RESEARCH ARTICLE
335
Proteoglycan synthesis is increased in cells with
impaired clathrin-dependent endocytosis
Alicia Llorente1,*, Kristian Prydz2,*, Mieke Sprangers2, Grethe Skretting1, Svein Olav Kolset3
and Kirsten Sandvig1,‡
1Department of Biochemistry, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
2Department of Biochemistry, University of Oslo, Box 1041 Blindern, 0316 Oslo, Norway
3Institute for Nutrition Research, University of Oslo, Box 1046 Blindern, 0316 Oslo, Norway
*Both authors contributed equally to this study
‡Author for correspondance (e-mail: [email protected])
Accepted 1 November 2000
Journal of Cell Science 114, 335-343 © The Company of Biologists Ltd
SUMMARY
Overexpression of a GTPase deficient dynamin mutant in
HeLa dynK44A cells causes a block in clathrin-dependent
endocytosis. When endocytosis is inhibited, these cells
incorporate higher levels of [35S]sulfate into both cellular
and secreted macromolecules and larger amounts of
proteoglycans such as syndecan and perlecan are
immunoprecipitated from [35S]sulfate-labelled lysates. Gel
filtration and ion-exchange chromatography revealed that
the increased [35S]sulfate incorporation into proteoglycans
was not due to significant differences in size or density of
negative charge of glycosaminoglycan chains attached
to proteoglycan core proteins. On the other hand,
measurements of the syndecan-1 mRNA level and of
[3H]leucine-labelled perlecan after immunoprecipitation
supported the idea that the increased [35S]sulfate
incorporation into proteoglycans was due to a selective
increase in the synthesis of proteoglycan core proteins.
Interestingly, the activity of protein kinase C was increased
in cells expressing mutant dynamin and inhibition of
protein kinase C with BIM reduced the differences in
[35S]sulfate incorporation between cells with normal and
impaired clathrin-dependent endocytosis. Thus, the
activation of protein kinase C observed upon inhibition of
clathrin-dependent endocytosis may be responsible for the
increased synthesis of proteoglycans.
INTRODUCTION
not only to attenuate signalling, but also for certain aspects of
the signalling process itself (Ceresa and Schmid, 2000).
We have previously observed that HeLa dynK44A cells
incubated with radioactive sulfate to label a modified ricin
containing a sulfation site (Llorente et al., 1998) incorporate a
larger amount of radioactive sulfate into high molecular mass
molecules, presumably proteoglycans (PG)s, when clathrindependent endocytosis is impaired. However, the incorporation
of sulfate into several newly synthetized proteins is unchanged.
PGs are formed by addition of one or more glycosaminoglycan
(GAG) chains to core proteins. The GAG chains are built of
repeating dissaccharide units and are classified according to the
nature of these units and by the degree and position of
sulfation. PGs seem to be synthesized by all vertebrate cell
types and have been found at cell surfaces, in vesicles, and in
the extracellular matrix (for review see Hardingham and
Fosang, 1992; Kjellén and Lindahl, 1991). These molecules
have been ascribed a large variety of functions that are often
mediated by electrostatic interactions of the GAG chains with
other molecules such us growth factors, extracellular matrix
molecules, or enzymes.
In this paper we have investigated the nature of the
high molecular mass molecules giving rise to increased
incorporation of radioactive sulfate upon inhibition of clathrindependent endocytosis. We show that the synthesis of PG core
proteins is increased when clathrin-dependent endocytosis is
Endocytosis of membrane, ligands and fluid occurs both by
clathrin-dependent and −independent mechanisms (for review
see Schmid, 1992; Smythe and Warren, 1991; Lamaze and
Schmid, 1995; Sandvig and van Deurs, 1994; van Deurs et al.,
1989). During the last years clathrin-dependent endocytosis
has been extensively characterized. Also several approaches
have been used to block this process, thus facilitating the study
of clathrin-independent endocytosis, the endocytic pathway
used by different molecules, and the role of clathrin-dependent
endocytosis for the physiology of the cells. Initially, K+depletion (Moya et al., 1985) and cytosol acidification
(Sandvig et al., 1987) were used to block clathrin-dependent
endocytosis. More recently the 100-kDa GTPase dynamin was
shown to play an important role in clathrin-dependent
endocytosis (for review see Damke, 1996; De Camilli et al.,
1995; Urrutia et al., 1997), and transiently and stably
transfected cell lines overexpressing GTPase defective
dynamin mutants are now commonly used to block this
endocytic mechanism (Damke et al., 1994; Herskovits et al.,
1993; van der Bliek et al., 1993). The role of endocytosis in
signal transduction has recently been investigated in stably
transfected HeLa cells where the overexpression of one of
these mutants, dynK44A, is regulated by tetracycline. The
results from these studies suggest that endocytosis is important
Key words: Clathrin-dependent endocytosis, Dynamin, Proteoglycan
synthesis, Protein kinase C
336
JOURNAL OF CELL SCIENCE 114 (2)
inhibited, whereas the total protein synthesis is not affected.
Protein kinase C (PKC) regulates PG synthesis in a number of
cell lines (Tao et al., 1997; Fagnen et al., 1999; Thiébot et al.,
1999), and this seems also to be the case in HeLa dynK44A
cells. Expression of mutant dynamin and reduction of
endocytosis in HeLa dynK44A cells leads to activation of PKC.
Evidence is presented that PKC activation is responsible for
the increased synthesis of PGs.
MATERIALS AND METHODS
Reagents
Tetracycline, bovine serum albumin (BSA), puromycin, guanidine,
Tris-HCl, bisindolylmaleimide (BIM), phorbol 12-myristate 13acetate (TPA) and Triton X-100 were from Sigma Chemical Co.,
St Louis, MO, USA. Chondroitinase ABC was purchased from
Seikagaku Corp., Tokyo, Japan. Ba(NO2)2 was from Merck,
Darmstadt, Germany. Na235SO4, [32P]dCTP, Na125I Sephadex G-50
Fine, Superose 6, and Protein A-Sepharose CL-4B were obtained
from Amersham Pharmacia Biotech, Uppsala, Sweden. Geneticin was
obtained from Saveen Biotech, Malmö, Sweden. [3H]Leucine was
from NEN Life Science Products, Boston, MA, USA. Econo-pac high
Q Cartridges were from Bio-Rad Laboratories, Hercules, CA. Acidic
fibroblast growth factor (FGF) was produced in bacteria, purified on
a heparin-Sepharose column (Wie˛ dĺocha et al., 1996), and iodinated
by the iodogen method (Fraker and Speck, 1978). Transferrin was
iodinated by the same method.
