The binding of epidermal growth factor (EGF) ()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, and 2 (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 -subunit, A and C, encoded by distinct but highly homologous genes(19) .

The large 2 subunit plus the medium and small subunits of AP-2 are very homologous to the corresponding subunits (1, µ1, and 1) of the Golgi clathrin-associated protein complex, AP-1(20, 21, 22, 23) . However, in place of an -subunit, AP-1 contains a -subunit (100 kDa) that has a relatively low level of similarity to the -subunit(24) . Studies of bovine brain-coated vesicles suggest that for AP-2 each -subunit is paired with a 2-subunit, whereas for AP-1, -adaptin is complexed with a 1-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 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 receptorAP 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 receptorAP-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 receptorAP-2 complexes.

EXPERIMENTAL PROCEDURES

Materials

Expression and Purification of the Carboxyl-terminal Portion of Rat C- and 2-Subunits

Cell Culture

Immunoprecipitation of APs

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 - and -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

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 C-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 C-ear-linker.

Potassium Depletion of Cells

Metabolic Labeling and Turnover of AP-2

Purification of EGF ReceptorAP-2 Complexes

To isolate AP-2 bound to receptors, 2 equal aliquots of the eluent from the EGF affinity column were immunoprecipitated with Ab31 to C-subunit in the absence or presence of recombinant C-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 C- and -Subunit Antibodies

()

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 C isoform. Consistently, we estimate that the C 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 1, that are considered characteristic of AP-1.

Time Course of Receptor Association with AP-2

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 C-ear-linker protein (lanes 6-10). The third aliquot was immunoprecipitated with antibody 986 to the EGF receptor (lanes 11-15). EGF receptor and -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 A- and C-subunit isoforms (typically 2-4% of total solubilized -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 C than A isoform is present. However, the A/C 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 A- or C-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 A/C was also similar in cell lysates and EGF receptor immunoprecipitates, although A was the predominant isoform relative to C in these cells(31) .

Immunoprecipitation of AP-2 from Metabolically Labeled Cells

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 C-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 C-ear-) and the radioactivity in the identical region of the gel of nonspecific precipitations (lane C-ear+). The 102-kDa band contained both C- and 2-subunits, as determined by immunoblotting of labeled proteins. The molar concentrations of µ2 and 2 relative to C/2 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 C(18) , and rat 2 subunits(23) . The molar concentration of 2 relative to C 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 C 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 2, µ2, and 2 together with the mouse C sequence (18, 20, 21, 22, 23) to normalize the radioactivity in the each band to the abundance of methionine residues. The molar ratio of C- and 2-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 (C/2), medium (µ2), and small (2) subunits was monitored. As seen in Fig. 3, quantitation indicated a similar rate of degradation, t 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 C/2, 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 ReceptorAP-2 Stoichiometry

The results of this experiment (Fig. 4) show that three bands corresponding to the mobilities C/2, 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 1 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 C- and -subunits was confirmed by Western blot analysis as described under ``Experimental Procedures'' (not shown).

Figure 4: Isolation of EGF receptorAP-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 receptorAP-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 C-ear-linker protein was used as a control for the nonspecific co-precipitation of labeled proteins. The migration position of radiolabeled EGF receptors, , , 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 180-kDa band, corresponding to phosphorylated EGF receptor was approximately twice that present in the C/2 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 C/2 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 8-9 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 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 -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 C-specific antibody showed that a relatively small pool of EGF receptors, equivalent to approximately 5-10 10 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 receptorAP-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 receptorAP-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 receptorAP-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. ()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 receptorAP-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. 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.

()

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.

()

W. Boll, K. Clairmont, and T. Kirchhausen, manuscript in preparation.

()

A. Sorkin, and G. Carpenter, unpublished data.

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