelleted by centrifugation, the supernatant was aspirated, and 0.5 ml of 1 µM bovine brain AP in IP buffer containing 10 mg/ml bovine serum albumin was added. Beads were incubated with AP for 1 h at 4 °C while agitating, washed three times with IP buffer, and boiled in SDS sample buffer. After electrophoresis and transfer to nitrocellulose filters, AP2 was detected using anti-alpha-adaptin Ab 100/2. Material bound to the beads is designated as AffinityPrecipitation. AP2 of the starting material is designated as AP-2. Note that in brain, the A isoform of alpha-adaptin with slower electrophoretic mobility is found in addition to the more ubiquitous C isoform(32) .



These results suggested that the AP2 recognition motif(s) was located in the carboxyl terminus of the receptor, but it remained possible that this interaction motif(s) was located in the kinase core domain and that deletion of the carboxyl terminus prevented it from being exposed. To obtain direct evidence for interaction of the carboxyl terminus of EGFR with AP2, the carboxyl terminus of EGFR (residues 975-1186) was expressed in E. coli as a fusion with glutathione S-transferase. GST-EGFR-CT was able to bind AP2 from bovine brain with high efficiency, while glutathione S-transferase alone was not (Fig. 6B). Because there was no tyrosine phosphorylation of GST-EGFR-CT (data not shown), this result confirmed the observation that AP2 and EGFR interacted directly without involvement of tyrosine phosphorylation.

alpha- and beta-Subunits of AP2 Each Interact with the Carboxyl Terminus of EGFR

To determine which subunits of the AP2 complex are capable of interacting with the carboxyl terminus of EGFR, the alpha- and beta-subunits of AP2 were individually expressed as fusion proteins in yeast and tested for interaction with GST-EGFR-CT. Both alpha- and beta-subunits of AP2 were specifically bound to GST-EGFR-CT (Fig. 7). Despite use of protease inhibitors in yeast lysis buffers, there was significant degradation of both fusion proteins, and some of the degradation products were associated with GST-EGFR-CT. The smallest detectable degradation product of the alpha-subunit that was bound to beads had an apparent molecular mass of 72 kDa, and the smallest product of the beta-subunit was 80 kDa, which correspond to 60 and 68 kDa of the alpha- and beta-adaptin portions of the fusion proteins, respectively. Because the HA-1 epitope tag of the fusion protein is located at the amino terminus, the interaction of the 60- and 68-kDa fragments with GST-EGFR-CT indicates that a region of interaction resides in the amino terminus, i.e. within the ``body'' of the adaptin subunits, which is separated from the 32-44-kDa ``head'' region by a protease-sensitive hinge(49) . Previous studies indicated that the asialoglycoprotein receptor and lysosomal acid phosphatase interact with the amino-terminal body domains of beta- and alpha-adaptins, respectively(21, 22) .


Figure 7: Binding of individual alpha- and beta-subunits of the AP2 complex to the recombinant EGFR carboxyl terminus. Epitope-tagged alphaC- and beta-subunits of AP2 were expressed in S. cerevisiae. Yeast lysate (0.5 ml), prepared as described under ``Experimental Procedures,'' was incubated with glutathione-agarose beads (10 µl) containing 30 µg of glutathione S-transferase (GST) or 10 µg of GST-EGFR-CT. Adaptins were detected on Western blots with antibody 12CA5 against the HA-1 amino-terminal epitope tag. Note that the molecular mass of adaptins appears larger because they are expressed as fusions with the 12-kDa leader. Material bound to the beads is designated as Affinityprecipitation. Glutathione S-transferase or GST-EGFR-CT was used for precipitation where indicated. The tworight lanes designated as alpha and beta represent aliquots of extracts from yeast expressing alpha- and betasubunits, respectively.




DISCUSSION

Two plausible mechanisms of EGF-induced association of EGFR with coated pit adaptins may be operative. EGF binding may translocate EGFR into coated pits, thus increasing the probability of their interacting with AP2 complexes. Alternatively, binding of EGF may increase the affinity of EGFR for AP2 complexes, resulting in anchoring of activated receptors whenever they enter coated pits. This study describes an autophosphorylation-regulated mechanism by which ligand-activated EGFR interact with AP complexes of coated pits, accounting in part for ligand-dependent retention in coated pits. The fact that in vivo interaction of EGFR with AP was impaired by inactivation of the receptor protein-tyrosine kinase and by mutations of the sites of autophosphorylation suggested that autophosphorylated receptors bound AP complexes with higher affinity than unphosphorylated EGFR. This was directly confirmed by analysis of interactions between isolated EGFR and AP complexes using the BIAcore instrument where receptor autophosphorylation significantly enhanced binding of AP complexes to EGFR. However, phosphorylated tyrosine residues were not directly involved in this interaction because the EGFRbulletAP complex once formed was not dissociated upon receptor dephosphorylation or by competition with phosphotyrosine, suggesting that the mechanism of association is different from tyrosine phosphorylation-mediated mechanisms involving SH2 domains(50) . Alternatively, autophosphorylation, which was shown to change the conformation of the intracellular domain of EGFR(17) , may expose surfaces in EGFR that interact with AP complexes. Indeed, denaturation dramatically enhanced binding to EGFR in the absence of receptor autophosphorylation and tyrosine kinase activity.

