(Received for publication, April 5, 1995)
From the
Ligand binding to the platelet-derived growth factor (PDGF)
receptor initiates a complex and diverging cascade of signaling
pathways. GTP-binding proteins with intrinsic GTPase activity
(G-proteins) frequently link cell surface receptors to intracellular
signaling pathways, but no close associations of the PDGF receptor and
any small G-proteins, nor any such associations activated by ligand
binding to the receptor have been previously reported. We demonstrate
that a small GTP-binding protein binds specifically to the murine and
human PDGF type-
Growth factor signal transduction through the platelet-derived
growth factor receptor consists of a complex cascade of events. The
platelet-derived growth factor (PDGF)(
GTP-binding proteins with
intrinsic GTPase activity (G-proteins) frequently link cell surface
receptors to intracellular signaling pathways. They function by cycling
between (inactive) GDP-bound and (active) GTP-bound states. The small
G-protein p21
In the present study we have investigated
the involvement of GTP-binding proteins in PDGF-mediated signaling in
normal fibroblasts. A technique reported recently for in situ protein labeling of nucleotide-binding proteins in permeabilized
cells was utilized (27) . The method involves the introduction
of [
In this report, we demonstrate
that a small GTP-binding protein associates with the PDGF type-
Figure 1:
Affinity labeling of nucleotide-binding
proteins in permeabilized Balb/c-3T3 and KBalb fibroblasts. A, [
To demonstrate that known low molecular weight GTP-binding proteins
could be specifically labeled in Balb fibroblasts with this in situ [
Figure 2:
Identification of known small GTP-binding
proteins labeled with
[
To further
study the PDGF type-
Figure 3:
Small
[
Efficient
detection of small G-proteins by the in situ GTP
Figure 4:
Ligand-stimulated autophosphorylation of
PDGF type-
Figure 5:
2-Dimensional gel analysis of the
GTP
Figure 6:
Proteolytic cleavage of immunoprecipitated
[
As expected,
Balb cell membranes were readily
[
Figure 7:
C3-ADP-ribosylation of Balb cell membrane
proteins. Purified recombinant C3 transferase was utilized to
ADP-ribosylate Rho G-proteins in Balb cell membranes. PanelA shows the
[
Ras proteins and related small GTPases play critical roles in
the control of normal and transformed cell
growth(47, 48, 49) . The Ras superfamily of
GTPases contains over 60 small (20-27-kDa) guanine-nucleotide
binding proteins that are involved in the control of a wide variety of
cellular functions(50) . Based on their sequences, Ras-like
proteins fall into five main families: Ras, Rho, Rab, Ran, and
Arf(40, 41) , each of whose members have similar
structural characteristics, with very distinct
functions(46, 48, 50, 51) .
We
report here that small GTP-binding proteins bind specifically to the
murine and human PDGF type-
Our results suggest that the
association of the Rho small G-protein with the PDGF type-
The Rho family of small
G-proteins consists of RhoA, RhoB, RhoC, Rac, Cdc42Hs, and TC10.
Proteins of this subgroup are involved in organization of the
cytoskeleton. Rac is required for membrane ruffling induced by growth
factors (a result of actin reorganization at the plasma
membrane)(51) , and the subsequent formation of actin stress
fibers requires Rho(46, 52) . PDGF and epidermal
growth factors have been shown to induce cytoskeletal changes in
several cell types. Studies carried out by microinjection of purified
Rho and Rac proteins indicated that there is a sequential involvement
of the Rac followed by the Rho small G-proteins in these cellular
responses(51) .
PDGF and other growth factors presumably
induce Rho-dependent responses by increasing the level of GTP-bound Rho
in cells, and this could be achieved either by activating proteins that
stimulate nucleotide exchange on Rho, or by inhibiting proteins that
enhance GTP hydrolysis, such as RhoGAP (GTPase-Activating
Proteins)(53, 54) . Several proteins have been
identified as candidates for GAPs, among them the cellular protein
p190, which forms a stable complex with Ras-GAP (the
upstream/downstream effector of Ras)(55) . This is of
particular interest, raising the possibility that the Ras and Rho
signaling pathways are coordinately controlled(56) . One of the
exchange factors for the Rho family is the human oncoprotein Dbl, which
catalyzes nucleotide exchange on Cdc42Hs (57) and on
Rho(53) . A family of proteins with a Dbl-like domain has been
identified(53) . Although some of them form in vitro complexes with several members of the Rho family of proteins, many
of them showed no exchange activity on Rho(58) . Whether these
proteins provide a link between growth factors receptors and Rho-like
GTPase remains to be determined. Another potential guanine dissociation
stimulator (GDS) has been identified for Rho proteins, smgGDS (small
GTP-binding protein GDS)(59) , but smgGDS appears not to be
completely specific for the Rho subfamily, since it is also active on
Ki-Ras and Rap1(60) .
