COMMUNICATION
High-affinity Binding of Epidermal Growth Factor (EGF) to EGF
Receptor Is Disrupted by Overexpression of Mutant Dynamin (K44A)*
Tove
Ringerike
,
Espen
Stang
,
Lene E.
Johannessen
,
Dagny
Sandnes§,
Finn Olav
Levy¶
, and
Inger Helene
Madshus
**
From the
Institute of Pathology,
¶ MSD Cardiovascular Research Center, and
Institute for
Surgical Research, University of Oslo, The National Hospital, N-0027
Oslo and the § Department of Pharmacology, University of
Oslo, P. O. Box 1057, N-0316 Oslo, Norway
 |
ABSTRACT |
Activation of the epidermal growth factor
receptor (EGFR) kinase was analyzed in cells conditionally defective
for clathrin-dependent endocytosis by overexpression of
mutant dynamin (K44A). EGF-induced autophosphorylation of the EGFR on
ice was strongly reduced in cells overexpressing mutant dynamin, and
consistently, binding analyses showed that high-affinity EGFRs were
lost. In the absence of mutant dynamin the cells displayed both high-
and low-affinity EGFR. At 4 °C EGF-EGFR localized mainly outside
coated pits regardless of expression of mutant dynamin. However, also
low-affinity EGFR efficiently moved to coated pits upon incubating
cells at 37 °C. Thus, expression of mutant dynamin disrupts
high-affinity binding of EGF, but not ligand-induced recruitment of
EGFR to clathrin-coated pits.
 |
INTRODUCTION |
Binding of epidermal growth factor
(EGF)1 to the EGF receptor
(EGFR) effects recruitment of EGFR to clathrin-coated pits, and eventually activated EGFR is delivered to lysosomes and degraded (for
review, see Refs. 1 and 2). Binding of EGF furthermore results in
autophosphorylation of the EGFR, and several authors have described
tyrosine-phosphorylated EGFR contained in endosomes (for review, see
Ref. 3). The adaptor proteins growth factor receptor-binding protein 2 (Grb2) and SH2-domain-containing
2 collagen-related protein (Shc),
as well as Son of Sevenless protein (SOS) (all involved in activation
of Ras) were shown to be enriched in the endosomal fraction of
EGF-treated hepatocytes (4). Based on these observations, several
authors have suggested that endocytosed EGFR is actively engaged in
signal transduction.
The GTPase dynamin is required for clathrin-coated vesicle formation
(for review, see Refs. 5 and 6), and a conditional defect in
endocytosis is imposed by the regulated expression of the K44A mutant
form of dynamin (7). HeLa cells expressing the K44A dynamin mutant are
conditionally and specifically defective in clathrin-mediated
endocytosis (7), and it was reported recently that EGF-induced
signaling was altered under these conditions (8). This raises the
possibility that trafficking might regulate specificity of signaling.
However, dynamin is a protein reported to interact with a number of
cellular proteins, among them proteins directly involved in signal
transduction (for review, see Ref. 9). We therefore set out to
investigate whether the altered signaling observed upon overexpression
of mutant dynamin was indeed caused by inhibition of
clathrin-dependent endocytosis directly or whether
overexpression of mutant dynamin per se affected signaling. To our surprise we found that upon overexpression of mutant dynamin high-affinity EGF binding was disrupted. Overexpression of mutant dynamin therefore directly affects signaling by EGF.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human recombinant EGF was from Bachem
Feinchemikalien AG, Budendorf, Switzerland. All chemicals were from
Sigma unless otherwise indicated.
Cells--
HeLa cells transfected with a plasmid encoding mutant
(K44A) or wild-type dynamin 1, where the promoter is negatively
controlled by tetracycline (7), were generously provided by Dr. Sandra L. Schmid, The Scripps Research Institute. The cells were grown in
Costar 3275 flasks (Costar Corp., Cambridge, MA), and the medium used
was Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, MD) containing 400 µg/ml geneticin (Life Technologies, Inc., Paisley, UK), 200 ng/ml puromycin, 2 mM L-glutamine
(BioWhittaker), and 1 × penicillin/streptomycin/fungizone mixture
(BioWhittaker) supplemented with 10% (v/v) fetal bovine serum
(BioWhittaker). Cells were seeded at a density of 15,000 cells/cm2 in disposable microtiter plates and grown for
24 h at 37 °C in the presence (uninduced cells) or absence
(induced cells) of 1 µg/ml tetracycline. For the next 24 h,
cells were serum-starved by incubation in the same medium with only
0.5% fetal bovine serum.