Cells
The HeLa cell lines stably transformed with the cDNAs for dynWT
or dynK44A were kindly provided by Dr S. L. Schmid, The Scripps
Research Institute, La Jolla, CA, USA (Damke et al., 1994). The cells
were grown in Falcon (Franklin Lakes, NJ, USA) or Nunc (Naperville,
IL, USA) flasks and maintained in DMEM (Flow Laboratories, Irvine,
Scotland) supplemented with 10% FCS, 100 units/ml penicillin, 100
µg/ml streptomycin, 2 mM glutamine, 400 µg/ml geneticin, 200 ng/ml
puromycin and 1 µg/ml tetracycline. BHK21 cells which in an
inducible manner produce antisense mRNA clathrin heavy chain
(CHC) (BHK21-tTa/anti-CHC) (G. Skretting, unpublished) were
grown in DMEM supplemented with 7.5% FCS, 100 units/ml
penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 200 µg/ml
geneticin, 200 ng/ml puromycin and 2 µg/ml tetracycline. For
experiments, these cells were grown with or without tetracycline for
the indicated times.
[35S]Sulfate and [3H]leucine labelling
Cells were washed with sulfate-free DMEM before [35S]sulfate (0.1
mCi/ml) was added to the cells in sulfate-free DMEM supplemented
with 5% FCS, 2 mM L-glutamine, 1% non-essential amino acids and
1 mM CaCl2. 4 hours or 10 hours later the medium fractions (approx.
1 ml) were briefly centrifuged to remove detached cells, and an equal
volume of 8 M guanidine/4% Triton X-100 in 0.2 M sodium acetate
buffer, pH 6.0 was added. The cells were washed with cold PBS on
ice, and then lyzed in 1 ml of 4 M guanidine/2% Triton X-100 in 0.2
M sodium acetate buffer, pH 6.0. To remove free [35S]sulfate, 1 ml of
each fraction was applied to 4 ml columns of Sephadex G-50 Fine in
0.05 M Tris-HCl, pH 8.0. Elution was carried out with a 0.05 M TrisHCl, pH 8.0, buffer containing 0.15 M NaCl. The first ml eluted after
application was discarded, and the next 1.5 ml was collected. The
radioactivity was then analyzed in a scintillation counter. The same
procedure was followed for [3H]leucine (50 µCi/ml) labelling, but
using leucine-free Hepes containing 5% FCS and 2 mM L-glutamine,
and for removal of free [3H]leucine. In some experiments, free
[35S]sulfate was removed from labelled cells by washing them with
PBS and then twice with TCA (5%) at room temperature for 10
minutes. Finally, the cells were solubilized in KOH (0.1 M). The
efficiency of the two methods to remove free [35S]sulfate was the
same.
Chondroitinase ABC and HNO2 treatment
To remove chondroitin sulfate chains from core proteins, samples
from medium and cell fractions pooled after Sephadex G-50 Fine
chromatography (approx. 10,000 cpm) were treated with
chondroitinase ABC (0.01 units) in 0.05 M Tris-HCl, pH 8.0,
containing 0.05 M sodium acetate and 0.1% BSA at 37°C overnight.
To break down heparan sulfate chains, equal volumes of 0.5 M H2SO4
and 0.5 M Ba(NO2)2 were mixed. After centrifugation, 15 µl of the
supernatant was added to approx. 10,000 cpm of sample (Shively and
Conrad, 1976). 10 minutes later the reaction was stopped by adding
1 M Tris-HCl (pH ≥5).
Immunoprecipitation
After removal of free [35S]sulfate or [3H]leucine, a polyclonal
antibody against mouse perlecan (a gift from Dr J. R. Hassell,
University of Pittsburg, PA, USA), a monoclonal antibody against
human versican (Seikagaku Corp., Tokyo, Japan), or a monoclonal
antibody against human syndecan (a gift from Dr M. Jalkanen, Center
for Biotechnology, Turku, Finland) (5 µg/ml) was incubated with
samples of medium and cell lysates overnight at 4°C in the presence
of 5 mM sulfate or 5 mM leucine. Protein A-Sepharose preblocked
with PBS containing 1% BSA was then added to the samples. After
2 hours at 4°C the beads were washed 5 times in 50 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 0.05% Triton X-100, 5 mM MgSO4 solution
with 1% BSA, and 3 times without BSA. The adsorbed material was
analyzed by SDS-PAGE.
Size-exclusion chromatography
Sephadex G-50 fine columns were used to remove free [35S]sulfate
from the medium and the cellular fractions. Then, similar volumes of
these fractions were treated with 0.5 M NaOH overnight at room
temperature to release GAG chains from core proteins. The reaction
was stopped by adding HCl until the pH was between 7 and 8. The
fractions were then subjected to Superose 6 gel chromatography with
a Pharmacia FPLC pump system, and eluted with 0.15 M NaCl in
0.05 M Tris-HCl with 0.5% Triton X-100, pH 8.0. Fractions (0.5 ml)
were collected with a flow rate of 0.5 ml/minute, and the radioactivity
was analyzed in a scintillation counter.
Ion-exchange chromato graphy
After removal of free [35S]sulfate or [3H]leucine, medium and cellular
samples (more than 15,000 cpm in 0.7 ml) were subjected to Econopac high capacity ion-exchange columns (5 ml) connected to a BioRad Econo System. Fractions (1 ml) were collected with a flow rate
of 2 ml/minute. Elution was carried out with a NaCl gradient (0.151.5 M) in 0.05 M Tris-HCl. The radioactivity in the fractions was
analyzed in a scintillation counter.
mRNA isolation and northern blot analysis
mRNA was isolated using the Dynabeads mRNA Direct Kit (Dynal,
Oslo, Norway), run on a 1% agarose gel with formaldehyde, and
finally blotted onto a Hybond-N membrane (Amersham Pharmacia
Biotech). cDNA probes for syndecan-1 (kindly provided by Dr Lars
Uhlin-Hansen, University of Tromsø, Norway) and glyceraldehyde3-phosphate dehydrogenase (Clonetech Laboratories, CA, USA) were
labelled with Redivue [α-32P]dCTP using RediprimeTM II (Amersham
Pharmacia Biotech). Hybridization was carried out overnight at 65°C.