The activation of the Rho subfamily of
G-proteins differs significantly with respect to rates of GTP exchange
and hydrolysis. Rho, like Ras, has a slow intrinsic rate of
hydrolysis(61) , but Rac and Cdc42 have much shorter half-lives
for bound GTP. Conversely, nucleotide exchange is much slower for Rac
than Ras(44, 62) . The increase in GTP labeling of the
receptor-associated Rho protein that we observe after ligand binding to
the PDGF type-
What downstream events might be activated through Rho
association with the PDGF type-
We thank Dr. T. Daniels for generously providing
antisera and Drs. A. Kazlauskas and J. Cooper for generously providing
the TRMP cells and the PDGF-receptor mutants.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
receptor. In response to PDGF-BB stimulation,
there is an increase in the amount of labeled small G-protein
associated with the PDGF type-
receptor. The GTP-binding protein
did not undergo ligand-induced association with a mutant receptor
protein that was unable to bind ATP. Proteolytic cleavage analysis,
together with two-dimensional separation techniques, identified the
small G-protein specifically associating with the PDGF type-
receptor after ligand binding as a member of the Rho family. This was
confirmed by demonstration that the small G-protein
co-immunoprecipitated by the anti-PDGF receptor antibody was a
substrate for the ADP-ribosyltransferase C3 exoenzyme. Thus, the PDGF
type-
receptor may form a complex with one or more small
G-proteins upon binding PDGF-BB, and the Rho small G-protein is likely
to be an important component of the proteins making up the multimeric
signaling complex of the PDGF type-
receptor.
)
is a
30-kDa homo- or heterodimer that binds to the PDGF receptors, a 180-kDa
glycoprotein homo- or heterodimer with at least two classes of
subunits: type-
and
type-
(1, 2, 3, 4, 5, 6) .
The PDGF type-
receptor possesses an intrinsic protein tyrosine
kinase activity, which is stimulated upon binding by
PDGF-BB(1, 7, 8) . A number of intracellular
activities occur rapidly after exposure of a cell to PDGF-BB, including
dimerization and autophosphorylation of the PDGF type-
receptor
and association and activation of specific proteins. Within the PDGF
type-
receptor, several tyrosine residues have been identified,
which function as high affinity binding sites for signaling molecules
containing the Src homology 2 domain sequences, such as phospholipase
C
, p21
GTPase-activating protein (Ras-GAP),
the p85 subunit of phosphatidylinositol 3-kinase, and
Syp(9, 10, 11, 12, 13) . In
addition, increases in phosphatidylinositol turnover and calcium
mobilization, activation of protein kinase C and microtubule-associated
protein kinase, and induction of a number of growth-related genes,
including c-myc, c-fos, JE, c-jun,
and egr-1 occur rapidly after ligand
binding(7, 14, 15, 16, 17) .
A causal or sequential relationship of these PDGF-induced and tyrosine
kinase-dependent phenomena to each other and to eventual DNA synthesis
is suspected but not well established. We have recently reported that
some signals, resulting in the induction of certain growth-related
genes, can be transmitted by the PDGF receptor in the absence of
tyrosine kinase activity(18) . Therefore, PDGF
receptor-activated signaling mechanisms and pathways other than those
characterized previously must exist.
has been implicated in mediating
signals from a number of tyrosine kinase-type growth factor receptors,
including the PDGF type-
receptor(19, 20, 21, 22, 23, 24) .
p21
may be indirectly linked to these receptors
via a pathway involving a number of intermediary adaptor or coupling
proteins (22, 24, 25, 26) . No close
associations of the PDGF receptor and any small G-proteins, however,
and no associations activated by ligand binding to the receptor, have
been previously reported.
