(Received for publication, November 2, 1994; and in revised form, January 9, 1995)
From the
We investigated the mechanism by which ligand-activated
epidermal growth factor receptors (EGFR) associate with coated pit
adaptor protein (AP) complexes. In vivo association, assayed
by coimmunoprecipitation of AP with mutant EGFR, required tyrosine
kinase activity, intact autophosphorylation sites, and the regulatory
carboxyl terminus of EGFR. The role of autophosphorylation of EGFR in
interaction with AP was examined in vitro using a
BIAcore instrument. Purified active EGFR, immobilized on
the biosensor surface, was reversibly autophosphorylated or
dephosphorylated by treatment with ATP or phosphatase.
Autophosphorylation of EGFR significantly increased AP binding. Once
formed, EGFR
AP complexes were resistant to disassembly by
dephosphorylation of EGFR or competition with phosphotyrosine,
indicating that phosphorylated tyrosine residues do not directly
participate in AP binding. Induction of conformational changes in EGFR
by treatment with urea increased AP binding up to 10-fold in the
absence of EGFR autophosphorylation. A recombinant EGFR carboxyl
terminus specifically bound the AP complex and each of the isolated
- and
-subunits of AP2. We conclude that tyrosine
autophosphorylation of EGFR exposes structural motif(s) in the carboxyl
terminus of EGFR that interact specifically with AP2.
Upon ligand binding, the diffusely distributed cell-surface
receptors for epidermal growth factor (EGFR()) and insulin
(InsR) are activated and redistribute to clathrin-coated pits that are
rapidly internalized into the cell(1) . Clearing receptors from
the cell surface (down-regulation) attenuates signaling by making
receptors unavailable for subsequent ligand binding and by degradation
of internalized ligand (2, 3) . The process of
ligand-induced internalization of EGFR and InsR requires intrinsic
protein-tyrosine kinase activity and specific sequence
motifs(4, 5, 6, 7, 8, 9) .
Endocytic ``codes'' in EGFR and InsR are 4-6-amino acid
sequences that contain obligatory tyrosine or phenylalanine residues (10, 11, 12) and are similar to those
identified for endocytosis of many cell-surface receptors(13) .
The requirement for ligand activation and intrinsic protein-tyrosine
kinase activity of EGFR and InsR contrasts with the independence of
these requirements for concentration of transferrin and low density
lipoprotein receptors in coated pits(14, 15) . Because
endocytic codes from EGFR can function in the context of the
transferrin receptor (12) and because deletion mutants of EGFR
that lack a kinase domain undergo high constitutive rates of
internalization(16) , the tyrosine kinase activity of EGFR has
been proposed to cause an autophosphorylation-dependent conformational
change that exposes masked endocytic codes located in the regulatory
carboxyl terminus(17) .
Sorkin and Carpenter (18) observed ligand- and temperature-dependent binding of EGFR
to coated pit adaptors in vivo, suggesting that this was
involved in receptor internalization. Previous in vitro studies identified interactions between adaptor complexes and the
cytoplasmic endocytic code-containing domains of the low density
lipoprotein receptor(19) , the mannose 6-phosphate receptor (20) , the asialoglycoprotein receptor(21) , and
lysosomal acid phosphatase(22) . Adaptor proteins (AP),
initially identified based upon their ability to promote the formation
of coat structures from clathrin(23) , are a major component of
coated pits. They consist of two 100-kDa polypeptides (adaptins)
and two smaller polypeptides. Adaptor complex 1 (AP1) is restricted to
the Golgi region, and AP2 is localized to plasma membrane coated pits;
each has a distinct subunit composition(24) . The specific
binding of liganddependent as well as constitutively internalized
receptors to AP may thus be essential for endocytosis of these
receptors.
One mechanism by which EGFR associates with cellular
proteins involves binding of SH2 domains to sites of tyrosine
autophosphorylation that are located in the regulatory carboxyl
terminus(25, 26) . However, adaptor proteins lack SH2
domains, so the mechanisms for their association with EGFR are likely
to be different. In this study, we demonstrate that autophosphorylation
of EGFR is required for binding adaptor complexes via a mechanism that
is distinct from binding to phosphotyrosine residues. Once formed,
EGFRAP complex stability is independent of tyrosine
phosphorylation. Moreover, adaptor complexes and individual
- and
-subunits of AP2 bind to the regulatory carboxyl terminus of EGFR
expressed as a glutathione S-transferase fusion protein
(GST-EGFR-CT). In vivo ligand- and temperature-dependent
interaction of EGFR with AP2 requires the regulatory carboxyl terminus
previously shown to be necessary for ligand-dependent endocytosis of
EGFR(8, 12) . These results support the hypothesis
that EGF- and autophosphorylation-dependent conformational changes in
EGFR allow interaction of the carboxyl terminus with AP2. Such a
mechanism could facilitate concentration of activated EGFR in coated
pits.