Binding of aFGF to cells
Cells were incubated for 2 hours on ice in Hepes medium containing
125I-aFGF (50 ng/ml). In some cases heparin (10 U/ml) was added.
The cells were then washed 3 times with ice-cold Hepes and lyzed in
0.1 M KOH. The radioactivity in the lysate was measured.
Endocytosis of 125I-transferrin
Endocytosis of 125I-transferrin was measured as described previously
(Ciechanover et al., 1983). In principle, cells were incubated with 125Itransferrin (50-150 ng/ml; 10,000-20,000 cpm/ng) at 37°C for 5
minutes, washed 3 times with ice-cold Hepes, and treated for 1 hour
at 0°C with Hepes containing 0.3% (w/v) pronase. The cells and the
medium were then transferred to Eppendorf tubes and centrifuged for
2 minutes. The radioactivity in the pellet and in the supernatant was
measured.
Measurements of cAMP
The content of cAMP in cells was measured with a [8-3H] cyclic AMP
assay system from Amersham Pharmacia Biotech. In principle, cells
were washed twice in PBS and then dissolved in ice-cold HCl (10
mM) in ethanol (96%). After a 5 minutes incubation at 0oC, the cells
were removed with a rubber policeman and the cell suspension
was centrifuged for 10 minutes in an Eppendorf centrifuge. The
supernatant was freeze-dried and the pellet was dissolved in KOH
(0.2 M). The OD (280 nm) of this solution was used as a measurement
of the amount of cells used. The freeze-dried supernatant was
dissolved in 250 µl Na-acetate (0.5 M, pH 6.2). This solution was
then used in the cAMP kit to measure the concentration of cAMP
according to the manufacturer’s instructions.
PKC and PKA assays
PKC and protein kinase A (PKA) activities were assayed according
to GibcoBRL instructions.
SDS-PAGE
SDS-PAGE was performed as described (Laemmli, 1970). Labelled
PGs were run in precast 4-20% Tris-HCl-glycine gels (Novex, San
Diego, CA, USA). The gels were then fixed in 4% acetic acid and
27% methanol for at least 30 minutes, and then treated with 1 M Nasalicylate, pH 5.8 in 2% glycerol for 15-30 minutes. Dried gels were
exposed for fluorography at −80°C.
RESULTS
[35S]Sulfate labelling of HeLa dynK44A cells
Removal of tetracycline from the growth medium of HeLa
dynK44A cells induces the overexpression of dynK44A, a
dynamin I molecule where lysine 44 has been substituted by
alanine (Damke et al., 1994). DynK44A is defective in GTP
binding and hydrolysis, and causes a selective block in
clathrin-dependent endocytosis, whereas clathrin-independent
endocytosis continues (Damke et al., 1994; Vieira et al., 1996;
Llorente et al., 1998). Initial experiments revealed a difference
in the level of [35S]sulfate incorporation into high molecular
mass molecules between HeLa dynK44A cells grown in the
presence or in the absence of tetracycline. To quantify this
difference, cells were metabolically labelled with [35S]sulfate
for 10 hours, and free sulfate was then removed from cell
lysates and medium fractions by Sephadex G-50 Fine
chromatography. Interestingly, the amount of [35S]sulfate
incorporated into both cellular and secreted molecules in cells
overexpressing mutant dynamin was increased (Fig. 1). The
largest increase was observed in the secreted molecules (Fig.
1). Furthermore, control experiments showed that the amount
of incorporated [35S]sulfate was unchanged by overexpression
of wild-type dynamin I in HeLa dynWT cells (Fig. 1). The
experiment shown in Fig. 1 was performed with cells grown
with or without tetracycline for 48 hours. High levels of
expressed dynK44A are obtained after this period of time
[35S]Sulfate incorporated (% of control)
Proteoglycans in HeLa dynK44A cells
337
200
150
100
50
Fraction:
Cells:
C M
C M
C M
dynK44A
dynWT
anti-CHC
Fig. 1. [35S]Sulfate incorporation in HeLa dynK44A, HeLa dynWT,
and BHK21-tTa/anti-CHC cells. After 2 days HeLa dynK44A cells,
HeLa dynWT cells, and BHK21-tTa/anti-CHC cells grown with or
without tetracycline were incubated with [35S]sulfate in sulfate-free
DMEM. As described in Materials and Methods, after 10 hours a
guanidine/Triton X-100 solution in sodium acetate buffer was added
to the cellular and medium fractions. Free [35S]sulfate was removed
by Sephadex G-50 Fine chromatography, and the radioactivity left in
the cellular (C) and in the medium (M) fraction was analyzed in a
scintillation counter. The bars show the cpm obtained in percent of
the control (cells grown with tetracycline). The figure shows the
deviation between duplicates of representative experiments.
(Damke et al., 1995). However, dynK44A expression is already
detected 4 hours after tetracycline removal (Damke et al.,
1995), and clathrin-dependent endocytosis is strongly inhibited
24 hours after removal of tetracycline, and it remains inhibited
after 48 and 72 hours (Fig. 2A). We therefore decided to
investigate the [35S]sulfate incorporation into macromolecules
in cells grown with or without tetracycline for shorter and
longer periods of time than 48 hours. As soon as 24 hours after
removal of tetracycline a small increase in cells with mutant
dynamin was observed (Fig. 2B, open bars). The increase was
more marked 48 hours after removal of tetracycline, and it was
maintained at the same level after 72 hours (Fig. 2B, open
bars). Control experiments showed that the differences in
[35S]sulfate incorporation were not due to differences in cell
numbers between cells grown with or without tetracycline for
24, 48 and 72 hours (Fig. 2B, hatched bars).
As already mentioned, dynK44A overexpression in HeLa
dynK44A cells causes a selective block in clathrin-dependent
endocytosis. However, it has been shown that dynK44A
overexpression may also alter other intracellular traffic steps
(Llorente et al., 1998; Nicoziani et al., 2000). To investigate
whether the effect of dynK44A overexpression on
macromolecules [35S]sulfate incorporation was in fact due to
a block in clathrin-dependent endocytosis, similar experiments
were performed with BHK21-tTa/anti-CHC cells. In a
tetracycline-inducible manner, these cells produce CHC
antisense mRNA, which inactivates endogenous mRNA and
prevents the synthesis of CHC. This results in an inhibition
of clathrin-dependent endocytosis, but not of fluid-phase
endocytosis. Also inhibition of clathrin-dependent endocytosis
in these cells caused an increased incorporation of [35S]sulfate
into cellular and released high molecular mass molecules
JOURNAL OF CELL SCIENCE 114 (2)
A
70
60
50
40
30
20
10
[
35
S]Sulfate incorporation
(% of +Tet.)