Western Blotting--
Cells in 12-well microtiter plates were
incubated as indicated in legends to figures and subsequently subjected
to Western blot analysis. The cells were lysed in lysis buffer (10 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, 2% (w/v)
sodium dodecyl sulfate (SDS) (Bio-Rad), 1% (v/v)
-mercaptoethanol, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 4% (v/v) glycerol, and 0.005% (w/v)
bromphenol blue) on ice for 10 min, heated at 95 °C for 10 min, and
centrifuged at 20 000 × g for 15 min before the
supernatant fraction was subjected to SDS-polyacrylamide gel
electrophoresis, and the proteins were electrotransferred to
nitrocellulose (NitroBind, Micron Separations Inc., Westborough, MA).
Antibodies used were sheep anti-EGFR (Life Technologies, Inc.),
peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch
Laboratories Inc., West Grove, PA), mouse anti-phosphotyrosine (Upstate
Biotechnology, Lake Placid, NY), and peroxidase-conjugated anti-sheep
IgG (Jackson ImmunoResearch Laboratories Inc.). The reactive proteins
were detected using an enhanced chemiluminescence (ECL) method
(Amersham Pharmacia Biotech, Buckinghamshire, UK).
Scatchard Plot--
Binding of 125I-EGF (Amersham
Pharmacia Biotech) to whole cells was measured by incubating cells in
24-well microtiter plates for 3 h on ice in 50 µl of minimal
essential medium without sodium bicarbonate (Life Technologies, Inc.)
with 0.1% bovine serum albumin (BSA) and increasing concentrations of
125I-EGF in the absence (total binding) or presence
(nonspecific binding) of unlabeled EGF (200 nM). The
incubation medium was carefully removed and transferred to tubes for
counting. The cells were washed three times with ice-cold
phosphate-buffered saline and hydrolyzed in 0.3 ml of 1 M
NaOH. The hydrolyzed cells were transferred to tubes for
counting.
The specific binding data (total binding minus nonspecific binding)
were analyzed by nonlinear curve fitting to equations describing one
(Y = Bmax × X/(Kd + X)) or two
(Y = Bmax1 × X/(Kd1 + X) + Bmax2 × X/(Kd2 + X)) binding sites using Prism 2.01 (GraphPad Software). For
the two-site model, the data were weighted by
1/Y2. The observed data and the theoretical
curves were plotted according to Scatchard (10) using Sigma-Plot
scientific graphing software (SPSS, Jandel Scientific, GmbH, Erkrath,
Germany).
Immunoelectron Microscopy--
Cells were grown in
25-cm2 flasks. To label endosomes with an electron-dense
marker, cells were incubated with BSA-coated 5-10 nm colloidal gold
(11) in the medium for 30 min at 37 °C. Following removal of the
BSA-gold-containing medium cells were chilled on ice and incubated with
10
8 M EGF on ice for 30 min. At the end of
the incubation period the cells were washed with ice-cold
phosphate-buffered saline, fixed, and processed for cryosections and
immunogold labeling (12) either immediately or after chase in BSA-gold-
and EGF-free medium at 37 °C. The sections were labeled using sheep
anti-EGFR antibodies (Life Technologies, Inc.) followed by goat
anti-sheep IgG-coated 18 nm colloidal gold (Jackson ImmunoResearch
Laboratories Inc.).
Determination of IP3 Mass--
Medium was removed
from cells grown in six-well disposable plates, and 600 µl of 0.4 M perchloric acid was added. The cells were scraped from
the plates with a rubber policeman, and the contents of each well was
transferred to glass tubes. The samples were left on ice for 30 min
before centrifugation and neutralization of the supernatant with 4 M KOH, 1 M Tris, 60 mM EDTA, in the presence of Universal Indicator. The IP3 content of the
neutralized supernatant was determined by a competitive radioligand
binding assay, using a binding protein prepared from bovine adrenal
glands (13). Results are presented as mean ± S.E. of three
experiments, each performed with three to six wells.
 |
RESULTS AND DISCUSSION |
EGF-dependent EGFR Kinase Activity in the Absence of
Endocytosis--
As it was reported that EGF-induced signaling was
altered by arrest of clathrin-dependent endocytosis (8), we
studied EGF-dependent EGFR kinase activity in the absence
of endocytosis. We chose to compare the same transfected cell line with
or without overexpression of mutant (K44A) dynamin 1 to avoid the risk
of clone variations. EGF was added to transfected HeLa cells with or
without tetracycline (without or with overexpression of K44A dynamin)
on ice, because endocytosis does not occur on ice. We found that cells
overexpressing K44A dynamin contained more EGFR than cells not
overexpressing K44A dynamin (Fig.