EGFR was immunopurified from A431 cells on a column containing immobilized monoclonal antibody 528, specific to the extracellular domain of EGFR(35) . Active EGFR was competitively eluted from the column with 0.1 mM EGF. To prepare denatured receptor, it was eluted with 6 M urea. Purity of proteins was verified by gel electrophoresis and Coomassie Blue staining to be >95%.
Clonal B82 cells expressing various EGFR constructs were prepared and maintained as described(12) . A431 cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% calf serum, 100 units/ml penicillin, 100 units/ml streptomycin, and 0.25 µg/ml amphotericin B. The medium was replaced with serum-free Dulbecco's modified Eagle's medium 18 h before experiments.
Proteins were resolved by electrophoresis,
transferred to nitrocellulose filters, and probed with one of the
following: rabbit polyclonal Ab 4516-8 or mouse monoclonal Ab PY20,
100/2, or AC1-M11. Both 100/2 and AC1-M11 recognized -adaptins
with approximately the same efficiency. Immunoreactive bands were
detected using peroxidase-conjugated donkey anti-rabbit or sheep
anti-mouse immunoglobulins and developed using enhanced
chemiluminescence. To reprobe the same filter with a different
antibody, it was then stripped for 30 min at 50 °C in 50 mM Tris-HCl, pH 7.5, in the presence of 2% SDS and 100 mM 2-mercaptoethanol.
To
autophosphorylate immobilized EGFR, 25 µl of 1 mM ATP in
BIAcore running buffer containing 5 mM MnCl was
injected. To dephosphorylate EGFR, 25 µl of dephosphorylation
buffer containing 250 units/ml calf intestinal alkaline phosphatase was
injected. Efficiency of both phosphorylation and dephosphorylation of
EGFR was estimated by injection of Ab PY20 in BIAcore running buffer at
a concentration of 75 µg/ml. In some experiments, 10 mM Tyr(P) in BIAcore running buffer was used as a competitor. AP
complexes were exchanged into BIAcore running buffer by gel filtration
and were spun for 15 min in a microcentrifuge prior to use. AP
complexes (35 µl) were injected at a concentration 0.3 mg/ml in
BIAcore running buffer. Although the highly purified adaptor
preparations used contained predominantly AP2(31) , the
specific AP bound to EGFR on the biosensor surface was not identified.
Both expression constructs were transformed into S.
cerevisiae strain EGY48 and selected on Trp medium. Expression of fusion proteins was induced by growth in 2%
galactose. Cells were then spun down, washed with water, and
resuspended in 50 ml of 100 mM Tris/SO
, pH 9.4,
containing 20 mM dithiothreitol. After incubation for 10 min
at room temperature, cells were pelleted by centrifugation and
resuspended in 5 ml of sorbitol buffer (1 MD-sorbitol, 20 mM Tris-HCl, pH 7.5, containing
6.7 g/liter yeast nitrogen base). 1 mg (100 lytic units) of zymolyase
was then added, and cells were incubated for 30 min at 30 °C with
continuous shaking. Spheroplasts were gently washed to remove zymolyase
by two centrifugations using 50 ml of ice-cold sorbitol buffer
containing 10 mM benzamidine and 1 mM EDTA.
Spheroplasts were solubilized in 1 ml of IP buffer for 20 min at 4
°C, and insoluble material was pelleted by centrifugation.
Figure 1:
Coimmunoprecipitation
of AP2 with EGFR in B82 cells. A, parental B82 cells (Par) and B82 cells expressing wild-type EGFR (WT),
enzymatically inactive EGFR (M), and EGFR lacking five
autophosphorylation sites (F5) were treated without or with 200 ng/ml
EGF, and EGFR were immunoprecipitated using anti-EGFR Ab 13A9. The
immunoprecipitated material was electrophoresed and transferred to
nitrocellulose, and the filter was cut in half. The top half was probed
with anti-Tyr(P) Ab PY20 and then reprobed with anti-EGFR Ab 4516-8,
while the bottom half was probed with anti-
-adaptin Ab AC1-M11. B, B82 cells expressing wild-type EGFR were incubated with 200
ng/ml EGF at 4 °C for 1 h and then placed at 37 °C for 0, 5,
10, 15, and 20 min. EGFR were immunoprecipitated, and
immmunoprecipitated material was then analyzed by electrophoresis and
Western blotting. The same filter was consecutively probed with
anti-EGFR Ab 4516-8 and Ab 100/2 to
-adaptin.
