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INTRODUCTION |
The trafficking and intracellular targeting of many transmembrane
proteins depend on motifs found within their cytosolic domains. One
such motif contains a critical tyrosine residue within the sequence
YXX
, where
represents a bulky hydrophobic residue (1,
2). Tyrosine-based motifs conforming to this consensus sequence can
interact directly with the medium chain (µ) subunits of
heterotetrameric adaptor complexes involved in several different intracellular trafficking pathways. The AP-2 adaptor complex
facilitates incorporation of transmembrane proteins containing
YXX
motifs into clathrin-coated vesicles at the cell
surface (1, 3-9), whereas AP-1, AP-3, and AP-4 adaptor complexes are
involved in other intracellular vesicular transport steps (reviewed in
Refs. 10-13). The µ subunits from all four adaptor complexes have
been shown to interact with the YXX
motif, with the
precise sequence and context of this motif determining the specificity
of the interaction (14, 15). Mutation of residues within and around the
YXX
motif affect interaction with µ subunits; but, in
all cases, mutation of the tyrosine residue in the motif
(e.g. to alanine) blocks interaction with the µ subunit
(4, 6, 8). Structural studies on peptides containing these motifs have
generated data that have been interpreted as showing that the
YXX
motif exists as part of a tight turn, an
-helix,
or a
-strand (16-20). However, whatever the predicted overall
structure, each of these studies has shown the side chain of the
critical tyrosine residue to project away from the peptide backbone.
The YXX
internalization motif is remarkably similar to
sequences in which the tyrosine residue can be phosphorylated and, once
phosphorylated, bind to Src homology 2 domains (21). It is now clear
that although both tyrosine kinases and medium subunits of adaptor
complexes recognize essentially the same motif, there is
discrimination, such that very few tyrosine-based motifs that have been
shown to interact with µ chains also act as substrates for tyrosine
kinases (22-24). Furthermore, although both µ chains and tyrosine
kinases accommodate the tyrosine side chain as part of their
interaction with the YXX
motif, there is no great
similarity between the YXX
-binding sites on µ chains and those on tyrosine kinases (20).
The similarity between motifs that interact with µ chains and those
that are recognized by some tyrosine kinases suggested that structural
analogs of the tyrosine side chain might disrupt the interaction
between µ chains and their target tyrosine motifs. Tyrphostins are
structural analogs of tyrosine. They were originally developed as
substrate-competitive inhibitors of the epidermal growth factor
tyrosine kinase (25-27). Tyrphostins have subsequently been used to
investigate the physiological roles of many different tyrosine kinases.
Some tyrphostins have also been reported to inhibit endocytosis and
autophagy (28) and vesicle formation from the trans-Golgi
network (29), thus implying a possible role for tyrosine kinases in
these processes.
Our previous studies using tyrphostins involved the type I integral
membrane protein TGN38, which cycles between the trans-Golgi network and the plasma membrane, but has a steady-state localization at
the trans-Golgi network (reviewed in Ref. 30). The cytosolic domain of TGN38 contains a consensus tyrosine-based motif, SDYQRL, which is essential for internalization of the protein (31-34). Both
this sequence and the complete cytosolic domain of TGN38 can
specifically interact with the µ2 subunit of the plasma
membrane-associated adaptor complex AP-2 (4, 5, 8). Furthermore, this
interaction is critically dependent on the tyrosine residue (4, 8). We
previously used an in vitro assay and yeast two-hybrid
growth assays to show that tyrphostins A23 and A46 inhibit the
interaction between TGN38 and µ2 (35). However, a range of other
tyrphostins did not disrupt the interaction, demonstrating that the
effect was independent of tyrosine kinase inhibition.
We have now modeled the structure of A23 and other tyrphostins into the
YXX
-binding cleft in µ2. This has provided us with a
structural explanation for the inhibition of the YXX
motif/µ2 interaction by A23.
If tyrphostin A23 were to block the interaction between
YXX
motifs and µ2 in intact cells, we would predict
that clathrin-mediated endocytosis would be inhibited. We have now
tested this by following the internalization of transferrin
(Tf)1 in cells incubated in
the presence or absence of various tyrphostins. Tf is internalized upon
binding to its receptor at the cell surface. This receptor is
internalized via a clathrin-mediated pathway by virtue of an
interaction between a tyrosine-based motif, YTRF, and µ2 (16, 36).