Autophosphorylation did enhance EGFRbulletAP complex formation both in vivo and in vitro, but a small amount of binding of AP2 to kinase-negative EGFR in an EGF-induced manner was also observed in the absence of any detectable receptor autophosphorylation. This observation correlates with the reported ability of enzymatically inactive EGFR (42) and other tyrosine kinase receptors (51, 52, 53, 54) to be internalized and/or down-regulated in a ligand-dependent manner, although at a low rate compared with their wild-type counterparts. Kinetic modeling of EGFR endocytosis suggested a two-step mechanism involving interactions necessary for redistribution of diffuse cell-surface receptors to coated pits and for binding of receptors to coated pits(12) . Interaction with AP2 likely reflects predominately the second step.

Interaction between EGFR and AP2 involved the receptor carboxyl terminus because mutant EGFR lacking the carboxyl terminus failed to interact in vivo, while the isolated carboxyl terminus fused to glutathione S-transferase bound AP2 in vitro. Sequences required for endocytosis are present in three regions of the EGFR carboxyl terminus(12) . Sequences required for EGFR interaction with AP2 complexes are also located in this regulatory domain, but more precise mapping will be required to determine the specific sequences that bind adaptins within this domain.

Interactions of AP2 with the cytoplasmic domains of receptors in vitro require tyrosine-containing motifs(19, 20, 21, 22) . However, many receptors compete with themselves for endocytosis, but do not compete with one another. That EGFR compete with themselves is implied from saturable high affinity endocytosis(55) . However, competition between EGFR and the transferrin receptor and between InsR and the mannose 6-phosphate receptor is not observed(9, 56) . This implies specificity in endocytosis despite the generality of aromatic residue tight turn endocytic code structures. Because a high percentage of AP2 was associated with activated EGFR in vivo(18) , a vast excess of AP2 appears unlikely. Together, these observations suggest that additional receptor-specific factors are likely to be required for endocytosis in addition to more general interactions with AP2. Specific recognition of a tyrosine-containing tight turn was demonstrated by interaction of one LIM domain of enigma with the strong endocytic code contained in exon 16 of InsR(57) .

Although competitor studies indicate that the affinity for AP2 interactions with lysosomal acid phosphatase, the polymeric immunoglobulin receptor, and the asialoglycoprotein receptor is low (21, 22, 58) , the localized concentration of AP2 in coated pits is likely high. In vivo interaction of EGFR with AP2 shown by coimmunoprecipitation suggests that this binding may be of higher affinity than that of other receptors, perhaps due in part to unique sites of interaction in the EGFR carboxyl terminus and in the adaptin subunits of the AP2 complex.

Where examined, the interaction of AP2 with cytoplasmic domains of receptors has involved the amino-terminal body domain of alpha- and beta-adaptin subunits of AP2(18, 21, 22) . In our experiments, both the alpha- and beta-subunits of AP2 interacted with the recombinant carboxyl terminus of EGFR, and this interaction included the amino-terminal body domain of adaptins. These results indicate that the carboxyl-terminal regulatory domain of EGFR interacts with the amino-terminal body domain of alpha- and beta-adaptins, but finer mapping will also be required to determine the precise sites of AP2 subunit interactions with EGFR in vivo.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant PO1 CA58689 and by the Markey Charitable Trust. 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.

§
To whom correspondence should be addressed: Dept. of Medicine 0650, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0650. Tel.: 619-534-4310; Fax: 619-534-8193.

(^1)
The abbreviations used are: EGFR, epidermal growth factor receptor(s); EGF, epidermal growth factor; InsR, insulin receptor; AP, adaptor protein; IP, immunoprecipitation; Ab, antibody.


ACKNOWLEDGEMENTS

We thank Dr. Margaret Robinson for the alpha-adaptin clone and AC1-M11 antibody, Dr. Thomas Kirchhausen for the beta-adaptin clone, Dr. Sandra Schmid for purified AP complexes, Dr. Roger Brent for the pJG4-5 expression vector, Dr. Quinn Vega for the GST-EGFR-CT expression plasmid, and Dr. Chia-Ping Chang for the F5 EGFR construct. We thank Dr. Thomas Kirchhausen for helpful discussions and sharing of information.


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