We first established that we could immobilize EGFR on the sensor
surface, that tyrosine kinase activity was retained, and that EGFR
could be reversibly autophosphorylated and dephosphorylated. Active
EGFR was immunoisolated from A431 cells using Ab 528, which competes
with EGF for receptor binding(47) . EGFR was eluted from the
528 IgG column with excess EGF and injected over the biosensor surface
containing immobilized Ab 13A9, which binds to the ectodomain of EGFR
but does not compete with EGF(27) . Due to the high affinity of
interaction, dissociation of EGFR from Ab 13A9 was negligible (Fig. 2). At the end of the EGFR injection, a base line was
established, and monoclonal antiphosphotyrosine antibody PY20 was
injected before and after injection of ATP/Mn. As
shown in Fig. 2, Ab PY20 bound the biosensor only after
injection of ATP, demonstrating the ability of captured EGFR to
autophosphorylate. Injection of Tyr(P) as a competitor completely
eluted bound Ab PY20, confirming the specificity of the interaction.
Calf intestinal alkaline phosphatase was then injected to
dephosphorylate immobilized EGFR. Phosphatase treatment abolished the
ability of subsequently injected Ab PY20 to bind to EGFR. Failure of Ab
PY20 to bind to EGFR was due to receptor dephosphorylation and not to
proteolysis or other reasons because a second injection of
ATP/Mn
restored binding of Ab PY20.
Figure 2:
Phosphorylation and dephosphorylation of
EGFR immobilized on the biosensor surface. Active EGFR was captured by
Ab 13A9 covalently linked to the carboxymethylated dextran layer on the
surface of the biosensor chip. Ab PY20 (Anti-P-Tyr),
ATP/Mn (ATP), Tyr(P) (P-Tyr), and
calf intestinal alkaline phosphatase (CIP) were injected over
the surface of the biosensor as indicated. Arrows point to the
beginning and the end of the injections. Plotted is the relative
response in resonance units (RU) versus time. Note
that the off-scale response observed when EGFR was injected was due to
the presence of glycerol in the injection buffer, but a stable base
line was then established.
The binding of
the adaptor complex isolated from bovine brain to unphosphorylated and
autophosphorylated EGFR was compared (Fig. 3). When AP was
injected over the surface of the biosensor after immobilization of Ab
13A9 but prior to injection of EGFR, no significant binding was
detected. EGFR was then captured on the biosensor, and AP was injected
before and after ATP. The amount of AP bound to EGFR was significantly
increased following injection of ATP/Mn, which
allowed autophosphorylation of immobilized EGFR. The gradually
decreasing base line reproducibly observed during injection of AP
presumably reflects dissociation of a substance weakly associated with
AP. Because Mn
is reported to cause aggregation of
the intracellular domain of EGFR, implying a conformational
change(48) , Mn
was also tested for effects
on AP binding to EGFR. Mn
(5 mM) injection
without ATP resulted in either no or a small positive effect on binding
of subsequently injected AP to immobilized EGFR (data not shown), in
contrast to the reproducibly increased AP binding following injection
of ATP/Mn
.
Figure 3:
Binding of adaptor complexes to EGFR
immobilized on the biosensor surface. Ab 13A9 was coupled to the
biosensor chip, and consecutive injections of bovine brain AP complexes
were performed before EGFR was captured, after active but
nonphosphorylated EGFR was captured, and after EGFR was
autophosphorylated by injection of ATP/Mn. RU, resonance units.
Figure 4:
Effect of EGFR dephosphorylation and
competition with Tyr(P) on preformed EGFRAP2 complexes. A, a complex between phosphorylated EGFR and AP was formed on
the biosensor chip. Tyr(P) (P-Tyr) and calf intestinal
alkaline phosphatase (CIP) were then consecutively injected.
The activity of the phosphatase was confirmed by injection of Ab PY20 (Anti-P-Tyr), and the activity of EGFR was demonstrated by
injection of ATP/Mn
(ATP) followed by
injection of Ab PY20. The specificity of Ab PY20 binding is
demonstrated by competitive elution with Tyr(P). Note that the
base-line resonance unit (RU) was set to that of the Ab
13A9
EGFR complex. B, the EGFR
AP2 complex was
coimmunoprecipitated from A431 cells treated without or with EGF using
anti-EGFR Ab 13A9. Immunobeads were treated with calf intestinal
alkaline phosphatase where indicated, and bound material was then
analyzed by electrophoresis and Western blotting. The same filter was
consecutively probed with anti-EGFR Ab 4516-8, Ab PY20, and Ab 100/2 to
-adaptin.