Thus, inhibition of clathrin-mediated endocytosis leads to a reduction
in internalized Tf (for example, see Ref. 37). We now show that
tyrphostin A23 inhibits the internalization of the Tf receptor (TfR)
from the plasma membrane. This effect is specific for A23; other
tyrphostins that act as inhibitors of tyrosine kinase activity do not
inhibit Tf internalization. Thus, this inhibition of Tf internalization
in intact cells correlates with the capacity of A23 to inhibit the
interaction between YXX
motifs and µ2 in
vitro.
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EXPERIMENTAL PROCEDURES |
Molecular Modeling--
The crystal structure (Protein Data Bank
Code 1i31) of the µ2 adaptin subunit (AP50) of the AP-2
clathrin adaptor complexed with the peptide FYRALM (20) was used as the
starting point for modeling. Hydrogen atoms were added to the structure
consistent with pH 7. All residues with an atom within 25 Å of lysine
203 were selected from the structure, and the rest were discarded. The
remaining volume in this sphere was filled with water, and all residues
in the concentric sphere between 20 and 25 Å were fixed in space
during subsequent calculations. Those residues in the 20-Å
sphere were energy-minimized to an average derivative of 0.01 kcal/Å.
Partial charges for the tyrphostin atoms were derived from Austin Model
1 semi-empirical molecular orbital calculations performed on the
respective anions in vacuum. The tyrphostin anions were docked simply
by superimposing their phenoxy groups onto the tyrosine ring of the
FYRALM peptide, removing this peptide, and resoaking the binding site
in water. Two conformations (P and Q) of tyrphostins A8 and A23 were
explored, distinguished by the exocyclic torsion between the aromatic
ring and the double bond.
Tissue Culture--
Heb7a cells were grown in Dulbecco's
modified Eagle's medium (Sigma) plus 10% fetal calf serum (Labtech
International) and 1% penicillin/streptomycin (Invitrogen) for at
least 2 days prior to use in experiments.
Tyrphostins--
Tyrphostins (Sigma and Calbiochem) were stored
as 350 mM (A23 and A63) or 8 mM (A51)
(1000-fold) stock solutions in Me2SO at
20 °C and
added to solutions just before use.
Radiolabeled Transferrin Internalization Assays--
The
kinetics of Tf internalization in Heb7a cells were measured following
the method described by McGraw and Subtil (38) using 0.5 µg/ml
125I-labeled human diferric Tf (PerkinElmer Life Sciences)
for labeling. Cells exposed to tyrphostins were incubated with the drug
for a total of 30 min (i.e. inclusive of the incubation time
for Tf uptake). The data were processed using Microsoft Excel and
plotted with the linear regression function in SigmaPlot 2001 (Version 7.1).
Fluorescence Microscopy--
Heb7a cells were grown on 22-mm
glass coverslips.
Internalization of Fluorescent Transferrin--
Cells were
washed with serum-free medium and labeled for 30 min with 25 µg/ml
human serum Tf conjugated to Alexa 594 (Molecular Probes, Inc.). The
cells were then fixed with methanol at
20 °C for 4 min.
Immunofluorescence--
Cells were fixed with methanol (as
described above) or 3% paraformaldehyde, 1 µM
CaCl2, and 1 µM MgCl2 in
phosphate-buffered saline (pH 7.4) at 20 °C for 20 min.
Paraformaldehyde-fixed cells were quenched with 30 mM
glycine in phosphate-buffered saline and permeabilized with 0.1%
Triton X-100 in phosphate-buffered saline for 4 min at 20 °C. All
cells were blocked with 3% bovine serum albumin in phosphate-buffered
saline for 15 min prior to incubation with primary antibodies (diluted
in the blocking buffer) for 1 h. Murine monoclonal antibodies
raised against LAMP-1 (CD107a) (clone H4A3, BD Biosciences), LAMP-2
(CD107b) (clone H4B4, BD Biosciences), CD63 (Biogenesis), and
EEA1 (clone 14, Transduction Laboratories) were used at 1:200
dilutions. Alexa 488-conjugated goat anti-mouse IgG (H + L) (Molecular
Probes, Inc.) diluted 1:800 in the blocking buffer was used as
secondary antibody. The coverslips were mounted with Mowiol (as
described by Harlow and Lane (39)) and examined on an inverted Leica
TCS NT (UV) confocal laser-scanning microscope with a ×100 plan apo
objective lens. Care was taken to ensure equal laser intensity,
photomultiplier tube setting, and pinhole size for Tf
internalization assays. Optical slices were taken at 0.5- or 1-µm
intervals through the cells and displayed as maximum projections in the
microscope software. The images were then imported to Adobe Photoshop
Version 6.0 to produce the final figures.