0
160
140
12
24
32
48
Hours after removal of Tet.
72
B
[35S]Sulfate
Cells
160
140
120
120
100
100
80
80
60
60
40
40
20
20
0
24
48
Cell numbers (% of +Tet.)
125
I-Transferrin endocytosed
(% of cell-associated)
338
Fig. 3. Proteoglycan characterization in HeLa dynK44A. HeLa
dynK44A cells grown in the presence (+Tet.) or in the absence
(−Tet.) of tetracycline for 48 hours were labelled with radioactive
sulfate for 10 hours. Then, after removing free [35S]sulfate, cellular
(C) and medium (M) fractions were treated or not (none) with
chondroitinase ABC (c-ABC) to break down chondroitin sulfate
chains, or with HNO2 to break down heparan sulfate chains.
[35S]Sulfate-labelled molecules were then subjected to SDS-PAGE
and autoradiography.
72
Hours after removal of Tet.
Fig. 2. Time-dependent effects of dynK44A overexpression. HeLa
dynK44A cells were grown with or without tetracycline for the
indicated periods of time. (A) 125I-Tranferrin endocytosis was
measured as described in Materials and Methods. The figure shows
deviations between duplicates of a representative experiment.
(B) Open bars: HeLa dynK44A cells were incubated with
[35S]sulfate in sulfate-free DMEM for the last 4 hours of the
indicated incubating times, and then washed twice with TCA (5%) at
room temperature for 10 minutes. The cells were then solubilized in
KOH (0.1 M) and the acid-precipitable radioactivity was measured.
The figure shows the deviation between two experiments. Hatched
bars: HeLa dynK44A cells were detached from their support and
then counted in a Coulter counter instrument (Coulter Electronics
Limited, UK). The figure shows the deviation between duplicates of
a representative experiments. The bars show cpm and cell numbers in
percent of the cpm and cell numbers obtained from cells grown with
tetracycline (+Tet.).
(Fig. 1), thus suggesting that the effect of dynK44A is due to
a block in clathrin-dependent endocytosis.
Characterization of [35S]sulfate-labelled
macromolecules in HeLa dynK44A cells
Sulfate groups can be linked to tyrosine residues in some
proteins, and to sugars in glycoproteins, glycolipids, and free
or core protein-linked GAG chains. In HeLa dynK44A cells,
[35S]sulfated-macromolecules mainly appear as smears on the
top of 4-20% gradient gels (Fig. 3), thus suggesting that most
of the [35S]sulfate is incorporated into the GAG chains of PGs.
To investigate the nature of the GAG chains, [35S]sulfatelabelled cellular and medium fractions were subjected to
depolymerization treatments before analysis by SDS-PAGE.
As shown in Fig. 3, the [35S]sulfate-labelled macromolecules
disappear almost completely from the cellular fraction upon
Fig. 4. Perlecan and syndecan immunoprecipitation in [35S]sulfatelabelled HeLa dynK44A cells. HeLa dynK44A cells grown in the
presence (+Tet.) or in the absence (−Tet.) of tetracycline for 48 hours
were labelled with radioactive sulfate for 10 hours. Cellular (C) and
medium (M) fractions where free [35S]sulfate has been removed
were incubated with antibodies against perlecan or syndecan
overnight at 4°C. Immunoprecipitated material was concentrated on
Protein A-Sepharose and analyzed by SDS-PAGE. Protein standards
are shown on the left.
treatment with nitrous acid, which degrades HSPGs and
heparin (Shively and Conrad, 1976). This result indicates that
the cellular fraction contains mainly heparan sulfate PGs
(HSPG)s. However, the medium fraction contains mainly
chondroitin sulfate PGs (CSPG)s, as suggested by the
disappearance of [35S]sulfate-labelled PGs after chondroitinase
ABC treatment (Fig. 3).
To further characterize the PGs present in HeLa dynK44A
cells, specific PGs were immunoprecipitated. For this purpose,
[35S]sulfate-labelled fractions were incubated with antibodies
against the HSPG perlecan, the CSPG versican, and syndecan1, an HS/CS hybrid PG. Both when perlecan and syndecan
immunoprecipated from the cellular fraction were run on SDSPAGE, a high molecular mass band was visible (Fig. 4, see
fraction C). Furthermore, the bands were stronger when
Proteoglycans in HeLa dynK44A cells
339
Fig. 6. [3H]Leucine labelling of HeLa dynK44A cells: [3H]leucine
incorporation into proteins and perlecan immunoprecipitation. Cells
grown with (+Tet.) or without (−Tet.) tetracycline for 48 hours were
labelled with [3H]leucine for 10 hours in Hepes medium without
unlabelled leucine containing 5% FCS and 2 mM L-glutamine for 10
hours and then lyzed. After removal of free [3H]leucine the
radioactivity in the cellular and in the medium fraction was analyzed
in a scintillation counter (A), and perlecan was immunoprecipitated
from the cell fraction as indicated in Materials and Methods (B). In
A the deviation between duplicates of a representative experiments is
shown.
Fig. 5. Determination of GAG density of negative charge in HeLa
dynK44A cells by ion-exchange chromatography. HeLa dynK44A
cells grown in the presence (+Tet.) or in the absence (−Tet.) of
tetracycline for 48 hours were labelled with radioactive sulfate for 10
hours. After removal of free [35S]sulfate, medium and cellular
samples (more than 15,000 cpm in 0.7 ml) were subjected to Econopac high capacity ion-exchange columns connected to a Bio-Rad’s
Econo System. Elution was carried out with a NaCl salt gradient
(0.15-1.5 M) in 0.05 M Tris-HCl. The amount of radioactivity in the
fractions was analyzed in a scintillation counter. A representative
experiment is shown.
dynK44A expression was induced (Fig. 4, lanes without tet).
No bands were visible when medium fractions were
immunoprecipitated with these antibodies (Fig. 4, see M).