Figure 5:
Binding of bovine brain adaptors to
denatured EGFR. A, EGFR was denatured by 6 M urea,
transferred to BIAcore running buffer by gel filtration on Sephadex
G-25, and captured by Ab 13A9 coupled to the biosensor surface,
followed by injection of AP. ATP/Mn (ATP)
and Ab PY20 (Anti-P-Tyr) were injected prior to injection AP
to confirm lack of enzymatic activity and consequent tyrosine
phosphorylation of the denatured receptor. RU, resonance
units. B, efficiency of AP binding to native unphosphorylated,
native phosphorylated, or urea-denatured receptors was determined. The
stoichiometry of stable complexes formed 5 min post-injection of AP was
calculated from the ratio of the relative responses corrected for the
difference in molecular mass of both species. Similar results were
obtained in two independent sets of
experiments.
Figure 6:
Binding of AP2 to the EGFR carboxyl
terminus. A, wild-type EGFR (wt) and EGFR lacking the
entire carboxyl terminus (c`973) were expressed in B82 cells. The cells
were treated without or with 200 ng/ml EGF, and EGFR were
immunoprecipitated with Ab 13A9. The immunoprecipitated material was
electrophoresed and transferred to nitrocellulose, and the filter was
cut in half. The top half was probed with anti-EGFR Ab 4516-8, while
the bottom half was probed with anti--adaptin Ab 100/2. B, glutathione-agarose beads (10 µl) containing 30 µg
of glutathione S-transferase (GST) or 10 µg of
glutathione S-transferase fused to residues 975-1186 of
human EGFR (GST-EGFR-CT) were first incubated in IP buffer with 10
mg/ml bovine serum albumin. Beads were pelleted by centrifugation, the
supernatant was aspirated, and 0.5 ml of 1 µM bovine brain
AP in IP buffer containing 10 mg/ml bovine serum albumin was added.
Beads were incubated with AP for 1 h at 4 °C while agitating,
washed three times with IP buffer, and boiled in SDS sample buffer.
After electrophoresis and transfer to nitrocellulose filters, AP2 was
detected using anti-
-adaptin Ab 100/2. Material bound to the beads
is designated as AffinityPrecipitation. AP2 of the
starting material is designated as AP-2. Note that in brain,
the A isoform of
-adaptin with slower electrophoretic mobility is
found in addition to the more ubiquitous C
isoform(32) .
These results suggested that the AP2 recognition motif(s) was located in the carboxyl terminus of the receptor, but it remained possible that this interaction motif(s) was located in the kinase core domain and that deletion of the carboxyl terminus prevented it from being exposed. To obtain direct evidence for interaction of the carboxyl terminus of EGFR with AP2, the carboxyl terminus of EGFR (residues 975-1186) was expressed in E. coli as a fusion with glutathione S-transferase. GST-EGFR-CT was able to bind AP2 from bovine brain with high efficiency, while glutathione S-transferase alone was not (Fig. 6B). Because there was no tyrosine phosphorylation of GST-EGFR-CT (data not shown), this result confirmed the observation that AP2 and EGFR interacted directly without involvement of tyrosine phosphorylation.
Figure 7:
Binding of individual - and
-subunits of the AP2 complex to the recombinant EGFR carboxyl
terminus. Epitope-tagged
C- and
-subunits of AP2 were
expressed in S. cerevisiae. Yeast lysate (0.5 ml), prepared as
described under ``Experimental Procedures,'' was incubated
with glutathione-agarose beads (10 µl) containing 30 µg of
glutathione S-transferase (GST) or 10 µg of
GST-EGFR-CT. Adaptins were detected on Western blots with antibody
12CA5 against the HA-1 amino-terminal epitope tag. Note that the
molecular mass of adaptins appears larger because they are expressed as
fusions with the 12-kDa leader. Material bound to the beads is
designated as Affinityprecipitation. Glutathione S-transferase or GST-EGFR-CT was used for precipitation where
indicated. The tworight lanes designated as
and
represent aliquots of extracts from
yeast expressing
- and
subunits,
respectively.