Mean pixel intensity measurements were determined over four quadrants
of cells at zoom ×1 and magnification ×63 using the Leica confocal
microscope software. The data were processed with Microsoft Excel and
plotted with SigmaPlot 2001(Version 7.1) using the linear regression function.
Transferrin Receptor Degradation Assay--
Heb7a cells
were incubated in the presence of 350 µM tyrphostin A23
for 0, 15, or 30 min. Post-nuclear cell lysates were prepared (40), and
equal amounts of protein, as determined by the Bradford assay (41),
were electrophoresed under reducing conditions on a 10%
SDS-polyacrylamide gel. The gel was immunoblotted with a murine
monoclonal antibody (H68.4) raised against the cytosolic domain of the
human TfR (a gift from C. R. Hopkins) (42).
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RESULTS |
Molecular Modeling of Tyrphostins into the µ2 Tyrosine-binding
Pocket--
We have previously shown that, in vitro, both
tyrphostins A8 and A23 can inhibit the interaction between
YXX
motifs and the µ2 subunit of the AP-2 adaptor
complex, with tyrphostin A23 doing so more efficiently (35). In
contrast, other tyrphostins failed to inhibit this interaction (35). In
an initial attempt to provide a molecular explanation for these
observations, we chose to model the structures of various tyrphostins
into the tyrosine-binding cleft in µ2. Tyrphostins A8, A23, and A46
have previously been shown to be efficient inhibitors of the
interaction between YXX
motifs and µ2 in recombinant
protein interaction assays, whereas tyrphostins A47, A25, and A51 are
not (35). We observed that the phenyl rings of tyrphostins A8, A23,
A46, and A47 were accommodated within the tyrosine-binding cleft in
µ2, whereas those of tyrphostins A25 and A51 were not. Tyrphostins A8
and A63 are monohydroxylated at position 4 of the phenyl ring in their
structure (i.e. the same as a tyrosine side chain);
tyrphostins A23, A46, and A47 are 3,4-dihydroxylated on their phenyl
ring; and tyrphostins A25 and A51 are 3,4,5-trihydroxyphenyl compounds
(Fig. 1). The energy-minimized complexes
of A8 and A23 in orientation/conformation P (see "Experimental Procedures") resulted in the tyrphostins binding snugly in the pocket
occupied by tyrosine in the FYRALM peptide complex. The O
atom forms a hydrogen-bonding network with Lys203,
Asp176, and Arg423 at the base of the binding
cleft, whereas the phenyl ring lies in the hydrophobic groove formed by
the side chain of Arg423, Phe174, and
Trp421, as shown in Fig. 2.
In contrast, the complexes in orientation/conformation Q (see
"Experimental Procedures") break this hydrogen-bonding network
because steric clashes between the dinitrile group and Val422 force the tyrphostins ~2 Å along the groove, away
from the O--binding site. In the P complex of tyrphostin A23, the
second ring oxygen abuts the guanidinium group of Arg423.
This probably accounts for the fact that this compound is more efficient than tyrphostin A8 as an inhibitor of the interaction between
YXX
motifs and µ2 (35). The reason for the failure of
tyrphostins with three-ring hydroxy groups (e.g. A25 and
A51) to inhibit the interaction between YXX
motifs and
µ2 also becomes evident from inspection of Fig. 2. The third hydroxy
group would necessarily be forced into the hydrophobic part of the
cleft (lined by Leu175) if the compound were to bind in a
similar fashion to that proposed for A8 and A23. Thus, it appears that
the tyrosine-binding cleft in µ2 can accommodate a 3,4-dihydroxy
derivative, but not a 3,4,5-trihydroxy derivative, of a phenyl ring.