-
P]GTP into permeabilized cells,
followed by in situ periodate oxidation. The result was the
generation by cross-linking of GTP
-labeled
G-proteins(28, 29) . The sensitivity and specificity
of this method allowed cross-linking of more than 50% of
p21
to
[
-
P]GTP
in transfected
fibroblasts. Heterotrimeric G-proteins (e.g. Gi
) can also be labeled by this
method(27) . This technique has recently been used to detect a
GTP/GDP binding site in the
-chain of the T-cell receptor
complex(30, 31) .
receptor upon PDGF-BB stimulation. We have identified the small
G-protein specifically associating with the PDGF type-
receptor
after ligand binding as a member of the Rho family of G-proteins. On
the basis of these results, this Rho G-protein is likely to be an
important component of the proteins making up the multimeric signaling
complex of the PDGF type-
receptor.
Cells
Balb/c-3T3 fibroblasts were obtained from
the American Type Culture Collection. Kirsten
v-ras-transformed Balb/c-3T3 fibroblasts (KBalb) were
described previously(32) . Cells were maintained in
Dulbecco's modified Eagle's medium (Life Technologies,
Inc.), supplemented with 10% heat-inactivated donor calf serum (Sigma).
When quiescent cells were required, monolayers were grown to confluence
and then starved for 48 h in Dulbecco's modified Eagle's
medium with 0.5% donor calf serum. Dog kidney epithelial cells (TRMP)
expressing wild type or a mutant human PDGF type- receptor (L635R)
were the generous gift of A. Kazlauskas and J. Cooper (33) .
The L635R mutation made the receptor unable to bind ATP and therefore
kinase-negative. These cells were maintained with a mixture 1:1 of
Dulbecco's modified Eagle's medium and F-12 medium
supplemented with 10% heat-inactivated fetal calf serum, including 400
mg/ml G418 (Geneticin, Life Technologies, Inc.). All media were
supplemented with 100 units/ml penicillin and 100 units/ml
streptomycin.
Antibodies and Reagents
Antiserum specific for the
human PDGF type- receptor, directed against amino acid sequence
1013-1025, and cross-reacting with mouse receptors was purchased
from Upstate Biotechnology, Inc. (UBI). Antibody 538, directed against
the PDGF type-
receptor, was a gift from Tom Daniel, Vanderbilt
University. Anti-p21
monoclonal antibody Y13-259
(rat mAb-IgG
) directed against residues 63-76 was
obtained from Oncogene Science. The following affinity-purified rabbit
polyclonal antibodies were purchased from Santa Cruz Biotechnology,
Inc.. Anti-Rap1A/B/Krev-1 was raised against a synthetic peptide
corresponding to residues 121-137, which are conserved between
Rap1A/B proteins. Anti-RhoA was raised against a synthetic peptide
corresponding to amino acids 119-132 of the RhoA protein.
Anti-RhoB was raised against a synthetic peptide corresponding to amino
acids 119-132 of the RhoB protein. Anti-phosphotyrosine mAb 4G10
was purchased from UBI. Affinity-purified rabbit anti-mouse Vectastain
ABC kit was from Vector Laboratories. Recombinant protein A- and
protein G-Sepharose were purchased from Sigma and Pharmacia Biotech
Inc. Recombinant human PDGF-BB homodimer (PDGFh
-BB) was
purchased from R& Systems. [
-
P]GTP was
purchased from DuPont NEN or Amersham Corp. (3000 Ci/mmol; final
specific activity 100-400 Ci/mmol).
[
-
P]NAD (1000 Ci/mmol) was purchased from
Amersham. Purified exoenzyme C3 fusion protein containing full-length
24.5-kDa Clostridium botulinum (D strain) C3, expressed in Escherichia coli, was purchased from UBI.
PDGF Receptor in Vivo Stimulation
Balb, Kbalb, and
TRMP cells were grown in 10-cm diameter dishes until confluence. They
were serum-starved in Dulbecco's modified Eagle's medium
plus 0.5% donor calf serum for 24-48 h. Starved cells were washed
twice with phosphate-buffered saline (PBS), at 37 °C, then twice
with the isotonic wash (120 mM KCl, 30 mM NaCl, 2
mM MnCl, 10 mM HEPES, pH 7.4), at 37
°C. For ``in vivo'' stimulation with PDGF-BB,
washed cells were overlaid with 3 ml of isotonic wash buffer at 37
°C in 5% CO
incubator, without or with
PDGFh
-BB (R& Systems), to a final concentration of 30
ng/ml for 2 or 5 min at 37 °C, as indicated.