Finally, since the PGs found in the medium fraction are CSPG
(Fig. 3), we considered the possibility that versican was one of
them. However, no bands were detected on SDS-PAGE
gels when [35S]sulfate-labelled medium fractions were
immunoprecipitated with an antibody against versican (data
not shown).
Analysis of GAG chain size and charge density in
HeLa dynK44A cells
Since sulfate is mainly incorporated into sugar units, the
increased [35S]sulfate incorporation in cells overexpressing
dynK44A could be due to alterations in the GAG length and/or
negative charge density (higher concentration of sulfate
groups) of PGs in these cells. To determine the size of the GAG
chains, [35S]sulfate-labelled cellular and medium fractions
were treated with NaOH to release GAG chains from core
proteins, and then analyzed by Superose 6 FPLC
chromatography. Only minor differences in the GAG
hydrodynamic volume (Kav) between cells with endogenous
dynamin and cells expressing dynK44A were found (data not
shown), thus suggesting that in both cases the GAG chains have
the same length.
To investigate if there are any differences in the density of
negative charge of the GAG chains from control cells and
cells expressing mutant dynamin, [35S]sulfate-labelled
cellular and medium samples were analyzed by ion-exchange
chromatography. If there is a difference in the GAG density
of charge, the concentrations of NaCl required to elute the
samples would be different (Safaiyan et al., 1999). Fig. 5
shows the elution pattern of medium and cellular samples
labelled with [35S]sulfate 2 days after tetracycline removal.
After the salt gradient starts (fraction 12) two peaks are
observed: one presumably containing sulfated proteins
(around fraction 19), and the other containing PGs (around
fraction 36). The PGs of the cellular and medium fractions
of cells grown with or without tetracycline were eluted with
the same concentration of NaCl, thus suggesting that they
have similar densities of negative charge. Furthermore, a
large amount of the total incorporated radioactive sulfate was
found in PGs both in cells grown with and without
tetracycline.
[3H]Leucine-labelled HeLa dynK44A cells
In an attempt to investigate whether the increase in sulfate
incorporation observed after dynK44A overexpression was due
to an increased synthesis of PG core proteins, HeLa dynK44A
grown with or without tetracycline for 48 hours were
metabolically labelled with [3H]leucine for 10 hours. After
removal of free [3H]leucine, similar amounts of [3H]leucinelabelled proteins were detected in both cellular and medium
fractions from HeLa cells with endogenous or mutant dynamin
(Fig. 6A). However, larger amounts of perlecan were
Fig. 7. Northern blot analysis of
syndecan-1 mRNA in HeLa dynK44A
cells. mRNA was isolated from HeLa
dynK44A cells grown in the presence
(+Tet.) or in the absence (−Tet.) of
tetracycline for 48 hours using the
Dynabeads mRNA Direct Kit, run on a
1% agarose gel in the presence of
formaldehyde, and blotted on to
Hybond-N membranes. Hybridization
was carried out at 65°C with labelled cDNA probes for syndecan-1
(SYN-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
as a control for the loaded material. A representative experiment is
shown.
immunoprecipitated from cellular [3H]leucine labelled
fractions of cells containing mutant dynamin (Fig. 6B).
Syndecan-1 mRNA levels are increased in cells with
dynK44A
The possibility that the increase in sulfate incorporation in
cells overexpressing dynK44A was due to an increased
synthesis of PG core proteins was further investigated by
northern blot analysis of the mRNA levels of syndecan-1. A
glyceraldehyde-3-phosphate dehydrogenase probe was used to
control the amount of mRNA applied in the different lanes.
Interestingly, cells grown without tetracycline for 48 hours
showed approx. 3-fold increased levels of syndecan-1 mRNA
(Fig. 7). This experiment, as the [3H]leucine-labelled perlecan
immunoprecipitation experiment (Fig. 6B), suggests that in
cells where clathrin-dependent endocytosis has been inhibited
by overexpressing dynK44A, the increased incorporation of
radioactive sulfate into macromolecules can be explained by
an increased synthesis of PG core proteins.
Binding of aFGF to HeLa dynK44A cells
PGs bind to several growth factors (Ruoslahti and Yamaguchi,
1991). It has been shown that HSPGs, the PGs found in the
cellular fraction of HeLa dynK44A (Fig. 3), bind FGFs
(Burgess and Maciag, 1989). Since cells overexpressing
mutant dynamin synthesize more PGs, we tested the possibility
that the binding of the acidic FGF was increased. When HeLa
dynK44A cells grown with or without tetracycline for 48 hours
were incubated with 125I-acidic FGF for 2 hours at 4°C, larger
amounts of acidic FGF bound to the plasma membrane of cells
overexpressing mutant dynamin (Fig. 8). Heparin strongly
inhibited acidic FGF binding to the cells (data not shown).
Furthermore, no significant difference in binding was observed
when cells were grown for 24 hours with or without
tetracycline (Fig. 8). This result is in agreement with the
observation that only minor differences in sulfate incorporation
into macromolecules in cells with or without mutant dynamin
are observed after 24 hours (Fig. 2B).
Protein kinase C activity is increased in cells
expressing dynK44A
Activation of PKC by phorbol esters leads to an increased PG
synthesis in several cell lines (Tao et al., 1997; Fagnen et al.,
1999; Thiébot et al., 1999). In particular, TPA has been shown
to upregulate the expression of syndecan in rat immature
Sertoli cells (Brucato et al., 2000), and of perlecan in the
erythroleukemia cell line K562 (Grassel et al., 1995). We found
I-acidic FGF bound (% of control)
JOURNAL OF CELL SCIENCE 114 (2)
125
340
250
225
200
175
150
125
100
75
50
25
24 h
Tet.:
+ _
48 h
+
_
Fig. 8. Binding of 125I-acidic FGF to HeLa dynK44A cells. Cells
grown with (+Tet.) or without tetracycline (−Tet.) for 24 hours or 48
hours were incubated with 125I-aFGF (50 ng/ml) for 2 hours on ice.