This is consistent with our previous in vitro observations
on the inhibition of YXX
motif/µ2 interactions by
different tyrphostins (35). In fact, addition of an extra hydroxyl
group in the 3-position of the phenyl ring is beneficial for the
interaction with µ2 (see above). Thus, a 3,4-dihydroxyphenyl compound
(such as tyrphostin A23 or A46) is predicted, by molecular modeling
studies, to both fit well in the tyrosine-binding cleft of µ2 and be
stabilized in that binding by hydrogen bonding and other interactions
(Fig. 2). This provides an explanation for our earlier observation (35)
that tyrphostin A23 inhibits the interaction between µ2 and a
YXX
motif more efficiently than tyrphostin A8 (which is
structurally identical to tyrphostin A23 apart from the fact that A8 is
a 4-monohydroxyphenyl compound and A23 is a 3,4-dihydroxyphenyl
compound) (Table I). However, these data
do not explain why tyrphostin A63 should not be equally as efficient as
tyrphostin A8 in inhibiting the interaction between µ2 and a
YXX
motif because both are 4-monohydroxyphenyl compounds (Fig. 1). In fact, the only difference between the structures of
tyrphostins A8 and A63 is that the former has a double bond linking the malononitrile group to the aromatic ring, whereas the
latter does not (Fig. 1). This led us to suspect that the pKa values for the hydroxyl groups on the phenyl
rings of the two molecules might be different. Measurements of the
tyrphostin pKa values showed this to be the case,
with the pKa for tyrphostin A8 being 6.73 and that
for A63 being 9.61 (Table I). This implies that the tyrphostin anion is
the species that binds into the µ2 active site and complements the
overall positive charge at the base of the cleft, which is composed of
the side chains of Lys203, Asp176, and
Arg423 (Fig. 2).

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Fig. 2.
Molecular modeling of tyrphostins A8 and A23
into the tyrosine-binding cleft in µ2.
Energy-minimized complexes of A8 and A23 (large ball and
stick) with µ2 adaptin (small ball and stick). All
protein residues within 5 Å of the ligands are shown. Oxygen and
nitrogen atoms are shown as dark gray spheres, and carbon
atoms as light gray spheres.
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Table I
pKa values for Trphostins
A buffer comprising sodium acetate, BisTris, Tris, Ches, and Chaps (20 mM each) was used for the pKa
measurements at 25 °C. For each compound, UV spectra were measured
between 250 and 500 nm at 0.5-pH unit intervals between pH 5 and 11 immediately after addition of the Me2SO stock solution (5 µl
of 2-10 mg/ml to 1 ml of buffer). An extra point for the titration of
tyrphostin A63 at pH 12 was obtained using 10 mM NaOH.
Spectra corresponding to the first ionization of each compound showed
clear isosbestic points, indicating clean conversion between the
neutral and mono-anionic species. Absorbances at the wavelengths shown
below, corresponding to the mono-anionic species, were plotted against
pH. These data were fitted to the equation AA + (AB AA) · (alog(pH pKa))/(1 + alog(pH pKa)) (where AA is the absorbance
at low pH, and AB is that at high pH) in Graft
Version 3.01 (Erithacus Software Ltd.) to determine
pKa
values.
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Tyrphostins A46 and A47 are structurally very similar, both being
3,4-dihydroxyphenyl compounds, and both can be modeled into the
tyrosine-binding cleft in µ2. Furthermore, the pKa values for A46 and A47 (7.13 and 6.87, respectively) (Table I) are such
that these compounds will be mostly ionized at physiological pH.
However, our previous in vitro binding studies showed that only tyrphostin A46 efficiently inhibits the interaction between µ2
and a YXX
motif (35). Because the structure and acidity of these tyrphostins are so similar, it was unclear why A47 was inactive, whereas A46 showed good activity. A possible explanation lies
in the relative stability of these tyrphostins toward hydrolysis. Tyrphostins A46 and A47 were incubated in the dark in pH 7 buffer at
37 °C, and the absorbance due to the anion was measured at regular
intervals. Although the signal due to A46 persisted, decaying <5% over 24 h, that due to the anion of A47 decayed
significantly over this period (Fig. 3).
