©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stoichiometric Interaction of the Epidermal Growth Factor Receptor with the Clathrin-associated Protein Complex AP-2 (*)

(Received for publication, August 25, 1994; and in revised form, October 31, 1994)

Alexander Sorkin (1)(§) Timothy McKinsey (1) William Shih (3) Tomas Kirchhausen (3) Graham Carpenter (1) (2)

From the  (1)Department of Biochemistry and (2)Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the (3)Department of Cell Biology, Harvard Medical School, and Center for Blood Research, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Plasma membrane clathrin-associated protein complexes (AP-2) have been shown to co-immunoprecipitate with the epidermal growth factor (EGF) receptor (Sorkin A., and Carpenter, G. (1993) Science 261, 612-615). Hence, we analyzed the stoichiometry of the EGF receptor interaction with AP-2 using a new antibody that efficiently immunoprecipitates native AP-2. EGF receptorbulletAP-2 complexes were isolated from S-labeled cells treated with EGF by EGF receptor affinity chromatography followed by precipitation with the antibody to AP-2. Quantitation of the relative molar concentrations of the proteins found in the complex revealed that 1 mol of AP-2 was associated with approximately 1.1 mol of EGF receptor. No other proteins were present in significant molar concentrations relative to AP-2, indicating that other proteins are not stoichiometrically involved in the interaction of EGF receptors and AP-2 in vivo. Co-immunoprecipitation experiments in cells expressing a mutant EGF receptor demonstrated that the cytoplasmic carboxyl-terminal 214 residues of the EGF receptor are essential for interaction with AP-2.


INTRODUCTION

The binding of epidermal growth factor (EGF) (^1)to its receptor results in the rapid disappearance of receptors from the cell surface(1) . Receptor down-regulation is due to the EGF-accelerated endocytosis and degradation of EGF receptors (reviewed in (2) and (3) ). It has been proposed that occupied EGF receptors are internalized severalfold faster than unoccupied receptors (4) and that the ligand-dependent acceleration of receptor internalization is likely the rate-limiting step in receptor down-regulation(2, 3, 4, 5) .

Morphological studies suggest that EGF increases receptor endocytosis by promoting receptor clustering into clathrin-coated pits on the plasma membrane which is followed by receptor internalization into clathrin-coated vesicles(6, 7, 8) . These observations together with similar analyses of other membrane receptors have lead to the view that plasma membrane-coated pits function as sorting organelles selectively recruiting receptors that contain internalization sequences or ``codes'' within their cytoplasmic domains (reviewed in Refs. 9 and 10).

A main structural component of coated pits is the clathrin lattice anchored to the cytoplasmic surface of the membrane by the associated protein complexes or adaptors (APs) (11, 12, reviewed in Refs. 9 and 13-15). AP-2 is the most ubiquitous of the associated proteins found in coated vesicles derived from the plasma membrane. It is a heterotetramer containing two large subunits, alpha and beta2 (100-115 kDa), a medium subunit µ2 (50 kDa), and a small subunit 2 (17 kDa) (16, reviewed in Refs. 9, 15, 17). In addition, there are two isoforms of the alpha-subunit, alphaA and alphaC, encoded by distinct but highly homologous genes(19) .

The large beta2 subunit plus the medium and small subunits of AP-2 are very homologous to the corresponding subunits (beta1, µ1, and 1) of the Golgi clathrin-associated protein complex, AP-1(20, 21, 22, 23) . However, in place of an alpha-subunit, AP-1 contains a -subunit (100 kDa) that has a relatively low level of similarity to the alpha-subunit(24) . Studies of bovine brain-coated vesicles suggest that for AP-2 each alpha-subunit is paired with a beta2-subunit, whereas for AP-1, -adaptin is complexed with a beta1-subunit that migrates with slightly slow mobility on SDS-polyacrylamide gel electrophoresis(19, 25) . The subunit and isoform composition of APs are poorly described in other cell types or cultured cell lines. The beta subunits of AP-2 and AP-1 are known to bind clathrin(26) ; however, the function of other subunits is still unclear.

Current data suggest that the interaction of AP-2 with the intracellular domain of transmembrane receptors mediates the selective recruitment of receptors into coated pits(27, 28, 29, 30) . However, the mechanism of receptorbulletAP interaction and the universality of this association with different classes of receptors are not yet understood. Previously published data have shown that AP-2 co-immunoprecipitates with EGF receptors from cells treated with EGF(31) . This study, however, did not establish the molecular composition of EGF receptorbulletAP-2 complexes. Here, we determine the stoichiometry of components in this complex and assess whether AP-2 interacts directly with the EGF receptor using a double affinity purification protocol to analyze metabolically labeled EGF receptorbulletAP-2 complexes.


EXPERIMENTAL PROCEDURES

Materials

EGF was purified from submaxillary glands as described previously(32) , and EGF-Affi-Gel 10 (3.5 mg/ml gel) was prepared according to Cohen et al.(33) . Polyclonal rabbit 986 antibody to EGF receptor was described elsewhere (34) while anti-EGF receptor rabbit serum 2913 (specific to intracellular domain) was kindly provided by Dr. L. Beguinot (S. H. Raffaele, Milano). Monoclonal antibody LA22 to the extracellular domain of human EGF receptor was obtained from Upstate Biotechnology, Inc. Monoclonal antibodies AC1-M11 and B1-M6 that recognize alpha- and beta-subunits of APs(35) , respectively, were a gift from Dr. M. S. Robinson (University of Cambridge). Polyclonal antibodies 31 (Ab31) to alphaC and 32 (Ab32) to beta-subunits were developed by injecting rabbits with soluble recombinant proteins corresponding to the carboxyl-terminal portion of rat alphaC- and beta2-subunits, respectively. Their specificity was confirmed by Western blot analysis of APs purified from coated vesicles of bovine brain. Polyclonal antibodies were used as the IgG fraction purified from the serum using Protein A-Sepharose (Sigma).