Cell Permeabilization
The cells were washed twice
with ice-cold PBS and twice with ice-cold labeling buffer (ICB buffer),
containing 20 mM Hepes, pH 7.8, 140 mM KCl, 10 mM NaCl, and 2.5 mM MgCl, and then permeabilized
with 50 µg/ml L-
-lysophosphatidylcholine (LPC)
(Sigma) for 5 min on ice. The cells were then dislodged with a cell
scraper and transferred to a microcentrifuge tube. Under these
conditions, 95% of the treated cells were permeable with trypan blue.
To remove excess LPC, permeabilized cells were centrifuged at 6,000 rpm
for 15 s in a microcentrifuge and resuspended in the labeling buffer.
In Situ Labeling with
[
Permeabilized cells in a final volume of 500
µl of labeling buffer were incubated with 1 µM [-
P]GTP
Nucleotides
-
P]GTP for 30 min at 37 °C, to
allow the added guanine nucleotide to bind or to exchange for
endogenous nucleotide, after which nucleotides were oxidatively cleaved
with 1 mM NaIO
(Sigma) for 1 min at 37 °C.
Condensation products were rapidly stabilized by reduction with 20
mM NaCNBH
(Sigma) for 1 min at 37 °C. To
remove free reactive GTP
nucleotides, cells were treated
with 20 mM NaBH
(Sigma) for 1 min at 37 °C,
then for 5 min on ice. After centrifugation for 3 min at 6,000 rpm in a
microcentrifuge, cells were lysed for 20-60 min on ice in lysis
buffer HTG (20 mM Hepes (pH 7.2), 1% Triton X-100, 10%
glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM Na
VO
, 5-10
µg/ml each aprotinin and leupeptin). The lysates were cleared by a
5-min centrifugation at 13,000
g, at 4 °C.
Immunoprecipitation and Electrophoresis
For
immunoprecipitation with rabbit antibodies, the lysates were precleared
with normal rabbit serum (NRS) previously coupled to protein A- or
protein G-Sepharose, as indicated, for 1-12 h at 4 °C;
preclearings were repeated at least three times. Precleared lysates
were then immunoprecipitated for periods ranging from 2 h to overnight
at 4 °C with specific antibodies. Protein-antibody complexes were
precipitated by incubation with protein A- or protein G-Sepharose for
90 min at 4 °C. p21 immunoprecipitation with
anti-p21
Y13-259 was performed by modifications
of the method described previously by Downward et
al.(34) . Permeabilized and labeled cells were lysed in
600 µl of 50 mM Hepes, pH 7.4, 100 mM NaCl, 5
mM MgCl
, 1% Triton X-100, 10 µg/ml aprotinin,
10 µg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl
fluoride. The lysates were cleared by centrifugation as described
above, and supernatant fractions were transferred to microcentrifuge
tubes with 75 µl of 4 M NaCl, 75 µl of 4% sodium
deoxycholate, and 0.4% SDS. Lysates were precleared three times with
rat IgG-agarose (Sigma), before the Y13-259 antibody was added. Samples
were incubated overnight at 4 °C. Y13-259-bound p21
was precipitated with anti-rat IgG-agarose (Sigma).
Immunoprecipitates were washed up to six times with lysis buffer. 40
µl of 3
Laemmli sample buffer was added to the washed
beads, and they were then heated at 90 °C for 5 min. Labeled
proteins were separated by SDS-PAGE in a 15% acrylamide gel, or as
indicated. The gels were fixed and dried. Dried gels were
autoradiographed using Kodak XAR-5 film, unless otherwise indicated.
Two-dimensional Gel Electrophoresis
A combination
of isolectric focusing (IEF) and SDS-polyacrylamide gel electrophoresis
(PAGE) was used to resolve proteins in two dimensions essentially as
described(35) . For IEF, samples were solubilized in 9.3 M urea, 50 mM dithiothreitol, 2.5% Triton X-100, 2%
ampholines (80% ampholines at pH 5-7 and 20% ampholines at pH
3-10 (Bio-Rad)). Tube gels for first dimension IEF were
11-12 cm long, with 1.5-mm internal diameter. As internal markers
for two-dimensional electrophoresis, a mixture of four proteins (pI
range: 3.8-7.6, in a range of 17-89 kDa) (Sigma) was added.