The hydrolysis followed first-order kinetics with a half-life of
3.4 h. Thus, the most likely explanation for the lack of activity
shown by A47 is that the compound is rapidly hydrolyzed in in
vitro interaction assays (performed in the light, a condition
that further reduces the stability of the compound).

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Fig. 3.
Stability of tyrphostins A46 and A47.
Shown is the evolution of the UV absorption signal of tyrphostin anions
at pH 7.2 and 37 °C with time. The buffer used is described in the
legend to Table I. The data for A46 ( ) were fitted to a straight
line, and there was <5% loss in the signal over 24 h. The data
for A47 ( ) were fitted to a single exponential decay with a
half-life of 230 min, resulting in >95% signal loss over 24 h.
Absorbance is given in arbitrary units.
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The preceding data demonstrate that specific tyrphostins inhibit the
interaction between tyrosine-based internalization motifs and µ2 by
binding to the tyrosine-binding cleft of µ2. This interaction is
stabilized by hydrogen (and other) bonds at the base of the cleft and
is optimal when a 3,4-dihydroxyphenyl tyrphostin, in mostly the anionic
form at physiological pH, is used. Tyrphostin A23 is such a tyrphostin,
previously shown to inhibit the interaction between tyrosine-based
internalization motifs and µ2 both in vitro and in yeast
two-hybrid interaction assays (35). This tyrphostin was selected
for use in further studies.
125I-Transferrin Internalization Assays--
Having
established the structural basis for the inhibition of the
YXX
motif/µ2 interaction by tyrphostin A23, it was
pertinent to investigate whether this inhibition occurs in
vivo to inhibit trafficking pathways in a mammalian system. To
address this question, we focused on the internalization of Tf from the
plasma membrane. Tf is internalized following binding to the TfR at the
cell surface, with the internalization of the TfR being dependent upon
interactions between µ2 and the tyrosine-based internalization motif
(YTRF) in the TfR cytosolic domain (16, 36, 44). A biochemical assay was used to probe the effects of selected tyrphostins on Tf
internalization. The assay followed the internalization of
125I-Tf into Heb7a cells incubated at 37 °C in the
presence or absence of different tyrphostins (Fig.
4). Incubation of cells in the presence
of 350 µM tyrphostin A23 (the concentration selected as a
result of observed effects in in vitro assays (35)) for 30 min led to a marked decrease in the rate (~4-fold) of
125I-Tf internalization compared with that observed in
control cells (Fig. 4A). Control tyrphostins (A51 and A63,
previously shown to have no effect on the interaction between µ2 and
the TGN38 YXX
internalization motif (35)) had no
significant effect on 125I-Tf internalization (Fig. 4,
B and C). Tyrphostins A51 and A23 (IC50 for inhibition of epidermal growth factor receptor
tyrosine kinase activity of 0.8 and 35 µM, respectively)
were both efficient inhibitors of tyrosine kinase activity, but only
A23 inhibited Tf internalization (Fig. 4, compare A
and B). Tyrphostin A63 (IC50 for inhibition of
epidermal growth factor receptor tyrosine kinase activity of 6500 µM) has been used as a negative control in assays of
inhibition of tyrosine kinase activity by tyrphostins (27); it was also
ineffective as an inhibitor of endocytosis (Fig. 4C).

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Fig. 4.
Radiolabeled transferrin internalization
assays. Heb7a cells were incubated with 0.5 µg/ml
125I-labeled human diferric Tf for varying times to assay
the kinetics of Tf internalization. Cells exposed to tyrphostins were
incubated with the drug for 30 min inclusive of the time point. The
graphs are plotted as linear regressions with error bars for
S.D. , drug-treated cells; , controls lacking tyrphostin.
A, treatment with tyrphostin A23 led to a marked decrease in
the rate (3.9-fold) of 125I-Tf internalization.
B and C, there was no significant change in
125I-Tf internalization rates for A51 (1.3-fold) and A63
(1.0-fold), respectively.