Expression and Purification of the Carboxyl-terminal Portion of Rat alphaC- and beta2-Subunits

The full-length cDNAs of the large subunits alphaC and beta2 of rat brain AP-2 previously characterized by us (23, 36) were used as polymerase chain reaction templates to generate the complete the carboxyl-terminal end portions spanning residues Ser-Phe and His-Asn of alphaC- and beta2-subunits, respectively. These fragments include the linker and ear domains of the large subunits(16, 23) . The polymerase chain reaction primers for alphaC were 5`-GG CAT ATG AGC ATC GAT GTG AAT GGG and 3`-CAT GGT GTG ACG TCT ATT CCT AGG GG and contained the unpaired bases for the NdeI cloning site (underlined) added to facilitate ligation into the NdeI cloning site of the bacterial expression vector pETa. The primers for beta2 were 5`-TG GCT CAC TTG CCA ATT CAC CAT and 3`-CGA CTA TAA TCG TGA GTG; ligation of the polymerase chain reaction product into the blunt-ended Ndel restriction site of pET5a recreated this site upstream of the insertion. In order to facilitate the purification of the protein fragments (see below), a histidine tag containing the sequence Met-Ser-Ala-Gly-6 times His was added in-frame immediately upstream of the starting methionine by insertion of an appropriate oligonucleotide cassette into the Ndel site. Sequence of the constructs was verified by DNA sequencing(38) . These constructs place the AP-2 fragments at the translational start site driven by T7 RNA polymerase whose expression is under control of the inducible lacUV promoter(37) . BL21(DE3) Escherichia coli cells were transformed with either one of the two expression vectors, and bacteria were grown at 37 °C until the cell density reached A approx 0.5 in 1 liter of LB medium supplemented with 100 µg of ampicillin. Cultures were cooled to room temperature in an ice-water bath, and expression of the fragments was induced by addition of 0.1 mM isopropyl beta-D-thiogalactopyranoside. Cells were harvested by centrifugation (JA-10, Beckman; 7000 rpm, 5 min, 4 °C), and the pellets were resuspended in 30 ml of sonication buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride). The cells were lysed by sonication (XL2020, Sonicator; 80% power setting) until they became slightly brown (5 times 30-s sonication bursts with 30-60-s cooling intervals in ice water). Lysates were clarified by centrifugation (JA17, 15,000 rpm, 30 min, 4 °C), and the supernatants, supplemented with 0.2% Triton X-100 and 10 mM beta-mercaptoethanol mixed with 0.5 ml of Ni-NTA agarose beads (Quiagen), prewashed with sonication buffer, by constant inversion (2-3 h, 4 °C). The beads were collected by gravity flow over an open-ended column (2.5-cm diameter) and washed five times by resuspension in 10 ml of sonication buffer supplemented with 10 mM imidazole and 0.2% Triton X-100. The histidine-tagged alphaC and beta2 proteins were eluted with five consecutive washes of 1 ml each (200 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl, pH 8.0). EDTA at 1 mM and phenylmethylsulfonyl fluoride at 0.25 mM were added to the pooled fractions, dialyzed overnight (300 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 4 °C), and concentrated in a Centriprep-10 (Millipore) to 5-10 mg/ml. After clarification by centrifugation (TL 100.4, Beckman; 85,000 rpm, 30 min, 4 °C), 0.5-ml aliquots were applied into a preparative Superdex 75 (Pharmacia) HR 16/50 sizing column (pre-equilibrated with 20 mM Hepes, pH 8.0, 300 mM NaCl, and 1 mM EDTA and running at a flow rate of 1.0 ml/min at room temperature). At least 90% of the alphaC and beta2 fragments were recovered as a monomeric species with a purity greater than 95% as determined by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining of the appropriate fractions. Aliquots were kept at -20 °C.

Cell Culture

Mouse NIH 3T3 cells expressing 4-8 times 10^5 human wild-type EGF receptors per cell, WT cells(34) , were used in most experiments. The EGF receptor mutant Dc214 in which 214 amino acid residues have been deleted from the carboxyl terminus was described previously(34) . NIH 3T3 cells expressing Dc214 displayed approximately 4.0-6.0 times 10^5 receptors per cell. Cells were grown in 100-250-mm dishes or trays (Costar) as described (34) and used for experiments when confluent. Cells were starved in 0.5% serum overnight prior to each experiment.

Immunoprecipitation of APs

Cells treated or not treated with EGF were washed with Ca, Mg-free phosphate-buffered saline (CMF-PBS) and solubilized in TGH buffer (1% Triton X-100, 10% glycerol, 50 mM NaCl, 50 mM Hepes, pH 7.3, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 544 µM iodoacetamide, 10 µg/ml aprotinin) by scraping the cells from the dish with a rubber policeman followed by gentle rotation for 10 min at 4 °C. Lysates were then centrifuged at 13,000 times g for 10 min. Approximately 50-60% of total cellular AP-2 pool was found in the supernatants after centrifugation. Supernatants were incubated with Ab31 or Ab32 for 3 h at 4 °C and then 30-60 min after the addition of Protein A-Sepharose. Most experiments were controlled for nonspecific immunoreactivity by immunoprecipitations with the specific anti-AP IgG in the presence of an excess of the corresponding recombinant fusion antigen protein. Also, preimmune rabbit serum or unrelated rabbit IgG (Sigma) were used for nonspecific control precipitations.

