(CANCER RESEARCH 48, 1105-1 109, March 1, 19881
Epidermal Growth Factor-induced Stimulation of Epidermal Growth Factor
Receptor Synthesis in Human Cytotrophoblasts and A431 Carcinoma Cells'
Louis DePalo2 and Manjusri Das@
Department ofBiochemistry and Biophysics, University ofPennsylvania, School ofMedicine, Philadelphia, Pennsylvania 19104
During our investigations on the human EGF-receptor, we
raised a monoclonal antibody that recognizes a polypeptide
Epidermalgrowthfactor(EGF)-receptoris a transmembraneglyco epitope present in the external EGF binding domain of the
protein whoseintracellular degradation is knownto be enhancedby EGF. receptor (14, 15). In the present study we have used this
We tested whetherthe receptoris replenishedduringthis processby an
antibody to investigate receptor synthesis in two cell prototypes:
enhancedrate ofsynthesis. Human A431 epidermoidcarcinomacells and
(a)
cytotrophoblasts isolated from human placenta, a repre
primary cukures of human placental cytotrophoblasts were usedin these
sentative normal cell type; and (b) the human A431 epidermoid
studies. Cells were labeled with I@Slmethianine, and EGF-receptor bio
carcinoma cell line. We find that both cell types display EGF
synthesis was quantitated by i@@p@dpitafion
using a monoclonal
anti-EGF-receptor antibody. EGF stimulated receptor biosynthesis at dependent receptor degradation, the effect being most pro
concentrationsof 0.1 to I aM. The effect was seen within 2 h of EGF nounced at saturating concentrations of EGF (@ 10 nM). How
addition. At high EGF concentrations the stimulatory effect was dimin ever, at low nanomolar concentrations EGF has an opposite
Ished. In contrast, the effect of EGF on receptor degradation in these effect. It simulates receptor biosynthesis. The stimulation is
cells was negligibleat lownanomolarconcentrationsand was prOnOunced EGF-receptor specific and not due to a general increase in
only at saturafing concentrations ( 10 n@i). These results show that
protein synthesis. These results demonstrate that EGF has two
occupationof the cell surface EGF-receptor by its ligand can lead to the
opposing effects on receptor metabolism, namely, stimulation
production of more receptor protein, thus counterbalancingthe negative
of
synthesis and enhancement of degradation. The effect on
effect on receptor degradation. At lownanomolar concentrationsof EGF
ABSTRACF
the stimulatory
synthesis
effect on receptor synthesis predominates over degrada
is seen at subsaturating
concentrations
of EGF,
whereas degradation is enhanced only at near-saturating doses.
Some implications of these results are discussed.
don, indicating a positive regulatory role of EGF in receptor action.
INTRODUCFION
MATERIALS
EGF'-receptor is a highly regulated Mr 170,000 transmem
brane glycoprotein. It is a multidomain protein with a cyto
plasmically facing tyrosine kinase site whose activity is regu
lated by EGF (1—4).One of the consequences of EGF binding
is down-regulation ofthe EGF-receptor. EGF accelerates ligand
and receptor internalization, leading to intracellular degrada
tion of EGF (5—8).A fraction of the internalized receptor
population is also degraded, the net effect being a decrease in
the number of receptors in the presence of its ligand (5, 9, 10).
Homologous EGF-receptor degradation (down-regulation) has
been found to occur in all receptor-positive cell types examined
so far, including those that display little or no mitogenic re
sponse to EGF. It has been established that enhanced receptor
degradation, rather than decreased synthesis, is causal for the
decrease in receptor number (1 1, 12).
One of the characteristics of the EGF-receptor system is that
the cells need a continuous 6- to 8-h exposure to EGF for the
expression of mitogenic response (13). If a continuing interac
tion of EGF with its receptor is needed for a critical tyrosine
phosphorylation event or the amassing of a critical mitogenic
signal, then a continuing availability of receptors is an impor
tant factor in determining cellular responsiveness. Yet EGF
induced receptor down-regulation will undermine the availabil
ity ofreceptors. Thus the role of receptor synthesis during EGF
action is an important consideration in understanding receptor
function.
AND METhODS
Materials. EGF was purified from mouse submaxillary glands (16).