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Tyrphostins A23 and A51 were both used at 10 times their
IC50 values for inhibition of epidermal growth factor
receptor tyrosine kinase activity in these assays. The fact that only
A23 inhibited Tf internalization indicates that it exerts its effect by
a mechanism other than inhibition of tyrosine kinase activity. Given
our previously published in vitro data (35), this mechanism
is most likely the inhibition of the interaction between µ2 and the
YTRF internalization motif in the cytosolic domain of the TfR.
Internalization of Fluorescently Labeled Transferrin--
Human
serum Tf conjugated to Alexa 594 (Alexa 594-Tf) was also used as a tool
to investigate any possible effects of tyrphostin A23 on trafficking
pathways (Fig. 5). Heb7a cells were
treated with 350 µM tyrphostin A23 for varying times and
allowed to internalize Alexa 594-Tf. Cells exposed to tyrphostin A23
for 30 min in total (Fig. 5B) showed reduced internalization
of the fluorescent probe compared with untreated cells (Fig.
5A), a result consistent with the 125I-Tf uptake
assays. Heb7a cells exposed to tyrphostin A23 for longer periods (Fig.
5, C-F) showed no significant morphological changes in the
distribution of the fluorescent probe compared with control cells (Fig.
5A). This demonstrates that, although the rate of Tf
internalization was inhibited by tyrphostin A23, this tyrphostin had no
significant effect on the distribution of Alexa 594-Tf throughout the
recycling pathway over the time course of the experiment. Alexa 594-Tf
uptake experiments were also performed on Heb7A cells incubated in the
presence of different concentrations of tyrphostin A23 for 30 min. The
inhibitory effect of tyrphostin A23 on Alexa 594-Tf uptake was found to
be dose-dependent (Fig.
6).

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Fig. 5.
Internalization of Alexa 594-conjugated
transferrin in Heb7a cells treated with tyrphostin A23 for varying
times. Heb7a cells were treated with 350 µM
tyrphostin A23 for 0 (A and B), 15 (C), 30 (D), 60 (E), or 90 (F) min. This was followed by further incubation with 25 µg/ml human serum Tf conjugated to Alexa 594 for 30 min in the
presence (B-F) or absence (A) of 350 µM tyrphostin A23. The cells were fixed with methanol,
mounted, and examined using an inverted Leica TCS NT (UV) confocal
laser-scanning microscope with a ×100 plan apo objective lens. Heb7a
cells exposed to tyrphostin A23 for 30 min in total (B)
showed reduced internalization of the fluorescent probe compared with
untreated cells (A). Heb7a cells exposed to tyrphostin A23
for longer periods (C-F) showed no significant
morphological changes in the distribution of the fluorescent probe
compared with control cells (A). Scale bar = 10 µm.
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Fig. 6.
Dose-response effect of varying tyrphostin
A23 concentrations on fluorescently labeled transferrin
internalization. Heb7a cells were incubated with tyrphostin A23 at
varying concentrations for 30 min together with 25 µg/ml human serum
Tf conjugated to Alexa 594. The concentrations of tyrphostin chosen
were multiples of the epidermal growth factor tyrosine kinase
IC50 (350 µM). The plot shows an inverse
relationship between increasing A23 concentration and mean pixel
intensity of internalized Tf over four quadrants of cells. The
error bars indicate S.D.
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Internalization of TGN38--
Because our original in
vitro studies focused on the effects of tyrphostins on the
interaction between µ2 and the tyrosine-based internalization motif
in the cytosolic domain of TGN38 (35), the effect of A23 on the
internalization of TGN38 was also assayed. This was done by an antibody
uptake assay using a monoclonal antibody that recognizes the extreme N
terminus of TGN38 (2F7.1), followed by detection of the internalized
antibody using a fluorescently labeled secondary antibody as described
previously (34). Normal rat kidney cells were incubated in the
presence or absence of 350 µM A23 for 5 min at 37 °C
and then with antibody 2F7.1 in the continued presence or absence of
350 µM A23 for 30 min at 37 °C prior to methanol
fixation, incubation with secondary antibody, and processing for
fluorescence microscopy. Mean pixel intensities were recorded from both
sets of cells under identical imaging conditions (as described under
"Experimental Procedures"). The mean pixel intensity (in arbitrary
units) in control cells was 121.25 ± 22.10 (n = 50 cells), and that in A23-treated cells was 38.09 ± 10.05 (n = 50 cells). Thus, in addition to inhibiting Tf
uptake, A23 also inhibited the internalization of TGN38. No other
tyrphostin tested had any effect on the internalization of TGN38.
