Clathrin coat proteins in endosomes by hkksew3563rd

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									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: alexander.sorkin@uchsc.edu)

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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