33S-labeledmethionine (1.5 Ci/smol) was purchasedfrom Amersham.
Monoclonal425 antibody(IgG2a),which is specific for a polypeptide
epitope of the human EGF-receptor, was raised in a BALB/c mouse
using human A431 carcinoma cells as the immunogen (14).
Isolation of Cytotrophoblasts. Cells were isolated from human term
placenta as described (17). The purified cytotrophoblasts were plated
in 35-mm dishes (106 cells/dish)
in DME medium, containing
25 mr@i
4-(2-hydroxyethyl)-1-piperazeneethanesulfonic acid, 25 mr@iglucose,
and 20% FBS. At 12 to 15 h after plating, the cells were used for the
biosynthetic labeling experiments described below.
Culture of A431 Cells. Human A431 vulval carcinoma cells were
maintained in DME medium containing 7% FBS and 10 sg/ml of
gentamycin. For biosynthetic labeling experiments, cells (—‘3
x 10')
were seeded into 16-mm dishes and were used for experiments 15 to
20 h later.
Method for Studying Degradationof EGF-Receptor.Cells were
washed with 4 MMmethionine containing DME medium and then
incubatedat 37C for 6 h in the same mediumcontainingI35Slmethio
nine (50 giCi/ml) and 2% dialyzed FBS. In experiments involving no
chase, the cells were lysedimmediately after labelingand then subjected
to immunoprecipitation with 425 monoclonal antibody as described
below. In other experiments, the labeled cells were washed with DME
medium and then chased for I S h at 3TC with unlabeled methionine
containing DME-2% dialyzed FBS medium in the presence of 0 to 30
nM EGF. The treated cells were lysed with a buffer containing
1%
TritonX-100, 2 mMphenylmethylsulfonylfluoride,2 m@ibenzamidine,
25
@g/ml of aprotinin,
0.15 M NaC1, and 10 mr@isodium
phosphate
buffer, pH 7.4. The lysates were subjected to immunoprecipitation with
Received9/9/87; revised I 1/23/87; accepted 12/2/87.
The costsof publicationof this article were defrayedin part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
I This
work
2 Present
was
address:
supported
by
NIH
Grants
Cardiovascular
CA-43787
Pulmonary
Division,
and
CA-15822.
Hospital
of
the
Uni
versity of Pennsylvania, Philadelphia, PA 19104.
3 To
4 The
whom
requests
abbreviations
for
reprints
used
are:
should
EGF,
be
epidermal
425 monoclonal
antibody as described below.
Method for StudyingEGF-inducedEGF-ReceptorSynthesis. Cells
were washed with 4 zM methionine containing DME-medium and
incubated at 37C for 0 to 3 h in the same medium with 0 to 30 JiM
EGF. Then I35Slmethionine was added to a final concentration
of 100
t@Ci/ml,and the incubation was continued at 3TC for an additional
addressed.
growth
factor,
DME-medium,
Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; TCA, trichloroa
cetic acid; SDS, sodium dodecyl sulfate; TGF-a, transforming growth factor-a.
hour. After this incubation, the cells were lysed as described in the
previous paragraph and subjected to immunoprecipitation with the 425
monoclonal
antibody.
1105
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@
@
,.@ —@
@!
-.@
EFFEC1@
OF EGF ON RECEPTORSYNTHESISAND DEGRADATION
@
________
16
Hours
Immunoprecipitation Technique.This was done as described(14, 18).
of chase
The 35S-labeledcell lysates(50 MI)wereincubatedat 4C for I h with 5
@l
of a l-mg/ml solution of 425 antibody (or nonimmune mouse IgG),
and the immunecomplexes were isolated by stirringthe mixturesat
0
Ni
Ab
NI
Ab
NI
Ab
30EGF(nM)
NiAb
4C for 30 mm with 1 mg of IgGSorb (The Enzyme Center, MA). The
IgGSOrb was washed, the bound radioactivity was eluted with SDS
-205
sample buffer, and the eluate was subjected to SDS-polyacrylamidegel
170 K@
electrophoresis and fluorography as described (14).