1A), but surprisingly we found
that the EGF-induced EGFR autophosphorylation in cells overexpressing
K44A dynamin was strongly reduced (Fig. 1B).
Immunoprecipitation experiments demonstrated that the phosphorylated
protein of molecular mass 170 kDa was indeed the EGFR (data not shown).
The specific reduction in EGFR autophosphorylation on ice in cells
overexpressing K44A dynamin is stronger than apparent from Fig.
1B, considering the increased amount of EGFR in cells
overexpressing K44A dynamin (Fig. 1A).

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Fig. 1.
EGF-dependent tyrosine
phosphorylation of EGFR on ice is inhibited in HeLa cells
overexpressing mutant dynamin (K44A). Serum-starved cells grown in
the presence or absence of tetracycline as described under
"Experimental Procedures" were treated with or without 3.5 × 10 9 M EGF for 10 min on ice, lysed, and
subjected to Western blotting as described under "Experimental
Procedures." A, EGF-treated uninduced cells (lane
1) and EGF-treated cells overexpressing K44A dynamin (lane
2) were Western blotted with an antibody to EGFR. B,
uninduced cells (lanes 1 and 2) and cells
overexpressing K44A dynamin (lanes 3 and 4)
incubated without (lanes 1 and 3) or with
(lanes 2 and 4) EGF were Western blotted with an
antibody to phosphotyrosine.
|
|
Analysis of EGF Binding in HeLa Cells with or without
Overexpression of K44A Dynamin--
We measured binding of
radiolabeled EGF to EGFR in cells with or without tetracycline and
analyzed the binding data by nonlinear curve fitting and Scatchard
analyses. As shown in Fig. 2A,
the data obtained from cells grown in the presence of tetracycline gave
a curvilinear Scatchard plot, demonstrating the presence of two
affinity classes of receptors. The high-affinity class of the EGFR
(4.2 ± 1.4% of total EGFR) had an apparent Kd of 8 ± 3 pM, and the low-affinity class of EGFR had
an apparent Kd of 400 ± 190 pM.
Analysis of EGF binding in cells overexpressing K44A dynamin, however,
revealed a linear Scatchard plot (Fig. 2B), indicating the
presence of only one receptor class. This class of receptors had an
apparent Kd of 700 ± 140 pM, not
significantly different from the low affinity class observed in
wild-type cells. Overexpression of wild-type dynamin did not reduce the
level of the high affinity class of the EGFR (data not shown).

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Fig. 2.
Scatchard analysis of binding of
125I-EGF to uninduced HeLa cells (A) and to
HeLa cells overexpressing K44A dynamin (B). The cells
were serum-starved and grown in the presence (A) or absence
(B) of tetracycline. Binding of different dilutions of
125I-EGF in the absence or presence of unlabeled EGF to
whole cells was measured and Scatchard analysis done as described under
"Experimental Procedures." The figure shows one representative
experiment of three, yielding similar results. Pooled results
(mean ± S.D., n = 3): A,
KdH = 8 ± 3 pM,
BmaxH = 3,000 ± 1000 sites/cell (4.2 ± 1.4%), KdL = 400 ± 190 pM,
BmaxL = 68,000 ± 16,000 sites/cell.
B, Kd = 700 ± 140 pM,
Bmax = 88,000 ± 20,000 sites/cell.