Immunoprecipitates were washed twice with TGH supplemented with 100 mM NaCl and then once with TGH. Typically, 7.5% SDS-polyacrylamide gels were used to separate proteins. In indicated experiments, 6 M urea was included in the separating gel as described elsewhere (19) to separate alpha- and beta-subunits. Transfer to nitrocellulose membrane and protein immunoblotting were carried out as described(34) . Sheep antibodies to mouse IgG (Cappel Inc.) or protein A (Zymed Inc.) conjugated with horseradish peroxidase and enhanced chemiluminescence (Amersham or DuPont NEN) were used to detect primary mouse or rabbit antibodies, respectively. Stripping of the antibodies from the membrane was performed according to the manufacturer's protocol (Amersham). Several films obtained after various lengths of exposure times with the same blot were analyzed to measure optical density within a linear range of sensitivity. A Bio-Rad densitometer was used for quantitation.

Co-immunoprecipitation Experiments

Cells in 150-mm dishes were incubated with 300 ng/ml EGF in binding medium (DMEM, 0.1% bovine serum albumin, 20 mM Hepes, pH 7.3) at 4 °C for 40-60 min to saturate receptors and then placed in a 37 °C water bath to allow endocytosis for the indicated times. Medium in the culture dishes reached 37 °C within 2 min. At the end of the 37 °C incubation, the cells were washed with CMF-PBS and solubilized in TGH, as described above. Lysates were then centrifuged at 110,000 times g for 20 min to minimize nonspecific associations and for removal of possible AP-2 aggregates. Supernatants, representing equal amounts of cells, were immunoprecipitated with a saturating amount of antibody 986 to the EGF receptor or Ab31 to the alphaC-subunit. Immunoprecipitation, electrophoresis, and transfer to nitrocellulose were performed as described above. The top (above the 116-kDa molecular mass marker) and bottom portions of the nitrocellulose membrane were blotted with antibody 2913 to the EGF receptor and antibody AC1-M11 to alpha-subunits, respectively. In experiments with Dc214 receptor, monoclonal antibody to EGF receptor (LA22) was used for blotting. Sheep antibodies to mouse IgG, conjugated with horseradish peroxidase, or I-Protein A (ICN Biochemical) was used to detect primary antibodies. To analyze radioactivity, blots were exposed to x-ray films and quantitated with a PhosphorImager (Molecular Dynamics). Chemiluminescence signals were detected and analyzed by densitometry.

The nonspecific precipitation of AP-2 by nonimmune rabbit IgG used in the same quantity as antibody 986 to EGF receptor was negligible (0.1-0.2% of total AP-2) compared to the specific association of AP-2 with the EGF receptor. Nonspecific association of EGF receptors with control IgG was in some experiments comparable with the specific co-immunoprecipitations with anti-AP IgG and varied with the source of control IgG used. Therefore, immunoprecipitation with Ab31 in the presence of alphaC-ear-linker peptide was used to control for nonspecific associations. To determine specific co-precipitation of the receptor with AP-2, the amount of EGF receptors in nonspecific immunoprecipitates was subtracted from that amount recovered in the immunoprecipitates in the absence of alphaC-ear-linker.

Potassium Depletion of Cells

In experiments comparing AP-2 association with wild-type and Dc214 EGF receptor mutant, potassium depletion of cells was performed as described (31, 39) to normalize AP-2 association with receptors. In brief, cells were subjected to hypotonic shock by incubation in DMEM/water (1:1) at 37 °C for 5 min. Cells were further incubated in buffer ``A'' (100 mM NaCl, 50 mM Hepes, pH 7.3) at 37 °C for 1 h and subsequently with EGF (300 ng/ml) in buffer ``B'' (100 mM NaCl, 50 mM Hepes, pH 7.3, 1 mM CaCl(2)) for 40 min at 4 °C. Finally, cells were placed in the 37 °C water bath for 15 min to allow receptorbulletAP-2 association (31) . Control cells were incubated for 5 min in DMEM instead of 50% DMEM and in buffer ``C'' (100 mM NaCl, 50 mM Hepes, pH 7.3, 1 mM CaCl(2), 10 mM KCl) instead of buffers A and B.

