Journal of Cell Science 112, 317-327 (1999) 317 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0099 Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors Tatiana Sorkina, Andrea Bild, Francesc Tebar and Alexander Sorkin* Department of Pharmacology, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262, USA *Author for correspondence (e-mail: firstname.lastname@example.org) Accepted 25 November 1998; published on WWW 13 January 1999 SUMMARY Activation of the epidermal growth factor receptor interaction of the EGFR with µ1 as well as µ2 subunits of (EGFR) by EGF results in binding of clathrin adaptor AP-1 and AP-2, correspondingly, was shown using the yeast protein complex AP-2 to the receptor cytoplasmic tail. two-hybrid assay. Brefeldin A, a drug that releases AP-1 The transient interaction with AP-2 is thought to be from the trans-Golgi membranes, had no effect on AP-1 responsible for the selective recruitment of the EGFR into association with endosomes and its co-precipitation with coated pits during endocytosis. In this study we found EGFR. Taken together, the data suggest that endosomal that EGF-induced EGFR/AP-2 association, measured EGFR-AP complexes make up a signiﬁcant portion of the by co-immunoprecipitation, persists after receptor total amount of these complexes detectable by co- internalization. Double-label immunoﬂuorescence of EGF- immunoprecipitation. It can be proposed that APs are treated A-431 and COS-1 cells revealed the presence of AP- capable of binding to the endosomal membrane via a 2, clathrin and eps15, another component of the plasma mechanism that requires AP interaction with the membrane coated pits, in the large perinuclear endosomes intracellular tails of multimeric receptors like activated loaded with EGFRs. By optical sectioning and image EGFR, which in turn allows recruitment of clathrin and deconvolution, the immunoreactivities were seen to be eps15. The hypothesis that the competition between distributed within vesicular and tubular elements of these adaptor complexes for binding to the receptor tails in endosomes. In addition, these compartments contained the endosomes may regulate of the sorting of receptors is transferrin receptors and a EEA.1 protein, markers of discussed. early endosomes. Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells. The direct Key words: EGF receptor, Clathrin, Adaptor INTRODUCTION dissociation of the clathrin lattice. Whether AP release from the membrane is a prerequisite for the vesicle fusion with the Clathrin coats function at the plasma membrane to promote endosomal membrane is not known. However, restricted rapid endocytosis of various receptors and other membrane cellular localization of APs and other coat proteins suggests proteins as well as soluble macromolecules and viruses that the membrane docking and speciﬁc targeting of APs are (reviewed in Schmid, 1997). Clathrin-coated pits located in the tightly regulated. trans-Golgi network (TGN) are essential for the receptor- Current data suggest that α/γ, µ and possibly σ subunits of mediated delivery of soluble enzymes to lysosomes (reviewed APs can be involved in the membrane-docking process in Traub and Kornfeld, 1997). The major components of coated (Gaidarov et al., 1996; Page and Robinson, 1995; Robinson, pits are the clathrin and the clathrin adaptor protein complexes 1993). The anchoring molecules speciﬁc for AP-2 and AP-1 or APs, AP-2 at the plasma membrane, and AP-1 in TGN recruitment to the plasma membrane or TGN, respectively, are (reviewed in Robinson, 1997). Each AP is a heterotetramer not identiﬁed. ADP-ribosylation factor, ARF1, is important for consisting of two 100-kDa subunits or adaptins (α/β2 in AP-2 binding of AP-1 to TGN membranes (Traub et al., 1993). ARF and γ/β1 in AP-1), one 47/50-kDa (µ2 and µ1) and one 17/19- requirement for AP-2 docking to plasma membrane has not kDa subunits (σ2 and σ1). Membrane-bound APs serve as been demonstrated (West et al., 1997). The latter study nucleation sites for the assembly of the clathrin lattice. The implicates phospholipase D-dependent production of clathrin-adaptor coats undergo rearrangements, resulting in phosphatidic acid in the recruitment of AP-2 at the plasma invagination of the coated membrane and pinching off the membrane. In addition, α-adaptins and possibly other subunits coated vesicle. The plasma membrane and TGN-derived coated of AP-2 bind to phosphotidylinositols, which might be vesicles fuse with endosomes, which requires at least a partial important for the membrane docking of AP-2 (Gaidarov et al., 318 T. Sorkina and others 1996; Rapoport et al., 1997). In neuronal cells AP-2 is thought AP-3 (Dell’Angelica et al., 1997; Simpson et al., 1996), which to anchor to the transmembrane protein synaptotagmin (Zhang is colocalized with clathrin buds in the peripheral endosomes et al., 1994). of A-431 cells (Dell’Angelica et al., 1998), suggests that AP- APs also interact with the cytoplasmic tails of membrane 3 can be involved in endosomal trafficking of EGF and other receptors and other integral membrane proteins via the µ receptors. subunits (for a review see Kirchhausen et al., 1997) and In this study the dynamics of EGFR-AP interactions and possibly β1 subunit (Rapoport et al., 1998). The importance of subcellular localization of clathrin-coat proteins was analyzed. the mannose-6-phosphate receptor for the high-affinity binding We report that EGF-induced EGFR association with APs, of AP-1 to TGN membranes has been demonstrated (Le clathrin and eps15, a component of the plasma membrane Borgne et al., 1996). However, the receptors capable of binding coated pits (Tebar et al., 1996; van Delft et al., 1997), is to APs are present in various cellular compartments, maintained in A-431 cells after receptor