Internalization of Fluorescent Dextran--
Our earlier in
vitro assays (35) and the modeling studies presented here suggest
that the inhibition of Tf uptake and of TGN38 internalization by A23 is
a result of inhibition of the interaction between µ2 and the
tyrosine-based internalization motifs in the cytosolic domains of the
TfR and TGN38. If this is the case, then internalization of a
fluid-phase marker should not be affected by incubation of cells in the
presence of A23. To address this, Heb7a cells were incubated in the
presence of 1 mg/ml fluorescently labeled 10-kDa dextran (as described
under "Experimental Procedures") for 30 min at 37 °C in the
presence or absence of 350 µM A23. Mean pixel intensities
were then recorded from both sets of cells under identical imaging
conditions. The mean pixel intensity (in arbitrary units) in control
cells was 34.0 ± 9.9 (n = 30 cells), and that in
A23-treated cells was 38.0 ± 11.0 (n = 30 cells).
Thus, incubation of cells in the presence of A23 for 30 min at 37 °C
had no effect on fluid-phase uptake of dextran. No other tyrphostin
tested had any effect on fluid-phase uptake of dextran.
Analysis of the Morphology of Endocytic Compartments--
The fact
that the distribution of internalized Alexa 594-Tf in cells that had
been incubated in the presence of tyrphostin A23 was indistinguishable
from that in control cells suggested that the compartments of the
endocytic recycling pathway were not perturbed by the
presence of tyrphostin A23. To confirm this and to address possible
effects of tyrphostin A23 on the morphology of other endocytic
compartments, we used immunofluorescence microscopy to localize a range
of endomembrane markers in both A23-treated (350 µM, 30 min) and untreated Heb7a cells. No significant changes in the
localization of the lysosomal/late endosomal markers LAMP-1 (Fig.
7, A and B), LAMP-2
(C and D), and CD63 (E and
F) and the early endosomal marker EEA1 (G and
H) were observed. Similar analysis using markers for the
Golgi apparatus and trans-Golgi network also failed to show
any difference between control cells and those that had been incubated
with tyrphostin A23 (data not shown).

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Fig. 7.
Immunofluorescence of endomembrane markers in
Heb7a cells treated with tyrphostin A23. Heb7a cells were
incubated in the presence (A, C, E,
and G) or absence (B, D, F,
and H) of 350 µM tyrphostin A23 for 30 min.
The cells were fixed with methanol (A-F) or
paraformaldehyde (G and H) and labeled with
antibodies raised against LAMP-1 (CD107a) (A and
B), LAMP-2 (CD107b) (C and D), CD63
(E and F), and EEA1 (G and
H). Alexa 488-conjugated secondary antibodies were used for
detection. Cells were imaged as described in the legend to Fig. 5.
Cells exposed to tyrphostin A23 for 30 min (A, C,
E, and G) showed no significant morphological
changes in the distribution of the endomembrane markers compared with
control cells (B, D, F, and
H). Scale bar = 10 µm.
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Transferrin Receptor Degradation Assay--
We considered it
possible that, although there were no gross changes in the morphology
of endocytic compartments, the presence of tyrphostin A23 might affect
the internalization/recycling of TfRs such that an elevated proportion
of internalized TfRs might be routed to late endosomes and lysosomes
for degradation. This possibility was investigated by immunoblot
analysis of post-nuclear supernatants from Heb7a cells that had been
incubated in the presence of 350 µM tyrphostin A23 for
different times (Fig. 8). The blot was
probed with a murine monoclonal antibody (H68.4) (42) raised against
the cytosolic domain of the human TfR. No reduction in the level of TfR
expression was detected during a 30-min incubation in the presence of
350 µM tyrphostin A23 (Fig. 8).

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Fig. 8.
Transferrin receptor degradation assay.