Metabolic Labeling and Turnover of AP-2

Subconfluent cells grown in 35-mm dishes were incubated for 24 h in methionine-free Modified Eagle's medium containing 1% dialyzed fetal calf serum and TranS-label (DuPont NEN) (80 µCi/ml). To measure the turnover of radiolabeled AP-2, the cells were washed with nonradioactive DMEM and further incubated in DMEM containing 1% calf serum and 2 mM methionine for 0-48 h at 37 °C. Under these conditions, the amount of total cellular protein usually increased by not more than 10-20% during the labeling period and 48-h ``chase.'' Incubation was terminated by washing the cells with cold CMF-PBS. The cells were then solubilized in TGH, and lysates were centrifuged at 13,000 times g for 10 min. AP-2 containing alphaC-subunits were immunoprecipitated from the supernatant with Ab31 to alphaC-subunit in the presence or absence of an excess alphaC-ear-linker fragment. 0.5 M NaCl was present in the immunoprecipitations, as well as in the two washes of the immunoprecipitates to decrease nonspecific radioactivity. Immunoprecipitates were analyzed on 7-11% gradient SDS-polyacrylamide gels to obtain maximum resolution in the 100-kDa region of the gel and to detect proteins migrating above the 14-kDa molecular mass marker. In some experiments, urea-containing gels were used as described (19) to separate alpha- and beta-subunits. Either gels were dried or proteins were transferred to PolyScreen transfer membrane (DuPont NEN Research Products). To detect radiolabeled proteins, gels and transfer membranes were exposed to PhosphorImager screens or x-ray films. The transfer membranes were also incubated with antibodies to the large subunits of APs, which were detected by chemiluminescence. S-Labeled bands were then matched with the immunoreactive bands detected by blotting.

Purification of EGF ReceptorbulletAP-2 Complexes

Confluent cells grown in 250-mm^2 dishes were labeled with [S]methionine (0.50-0.75 mCi/dish, 20-25 µCi/ml) for 24 h as described above. Cells were rinsed with nonradioactive DMEM and incubated in DMEM for 15 min at 37 °C. To saturate surface receptors, the cells were incubated at 4 °C for 40 min in binding medium supplemented with 300 ng/ml EGF. To initiate AP-2 association with EGF receptors, the cells were subsequently incubated for 7 min at 37 °C. Cells were then washed with CMF-PBS, scraped in 2 ml of ice-cold TGH, and transferred into tubes. Tubes were gently rotated to solubilize membranes for 10 min at 4 °C and centrifuged at 110,000 times g for 20 min. To isolate EGF receptors and receptor-associated proteins, supernatants from two dishes were combined, mixed with 80 µl of EGF-Affi-Gel, and incubated for 2 h at 4 °C. The EGF-Affi-Gel was then washed in TGH, incubated for 10 min in TGH, and washed again with TGH. The absorbed EGF receptors (and proteins associated with the receptors) were eluted from the EGF-Affi-Gel by incubation in 0.8 ml of a 1:1 mixture of 1 mg/ml EGF and TGH for 1 h at 4 °C.

To isolate AP-2 bound to receptors, 2 equal aliquots of the eluent from the EGF affinity column were immunoprecipitated with Ab31 to alphaC-subunit in the absence or presence of recombinant alphaC-ear-linker protein (300 ng). Protein A-Sepharose immunoprecipitates were washed twice with TGH containing 100 mM NaCl and twice in TGH without NaCl. The immunoprecipitates were then separated on 7-11% gradient SDS-gels. In some experiments, 3-15% gels were used to enable detection of a larger molecular weight range of proteins. After the gels were fixed and dried, radiolabeled proteins were analyzed with a PhosphorImager or exposed to x-ray film. In some experiments, proteins were transferred to PolyScreen transfer membrane. To detect radiolabeled proteins, transfer membranes were exposed to the PhosphorImager screens or x-ray films. Then the transfer membranes were incubated with various antibodies to APs and EGF receptor, which were detected by chemiluminescence. S-Labeled bands were then matched with the bands detected by blotting.


RESULTS

Characterization of alphaC- and beta-Subunit Antibodies

A previous study demonstrated the presence of AP-2 in immunoprecipitates of EGF receptors from cells treated with EGF at 37 °C(31) . To further investigate the nature of receptor interaction with AP-2, antibodies capable of immunoprecipitating native AP-2 complexes from cell extracts were required. Therefore, recombinant proteins corresponding to the carboxyl-terminal portions of the alphaC and beta2 subunits of rat brain AP-2 were employed as antigens to generate Ab31 and Ab32, respectively. Results of Western blotting analysis and immunoprecipitation of APs from mouse NIH 3T3 cells are summarized in Table 1. Ab31 recognizes alphaC (approx102 kDa), but not the related alphaA isoform. Ab32 recognizes both beta1 (approx105 kDa) and beta2 (approx102 kDa) isoforms. These results were confirmed with monoclonal antibody AC1-M11 that in blotting analysis detects both alphaA (approx105 kDa) and alphaC, and with monoclonal antibody B1-M6 that recognizes beta1 and beta2(35) . AP-2 complexes precipitated by Ab31 were composed exclusively of alphaC and predominantly beta2 subunits. A small amount of the beta1 subunit was detected in Ab31 immunoprecipitates. Presumably, a minor fraction of AP-2 may incorporate the beta1 subunit instead of beta2. (^2)



Ab32 was effective in immunoprecipitating a random sample of all available APs. Comparative blotting of Ab32 precipitates and cellular lysates with AC1-M11 indicates that in NIH 3T3 cells approximately 60-70% of the solubilized AP-2 pool contains the alphaC isoform. Consistently, we estimate that the alphaC antibody (Ab31) precipitates approximately 60-70% of the total solubilized AP-2 pool in NIH 3T3 cells. In subsequent experiments, Ab31 was used to minimize analysis of large subunits of AP-2 components, such as beta1, that are considered characteristic of AP-1.