internalization. The particularly in endosomes. In contrast, no signiﬁcant data suggest that the association of EGFRs and APs might be accumulation of AP-2 in endosomes containing internalized stabilized by multivalent interactions during endocytosis, receptors has been reported. Relocalization of activated FcεRI which may result in the assembly of clathrin-adaptor coats in receptors to restricted membrane domains did not result in endosomes. corresponding redistribution of AP-2 (Santini and Keen, 1996). Thus, the importance of cargo for the speciﬁc adaptor recruitment to the membrane remains a controversial issue, and MATERIALS AND METHODS the regulatory mechanisms of this transient receptor-adaptor interaction need to be characterized. Reagents The endocytosis of the receptor for epidermal growth factor Human recombinant EGF was obtained from Collaborative Research (EGFR) served as model system to study ligand-dependent Inc. Iron-saturated transferrins conjugated with ﬂuorescein (TRF- receptor trafficking for many years (reviewed in Sorkin and FITC) or Texas Red (TRF-TR) were purchased from Molecular Waters, 1993). The endocytic pathway is particularly well Probes. Polyclonal rabbit 986 and 451 antibodies to EGFR (anti- studied in human epidermoid carcinoma A-431 cells which EGFR) were a gift from Dr G. Carpenter (Vanderbilt University, Nashville). Rabbit serum Ab2913 speciﬁc to the intracellular domain express very high levels of EGFR. Activation of EGFRs by of EGFR was a gift of Dr L. Beguinot (DIBIT Rafaele, Milan, Italy). epidermal growth factor (EGF) results in internalization of Monoclonal antibodies AC1-M11 that recognize α-subunits of AP-2 EGF-receptor complexes via a rapid clathrin-coated-pit were a gift from Dr M. S. Robinson (University of Cambridge, pathway and a slower clathrin-independent mechanism England). Polyclonal antibodies 32 (Ab32) to β-subunits of AP-2 and (Haigler et al., 1979; Hopkins et al., 1985; Lamaze et al., 1993; AP-1 were characterized in our previous studies (Sorkin et al., 1995). Miller et al., 1986; Wiley, 1988). Both internalization pathways Dr P. P. Di Fiore (European Institute of Oncology, Milan, Italy) kindly lead to the same early endosomal compartment and provided polyclonal antibody to eps15 (Ab577). A monoclonal subsequently to multivesicular endosomes (MVEs) (Hopkins antibody to the clathrin heavy chain (X-22) and the α-subunit (AP.6) et al., 1985; Miller et al., 1986). Although EGF-EGFR (Brodsky, 1985) were obtained from ATCC, whereas monoclonals complexes are rapidly recycled back from endosomes to the 100/3 speciﬁc to γ-adaptin were from Sigma. A monoclonal antibody to EEA.1 protein was from Transduction Laboratories. Polyclonal cell surface (Sorkin et al., 1991a), a substantial fraction of these antibodies were used as the IgG-fraction puriﬁed from serum using complexes are sorted to the lysosome-degradation pathway Protein A-Sepharose (Sigma) or affinity-puriﬁed. after each round of internalization, which results in down- regulation of EGFRs (Stoscheck and Carpenter, 1984). The Cells proteolytic degradation of EGFRs appears to be a consequence Human epidermoid carcinoma A-431 cells (2-4×106 EGFR/cell) were of the direct fusion of MVEs with lysosomes (Futter et al., maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM) 1996). containing 10% calf serum with antibiotics and glutamine. Green The molecular mechanisms of EGF-induced receptor monkey kidney COS-1 cells (4×105 EGFR/cell) were grown in internalization and intracellular sorting are not well DMEM containing 10% newborn calf serum, antibiotics and understood. It has been demonstrated that EGF-activated glutamine. Cells were grown to about 90% or 50% conﬂuency for co-immunoprecipitation or immunoﬂuorescence experiments, receptors interact with AP-2 (Sorkin and Carpenter, 1993). respectively. This observation is consistent with the general dogma that receptors are recruited into coated pits by means of selective Immunoprecipitation of EGF receptors and AP-2 recognition by APs. This theory, however, did not survive Cells grown on 35 mm dishes (A-431) or 100 mm dishes (COS-1) testing in functional experiments in vivo: EGFR mutants were treated or not with EGF (500 ng/ml) in binding medium lacking the major AP-2 binding site have been shown to (DMEM, 0.1% bovine serum albumin, 20 mM Hepes, pH 7.3). In internalize via clathrin-dependent pathways (Nesterov et al., some experiments cells treated with EGF were incubated with 0.2 M 1995; Sorkin et al., 1996). Thus, although the existence of sodium acetate, 0.5 M NaCl (pH 4.5) to remove surface-bound EGF multiple weak AP binding sites in the EGFR is possible, the (Sorkin and Carpenter, 1991). Cells were then washed with Ca2+-, role of EGFR-AP interactions remains unclear. Furthermore, Mg2+-free phosphate-buffered saline (CMF-PBS) and solubilized by scraping with a rubber policeman in TGH buffer (1% Triton X-100, in vitro studies on broken cells suggested that additional factors 10% glycerol, 50 mM NaCl, 50 mM Hepes, pH 7.3, 5 mM EDTA, 1 other than AP-2 are required for efficient sequestration of mM sodium orthovanadate, 1 mM phenylmethylsulfonyl ﬂuoride, 10 EGFRs in coated pits (Lamaze et al., 1993). µg/ml leupeptine, 544 µM iodacetamide, 10 µg/ml aprotinin) The protein interactions that are responsible for the sorting followed by gentle rotation for 10 minutes at 4°C. Lysates were then of EGFRs in endosomes to recycling or lysosomal pathways centrifuged at 100,000 g for 20 minutes. Approximately 50% and 95- are also not identiﬁed. The discovery of a new adaptor complex 99% of the total cellular pools of AP-2 and AP-1, respectively, and at Clathrin coat proteins in endosomes 319 least 95% of the EGFR pool, were found in the supernatants after mg/ml