TCA Precipitation of Labeled Cell Lysates. Aliquots (10 @il)
of 35Slabeled cellular lysates were incubated at 4C for 30 mm with I ml of
25% TCA and 2% casein hydrolysate. The precipitated proteins were
-116
.
filtered over HA Millipore filters (0.45-tim pore size), washed twice
‘@:@
with 8% TCA, and then measuredfor radioactivityusing a toluene
@ri@
RESULTS
Biosynthesis and Degradation of the EGF-Receptor in Cyto
trophoblasts. In previous studies we found that trophoblastic
membranes from human placenta contain a Mr 170,000 protein
that can be affinity labeled with ‘251-EGF
and which displays
EGF-stimulated autophosphorylating activity (14, 15). Here we
show that isolated cytotrophoblasts biosynthesize a M@170,000
protein that is immunoprecipitated by an EGF-receptor-specific
monoclonal antibody (Fig. 1). The antibody is directed to a
polypeptide epitope present in the external domain of the
receptor (14). To test whether the synthesized receptor under
goes EGF-dependent degradation, the 35S-labeled cells were
changed into unlabeled medium and then incubated at 37°Cfor
16 h in the presence or absence of EGF (Fig. 1). In the absence
of EGF there was 49% of the original labeled receptors remain
ing after 16 h. In the presence of EGF, degradation of receptor
was enhanced (Fig. 1). After 16-h treatments with 1 nM and 10
nM EGF,
38% and
66
.,4―
based scintillation fluid.
@
-93
@-.
15%, respectively,
of the original
labeled
-29
%Control
receptor
100
47
42
15
Fig. 2. EGF concentrationdependenceof receptor degradationin human
A431 carcinomacells.Cellsin 16-mmdisheswereincubatedat 3TC for 6 h with
[35Slmethionine
andthensubjectedto 0- or 16-hchasewith unlabeledmethionine
in the presenceof the indicated amounts of EGF. The experimental details are
describedin “Materials
and Methods.@
The resultsof immunoprecipitation,
electrophoresis, and fluorography with antibody (Ab) and nonimmune lgG (N:)
are shown. The percentageof control receptors remaining after the chase was
determined by densitometric analysisof the fluorograms.
A similar analysis of the EGF-receptor in A431 cells showed
a somewhat different EGF concentration dependence of recep
tor degradation (Fig. 2). For effecting the same fractional extent
of receptor degradation, the A431 cells require 3- to 10-fold
higher concentrations of EGF than that required by cytotroph
oblasts. This indicates that, in comparison
with cytotropho
receptors remained intact. These results indicate that cytotroph
oblasts can mediate endocytosis and degradation of EGF-recep
tor. However, it should be noted that the effect of EGF is
blasts, the A43 1 cells have a somewhat reduced ability for
ligand-mediated receptor endocytosis and degradation.
EGF Stimulates Receptor Biosynthesis in Cytotrophoblasts
insignificant
and A431 Cells. To test the effect of EGF on receptor biosyn
at 1 nM and pronounced
only at a saturating
concentration (10 nM).
Hours of
@
chase
@
thesis in cytotrophoblasts, the cells were pretreated with EGF
and then labeled with [35Slmethionine for 1 h. The results of
immunoprecipitation show an increase in 35Slabel in the Mr
_____ 16____
______
NIAb
0
NIAb
1
NiAb
10
___
nM EGF
NIAb
160,000-170,000
EGF-receptor
band
(Fig.
3A).
A
maximal
increase (2- to 3-fold) was obtained at I nM EGF. At higher
-205
concentrations
ofEGF there
wasa decrease
inthestimulatory
effect. Studies on time-course ofthe EGF-induced effect showed
that a 2-h pretreatment with EGF was sufficient for stimulation
@
of receptor biosynthesis (Fig. 3B).