Time Course of Receptor Association with AP-2

The time course of AP-2bulletEGF receptor association after EGF stimulation was examined using mouse NIH 3T3 cells expressing wild-type human EGF receptors (WT cells). The cells were incubated with a saturating concentration of EGF at 4 °C for 40 min and then transferred to 37 °C to permit endocytosis. At the indicated times, cells were solubilized, and AP-2 or EGF receptors were immunoprecipitated, respectively, with Ab31 to alphaC subunit or antibody 986 to EGF receptor (Fig. 1). After electrophoresis, the primary antigen and the co-precipitating proteins were analyzed by Western blotting. As shown in Fig. 1A (lanes 1-10), the pool of EGF receptors co-immunoprecipitated with alphaC-subunit was relatively small, typically 0.3-0.6% of the total cellular pool of EGF receptors. Immunoprecipitation with Ab31 in the presence of excess recombinant alphaC-ear-linker antigen (Fig. 1A, lanes 6-10) was used to demonstrate specificity of the primary antibody. In Fig. 1B, quantitation of the data shown in Fig. 1A, lanes 1-10, indicates that co-immunoprecipitation of EGF receptors with AP-2 reached a maximum at 6-8 min following the shift to 37 °C and subsequently declined at more extended incubation times (Fig. 1B, closed circles). These results are consistent with the time course of EGF internalization (34) and with the kinetics of EGF appearance in clathrin-coated vesicles(7) .


Figure 1: Time course of EGF receptor association with AP-2. NIH 3T3 (WT) cells were incubated with EGF (300 ng/ml) at 4 °C, and the temperature was then shifted to 37 °C for the indicated times. Cell lysates for each time point were divided into three equal aliquots. The first and second aliquots were immunoprecipitated with Ab31 to AP-2 alone (lanes 1-5) or in the presence of an excess alphaC-ear-linker protein (lanes 6-10). The third aliquot was immunoprecipitated with antibody 986 to the EGF receptor (lanes 11-15). EGF receptor and alpha-subunits of AP-2 were detected, respectively, by immunoblotting with antisera 2319 to the EGF receptor and AC1-M11. The amount of EGF receptors co-precipitated with AP-2 (closed circles) and AP-2 co-precipitated with EGF receptors (open circles) is expressed in arbitrary units (a.u.), as described under ``Experimental Procedures.'' Results are representative of three independent experiments.



The data presented in Fig. 1A (lanes 11-15) demonstrate the converse, i.e. that AP-2 co-immunoprecipitates with EGF receptors. Both alphaA- and alphaC-subunit isoforms (typically 2-4% of total solubilized alpha-subunit pool) were co-immunoprecipitated with EGF receptors. A similar amount of AP-2 was associated with EGF receptors when cells were exposed to EGF at 37 °C without preincubation at 4 °C (data not shown). Co-immunoprecipitation of AP-2 with EGF receptors also reached a maximum at 6-8 min following the shift to 37 °C (Fig. 1B, open circles).

These results demonstrate that association of EGF receptors with AP-2 had similar kinetics when monitored by either EGF receptor or AP-2 immunoprecipitation. In mouse cells, more alphaC than alphaA isoform is present. However, the alphaA/alphaC ratio in EGF receptor co-immunoprecipitates was similar to the ratio of these isoforms in cell lysates. This suggests that both types of AP-2, containing alphaA- or alphaC-subunits, associate with the EGF receptor following the same proportion relative to the cellular abundance of each isoform. Previous data showed that in human fibroblasts and epidermoid carcinoma cells, the ratio alphaA/alphaC was also similar in cell lysates and EGF receptor immunoprecipitates, although alphaA was the predominant isoform relative to alphaC in these cells(31) .

Immunoprecipitation of AP-2 from Metabolically Labeled Cells

To examine the qualitative and quantitative subunit composition of native AP-2, cells were metabolically labeled with [S]methionine for 24 h and AP-2 was immunoprecipitated from cell lysates with Ab31 in the absence or presence of alphaC-ear-linker protein. As shown in Fig. 2, the most intensely radiolabeled band was detected at approx102 kDa, corresponding to the co-migration of alphaC- and beta2-subunits. The presence of both subunits was confirmed by SDS-urea gels and Western blotting (data not shown). In addition, a small amount of the approx105-kDa beta1 subunit (2-10% relative to beta2) was detected in alphaC immunoprecipitates. The other major radiolabeled proteins detected in the autoradiogram include 50-kDa and 17-kDa species corresponding, respectively, to the µ2- and 2-subunits of AP-2.


Figure 2: Analysis of metabolically labeled AP-2 subunits. WT cells were metabolically labeled with [S]methionine for 24 h, and, following cell lysis in TGH, AP-2 was precipitated with Ab31 in the presence or absence of alphaC-ear-linker. Immunoprecipitates were electrophoresed, and radiolabeled proteins were detected using x-ray film (A). The amount of radioactivity in the bands corresponding to AP-2 subunits (arrows) was quantitated with a PhosphorImager. The amount of the protein specifically precipitated with Ab31 was calculated as a difference between the radioactivity in the band in the Ab31 immunoprecipitate (lane alphaC-ear-) and the radioactivity in the identical region of the gel of nonspecific precipitations (lane alphaC-ear+). The 102-kDa band contained both alphaC- and beta2-subunits, as determined by immunoblotting of labeled proteins. The molar concentrations of µ2 and 2 relative to alphaC/beta2 adaptins (B) were determined by normalizing the specific radioactivity of the bands to the number of methionine residues in each protein. These numbers were obtained from the sequences of rat µ2(21) , rat 2(20) , mouse alphaC(18) , and rat beta2 subunits(23) . The molar concentration of beta2 relative to alphaC was quantitated from separate experiments in which these subunits were resolved on SDS-urea gels (data not shown). Data are expressed as percent of the amount of the alphaC subunit.