para-phenylenediamine. The samples were analyzed using centrifugation. Supernatants were incubated with anti-EGFR conventional or digital deconvolution microscopy. A Nikon Diaphot (Ab986/451) for 3-15 hours at 4°C and then 30-60 minutes after the 300 microscope equipped with 100×1.4 NA oil immersion objective addition of Protein A-Sepharose (Sigma). Normal rabbit IgG (Zymed lens, and the single ﬂuorochrome ﬁlter sets for either Texas Red, Inc.) were used for non-speciﬁc controls. ﬂuorescein or simultaneous Texas Red/ﬂuorescein ﬂuorescence Immunoprecipitates were washed twice with TGH supplemented (Chroma Inc.), were used for visualization and recording the images. with 100 mM NaCl and then once without NaCl. 7.5% SDS- To obtain high resolution three-dimensional images of cells, polyacrylamide gels were used to separate proteins. Transfer to DeltaVision workstation (Applied Precision, Inc.), which includes an nitrocellulose membranes and protein immunoblotting were carried Olympus ﬂuorescent microscope, was employed. Typically 20-30 out as described (Sorkin et al., 1996). The top (above the 116 kDa serial two-dimensional images were recorded at 200 nm intervals molecular mass marker) and bottom portions of the nitrocellulose using a thermoelectrically cooled charged-coupled device (CCD) membrane were probed, respectively, with anti-EGFR Ab2913 and camera (PXL, Photometrics Ltd, Tucson, AZ). In some experiments adaptin antibody (AC1-M11 to α-subunits or 100/3 to γ-subunit). a QED Imaging workstation equipped with a Nikon Diaphot Sheep antibodies to mouse IgG (Cappel Inc.) or protein A (Zymed microscope and Micromax CCD camera with a Sony Interline area Inc.) conjugated with horseradish peroxidase and with enhanced array (Princeton Instruments) with high sensitivity within the blue- chemiluminescence (Amersham or NEN) were used to detect primary green range of the spectrum was used. A Z-stack of images obtained mouse or rabbit antibodies, respectively. on DeltaVision or QED workstations were deconvoluted using a modiﬁcation of the constrained iteration method. Final analysis of all Co-immunoprecipitation of AP-2 and eps15 in cellular images was performed using AdobePhotoshop 4.03. fractions To separate the cytosolic and membrane fractions, cells treated or not Two-hybrid analysis treated with EGF were mildly permeabilized by incubation in CMF- The yeast two-hybrid protein-protein interaction assay protocol PBS, containing 0.02% saponin, 1 mM EGTA, 5 mM EDTA, 1 mM followed the MatchMaker Two-Hybrid System 2 manual (Clontech, sodium orthovanadate, 10 mM sodium ﬂuoride, 1 mM PMSF and Palo Alto, CA). A fusion protein of a GAL4 transcription factor protease inhibitors, for 30 minutes at 4°C. After removal of the binding domain (GAL4bd) in the vector pAS2-1 with various saponin (cytosolic) fraction, the permeabilized cells containing the fragments of EGFR was constructed. EGFR fragments were generated membrane proteins were washed with CMF-PBS and solubilized by by PCR, and cloned into NcoI and SalI restriction sites. All clones scraping the cells away from the dish with a rubber policeman in TGH were veriﬁed by restriction analysis and sequencing. Full-length µ1 containing 1% sodium deoxycholate (TGH-DOC), followed by gentle and µ2, as well as ∆µ2, in which the ﬁrst 120 amino acid residues are rotation for 10 minutes at 4°C. Sodium deoxycholate was added to deleted, cloned in pACT2 vector which contained the GAL4 release all membrane-bound forms of AP-2 and eps15 (Tebar et al., activation domain (GAL4ad) (Ohno et al., 1995), were generously 1996). donated by Dr J. S. Bonifacino (NIH). Transformations of plasmids The saponin and TGH-DOC fractions were centrifuged at 100,000 into yeast strains Y187 and CG1945 were performed by the g for 20 minutes at 4°C and incubated with Ab32 to β-adaptins for 3 PEG/LiAc method. To test for positive interactions of EGFR with the hours at 4°C and then for 1 hour after addition of Protein A-Sepharose. µ subunits, Y187 transformations were used for β-galactosidase (β- Pre-immune rabbit serum or unrelated rabbit IgG (Zymed) were used gal) assays while CG1945 transformations were used in the growth to control for non-speciﬁc immunoprecipitations. Immunoprecipitates assay. Single colonies were picked and grown on synthetic dropout were washed twice with cold CMF-PBS or TGH supplemented with medium (SD) lacking Trp and Leu amino acids (−Trp,−Leu) plates at 100 mM NaCl and then once without NaCl. The electrophoresis, 30°C for 2-3 days, and then measured for β-gal activity using the transfer to nitrocellulose membranes and western blot analysis were colony-lift ﬁlter assay. Blue colonies were analyzed for up to 8 hours carried out as described above. The top (above the 116 kDa molecular following addition of X-gal. Concurrently, CG1945 transformations mass marker) and bottom portions of the nitrocellulose membrane were streaked onto SD −Leu,−Trp,−His plates with 5 mM 3-amino- were blotted with antibody to eps15 (Ab577) and α-subunits (AC1- 1,2,4-triazole (3-AT; Sigma). After growth at 30°C for 48 hours to M11), respectively. The detection of primary antibodies was allow for depletion of histidine, cells were picked from these plates performed as described above. and restreaked onto the fresh −His,−Leu,−Trp, 5 mM 3-AT plates. Cells were grown for additional 3-4 days and were scored for growth. Immunoﬂuorescence staining Several controls to test for autonomous activation and other caveats Cells grown on coverslips and incubated with EGF or labeled ligands were used with all experiments according to the manual. GAL4bd- (EGF-TR, TRF-FITC and TRF-TR) were ﬁxed with freshly prepared fusion constructs of the peptide containing the three repeats of the 4% para-formaldehyde (Electron Microscopy Sciences) for 12 