Two plausible mechanisms of EGF-induced association of EGFR
with coated pit adaptins may be operative. EGF binding may translocate
EGFR into coated pits, thus increasing the probability of their
interacting with AP2 complexes. Alternatively, binding of EGF may
increase the affinity of EGFR for AP2 complexes, resulting in anchoring
of activated receptors whenever they enter coated pits. This study
describes an autophosphorylation-regulated mechanism by which
ligand-activated EGFR interact with AP complexes of coated pits,
accounting in part for ligand-dependent retention in coated pits. The
fact that in vivo interaction of EGFR with AP was impaired by
inactivation of the receptor protein-tyrosine kinase and by mutations
of the sites of autophosphorylation suggested that autophosphorylated
receptors bound AP complexes with higher affinity than unphosphorylated
EGFR. This was directly confirmed by analysis of interactions between
isolated EGFR and AP complexes using the BIAcore instrument where receptor autophosphorylation significantly
enhanced binding of AP complexes to EGFR. However, phosphorylated
tyrosine residues were not directly involved in this interaction
because the EGFR
AP complex once formed was not dissociated upon
receptor dephosphorylation or by competition with phosphotyrosine,
suggesting that the mechanism of association is different from tyrosine
phosphorylation-mediated mechanisms involving SH2 domains(50) .
Alternatively, autophosphorylation, which was shown to change the
conformation of the intracellular domain of EGFR(17) , may
expose surfaces in EGFR that interact with AP complexes. Indeed,
denaturation dramatically enhanced binding to EGFR in the absence of
receptor autophosphorylation and tyrosine kinase activity.
Autophosphorylation did enhance EGFRAP complex formation both in vivo and in vitro, but a small amount of binding
of AP2 to kinase-negative EGFR in an EGF-induced manner was also
observed in the absence of any detectable receptor autophosphorylation.
This observation correlates with the reported ability of enzymatically
inactive EGFR (42) and other tyrosine kinase receptors (51, 52, 53, 54) to be internalized
and/or down-regulated in a ligand-dependent manner, although at a low
rate compared with their wild-type counterparts. Kinetic modeling of
EGFR endocytosis suggested a two-step mechanism involving interactions
necessary for redistribution of diffuse cell-surface receptors to
coated pits and for binding of receptors to coated pits(12) .
Interaction with AP2 likely reflects predominately the second step.
Interaction between EGFR and AP2 involved the receptor carboxyl terminus because mutant EGFR lacking the carboxyl terminus failed to interact in vivo, while the isolated carboxyl terminus fused to glutathione S-transferase bound AP2 in vitro. Sequences required for endocytosis are present in three regions of the EGFR carboxyl terminus(12) . Sequences required for EGFR interaction with AP2 complexes are also located in this regulatory domain, but more precise mapping will be required to determine the specific sequences that bind adaptins within this domain.
Interactions of AP2 with the cytoplasmic domains of receptors in vitro require tyrosine-containing motifs(19, 20, 21, 22) . However, many receptors compete with themselves for endocytosis, but do not compete with one another. That EGFR compete with themselves is implied from saturable high affinity endocytosis(55) . However, competition between EGFR and the transferrin receptor and between InsR and the mannose 6-phosphate receptor is not observed(9, 56) . This implies specificity in endocytosis despite the generality of aromatic residue tight turn endocytic code structures. Because a high percentage of AP2 was associated with activated EGFR in vivo(18) , a vast excess of AP2 appears unlikely. Together, these observations suggest that additional receptor-specific factors are likely to be required for endocytosis in addition to more general interactions with AP2. Specific recognition of a tyrosine-containing tight turn was demonstrated by interaction of one LIM domain of enigma with the strong endocytic code contained in exon 16 of InsR(57) .
Although competitor studies indicate that the affinity for AP2 interactions with lysosomal acid phosphatase, the polymeric immunoglobulin receptor, and the asialoglycoprotein receptor is low (21, 22, 58) , the localized concentration of AP2 in coated pits is likely high. In vivo interaction of EGFR with AP2 shown by coimmunoprecipitation suggests that this binding may be of higher affinity than that of other receptors, perhaps due in part to unique sites of interaction in the EGFR carboxyl terminus and in the adaptin subunits of the AP2 complex.
Where examined, the interaction
of AP2 with cytoplasmic domains of receptors has involved the
amino-terminal body domain of - and
-adaptin subunits of
AP2(18, 21, 22) . In our experiments, both
the
- and
-subunits of AP2 interacted with the recombinant
carboxyl terminus of EGFR, and this interaction included the
amino-terminal body domain of adaptins. These results indicate that the
carboxyl-terminal regulatory domain of EGFR interacts with the
amino-terminal body domain of
- and
-adaptins, but finer
mapping will also be required to determine the precise sites of AP2
subunit interactions with EGFR in vivo.