The molar ratio of individual AP-2 subunits in the immunoprecipitate was determined by measuring the amount of radioactivity in bands corresponding to AP-2 subunits. Since the sequences of known AP-2 subunits are almost identical among mammalian species, we used the methionine content of the cloned rat beta2, µ2, and 2 together with the mouse alphaC sequence (18, 20, 21, 22, 23) to normalize the radioactivity in the each band to the abundance of methionine residues. The molar ratio of alphaC- and beta2-subunits was estimated from SDS-urea gels. As summarized in Fig. 2B, the four subunits of AP-2 were present in approximately equimolar amounts.

Because the extent of protein labeling in vivo is influenced by the rate of protein turnover, we also measured individual degradation rates of the AP-2 subunits. Cells incubated with [S]methionine were ``chased'' in a medium containing unlabeled methionine, and AP-2 was immunoprecipitated by Ab31 as described above. The amount of radioactivity in the bands corresponding to the large (alphaC/beta2), medium (µ2), and small (2) subunits was monitored. As seen in Fig. 3, quantitation indicated a similar rate of degradation, t approx 30-36 h, for each of the AP-2 subunits. These results suggest that all subunits are labeled to a similar extent during a 24-h incubation with [S]methionine, and that the molar ratios of individual proteins in the AP-2 complex, as calculated from the radioactivity in the gel bands (Fig. 2B) are correct and do not need to be adjusted for different subunit turnover rates. In contrast, the half-life of EGF receptors in the same experiment was calculated at approximately 8-9 h, consistent with previous measurements in similar cells(34) .


Figure 3: Degradation rate of AP-2 subunits and EGF receptors. WT cells were metabolically labeled with [S]methionine for 24 h and then were incubated for the indicated times in the presence of unlabeled methionine as described under ``Experimental Procedures.'' AP-2 and EGF receptors were immunoprecipitated from TGH extracts with Ab31 and antibody 986, respectively. The amount of radioactivity in bands corresponding to the large AP-2 subunits alphaC/beta2, 102 kDa (closed circles), medium subunit (µ2) (open circles), and small subunit (2) (closed triangles) at each time point was quantitated as described in Fig. 2. The amount of radiolabeled EGF receptor (open triangles) was determined from the radioactivity in the major 175-kDa band from the anti-EGF receptor precipitates as described previously(34) . The amount of each protein is expressed as percent of the initial amount of that protein recovered from the cells at time 0.



Finally, other unidentified radiolabeled proteins, for example a 65-kDa and a 250-kDa species, co-precipitated with AP-2 in a specific manner (see Fig. 2A). The molar concentration of these molecules, calculated from the average content of methionine residues in proteins (40) , was, however, less than 0.5 mol/mol of AP-2.

Measurement of EGF ReceptorbulletAP-2 Stoichiometry

EGF receptorbulletAP-2 complexes were isolated from metabolically prelabeled and EGF-stimulated WT cells to quantitate the stoichiometry of EGF receptorbulletAP-2 interaction and to determine whether other proteins may also be present in similar proportions in this complex. WT cells were incubated with [S]methionine for 24 h and then incubated with EGF at 4 °C for 40 min followed by a temperature shift to 37 °C for 7 min to induce maximal EGF receptor association with AP-2. Subsequently, EGF receptors and any proteins associated with EGF receptors were isolated from cell lysates by EGF-Affi-Gel affinity chromatography. After washing, EGF receptors were eluted by the addition of free EGF. AP-2bulletreceptor complexes were then isolated from this eluate using Ab31 to immunoprecipitate AP-2 and associated proteins. This protocol was designed to eliminate free EGF receptors and free AP-2 as well as complexes of EGF receptor or of AP-2 with other proteins.

The results of this experiment (Fig. 4) show that three bands corresponding to the mobilities alphaC/beta2, µ2, and 2 subunits of AP-2, plus one band corresponding to the EGF receptor were recovered in the specific AP-2 immunoprecipitates derived from the EGF eluent. In addition, a faint beta1 band was also observed. As expected, the pattern and relative molar concentrations of AP-2 subunits immunoprecipitated after EGF receptor purification was similar to that obtained by direct immunoprecipitation from cell lysates (compare Fig. 2A with Fig. 4). The identity of alphaC- and beta-subunits was confirmed by Western blot analysis as described under ``Experimental Procedures'' (not shown).


Figure 4: Isolation of EGF receptorbulletAP-2 complexes. Metabolically prelabeled (24 h) WT cells were incubated with EGF at 4 °C for 40 min, then shifted to 37 °C for 7 min, and subsequently lysed in TGH. EGF receptorbulletAP-2 complexes were isolated by affinity chromatography on EGF-Affi-Gel followed by immunoprecipitation with Ab31 as described under ``Experimental Procedures.'' Immunoprecipitation with Ab31 in the presence of the excess of alphaC-ear-linker protein was used as a control for the nonspecific co-precipitation of labeled proteins. The migration position of radiolabeled EGF receptors, alpha, beta, µ2, and 2 AP-2 subunits are indicated by arrows. EGF receptors and large AP-2 subunits were matched with the bands detected by blotting with the corresponding antibody, as described under ``Experimental Procedures.'' The lane labeled Lysate represents 0.5% of the total cell lysate used for the EGF receptor affinity purification with the same exposure time.