internalization motif of TGN38 or its mutated version (YG mutation) minutes at room temperature, and mildly permeabilized using two in pAS2-1 vector were kindly provided by Dr Bonifacino and used as techniques. The ﬁrst technique allows better detection of the coated positive or negative controls of the interaction with µ1/2. pit proteins and uses a 3-minute permeabilization in CMF-PBS containing 0.1% Triton X-100, 0.1% BSA at room temperature. Coverslips were then incubated in the same buffer, in which Triton X- 100 was omitted, at room temperature for 1 hour with the primary RESULTS antibody, washed intensively and then incubated with the secondary donkey anti-mouse IgG and anti-rabbit IgG labeled with Texas Red EGFR association with AP-2 during endocytosis or ﬂuorescein (Jackson Tech.). Both primary and secondary antibody In our previous studies EGF-induced association of EGFR with solutions were precleared by centrifugation at 100,000 g for 10 AP-2 was demonstrated in A-431 and other cells (Sorkin and minutes. The second technique is useful to prevent the loss of labeled ligands from endosomes. Fixed cells were incubated in binding Carpenter, 1993; Sorkin et al., 1995). Apparently, this medium for 10 minutes, and permeabilized in CMS-PBS containing interaction may occur at the plasma membrane during 0.05% saponin and 1% BSA for 30 minutes. Subsequent incubations recruitment of the activated receptors into coated pits. with the primary and secondary antibodies were carried out in CMS- However, EGFR remain EGF-occupied and, therefore, PBS containing 0.01% saponin and 1% BSA. After staining the dimerized and phosphorylated after internalization (Carpentier coverslips were mounted in Fluoromount-G (Fisher) containing 1 et al., 1987; Lai et al., 1989; Sorkin and Carpenter, 1991). 320 T. Sorkina and others large (up to 1-2 µm) endosome-like structures was seen in the perinuclear region of the cells (Fig. 3A,D). Surprisingly, α- adaptin as well as clathrin heavy chains were clearly detected in most of these large endosomes loaded with EGFR (Fig. 3B,C,E,F). As expected, a punctate staining of clathrin and AP-2, which did not overlap with EGFR and that corresponds to the plasma membrane and TGN (for clathrin) coated pits, could be seen on the sections through the middle of the cell (Fig. 3), and much more strongly on the sections close to the cell surface (not shown). Optical sectioning and image correction by deconvolution revealed that EGFR Fig. 1. Time course of AP-2 co-immunoprecipitation with EGFR in immunoreactivity is associated with the small vesicles A-431 cells. Cells were incubated with the saturating concentration connected by tubular elements that often bend around each of EGF (500 ng/ml) for indicated periods of time (minutes) at 37°C, other. EGFR staining only partially overlapped with the and then the EGFRs were immunoprecipitated from TGH lysates of clathrin and AP-2 immunoreactivity, indicating that a limited the cells. EGFRs and α-subunits of AP-2 (αA and αC) were detected pool of endosomal EGFRs are associated with coated pit in immunoprecipitates by western blotting with anti-EGFR Ab2931 proteins. and AC1-M11, respectively. The time course was similar in four The immunoﬂuorescence labeling of COS-1 cells, treated independent experiments. with EGF for 30 minutes at 37°C, revealed EGF-dependent co- localization of EGFR and clathrin in perinuclear endosomes, although the extent of clathrin accumulation in endosomes was These observations prompted us to test whether EGFR/AP-2 less dramatic compared to that in A-431 cells (Fig. 3G-I). association is also retained in endosomes. The time course of Surprisingly, very little AP-2 was found in endosomes of EGF- AP-2 co-immunoprecipitation with EGFR showed that treated COS-1 cells, although EGFR/AP-2 co- association of AP-2 with EGFR reaches a maximum at 15 immunoprecipitation was detected in COS-1 cells treated with minutes and is maintained at a level that is slightly lower than maximal for at least 45 minutes (Fig. 1). The persistence of EGFR/AP-2 association during endocytosis suggested that endosomal EGFR may be complexed with AP-2 and may, therefore, contribute to the total pool of these complexes detected by co-immunoprecipitation. In order to distinguish between the surface and internalized EGF/EGFR/AP-2 complexes, the mild acid-wash treatment was employed. The cells were ﬁrst incubated with EGF at 4°C, and then endocytosis was initiated by placing cells at 37°C. At the end of the incubation, cells were either treated or not with the acidic buffer to remove surface-bound EGF. Such treatment removes at least 90-95% of surface-bound EGF and leads to immediate monomerization, deactivation of the kinase and dephosphorylation of surface EGFR, whereas internalized EGFRs are not affected (Nesterov et al., 1990; Sorkin and Carpenter, 1991; Sorkin et al., 1991a). As shown in Fig. 2, a 37°C-incubation resulted in the binding of AP-2 to EGFR, revealed by co-immunoprecipitation. Acid wash had only a moderate effect (about a twofold decrease of the Fig. 2. Time course of AP-2 co-immunoprecipitation with EGFR treated or not treated with acid wash. A-431 cells were incubated speciﬁc signal) on the extent of AP-2 co-immunoprecipitation with EGF for 1 hour at 4°C, and then for the indicated periods of with EGFR at the early stages of endocytosis. At later times, time (minutes) at 37°C. At the end of a 37°C incubation, surface acid-resistant (intracellular) EGF-EGFR complexes were EGF was stripped (+) or not stripped (−) by the mild acid wash. The solely responsible for the association with AP-2 detected by EGFRs were immunoprecipitated from TGH lysates of the cells. co-immunoprecipitation. Thus, the maintenance of acid- EGFRs and α-adaptins (αA and αC) were detected