Next we tested the effect of EGF on receptor biosynthesis in
j±@
-66
A431
cells. In previous
similar
studies
on A431,
the relatively
high concentrations of EGF used (30 to 50 nM) had no detect
able effect on the rate of receptor biosynthesis (1 1, 12). To test
@
@-
_45
the
.-@
A431
effect
cells
of
lower
with
concentrations
0.3
to
30
of
nr@i
EGF
and
EGF,
then
we
labeled
pretreated
the
with
(35SJ
methionine. The results in Fig. 4A show that, in the low
-29
concentration
range (0.3 to 3 nM), EGF has a stimulatory
effect
on receptor biosynthesis. The amount of immunoprecipitable
%Control
receptor
1
3
15
35S-labeled
Mr 160,000170,000
receptorincreased‘@-2-fold
Fig. I. Biosynthesisand degradationof the EGF-receptorin cytotrophoblasts
fromtermhumanplacenta.Cytotrophoblasts
in 35-mmdisheswereincubatedat
37C for 6 h with l35Slmethionine
asdescribedin “Materials
and Methods.―
After
the incubation, a set ofdishes (0-h chase)was washedand immediately processed
for immunoprecipitation.The otherdisheswerewashed,transferredto unlabeled
methionine medium, chased for 16 h at 3TC in the presenceof the indicated
amounts of EGF, and then lysed and processedfor immunoprecipitation. The
results of immunoprecipitation, electrophoresis, and fluorography with antibody
(4b)
and nonimmune
lgG (N:)
are shown.
The position
of the EGF-receptor
is
indicated by the arrowhead. The percentagecontrol of receptors remaining after
the chasewasdeterminedby densitometricanalysisof the fluorograms.
after treatment with 1 nM EGF (Fig. 4A). A 2-h pretreatment
with EGF was sufficient for the induction of stimulation (Fig.
4B@.
An examination of the total 35S-labeled A431 cellular pro
teins by electrophoresis
and fluorography
showed no obvious
increase in overall protein synthesis after a 2-h EGF treatment
(Fig. 5). Trichloroacetic acid precipitation of the labeled A431
proteins also failed to show any significant EGF-induced effect
on general protein synthesis (Fig. 5). In the cytotrophoblasts
1106
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EFFECI OF EGF ON RECEPTOR SYNTHESIS AND DEGRADATION
Cl)
Cl)
OF
a.'>
LL.(1)
@Cl)
ZW
I-.
-J
>-
—0
SD
-@LLJ
Oa:
—Jo
OW
LI. LI'.
LU
EGF(nM)@
0
0.3
I
3
30
EGF(nM) c:@Q
0.3
I
3
30
LI.C1)
O@5
LI.(/)
ZW
O@5
Ox
-J
ZW
0I
>-
•a.
-Jo
@LLJ
SQ.
OW
LI.@
-Jo
OW
HOURSOFEGF@
TREATMENT
@
0
24
Fig. 3. EGF stimulates EGF-receptor synthesis in cytotrophoblasts. A, EGF
concentration dependenceof stimulation; B, time-course of EGF-induced stimu
lation. For the experiment in A, the cells were incubated at 3TC for 2 h with the
indicated amounts of EGF in 2% methionine-containing medium, then kept for
an additional hour at 3TC with [35SJmethioninein the same medium, and
subjectedto immunoprecipitation, electrophoresis, and fluorography asdescribed
in “Materials
and Methods.@For the experiment in B, the cells were incubated
at 37T for 4 h in 2% methionine-containingmedium. EGF (0.3 nM) wasadded
to the cells for the indicated times (0 representsa 4-h incubation without EGF).
l33SlMethionine was present for the last I h in all samples.lmmunoprecipitation,
electrophoresis, and fluorography were conducted as describedin “Materials
and
Methods.―
Fluorographyof the 35S-labeled
receptorbandanddensitometricplots
of the data are depicted.
there was a 20 to 30% increase in total protein synthesis after
2 h ofEGF treatment (Fig. 5), but this increase was insignificant
in comparison with the 200 to 300% stimulation of receptor
biosynthesis.
Overall the results in Figs. 1 to 5 show that EGF has a
specific stimulatory effect of EGF-receptor biosynthesis in at
least two different cell types, and that the concentrations of
EGF which produce maximal effects on synthesis have only
insignificant
effects on receptor
DISCUSSION
degradation.