Then, the radioactivity in the lysate was measured. The figure shows
the deviation between two experiments.
that addition of TPA (0.1 µM) increased the incorporation of
[35S]sulfate in HeLa dynK44A cells (data not shown), thus
suggesting that also in these cells activation of PKC leads to
an increased PG synthesis. We therefore decided to measure
the levels of activated PKC in cells with or without mutant
dynamin. Interestingly, cells grown without tetracycline for 48
hours showed higher levels of activated PKC than cells grown
with tetracycline (Fig. 9A). Since differences in PKC activity
may be responsible for the differences in PG synthesis between
cells with or without dynK44A, we measured the sulfate
incorporation into PGs in the presence of BIM, a PKC inhibitor
(Toullec et al., 1991). BIM (10 µM) was added 24 hours after
removal of tetracycline, when the activity of PKC is only
partially stimulated (Fig. 9A). 24 hours later the incorporation
of radioactive sulfate into macromolecules was measured. As
shown in Fig. 9B, the differences between cells with or without
mutant dynamin were reduced by 50%. These results clearly
indicate that the activity of PKC is important for the level of
PG synthesis. Finally, we could not detect changes in the
activity of PKA or the levels of cAMP when clathrindependent endocytosis was inhibited (data not shown).
DISCUSSION
The main finding in the present work is that overexpression of
the dynamin mutant dynK44A and the subsequent inhibition
of clathrin-dependent endocytosis leads to a selective increase
in PG synthesis, probably due to activation of PKC.
The increased incorporation of [35S]sulfate into cellular and
secreted PGs of cells with impaired clathrin-dependent
endocytosis was not due to differences in cell growth.
Moreover, it was shown by gel filtration and ion-exchange
chromatography that cells with or without dynK44A have PGs
with GAG chains of similar size and density of negative
charges, thus suggesting that the core proteins of PGs in cells
with endogenous dynamin II or expressing the mutant of
dynamin I, dynK44A, are modified in an identical manner with
respect to these criteria in the Golgi apparatus.
A
175
150
125
100
75
50
25
24 h
Tet.:
Characterization of the PGs present in HeLa dynK44A by
immunoprecipitation of lyzed [35S]sulfate-labelled cells
revealed the presence of perlecan and syndecan in the cellular
fraction but not in the secreted fraction. Indeed, although
syndecan has been found in the medium (Jalkanen et al., 1987),
it is usually present as a plasma membrane PG (for review see
Carey, 1997). Concerning perlecan, it is found both in the
cellular fraction and in the medium of MDCK cells (Svennevig
et al., 1995), whereas in colon carcinoma cells it is closely
associated with the plasma membrane (Iozzo et al., 1994). The
fact that in HeLa dynK44A cells perlecan was not
immunoprecipitated from the medium fraction is in agreement
with our results that HSPGs are not found in the medium of
HeLa dynK44A cells.
Interestingly, our results suggest that the increased amount
of radioactive sulfate found in cells overexpressing mutant
dynamin may be due to a selective increase in the synthesis of
PG core proteins. Higher amounts of [3H]leucine-labelled
perlecan were immunoprecipitated from cells containing
mutant dynamin, although the overall protein synthesis was
unchanged. Furthermore, the mRNA level of the PG syndecan1 was higher in cells overexpressing mutant dynamin. In
contrast, the mRNA level of glyceraldehyde-3-phosphate
dehydrogenase was not increased, thus indicating that
inhibition of clathrin-dependent endocytosis does not increase
the levels of mRNA expression in general.
Dynamin II has been localized on the plasma membrane
(Damke et al., 1994) and at the Golgi apparatus (Cao et al.,
1998), and in vitro studies show that dynamin seems to be
required for the formation of clathrin-coated and constitutive
secretory vesicles from the TGN (Jones et al., 1998).
Furthermore, a dynamin-related protein in yeast, Vsp1p, has
been implicated in trafficking from the Golgi apparatus to
the vacuole (Rothman et al., 1990). Overexpression of
mutant dynamin I clearly changes the distribution of the
mannose-6-phosphate receptor intracellularly, suggesting a
role for dynamin or a dynamin-like molecule in intracellular
transport (Llorente et al., 1998; Nicoziani et al., 2000).
Furthermore, we have previously shown that in HeLa
dynK44A cells overexpressing mutant dynamin not only
clathrin-dependent endocytosis, but also transport of
internalized ricin to the Golgi apparatus is inhibited
(Llorente et al., 1998). However, experiments performed in
BHK21-tTa/anti-CHC cells where clathrin-dependent
endocytosis had been inhibited supported the idea that the
increase in the synthesis of PG core proteins results from a
+ _
150
125
100
75
50
25
None
48 h
+ _
341
B
175
[35S]Sulfate incorporation
(% of +Tet.)
Fig. 9. PKC activity and effect of BIM on
[35S]sulfate incorporation in HeLa dynK44A cells.
(A) Cells were grown with (+Tet.) or without
(−Tet.) tetracycline for 24 hours or 48 hours and
then PKC activity was measured following
manufacturer’s instructions. The bars show the cpm
obtained in percent of the control (+Tet.). The
figure shows the deviation between two
experiments. (B) BIM (10 µM) was added to cells
grown with (+Tet.) or without (−Tet.) tetracycline
for 24 hours. 20 hours later the cells were incubated
with [35S]sulfate in sulfate-free DMEM for 4 hours,
washed twice with TCA, and solubilized in KOH
(0.1 M). The figure shows the deviation between
duplicates of a representative experiment.
PKC activity (% of +Tet.)
Proteoglycans in HeLa dynK44A cells
Tet.:
+ _
BIM
+ _
block in clathrin-dependent endocytosis. Although both
dynamin and clathrin affect uptake from clathrin-coated pits
at the cell surface, they do not always act together. For
instance, dynamin but not clathrin is important for caveolae
function (Henley et al., 1998).
Why do cells synthesize more PGs when clathrindependent endocytosis is inhibited? It has recently been
shown that clathrin-dependent endocytosis is necessary for
the activation of kinases such as phosphatidyl inositol 3kinase and ERK 1/2 (Vieira et al., 1996; Ceresa et al., 1998).
However, clathrin-dependent endocytosis can also serve as a
mechanism for signal transduction attenuation since it
removes receptors from the cell surface (Wells et al., 1990;
Vieira et al., 1996). PKC has been shown to be involved in
PG synthesis (Tao et al., 1997; Fagnen et al., 1999; Thiébot
et al., 1999), and inhibition of endocytosis and degradation
of activated receptors could result in an increased and
prolonged activity of this enzyme. We find that the PKC
activity is increased when clathrin-dependent endocytosis is
inhibited, and that PKC also seems to regulate PG synthesis
in HeLa dynK44A cells. Moreover, in the presence of a PKC
inhibitor the differences in sulfate incorporation between
cells with and without mutant dynamin are reduced.