in resistant EGFR/AP-2 association during endocytosis suggests immunoprecipitates by western blotting with anti-EGFR Ab2931 and that a substantial pool of these complexes are located AC1-M11, respectively. Top, western blot detection of α-adaptins in intracellularly. EGFR immunoprecipitates; Bottom, quantitation of the amount of AP-2 present in the EGFR immunoprecipitates normalized to the Localization of AP-2 and clathrin in EGF-treated amount of EGFRs detected by immunoblotting (a.u., arbitrary units). cells The average amount of AP-2 detected in non-speciﬁc immunoprecipitates with rabbit IgG is indicated by the dashed line. The localization of AP-2 relative to EGFR was inspected The experiment is representative of three similar experiments. Note using a double-label immunoﬂuorescence technique. Cells that a 20-minute incubation at 37°C after pre-occupying the receptors were incubated with EGF for 30 minutes at 37°C, ﬁxed and with EGF at 4°C roughly corresponds on the time scale of processed for immunostaining using a Triton X-100 endocytosis to a 30-minute incubation with EGF at 37°C without permeabilization protocol. The accumulation of EGFR in 4°C-preincubation. Clathrin coat proteins in endosomes 321 Fig. 3. Localization of clathrin and AP-2 in endosomes of A-431 and COS-1 cells. A-413 (A-F) and COS-1 (G-I) cells were incubated with, respectively, 500 ng/ml and 200 ng/ml EGF for 30 minutes at 37°C, ﬁxed and stained with polyclonal antibodies to EGFR Ab2931 (green; A,D,G) and monoclonal clathrin heavy chain antibody X-22 (red; E,H) or α- adaptin antibody AP.6 (red; B) using a Triton X-100 permeabilization protocol. After data acquisition on the DeltaVision workstation, the ﬂuorescein and Texas Red channels were merged (C,F,I) after adjustment of both ﬂuorescence signals to similar levels. ‘Yellow’ indicates the overlap of Texas Red and ﬂuorescein ﬂuorescence. Higher magniﬁcation images of the overlay images of the individual endosomes are presented on the bottom. Note the absence of image pixelation indicates that the staining is within the resolution range of the CCD camera. All images comprise an individual optical section from the middle of the cell, where the most intense signal for EGFR was observed. Arrows point to examples of co- localization of clathrin and EGFR in endosomes. EGF for 30 minutes at 37°C (data not shown). The extent of eps15 is associated with the α-subunit of AP-2 in NIH 3T3 AP-2 co-immunoprecipitation with EGFR was, however, much cells (Benmerah et al., 1995; Tebar et al., 1996). Fig. 4A shows less than in A-431 cells, which might explain the poor AP-2 that EGF treatment does not affect the extent of AP-2/Eps15 detection in endosomes of COS-1 cells. It is also possible that co-immunoprecipitation in cytosolic and membrane fractions in COS-1 cells, other adaptor complexes may be responsible in A-431 cells. Interestingly, the relative size of the cytosolic for EGF-dependent clathrin recruitment onto endosomes. pool of AP-2 is smaller in A-431 cells (Fig. 4A) compared to To conﬁrm that AP-2 is associated with the endosomes but NIH 3T3 cells (Tebar et al., 1996). Only a limited pool of not with the surface aggregates of EGFRs, A-431 cells were membrane-bound AP-2 is associated with eps15, which is allowed to internalize Texas Red-conjugate of EGF (EGF-TR) consistent with the restricted localization of eps15 at the and then treated with the mild acidic buffer to remove non- periphery of the coat (Tebar et al., 1996). internalized EGF-TR. Staining with antibody to α-adaptin In A-431 and COS-1 cells eps15 is also co-localized with performed using a saponin-permeabilization protocol revealed the markers of plasma membrane coated pits (data not shown). co-localization of AP-2 with acid-resistant and, therefore, However, in contrast to what was observed in NIH 3T3 cells internalized EGF-TR (data not shown). (Tebar et al., 1996; van Delft et al., 1997), EGF induces a signiﬁcant re-distribution of eps15 to endosomes containing Eps15 is bound to AP-2 and follows EGFR/AP-2 to EGFR in A-431 cells (Fig. 4B,C). The extent of eps15 co- endosomes localization with EGFR in endosomes stained with anti-EGFR The data of Fig. 3 demonstrated the presence of clathrin and was comparable to that observed for AP-2. In COS-1 cells, AP-2 in endosomes. Another component of plasma membrane EGF-induced accumulation of eps15 in endosomes was much clathrin-coated pits is a protein called eps15 (Tebar et al., 1996; less dramatic. The data suggest that eps15 distribution to van Delft et al., 1997). A large fraction of the cellular pool of endosomes emulates the distribution of AP-2. 322 T. Sorkina and others Cells were incubated with TRF-TR or TRF-FITC in the absence or presence of EGF, and then stained with antibodies to eps15 or clathrin. Fig. 5A-C demonstrates that endocytosis of TRF-TR alone did not lead to signiﬁcant accumulation of eps15 in labeled endosomes. Simultaneous internalization of EGF and TRF-TR is known to result in co-localization of two ligand-receptor complexes in the early endosomal compartments (Hopkins and Trowbridge, 1983). As shown in Fig. 5D-F, EGF causes the accumulation of TRF-TR in large perinuclear endosomes that also contain eps15. Essentially similar results were obtained with co-staining of TRF-TR and α-adaptin (data not shown). In contrast, a pool of clathrin was seen associated with TRF-FITC-containing compartments in the absence of EGF (Fig. 5G-I), albeit the amount of endosomal clathrin was substantially increased in cells treated with EGF (Fig. 5J-L). In summary, the data presented in Fig. 5 indicate that the recruitment of clathrin-coat protein to endosomes is the speciﬁc feature of the endocytosis of EGF- occupied EGFR. The accumulation of the internalized transferrin in these