Heb7a cells were treated with 350 µM tyrphostin A23 for
0, 15, 30, or 60 min. Post-nuclear cell lysates were prepared, and
equal amounts of protein were electrophoresed under reducing conditions
on a 10% SDS-polyacrylamide gel prior to transfer to nitrocellulose
and incubation with a murine monoclonal antibody (H68.4) raised against
the cytoplasmic domain of the human TfR. Antibody binding was detected
by ECL as described under "Experimental Procedures." Antibody H68.4
detected a 95-kDa band of equal intensity in each of the lanes. This
shows that the number of TfRs remained constant throughout the
experiment and that they were not degraded in response to treatment
with tyrphostin A23.
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DISCUSSION |
We had previously shown that, in vitro and in yeast
two-hybrid interaction assays, certain tyrphostins are capable of
inhibiting the interaction between µ2 and tyrosine-based
internalization motifs (35). Molecular modeling of different
tyrphostins into the tyrosine-binding cleft in µ2 has now provided a
structural explanation for the finding that certain tyrphostins inhibit
this interaction, whereas others do not. It is clear that, although mono- and dihydroxylated phenyl rings can be accommodated in the tyrosine-binding cleft in µ2, trihydroxylated phenyl rings cannot. Thus, certain tyrphostins are excluded. Furthermore, tyrphostins with
dihydroxylated phenyl rings are likely to bind to µ2 with higher
affinity than those with monohydroxylated phenyl rings because the
dihydroxylated derivatives are capable of a more extensive bonding
network at the base of the cleft than their monohydroxylated counterparts. We also observed that the pKa values
for the hydroxyl group(s) on the phenyl ring of the tyrphostins can influence the interaction with the tyrosine-binding cleft in µ2; those with a pKa of ~7 effectively inhibit the
interaction, whereas those with a higher pKa (~9)
do not. This is consistent with the anionic form of the tyrphostin
being the active species, interacting with the overall positive charge
at the base of the binding cleft in µ2.
Having provided a structural explanation for the inhibition of the
interaction between µ2 and tyrosine-based internalization motifs by
specific tyrphostins, we went on to address whether tyrphostin A23, a
known inhibitor of the tyrosine motif/µ2 interaction in
vitro, has any effect on endocytosis in mammalian cells.
Consistent with both the in vitro data and the modeling
studies, tyrphostin A23 significantly reduced (~4-fold) the
endocytosis of Tf in Heb7a cells. Control tyrphostins, known to have no
effect on the tyrosine motif/µ2 interaction in vitro, had
no effect on Tf uptake. These results are consistent with tyrphostin
A23 inhibition of the interaction between µ2 and tyrosine-based
internalization motifs in intact cells. It is interesting to note that
Nesterov et al. (43) made a dominant-negative
epitope-tagged mutant of µ2 and assessed its effect on AP-2 and
endocytosis. Elevated expression of this D176A/W421A µ2 mutant
resulted in metabolic replacement of endogenous µ2 by the mutant in
AP-2 complexes. This led to complete abrogation of AP-2 interaction
with the tyrosine-based internalization motifs because the D176A/W421A
mutations lie within the tyrosine-binding pocket of µ2 (20, 43).
Furthermore, expression of D176A/W421A µ2 led to a 4-fold decrease in
the internalization of Tf from the cell surface (43). This decrease in
Tf internalization matches that which we observed following incubation
of cells in the presence of tyrphostin A23.
The data presented here show that tyrphostin A23 reduced the rate of Tf
internalization by ~4-fold. However, there was no effect on the gross
morphology of the endomembrane system over the time course of the
experiment. These results are consistent with tyrphostin A23 inhibition
of the interaction between the tyrosine-based internalization motif on
the TfR and the µ2 medium chain subunit of the AP-2 adaptor complex.
These data show that tyrphostin A23 can be used as an inhibitor of
endocytosis in mammalian cells and that, when using tyrphostins as
inhibitors of tyrosine kinases, any observed effects on membrane
trafficking events should be interpreted with caution due to possible
interaction with µ chains. It is possible that different structural
analogs of tyrosine could inhibit the interaction between
tyrosine-based motifs and other adaptor complex medium chains. Future
molecular modeling could potentially identify new drugs designed to
inhibit other membrane trafficking pathways. These drugs could target
different adaptor complex medium chains or their as yet unidentified
homologs. Thus, specifically designed tyrphostins may become useful
tools for dissecting membrane trafficking pathways in eukaryotic cells.