Therefore, we suggest that the continuous presence at the cell
surface of activated receptors signalling through the PKC
pathway, leads to an increase in PKC activity that causes the
stimulation of PG core protein synthesis. Growth factors
present in the serum may be potential ligands for these
receptors since it is known that these molecules regulate PG
expression (Elenius et al., 1992).
Phospholipase Cγ, an enzyme that produces the PKC
activator diacylglycerol, is hyperphosphorylated in cells
overexpressing dynK44A (Vieira et al., 1996). However,
phospholipase Cγ is probably not responsible for the activation
of PKC here shown since it has been recently reported that its
activity is not increased in these cells in spite of the change in
phosphorylation (Ringerike et al., 1998).
PGs are involved in the binding and internalization of
several growth factors (Ruoslahti and Yamaguchi, 1991).
Therefore, the increased PG synthesis in the cellular fraction
observed in cells overexpressing dynK44A was likely to result
in an increased binding of growth factors to the cell surface.
As here shown, more acidic FGF was bound to cells
overexpressing mutant dynamin than to cells with endogenous
dynamin. Since HeLa dynK44A cells expressing mutant
dynamin are used to study endocytosis and its relevance for
342
JOURNAL OF CELL SCIENCE 114 (2)
signalling of different ligands, it is important for the
interpretation of such results to be aware of the change in PG
expression. A shift in endocytosis or a shift in response to a
certain ligand is not necessarily caused by the inhibition of
clathrin-dependent endocytosis of the ligand. PGs can be
important for signalling (Spivak-Kroizman et al., 1994;
Rapraeger et al., 1994), and a change in the subset of these
molecules might also affect the pathway of endocytosis
followed by a ligand.
We thank Jorunn Jacobsen, Anne-Grethe Myrann, Mette Sværen
and Klaus Magnus Johansen for expert technical assistance. This work
was supported by the Norwegian Research Council for Science and
the Humanities (NAVF), The Norwegian Cancer Society, the NovoNordisk Foundation, Blix legacy, Torsteds legacy, the Jahre
foundation, Jeanette and Søren Bothners legacy. Mieke Sprangers was
a Leonardo programme student from the University of Professional
Education in Rotterdam.
REFERENCES
Brucato, S., Harduin-Lepers, A., Godard, F., Bocquet, J. and Villers, C.
(2000). Expression of glypican-1, syndecan-1 and syndecan-4 mRNAs
protein kinase C-regulated in rat immature Sertoli cells by semi-quantitative
RT-PCR analysis. Biochim. Biophys. Acta 1474, 31-40.
Burgess, W. H. and Maciag, T. (1989). The heparin-binding (fibroblast)
growth factor family of proteins. Annu. Rev. Biochem. 58, 575-606.
Cao, H., Garcia, F. and McNiven, M. A. (1998). Differential distribution of
dynamin isoforms in mammalian cells. Mol. Biol. Cell 9, 2595-2609.
Carey, D. J. (1997). Syndecans: multifunctional cell-surface co-receptors.
Biochem. J. 327, 1-16.
Ceresa, B. P., Kao, A. W., Santeler, S. R. and Pessin, J. E. (1998). Inhibition
of clathrin-mediated endocytosis selectively attenuates specific insulin
receptor signal transduction pathways. Mol. Cell Biol. 18, 3862-3870.
Ceresa, B. and Schmid, S. (2000). Regulation of signal transduction by
endocytosis. Curr. Opin. Cell Biol. 12, 204-210.
Ciechanover, A., Schwartz, A. L., Dautry-Varsat, A. and Lodish, H. F.
(1983). Kinetics of internalization and recycling of transferrin and the
transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic
agents. J. Biol. Chem. 258, 9681-9689.
Damke, H., Baba, T., Warnock, D. E. and Schmid, S. L. (1994). Induction
of mutant dynamin specifically blocks endocytic coated vesicle formation.
J. Cell Biol. 127, 915-934.
Damke, H., Gossen, M., Freundlieb, S., Bujard, H. and Schmid, S. L.
(1995). Tightly regulated and inducible expression of dominant interfering
dynamin mutant in stably transformed HeLa cells. Meth. Enzymol. 257,
209-220.
Damke, H. (1996). Dynamin and receptor-mediated endocytosis. FEBS Lett.
389, 48-51.
De Camilli, P., Takei, K. and McPherson, P. S. (1995). The function of
dynamin in endocytosis. Curr. Opin. Neurobiol. 5, 559-565.
Elenius, K., Maatta, A., Salmivirta, M. and Jalkanen, M. (1992). Growth
factors induce 3T3 cells to express bFGF-binding syndecan. J. Biol. Chem.
267, 6435-6441.
Fagnen, G., Phamantu, N. T., Bocquet, J. and Bonnamy, P. J. (1999).
Activation of protein kinase C increases proteoglycan synthesis in immature
rat Sertoli cells. Biochim. Biophys. Acta 1472, 250-261.
Fraker, P. J. and Speck, J. C. Jr (1978). Protein and cell membrane
iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,
6a-diphrenylglycoluril. Biochem. Biophys. Res. Commun. 80, 849-857.
Grassel, S., Cohen, I. R., Murdoch, A. D., Eichstetter, I. and Iozzo, R. V.
(1995). The proteoglycan perlecan is expressed in the erythroleukemia cell
line K562 and is upregulated by sodium butyrate and phorbol ester. Mol.
Cell Biochem. 145, 61-68.
Hardingham, T. E. and Fosang, A. J. (1992). Proteoglycans: many forms
and many functions. FASEB J. 6, 861-870.
Henley, J. R., Krueger, E. W., Oswald, B. J. and McNiven, M. A. (1998).
Dynamin-mediated internalization of caveolae. J. Cell Biol. 141, 85-99.
Herskovits, J. S., Burgess, C. C., Obar, R. A. and Vallee, R. B. (1993).
Effects of mutant rat dynamin on endocytosis. J. Cell Biol. 122, 565-578.
Iozzo, R. V., Cohen, I. R., Grässel, S. and Murdoch, A. D. (1994). The
biology of perlecan: the multifaceted heparan sulphate proteoglycan of
basement membranes and pericellular matrices. Biochem. J. 302,
625-639.
Jalkanen, M., Rapraeger, A., Saunders, S. and Bernfield, M. (1987). Cell
surface proteoglycan of mouse mammary epithelial cells is shed by cleavage
of its matrix-binding ectodomain from its membrane-associated domain. J.