endosomes indicates that these compartments represent early and/or recycling endosomes. Furthermore, the endosomes containing EGFRs and clathrin- coat proteins were also positive for protein EEA.1, the marker of early and ‘intermediate’ endosomes (Mu et al., 1994) (data not shown). AP-1 in endosomes Based on visual analysis of the large number of experiments, the extent of clathrin accumulation in endosomes of A-431 cells is higher compared to that of AP-2. This observation prompted us to test whether another adaptor complex, AP-1, also docks on EGFR-containing endosomes and contributes to the clathrin recruitment. Immunoﬂuorescence labeling of A- 431 cells with anti-γ-adaptin showed that although the main Fig. 4. Association of AP-2 with eps15, and the eps15 localization in region of AP-1 localization is TGN, a punctate staining of AP- A-431cells. (A) Cells were incubated with or without 500 ng/ml EGF 1 can be seen at a distance from TGN, especially in cells treated for 30 minutes at 37°C, permeabilized with saponin, and then with EGF (Fig. 6B). Double-label staining showed that AP-1 solubilized in TGH-DOC buffer. Equal portions of the cytosolic immunoreactivity overlaps with EGFR in perinuclear (saponin eluent) and membrane fraction were incubated with saturative amounts of Ab32 (anti-β) to immunoprecipitate APs or endosomes (Fig. 6A,B), similar to that overlap observed for with a corresponding amount of rabbit IgG. Eps15 and α-subunits of EGFRs and AP-2, clathrin and eps15 (Figs 3, 4). AP-2 were detected in immunoprecipitates by western blotting with To test whether EGFRs interact with AP-1, the co- Ab577 and AC1-M11, respectively. (B,C) Cells were incubated with immunoprecipitation assay was employed. Fig. 7 shows that 500 ng/ml EGF for 30 minutes at 37°C, and processed for double- AP-1 can be readily detected in EGFR immunoprecipitates label immunoﬂuorescence staining using mouse monoclonal recovered from EGF-stimulated cells. The time course of antibodies to EGFR (B) and rabbit antibodies Ab577 to eps15 (C), EGFR/AP-1 association measured by co-immunoprecipitation using a Triton X-100 permeabilization protocol. Rabbit and mouse was similar to that of AP-2 (Fig. 7B). The extent of AP-1 primary antibodies were detected with corresponding secondary IgGs binding to EGFRs was, however, smaller than that of AP-2. labeled with ﬂuorescein or Texas Red. Cells were visualized using a Whereas up to 20-25% of the total cellular AP-2 (40-50% of conventional Nikon microscope. Note the strong co-localization of EGFR and esp15 in large endosomes at the focal plane Triton X-100-extractable pool) could be co- corresponding to the best staining of EGFRs. immunoprecipitated with EGFR, about 5% of cellular AP-1 was associated with EGFR. It has been demonstrated that the association of AP-1 with TGN membranes can be disturbed by Brefeldin A (BFA) Movement of coat proteins to early/intermediate (Robinson and Kreis, 1992). To examine whether AP-1 binding endosomes is EGF-dependent to endosomes is also sensitive to BFA, we inspected the To prove that the recruitment of coat components to endosomes localization of AP-1 in cells treated with BFA prior to, and is EGF-dependent, the localization of clathrin-coat proteins during the EGF stimulation. As seen in Fig. 6C,D, BFA caused was compared to that of transferrin in A-431 cells treated or dispersion of γ-adaptin staining associated with TGN, whereas not treated with EGF. Transferrin receptor is expressed at high AP-1 staining of EGF-containing endosomes has not been levels in A-431 cells, and the addition of ﬂuorescent transferrin disturbed. In fact, endosomal staining of AP-1 was seen more results in accumulation of the label in the early endosomes. clearly in cells treated with BFA, because of the diffusion of Clathrin coat proteins in endosomes 323 TGN staining. Furthermore, BFA had no effect on the extent DISCUSSION of co-immunoprecipitation of AP-1 with EGFR (Fig. 7). Thus, data of Figs 6 and 7 suggest that AP-1 is bound to the Interactions of EGFR with APs in endosomes endosomal membrane via the BFA-insensitive mechanism The EGF- and temperature-dependent interaction of AP-2 requiring stable association with EGFRs. with EGFR was initially demonstrated using a co- immunoprecipitation assay (Sorkin and Carpenter, 1993) and EGFR binds µ1 and µ2 attributed to the function of AP-2 in recruiting activated The simplest explanation of the immunolocalization and EGFRs into the plasma membrane coated pits. However, there immunoprecipitation studies in A-431 cells is that AP-1 as is no obvious restraint to prevent AP-2 binding to internalized well as AP-2 is recruited to endosomes, due to direct EGFR that remain largely dimerized, active and tyrosine association with activated EGFR. To conﬁrm that EGFRs are phosphorylated (Carpentier et al., 1987; Lai et al., 1989; capable of binding to AP-1 and AP-2 in vivo, we performed Nesterov et al., 1990; Sorkin and Carpenter, 1991). Here we a protein-protein interaction analysis using the yeast two- show that EGF-dependent EGFR/AP-2 association is hybrid system. Such an approach has been used to maintained during continuous endocytosis in A-431 cells (Fig. demonstrate that polypeptides corresponding to intracellular 1) and is not sensitive to the removal of EGF from the surface domains of several integral membrane proteins, which possess receptors (Fig. 2). Together with the results of digital tyrosine-containing internalization signals, for instance deconvolution microscopy, the data strongly suggest that a TGN38, bind to the µ subunits of APs (Ohno et al., 1995). pool of EGFR/AP-2 complexes are preserved after Therefore, we tested whether the carboxyl terminus of EGFR, internalization. which contains multiple internalization motifs (Chang et