|
|
Subcellular Localization of EGFR with or without Overexpression of
K44A Dynamin--
Immunoelectron microscopy studies (9) showed that in
the absence of tetracycline the cells contained more EGFR at the plasma membrane than did uninduced cells (data not shown), consistent with the
results in Fig. 1A and in Fig. 2. When EGF was added to
cells on ice, the EGFR localized to smooth, uncoated, uninvaginated regions of the plasma membrane regardless of whether K44A dynamin was
expressed or not (Fig. 3, A,
F, and G). Only rarely did the EGFR localize to coated
pits. Chasing at 37 °C, however, caused a rapid relocalization of
the EGFR. After 10 min at 37 °C the receptor in uninduced cells had
moved into coated pits (Fig. 3B) and been internalized into
coated vesicles Fig. 3C and BSA-gold containing early
endosomes (Fig. 3D) as well as later endosomes with the
morphology of multivesicular bodies (Fig. 3E). Some
labeling, probably representing newly synthesized EGFR, was observed in the Golgi region of the cells (Fig. 3D). Expression of K44A
dynamin prevents the formation of clathrin-coated vesicles, but not the initial formation of clathrin-coated pits (7). In cells overexpressing K44A dynamin no labeling was found in endosomes, instead the EGFR localized to the blind bulb-shaped end of long, narrow tubular invaginations of the plasma membrane (Fig. 3, H and
I). At the end of the invaginations, where the EGFR
localized, there was a clear cytoplasmic coat previously shown to
consist of clathrin (14). Some labeling was also found in what looked
like clathrin-coated vesicles (Fig. 3, J and K).
However, we interpret these structures to be clathrin-coated pits,
because the plane of sectioning will not always allow visualization of
the narrow, tubular connection with the plasma membrane. These data
clearly show that even the low-affinity class of EGFR is efficiently
recruited to clathrin-coated pits. The observation that in contrast to
in uninduced cells the EGFR in cells overexpressing K44A dynamin could
not be detected in endosomes is consistent with the reported inhibition
of EGFR endocytosis in these cells (7) and with our observation of efficient arrest of clathrin-dependent endocytosis as
measured with radiolabeled transferrin (data not shown). The general
impression, when comparing cells with or without overexpression of K44A
dynamin, was that the number of coated structures was highly increased in cells overexpressing K44A dynamin. In addition to their long, tubular neck these coated pits often showed a more irregular shape compared with coated pits/vesicles seen in wild-type cells. Coated structures were often found in groups, and often two or more coated structures seemed interconnected, seen as figure eight-shaped patterns (Fig. 3, I and K). More precise
morphological studies are required to elucidate the reason for this,
but our observations indicate that more than one coated pit can exist
at the end of each tubule.

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Fig. 3.
Immunocytochemical localization of EGFR.
HeLa cells grown in the presence of tetracycline (A-E) or
in the absence of tetracycline (F-K) had endocytosed 5-10
nm BSA-gold before incubation with EGF on ice. Following binding of
10 8 M EGF the cells were either fixed
immediately (A, F, and G) or chased
for 10 min in EGF-free prewarmed medium (B-E and
H-K). Immunocytochemical labeling, as described under
"Experimental Procedures," showed that immediately after binding of
EGF on ice the EGFR (large arrowheads) localized to smooth
regions of the plasma membrane both in the presence of tetracycline
(A) and in the absence of tetracycline (F and
G). Note the atypical morphology of the coated pits with
long tubular plasma membrane connections (small arrowheads)
in K44A dynamin expressing cells (F-I). After 10-min chase
at 37 °C the EGFR in uninduced cells had moved into coated pits
(cp) (B) and been internalized into coated
vesicles (C) and BSA-gold containing early endosomes
(e.e.) (D) as well as multivesicular bodies
(mvb) (E). Some labeling was observed in the
Golgi region (g) of the cells (D). In cells
expressing K44A dynamin the EGFR localized to the coated end of the
long tubular invaginations (H and I). Some
labeling was also found in what look like clathrin coated vesicles
(J and K); note the irregular morphology of the
coated pits, vesicles shown (outlined) in I and
K. The large discontinuity in the outlining indicates the
possible position of the tubular plasma membrane connection.
Bars represent 100 nm.
|
|
Activity of Phospholipase C
(PLC
) in Uninduced Cells and in
Cells Overexpressing K44A Dynamin--
A possible way whereby the
specific kinase activity as well as the affinity of the EGFR could be
negatively modulated is by phosphorylation on Thr654 in the
cytoplasmic part of the EGFR, a substrate of protein kinase C (PKC)
(15). PKC can be activated by diacylglycerol, produced by
activation of PLC
. As PLC
in the absence of EGF was reportedly more tyrosine-phosphorylated in cells overexpressing K44A dynamin than
in uninduced cells (8), we determined IP3 mass in HeLa cells with or without overexpression of K44A dynamin. The
IP3 mass in the presence of tetracycline was 34.1 ± 2.8 pmol/mg of protein, and in the absence of tetracycline the
IP3 mass was 35.0 ± 0.5 pmol/mg of protein. The
hyperphosphorylation of PLC
did therefore not result in increased
IP3 production, thus excluding the possibility that the
hyperphosphorylation of PLC
caused increased formation of
diacylglycerol and thereby increased activation of PKC. We cannot
exclude the possibility that other phospholipases are activated in
cells overexpressing K44A dynamin.