Cell Biol. 105, 3087-3096.
Jones, S. M., Howell, K. E., Henley, J. R., Cao, H. and McNiven, M. A.
(1998). Role of dynamin in the formation of transport vesicles from the
trans-Golgi network. Science 279, 573-577.
Kjellén, L. and Lindahl, U. (1991). Proteoglycans: structures and
interactions. Annu. Rev. Biochem. 60, 443-475.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 227, 680-685.
Lamaze, C. and Schmid, S. L. (1995). The emergence of clathrin-independent
pinocytic pathways. Curr. Opin. Cell Biol. 7, 573-580.
Llorente, A., Rapak, A., Schmid, S. L., van Deurs, B. and Sandvig, K.
(1998). Expression of mutant dynamin inhibits toxicity and transport of
endocytosed ricin to the Golgi apparatus. J. Cell Biol. 140, 553-563.
Moya, M., Dautry-Varsat, A., Goud, B., Louvard, D. and Boquet, P.
(1985). Inhibition of coated pit formation in Hep2 cells blocks the
cytotoxicity of diphtheria toxin but not that of ricin toxin. J. Cell Biol. 101,
548-559.
Nicoziani, P., Vilhardt, F., Llorente, A., Hilout, L., Courtoy, P. J., Sandvig,
K. and van Deurs, B. (2000). Role for dynamin in late endosome dynamics
and trafficking of the cation-independent mannose 6-phosphate receptor.
Mol. Biol. Cell 11, 481-495.
Rapraeger, A. C., Guimond, S., Krufka, A. and Olwin, B. B. (1994).
Regulation by heparan sulfate in fibroblast growth factor signaling. Meth.
Enzymol. 245, 219-240.
Ringerike, T., Stang, E., Johannessen, L. E., Sandnes, D., Levy, F. O. and
Madshus, I. H. (1998). High-affinity binding of epidermal growth factor
(EGF) to EGF receptor is disrupted by overexpression of mutant dynamin
(K44A). J. Biol. Chem. 273, 16639-16642.
Rothman, J. H., Raymond, C. K., Gilbert, T., O’Hara, P. J. and Stevens,
T. H. (1990). A putative GTP binding protein homologous to
interferon-inducible Mx proteins performs an essential function in yeast
protein sorting. Cell 61, 1063-1074.
Ruoslahti, E. and Yamaguchi, Y. (1991). Proteoglycans as modulators of
growth factor activities. Cell 64, 867-869.
Safaiyan, F., Kolset, S. O., Prydz, K., Gottfridsson, E., Lindahl, U. and
Salmitirva, M. (1999). Selective effects of sodium chlorate treatment on the
sulfation of heparan sulfate. J. Biol. Chem. 274, 36267-36273.
Sandvig, K., Olsnes, S., Petersen, O. W. and van Deurs, B. (1987).
Acidification of the cytosol inhibits endocytosis from coated pits. J. Cell
Biol. 105, 679-689.
Sandvig, K. and van Deurs, B. (1994). Endocytosis without clathrin. Trends
Cell Biol. 4, 275-277.
Schmid, S. L. (1992). The mechanism of receptor-mediated endocytosis: more
questions than answers. BioEssays 14, 589-596.
Shively, J. E. and Conrad, H. E. (1976). Formation of anhydrosugars in the
chemical depolymerization of heparin. Biochemistry 15, 3932-3942.
Smythe, E. and Warren, G. (1991). The mechanism of receptor-mediated
endocytosis. Eur. J. Biochem. 202, 689-699.
Spivak-Kroizman, T., Lemmon, M. A., Dikic, I., Ladbury, J. E., Pinchasi,
D., Huang, J., Jaye, M., Crumley, G., Schlessinger, J. and Lax, I.
(1994). Heparin-induced oligomerization of FGF molecules is responsible
for FGF receptor dimerization, activation, and cell proliferation. Cell 79,
1015-1024.
Svennevig, K., Prydz, K. and Kolset, S. O. (1995). Proteoglycans in polarized
epithelial Madin-Darby canine kidney cells. Biochem. J. 311, 881-888.
Tao, Z., Smart, F. W., Figueroa, J. E., Glancy, D. L. and Vijayagopal, P.
(1997). Enhanced synthesis of proteoglycans by vascular endothelial cells
treated with phorbol ester. Life Sci. 61, 723-738.
Thiébot, B., Langris, M., Bonnamy, P. J. and Bocquet, J. (1999). Activation
of protein kinase C pathway by phorbol ester results in a proteoglycan
synthesis increase in peritubular cells from immature rat testis. Biochim.
Biophys. Acta 1426, 151-167.
Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T.,
Ajakane, M., Baudet, V., Boissin, P., Boursier, E. and Loriolle, F. (1991).
The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of
protein kinase C. J. Biol. Chem. 266, 15771-15781.
Urrutia, R., Henley, J. R., Cook, T. and McNiven, M. A. (1997). The
Proteoglycans in HeLa dynK44A cells
dynamins: Redundant or distinct functions for an expanding family of
related GTPases? Proc. Nat. Acad. Sci. USA 94, 377-384.
van der Bliek, A. M., Redelmeier, T. E., Damke, H., Tisdale, E. J.,
Meyerowitz, E. M. and Schmid, S. L. (1993). Mutations in human
dynamin block an intermediate stage in coated vesicle formation. J. Cell
Biol. 122, 553-563.
van Deurs, B., Petersen, O. W., Olsnes, S. and Sandvig, K. (1989). The ways
of endocytosis. Int. Rev. Cyt. 117, 131-177.
Vieira, A. V., Lamaze, C. and Schmid, S. L. (1996). Control of EGF
343
receptor signaling by clathrin-mediated endocytosis. Science 274,
2086-2089.
Wells, A., Welsh, J. B., Lazar, C. S., Wiley, H. S., Gill, G. N. and Rosenfeld,
M. G. (1990). Ligand-induced transformation by a noninternalizing
epidermal growth factor receptor. Science 247, 962-964.
Wie˛dĺocha, A., Falnes, P. Ø., Rapak, A., Muñoz, R., Klingenberg, O. and
Olsnes, S. (1996). Stimulation of proliferation of a human osteosarcoma cell line
by exogenous acidic fibroblast growth factor requires both activation of receptor
tyrosine kinase and growth factor internalization. Mol. Cell Biol. 16, 270-280.