al., The following working model of EGFR/AP interactions 1993), interacts with µ1 or µ2 in yeast. The results of growth during internalization is proposed. EGF binding elevates the and β-galactosidase assays of EGFR/µ interactions in affinity of EGFR interaction with AP-2, leading to increased comparison with that interaction of the internalization motif recruitment of receptors into coated pits. That EGFR can bind of TGN38 are presented in Table 1. The fragment of EGFR AP-2 at the cell surface is suggested by the detection of corresponding to residues 908-1186 showed interaction with EGFR/AP-2 co-immunoprecipitation in K+-depleted cells the full-length µ1 and µ2, as well as with ∆µ2. The EGFR when clathrin-dependent endocytosis is blocked (Sorkin and interaction with µ2 was, however, signiﬁcantly weaker than Carpenter, 1993). However, we propose that it is the endosomal that of the ‘positive control’, a peptide containing three repeats EGFR/AP-2 complexes that constitute a substantial fraction of of the internalization motif of TGN38. The strength of these complexes detected by co-immunoprecipitation under interaction of EGFR fragment 908-1186 with µ1 was conditions of normal endocytosis. In fact, the EGFR family are comparable with that of TGN38 peptide when estimated using the only receptors, except for inﬂuenza virus hemagglutinin growth (Table 1) or liquid β-galactosidase assay (data not (Fire et al., 1997), for which co-immunoprecipitation with AP- shown). To map µ-binding regions of EGFR, several small 2 is documented (Gilboa et al., 1995; Sorkin and Carpenter, fragments of EGFR carboxyl terminus were prepared. 1993). For instance, the co-immunoprecipitation of transferrin Fragment 908-1022 did not show any interaction with µ receptor with AP-2 has not been demonstrated. Perhaps the subunits (Table 1). However, other fragments (including 973- transient receptor-AP interactions during internalization do not 1022, 972-1186 and 1020-1186) showed strong result in an accumulation of receptor-adaptor complexes that transactivation activity in control experiments in the absence is sufficient for detection by co-immunoprecipitation. of GAL4-ad and despite the presence of 3-AT, and could not However, if this interaction is not transient and sustained in be used for mapping. Nevertheless, the data of two-hybrid endosomes, as observed for EGFRs in A-431 cells, it can be experiments demonstrated that EGFR can directly bind AP-1 readily detected by co-immunoprecipitation. Correspondingly, and that it binds to µ1 with an affinity comparable to that of AP-2 does not follow transferrin receptor to endosomes in A- its interaction with µ2. 431 cells. Presumably, prolonged EGFR/AP-2 association in Table 1. Interaction of EGFR fragments with µ subunits in yeast two-hybrid system GAL4bd-fusion constructs EGFR fragments TGN38 (amino acid residues) GAL4ad-fusion constructs Assay* SDYQRL SDGQRL 908-1022 908-1186 972-1186 972-1002 1020-1186 None β-gal −‡ − − − ++ ++ ++ growth − − − − ++ ++ ++ µl β-gal ++ − − ++ +++ +++ +++ growth ++ − − ++ +++ +++ ++ µ2/∆µ2 β-gal +++ − − ++ +++ +++ +++ growth +++ − − + +++ ++ +++ *The relative intensity of β-galactosidase reaction and cell growth is ranged from the maximal (+++) observed for TGN38/µ2 interaction to the minimal (+) for EGFR 908-1186/µ2 interaction. ‡(−) No blue staining or cell growth. 324 T. Sorkina and others vivo is enforced by the multivalent interactions of EGFR AP-1 accumulation in endosomes and association with the multimers. Another possibility is that EGFR signaling in EGFR is BFA-independent, these processes might be regulated endosomes results in production of lipids, such as phosphatidic by the same mechanism as AP-2 binding to the EGFR and to acid or phosphatidylinositol-3-phosphate, that might be the plasma membrane (which is BFA-insensitive). The results important for stabilization of the AP binding to the membrane of the two-hybrid assay, which demonstrate binding of the (Gaidarov et al., 1996; West et al., 1997). carboxyl terminus of EGFR to µ subunits of APs, support the The surprising observation is the detection of AP-1 in EGFR possibility of the direct receptor binding to AP-1. The immunoprecipitates and in endosomes of A-431 cells. Because interaction of the EGFR with µ1 was slightly stronger than Fig. 5. Co-localization of transferrin, eps15 and clathrin in endosomes of A-431 cells. (A-F) Cells were incubated with 5 µg/ml TRF-TR in the absence (A-C) or presence of 500 ng/ml EGF (D-F) for 30 minutes at 37°C, ﬁxed and stained with the Ab577 to eps15 followed by secondary IgGs labeled with ﬂuorescein (B,E). (G-L) Cells were incubated with 5 µg/ml TRF-FITC in the absence (G-I) or presence of 500 ng/ml EGF (J- L) for 30 minutes at 37°C, ﬁxed and stained with the monoclonal X-22 antibody to clathrin followed by the secondary IgG labeled with Texas Red (H,K). The saponin permeabilization protocol was used. The serial optical sections were acquired and deconvoluted using a QED Imaging system and deconvoluted as described Materials and methods. The ﬂuorescein (green) and Texas Red (red) channels were merged (C,F,I,L) after adjustment of both ﬂuorescence signals to similar levels. ‘Yellow’ indicates the overlap of Texas Red and ﬂuorescein ﬂuorescence. All images comprise an individual optical section (0.2 µm) from the middle of the cell, where the most intense signal for transferrin was observed. Bars, 5 µm. Clathrin coat proteins in endosomes 325 Fig. 7. AP-1 co-immunoprecipitation with EGFR in A-431 cells. (A) Cells were incubated for 15 minutes with or without 10 µg/ml BFA at 37°C, and then in the same media with 500 ng/ml EGF for 30 minutes at 37°C. EGFRs were immunoprecipitated from TGH lysates of the cells, and detected by western blotting with antibodies