Our data show that HeLa cells overexpressing K44A dynamin have lost
high affinity binding of EGF to the EGFR. Because it has been reported
that high-affinity receptors are required and sufficient for all
EGF-induced responses (16, 17), some of the previously reported changes
in EGF-dependent signaling in cells overexpressing mutant
dynamin (8) can be explained by the lack of high-affinity EGF-binding.
The molecular background for the high- and low-affinity state of the
EGFR is still unclear. The observation that activation of PKC by
phorbol esters converted high-affinity EGFR to low-affinity EGFR (18)
indicated that the intracellular part of the EGFR regulates its
affinity for EGF. Cells expressing EGFR lacking the entire
intracellular domain or the C-terminal 63 amino acids possessed only
low-affinity receptors (19, 20). Deletion of the major
autophosphorylation sites (21) or PKC phosphorylation sites (22) did
not alter the affinity of the receptor, neither did inactivation of the
tyrosine kinase by the introduction of a point mutation (19, 20) or by
an insertion of 4 amino acids (23, 24). High-affinity EGFR have been
demonstrated to be cytoskeleton-associated, and the EGFR binds
specifically to actin (25-29). However, it was recently demonstrated
that removal of the actin-binding site did not affect the affinity of
EGFR for EGF (30). Instead, another domain of the intracellular part of
the receptor located within the kinase domain was found to regulate the
affinity for EGF, and the possibility of an interaction between the
intracellular part of the EGFR and an affinity-modulating protein was
suggested recently (30). A potential affinity-modulating protein might
behave differently in cells overexpressing wild-type and mutant (K44A)
dynamin, because overexpression of wild-type dynamin did not reduce
high-affinity EGF-EGFR interaction (data not shown).
We could not detect direct interaction between overexpressed mutant or
wild-type dynamin and the EGFR (data not shown). A possible explanation
is therefore that mutant dynamin affects the EGFR affinity by
sequestration of an affinity-modulating molecule. Such a molecule could
be one of a variety of macromolecules demonstrated to bind dynamin,
including microtubules (31), acidic phospholipids (32), and several SH3
(Src homology domain-3) domain-containing intracellular
signal-transducing proteins (9).
Vieira et al. (8) observed that ligand-induced EGFR
signaling is attenuated in cells overexpressing K44A dynamin. They explained this finding by the arrested endocytosis of the ligand-bound EGFR under these conditions, thus implying that the EGFR must traffic
to endosomes for proper signaling to occur. Although confirming the
finding that ligand-induced signaling is attenuated in cells overexpressing K44A dynamin, we have demonstrated that the EGF-induced kinase activity is strongly reduced in cells overexpressing K44A dynamin compared with in uninduced cells, even when no endocytosis occurs in either cell line. The reason for this is the fact that overexpression of mutant dynamin per se affects the affinity
state of the EGFR. Thus, in conclusion, other factors than the arrested endocytosis might explain the reduced EGFR signaling in cells overexpressing K44A dynamin.
 |
ACKNOWLEDGEMENT |
We thank Sandra L. Schmid for kindly providing
HeLa cells transfected with wild-type or mutant (K44A) dynamin.
 |
FOOTNOTES |
*
This work was supported by The Norwegian Cancer Society, The
National Council for Science and the Humanities, Medinnova, Nordic Insulin Foundation Committee, The Anders Jahre's Foundation for the
Promotion of Science, Blix Legacy and Bruuns Legacy.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Institute of Pathology,
The National Hospital, N-0027 Oslo, Norway. Tel.: 47-22868609; Fax:
47-22112261; E-mail: i.h.madshus{at}labmed.uio.no.
1
The abbreviations used are: EGF, epidermal
growth factor; EGFR, EGF receptor; BSA, bovine serum albumin; PLC
,
phospholipase C
; PKC, protein kinase C; IP3, inositol
1,4,5-trisphosphate.
 |
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