HOURSOFEGF
TREATM ENT
0
2
4
Fig. 4. EGF-inducedstimulationof receptorsynthesisin A431 cells.A, EGF
concentrationdependenceof stimulation; B, time-courseof the EGF-induced
effect. The experiments were conducted as describedin the Fig. 3 legend and in
@‘Materials
and Methods.―
Fluorographyof the 3'S-labeledreceptorband and
densitometric plots of the data are depicted.
internalized receptors are known to retain their tyrosine kinase
activity for some time period (19). A large fraction of the
internalized receptors recycle to the cell surface (20), thus
allowing the reactivation of inactivated receptor-kinases by
rebinding to EGF. These dual processes of internalization and
recycling are rapid (t,/2 < 15 mm) and are accompanied by the
somewhat slower process of receptor degradation (20). The
cumulative effeci of receptor degradation is seen as down
regulation, i.e., reduction in receptor number. The results in
Figs. 1 and 2 show that relatively
high concentrations
of EGF
are required to effect sizable receptor degradation. The half-life
of the receptor in cytotrophoblasts (as extrapolated from the
data in Fig. 1, assuming that degradation is a first-order proc
ess) is 15.5 h in the absence of EGF and 11.5 h and 6 h,
respectively, in the presence of 1 nM and 10 nM EGF. In A431
As with many other ligand-receptor systems, the EGF-recep
toT is known to be internalized after binding to its ligand. The cells (Fig. 2) the extrapolated half-life values are 14.5 h, 12.5
1107
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EFFECI OF EGF ON RECEPTORSYNTHESISAND DEGRADATION
Cytotrophoblasts
@
5
6
0
0.3
A431 cells
TCA-pptd.
.@cpm x IOi.@25
1
3
24
26
25
@.eEGF(nM)@
0
—.4
@
.,,$
-@ti
1111
— 2@
‘@
•‘
—
doses, the stimulatory effect of EGF is abolished (Fig. 4).
Ligand-directed increases in receptor number have been
noted for the receptors for interleukin-2 (30), growth hormone
(31), prolactin (32), and insulin (33). Thus homeostatic auto
regulation of receptor synthesis by ligand action appears to be
a wide-spread phenomenon. In the case of the EGF-receptor
the phenomenon is interesting because of its cancer connec
tions. The EGF-receptor is the product of the c-erb-B protoon
cogene (2), which is amplified in many cancer cells (34, 35). Its
ligand, TGF-a, an EGF family protein, is expressed by many
transformed cells (36). Production of both TGF-ce and EGF
receptor within the same cell may not only lead to autocrine
stimulation ofoncogenesis, but also to aberrant overproduction
of EGF-receptors that is characteristic of many human cancers.
ACKNOWLEDGMENTS
We thank Dr. A. Basu and Dr. P. Das for experimental assistance.
REFERENCES
Fig. 5. Effectof EGF on total proteinsynthesisin cytotrophoblasts
and A431
cells. Cells in 16-mm dishes were incubated at 3TC for 2 h in 2% methionine
medium containing the indicated amounts of EGF and then kept for an additional
hourat 3TC with 13'Slmethioninein the samemedium.Cells werelysedwith 30
,d of lysis buffer. Aliquots (10 @zl)
of labeledcell lysateswere subjectedto
electrophoresisand fluorography. The amount of trichloroacetic acid-precipitable
counts (TCA pptd. cpm) present in the lO-Mlaliquots oflysates is indicated above.
Fluorographic exposuretimes were I 5 h and 3 h, respectively,for cytotrophoblasts
and A43 I cells.
h, and 6 h, respectively, in the presence of 0, 10 nM, and 30 n@i
EGF.
One of the questions that is raised by these and other studies
on receptor degradation is how does EGF continue to be
mitogenically active in the face of such receptor loss. It can be
noted that, at low concentrations of EGF that are mitogenic (@
1 nM), the extent of receptor degradation is small (Figs. 1 and
2). Moreover, our results (Figs. 3 and 4) demonstrate that
receptor degradation can be counterbalanced by a positive effect
of EGF on receptor synthesis. Thus, at low subnanomolar
concentrations of EGF, an enhanced rate of receptor biosyn
thesis can help to maintain the continued availability of recep
tors that is essential for EGF action.
Our results on EGF-induced
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synthesis
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MDA
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29). The EGF-receptor appears to be yet another species that
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workers (1 1, 12) studied receptor synthesis using high concen
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1988 American Association for Cancer Research.
Epidermal Growth Factor-induced Stimulation of Epidermal
Growth Factor-Receptor Synthesis in Human Cytotrophoblasts
and A431 Carcinoma Cells
Louis DePalo and Manjusri Das
Cancer Res 1988;48:1105-1109.
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