The quantity of all bands in Fig. 4detectable by PhosphorImager analysis was calculated as the difference between the amount of radioactivity in identical regions of the gel corresponding to the specific and nonspecific immunoprecipitates. The specific radioactivity in the approx180-kDa band, corresponding to phosphorylated EGF receptor was approximately twice that present in the alphaC/beta2 band. The amount of radioactivity present in these bands was then normalized to the number of methionine residues in each protein and also to the percentage of labeled protein. Since the t of alphaC/beta2 is approximately 34 h (Fig. 2), approximately 40% of solubilized pool of these subunits was labeled during the 24 h incubation with [S]methionine. In contrast, the t of EGF receptors was approx8-9 h (Fig. 3), indicating that in the same labeling period approximately 85% of the total pool of EGF receptors was labeled with [S]methionine. The band intensities, therefore, were also normalized to correct for the resultant differences in the specific radioactivities of EGF receptors and AP-2 subunits. Using these corrections, an average stoichiometry of 1.1 ± 0.2 mol of EGF receptor per mol of AP-2 was estimated for five independent experiments.

The EGF receptor was the only co-precipitating protein found in specific AP-2 immunoprecipitates in a significant amount, i.e. more than 0.2 mol/mol of AP-2. Control experiments using 3-15% gels did not reveal the presence of additional bands with relative mobilities of greater than 10 kDa or less than 500 kDa. Thus, we suggest that no other proteins are necessary in stoichiometric amounts to maintain the equimolar interaction of AP-2 and EGF receptor. It is possible that other proteins were not detected because of their low content of methionine and/or low rate of biosynthesis. However, the molar concentration of such proteins would have to be smaller than that of the 2 subunit which has a long half-life (36 h) and which contains only 4 methionine residues.

Interaction of AP-2 with an EGF Receptor Mutant

The experiments described above demonstrate the direct interaction of transfected wild-type human EGF receptors with mouse AP-2. This allowed us to examine the interaction of AP-2 with mutant EGF receptors expressed in NIH 3T3 cells. It has been previously proposed that regions and/or sequences in the carboxyl-terminal domain of the EGF receptor are essential for rapid internalization of the EGF receptor and may contain one or more putative internalization motifs(5, 41) . Therefore, to test whether there is a correlation between the endocytic rate of the EGF receptor and its ability to bind AP-2, the interaction of AP-2 with the EGF receptor mutant Dc214, in which 214 carboxyl-terminal residues are deleted, was studied.

The data in Fig. 5show that in the absence of EGF AP-2 does not interact with WT or Dc214 receptors. When cells were incubated with EGF at 4 °C and then allowed to internalize EGF at 37 °C, AP-2 co-immunoprecipitated with WT receptor, but not with the Dc214 receptor mutant.


Figure 5: Interaction of AP-2 with wild-type and Dc214 EGF receptors. NIH 3T3 cells expressing wild-type (WT) or Dc214 truncated EGF receptors were subjected as indicated to K-depletion, incubated with EGF (300 ng/ml) at 4 °C in buffer B, and placed at 37 °C for 15 min as described under ``Experimental Procedures.'' Control cells, i.e. no K depletion, were incubated at 4 °C in buffer C in the presence or absence of EGF as indicated. EGF-treated cells were further incubated at 37 °C for 9 min. After incubations, cells were solubilized in TGH, and EGF receptors were immunoprecipitated with EGF receptor antibody. Aliquots of lysates corresponding to 5% of the amount used for immunoprecipitation were electrophoresed to compare with immunoprecipitates. The alpha-subunits of AP-2 were detected in receptor immunoprecipitates (panel B) and lysates (panel C) by immunoblotting with AC1-M11. EGF receptor was probed with monoclonal antibody LA22 (panel A).



In cell cultures, I-EGF is poorly internalized by Dc214 receptors(5, 34) , indicating that this mutant EGF receptor may not enter coated pits. To test whether the failure of the Dc214 mutant to associate with AP-2 was due to its inability to enter coated pits, we used K depletion conditions to compare AP-2 association with wild-type and Dc214 receptors in the absence of coated pits(31) . In WT cells, K depletion resulted in a significant increase in AP-2 co-immunoprecipitated with the EGF receptor, similar to results obtained with human cells(31) . The data in Fig. 5A demonstrate, however, no significant increase of AP-2 association with the Dc214 mutant receptor under the same conditions. Therefore, residues 973-1186 in the cytoplasmic domain of the EGF receptor are critical for receptor interaction with AP-2. These results also indicate that the low internalization efficiency of the Dc214 receptor may be attributable to its inability to associate with AP-2.


DISCUSSION

AP-2 is implicated in functions essential for the dynamic cycle of clathrin-coated pits and vesicles. One of these functions is the selective recruitment of membrane proteins into coated pits by the recognition of internalization ``codes'' located within the cytoplasmic domains of receptors (reviewed in Refs. 9, 10, and 15). Initial evidence for this model was based on the in vitro binding of purified AP-2 to the intracellular domains of transmembrane proteins known to be capable of efficient clustering in coated pits, such as low density lipoprotein, mannose 6-phosphate, asialoglycoprotein receptors, and lysosomal acid phosphatase(27, 28, 29, 30) . These experiments showed that although the in vitro interaction was apparently specific and required an internalization motif, the affinity and stoichiometry of the interaction was very low (29, 30) . More recent experiments have shown that EGF receptors associate in vivo with AP-2 following EGF addition to intact human cells at 37 °C(31) . Although the association was stable, the experimental design did not allow a determination of the stoichiometry interaction in vivo, nor did it allow a test of whether there was direct interaction between receptors and AP-2.