IEF gels were prerun for 15 min at 200 V, 30 min at 300 V, and 30 min
at 400 V. Samples were run for 15-17 h at 800 V, followed by 1 h
at 1000 V. The pH gradient after electrophoresis ranged from 4.5 to
6.5. For the second dimension, a 15% SDS-PAGE was used.
Immunoblot Analysis
GTP-labeled
small G-protein-antibodies complex immunoprecipitates were eluted from
the beads with Laemmli sample buffer, separated on a 5-15%
gradient SDS-PAGE gel, and electrophoretically transferred to
nitrocellulose (0.45 µm, Schleicher & Schuell) using a semi-dry
transfer apparatus (Hoeffer Scientific, CA), in a continuous buffer
system (39 mM glycine, 48 mM Tris, 0.0375% SDS, 20%
methanol), for 1 h at a constant current of 0.8 mA/cm
,
unless otherwise indicated. Filters were blocked for 2 h at 37 °C
or overnight at 4 °C, with 3% bovine serum albumin, 10 mM Tris (pH 8), 150 mM NaCl, 0.05% Tween 20. Filters were
incubated with a mouse mAb against phosphotyrosine residues, 4G10
(IgG
; UBI). As a secondary antibody, a biotinylated goat
anti-mouse antibody (Vector Laboratories) was used, and the immunoblot
was developed with an ABC amplification kit (Vector), coupled to
alkaline phosphatase, following the manufacturer's protocol. A
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate
premixed solution was used (Sigma).
Proteolytic Cleavage Analysis
Small G-protein
immunoprecipitates derived from GTP-labeled Balb cells
were washed up to six times with lysis buffer as described above. They
were desalted by washing twice with distilled water and resuspended in
50 mM ammonium acetate (pH 4.4) and treated with 3-5
µg of Staphylococcus aureus V8 endoproteinase Glu-C
(Promega) for 20 h at 37 °C. The reaction was stopped by addition
of 40 µl of 3 X sample buffer (4% SDS, 12% glycerol, 50 mM Tris, 2%
-mercaptoethanol, 0.01% Serva Blue)(36) ,
and incubating for 30 min at 40 °C. Labeled peptides resulting from
cleavage were separated electrophoretically using a high resolution
Tris-Tricine buffer SDS-PAGE system (16.5% T, 3% C, containing
glycerol)(36) . The gels were either fixed for 1 h in 50%
methanol, 10% acetic acid and dried, or transferred onto Immobilon-PVDF
membranes in a continuous buffer system without SDS (25 mM Tris, pH 8, 192 mM glycine, in 20%
methanol)(37) . Dried gels and filters were analyzed in a
PhosphorImager (Molecular Dynamics) or in a InstantImager (Packard),
respectively. In addition gels and filters were exposed to Kodak-XAR
film or Hyperfilm-MP (Amersham).
Cellular Membrane Preparation
Balb fibroblasts
were grown and rendered quiescent as described above. After PDGF-BB in vivo stimulation, cells were washed twice in cold PBS,
scraped from plates, and centrifuged at 1800 g at 4
°C for 5 min. Cells were resuspended in 1.0 ml of ice-cold TMSDE
buffer (50 mM Tris, pH 7.6, 75 mM sucrose, 6 mM MgCl
, 1 mM dithiothreitol, 1 mM
EDTA, with 10 µg/ml aprotinin, 10 µg/ml leupeptin added prior
to use) and incubated at 0 °C for 10 min. To disrupt the cells,
they were frozen at -70 °C and then thawed. Broken cells were
homogenized by shearing through a 27-gauge needle with a tuberculin
syringe, 8-10 times, on ice. The particulate suspension was
centrifuged in a Sorvall Superspeed centifuge at 20,000
g at 4 °C for 30 min, and the pellet containing membranes was
then resuspended in 0.1-0.2 ml of TMSDE supplemented with
protease inhibitors(38) . Protein concentration was quantitated
using the Bio-Rad assay standardized against a bovine serum albumin
standard curve. Membranes were stored at -70 °C until use.