Ab2931, while γ-subunit of AP-1 and α-subunits of AP-2 were probed with the mixture of antibodies AC1-M11 and 100/3. (B) A- 431 cells were incubated with EGF (500 ng/ml) for the indicated periods of time (minutes) at 37°C, and then the EGFRs were immunoprecipitated from TGH lysates of the cells. The γ-subunits of Fig. 6. Localization of AP-1 in A-431 cells treated with EGF. Cells AP-1 were detected in immunoprecipitates as described for A. The were incubated for 15 minutes with (C,D) or without 10 µg/ml BFA time course was similar in three independent experiments. (A,B) at 37°C, and then in the same medium with 500 ng/ml EGF for 30 minutes at 37°C. Formaldehyde-ﬁxed cells were processed for double-label immunoﬂuorescence microscopy with rabbit anti-EGFR data). One possible explanation is the limited sensitivity of the Ab2913 (A,C) and mouse antibodies 100/3 to γ-adaptin (B,D) using co-immunoprecipitation assay for the detection of indirectly a Triton X-100 permeabilization protocol. Rabbit and mouse primary associated proteins in detergent solutions. In addition, the antibodies were detected with corresponding secondary IgGs labeled existence of another EGF-dependent mechanism of the with ﬂuorescein or Texas Red. Cells were visualized using a conventional Nikon microscope (see Materials and methods). Arrows membrane docking of eps15 cannot be ruled out. indicate examples of co-localization of the endosomes containing Clathrin has been previously found in the peripheral EGFR (A,C) and AP-1 (B,D). Note, the dispersion of TGN staining endosomes of A-431 cells by whole-cell-mount electron of γ-adaptin in the presence of BFA (D). microscopy (Stoorvogel et al., 1996). Our experiments also demonstrated the presence of clathrin in endosomes containing transferrin receptor, and the increase of the endosomal clathrin with µ2 when estimated by the growth assay (Table 1). This is pool in the presence of EGF. It can be proposed that clathrin in contrast to the much stronger interactions of internalization is constitutively associated with endosomes due to its signals of other proteins with µ2 compared to µ1 in the two- anchoring to AP-3 (Dell’Angelica et al., 1998), whereas an hybrid system (Ohno et al., 1996). Interestingly, unc-101 gene, additional recruitment of clathrin to endosomes may result encoding a homolog of mammalian µ1, negatively regulates from the EGF-induced accumulation of AP-2 and AP-1 in the let-23 (EGFR) signaling pathway in C. elegans (Lee et al., these endosomes. 1994), possibly by affecting the degradation of the receptor. Thus, it can be hypothesized that in mammalian cells AP-1 Coated pit proteins are associated with the might be also involved in the sorting of EGFRs to the early/intermediate endosomes lysosomal pathway. The EGF-dependent appearance of clathrin, AP-2, AP-1 and eps15 in endosomes of A-431 cells is the ﬁrst demonstration, Eps15 and clathrin in endosomes to our knowledge, of the massive re-distribution of coated pit Eps15 is constitutively associated with AP-2 in A-431 (Fig. 4) proteins to endosomes in intact cells. The large perinuclear and other cells (Benmerah et al., 1995; Iannolo et al., 1997), endosomes might correspond to the classical MVEs that are and appears to follow the intracellular distribution of AP-2 often seen in A-431 and other cells and contain transferrin induced by EGF (Figs 4-8). Eps15 was not, however, found in receptors and EEA.1 protein (Beguinot et al., 1984; Gu et al., EGFR immunoprecipitates, obtained under mild conditions, in 1997; Haigler et al., 1979; Miller et al., 1986). Previous studies any signiﬁcant amount (Fazioli et al., 1993; our unpublished did not detect a clathrin lattice in MVE-like structures; 326 T. Sorkina and others however, its absence could be due to the high sensitivity of receptor interaction with AP-1, which would result in a slow these coats to the sample preparation procedures used in turnover of the EGFRs. Finally, we propose that A-431 cells electron microscopy and subcellular fractionation experiments. represent a case of exaggeration of transient interactions of For instance, clathrin coats on the peripheral endosomes in A- EGFRs in endosomes, and can serve as a model system to 431 cells could only be seen when the cells were saponin- study the biogenesis, morphology and the function of the permeabilized prior to ﬁxation, and the endosomes were tubular-vesicular endosomes and their clathrin-adaptor coats. stabilized by the horseradish peroxidase reaction product (Stoorvogel et al., 1996). The ability of AP-2 and clathrin to The authors are thankful to Drs G. Carpenter, L. Beguinot, P. P. Di dock on lysosome-like organelles in permeabilized cells has Fiore and M. S. Robinson for the gifts of antibodies, and to Dr been demonstrated (Traub et al., 1996). Immunoﬂuorescence Bonifacino for the yeast expression plasmids. We are grateful to Dr Royston Carter for help with the two-hybrid studies and critical and electron microscopy studies revealed the presence of a pool reading of the manuscript, and Steven Fedul for help with of AP-1 in endosome-like vesicles located at some distance immunoﬂuorescence imaging on the DeltaVision workstation that is from TGN (Le Borgne et al., 1996). All these data suggest that supported by the NIH grant SIO RR 12043-01. This work was the localization of AP-2 and AP-1 is not restricted to the supported by NIH grant DK46817 and UCHSC/HHMI grant to A.S., plasma membrane or TGN, respectively, and that these and ACS/University of Colorado Cancer Center grant to F.T. Cancer adaptors together with other coat elements can function in Center Core Services of University of Colorado are supported by divergent compartments of the endocytic pathway. Grant CA46934. 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