The data in this report confirm, in NIH 3T3 cells expressing human EGF receptors, the EGF- and temperature-dependent interaction of EGF receptors with AP-2 in mouse cells (Fig. 2) similar to that previously observed in human cells(31) . The amount of AP-2 co-immunoprecipitated with EGF receptors in NIH 3T3 cells (Fig. 1) was significantly smaller, however, than that detected in A-431 cells. Moreover, immunoprecipitation of AP-2 using an alphaC-specific antibody showed that a relatively small pool of EGF receptors, equivalent to approximately 5-10 times 10^3 receptors per cell, is associated with AP-2 at any one time under these experimental conditions. The small fraction of receptors and AP-2 detected in these complexes is likely due to the transient nature of receptorbulletAP-2 association during internalization through coated pits. Also, it is possible that the rate of receptor transition through the early stages of endocytosis is dependent on the particular receptor and cell type. For example, the maximal rate constant for EGF internalization is approximately 50% higher in mouse fibroblasts (5, 34) than in A-431 cells (42) or human fibroblasts(42, 43) . This may partially explain the quantitative differences in the extent of association between the EGF receptor and AP-2 in NIH 3T3 and A-431 cells.

Interaction of EGF receptor with proteins that have src homology 2 (SH2) domains is mediated by phosphotyrosine-containing motifs in the carboxyl-terminal domain of the receptor(44) . Some SH2-containing proteins, termed adaptors, are known to mediate association with other proteins; for example, GRB-2 mediates association of the ras guanine nucleotide exchanger (Sos) with the EGF receptor(45) . Since AP-2 subunits do not have SH2 domains, it seemed plausible that AP-2 interaction with the activated EGF receptor might be mediated by an SH2-containing adaptor protein. However, in preparations of metabolically labeled EGF receptorbulletAP-2 complexes, we did not detect other radiolabeled proteins in significant amounts. This indicates that other proteins do not mediate the stoichiometric association of EGF receptor and AP-2 which is consistent with a direct interaction of EGF receptor and AP-2. Our data do not rule out the involvement of additional regulatory proteins which may have a catalytic function in complex formation.

The novel two-step isolation of receptorbulletAP-2 complexes indicates the relative stability of these complexes and demonstrates that an average of one AP-2 tetramer is associated with one EGF receptor monomer. Although EGF receptors (46, 47) and AP-2 (48) are both capable of aggregation, we suggest that predominantly monomeric forms of each comprise the complex in TGH lysates for the following reasons. First, the stability of receptor dimers in Triton X-100 is low due to reduced EGF binding affinity(47, 49) . Glycerol gradient centrifugation of TGH lysates reveals that more than 95% EGF receptors are monomers. (^3)Second, AP-2 tends to aggregate at a much higher concentration than the concentration of AP-2 in either the TGH lysates or the EGF affinity column eluent(48, 50) . Lastly, in these experiments, large aggregates were removed by high speed centrifugation. It is possible, though, that EGF receptor dimerization is important for the formation of EGF receptorbulletAP-2 complexes in vivo.

Finally, the working model developed here and in previous studies (31, 34) suggests the direct interaction of AP-2 with the activated EGF receptors at an early step in endocytosis. EGF-induced receptor tyrosine kinase activity has been shown to be necessary for rapid internalization of EGF receptors(5, 34, 43) . Activation of the receptor kinase leads to autophosphorylation and conformational changes in the intracellular domain of the receptor which may make receptor internalization motifs accessible to AP-2(34) . This is consistent with preliminary results showing that the tyrosine kinase inhibitor genistein prevents EGF receptor interaction with AP-2.^3 Although several sequences within the carboxyl terminus of the EGF receptor can serve as internalization codes(41) , the identification of the exact binding site(s) for AP-2 in the native EGF receptor remains to be completed. Studies of the influence of point mutations within the putative EGF receptor internalization motifs on the receptor association with AP-2 are in progress.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA24071 (to G. C.), DK46817 (to A. S.), and GM36548 (to T. K.). 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: Dept. of Pharmacology, University of Colorado Health Science Center, 4200 East Ninth Ave., Denver, CO 80262.

(^1)
The abbreviations used are: EGF, epidermal growth factor; DMEM, Dulbecco's modified Eagle's medium, AP-2, plasma membrane clathrin-associated protein complex; AP-1, Golgi clathrin-associated protein complex; CMF-PBS, Ca- and Mg-free phosphate-buffered saline; TGH, Triton X-100 solubilization buffer.

(^2)
W. Boll, K. Clairmont, and T. Kirchhausen, manuscript in preparation.

(^3)
A. Sorkin, and G. Carpenter, unpublished data.


ACKNOWLEDGEMENTS

We greatly appreciate the excellent technical assistance of Tatiana Sorkina and Usha Barnela. We are grateful to Dr. M. S. Robinson (University of Cambridge) for the gift of monoclonal antibodies AC1-M11 and B1-M6 to APs and Dr. L. Beguinot for EGF receptor serum 2913 and transfected cells.


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