[
[P]NAD-ribosylation of Balb
Cell Membrane
Proteins
P]ADP-ribosylation of Balb
cell membranes (80-200 µg of protein) by botulinum C3
ADP-ribosyltransferase was performed in a reaction buffer containing 50
mM triethanolamine-HCl, pH 7.4, 2 mM EDTA, 1
mM dithiothreitol, 0.5 mM ATP, 2.5 mM MgCl
, 10 mM thymidine, 0.2% Triton X-100,
[
P]NAD (1 µCi/tube), and 6-7 µg/ml
purified recombinant C3 exoenzyme, in a total volume of 80-200
µl, for 60 min at 37 °C(39) . Reaction was terminated
by addition of 3
Laemmli sample buffer and boiling for 5 min.
In other cases, the reaction was terminated by addition of 900 µl
of HTG lysis buffer. In this case, the membranes were incubated for 60
min at 4 °C. The lysates were cleared by a 5-min centrifugation at
13,000
g at 4 °C prior to the immunoprecipitation,
as described above. Proteins were resolved in a 12.5% SDS-PAGE gel; the
gel was transferred to Immobilon-PVDF following the procedure described
above. The radioactivity in the gels and filters were quantified in an
InstantImager (Packard). Dried gels and filters were autoradiographed
using Hyperfilm-MP (Amersham).
Affinity Labeling of GTP-binding Proteins in
Permeabilized Balb Cells
To identify potential GTP-binding proteins involved in signal
transduction through the PDGF receptor, a recently developed technique
for labeling of nucleotide binding proteins was utilized. This
technique involves the introduction of
[-
P]GTP into permeabilized cells, followed
by ``in situ'' periodate
oxidation(30, 31) . This method has recently
established that the T cell receptor
chain has the capacity to
bind GTP and GDP(31) . This technique permits specific labeling
of such different types of G-proteins as the
-subunits of the
heterotrimeric G
and G
proteins, as well as
small G-proteins such as p21
protein(27) . In the present study, this technique
was used to label GTP-binding proteins in Balb cells and Balb cells
expressing activated
(v-K
)-p21
.
Confluent cells were serum-starved and in vivo stimulated with
30 ng/ml PDGF-BBh
for 5 min at 37 °C or left untreated.
The cells were then permeabilized with 50 µg/ml LPC for 5 min on
ice. The cells were scraped from the plates into microcentrifuge tubes,
where they were GTP
-labeled after incubation with 1
µM [
-
P]GTP for 30 min at 37
°C. Using this in situ cross-linking method, we were able
to selectively label several G-proteins in the range of 21-27 kDa
with [
-
P]GTP
, as seen in
whole lysates of Balb or v-ras-expressing Balb cells, resolved
on a 15% SDS-PAGE gel (Fig. 1A).
-
P]GTP
labeling of cellular GTP-binding proteins. Confluent Balb (lanes1 and 2) and
v-ras-containing Balb (Kbalb) cells (lanes3 and 4) were serum-starved (0.5%) for 24-48 h, and in vivo stimulated with 30 ng of PDGF-BBh
/ml for 5
min at 37 °C (+), or left untreated (-). Cells were then
permeabilized with 50 µg/ml LPC for 5 min on ice. Cells were then
scraped from the plates into tubes, where they were
GTP
-labeled after incubation with 1 µM [
-
P]GTP for 30 min at 37 °C. Whole
cell lysates were then resolved by 15% SDS-PAGE. An autoradiogram of
the dried gel is shown here. The position of molecular mass markers is
indicated to the left of the figure. Using this method,
several G-proteins in the range of 21-27 kDa could be selectively
labeled with [
-
P]GTP
. B, competition of insitu affinity
labeling of GTP-binding proteins. Dog kidney epithelial cells (TRMP)
expressing wild type human PDGF type-
receptor were serum-starved
for 48 h, then in vivo stimulated with 30 ng of
PDGF-BBh
/ml for 5 min at 37 °C. The cells were then
permeabilized with LPC, and labeled with
[
-
P]GTP after 30 min at 37 °C, as
described, in the absence or presence of 100 µM cold
deoxynucleotides as competitors. The labeling reaction was inhibited by
the addition of cold dGDP or dGTP, but not dCTP or dATP. The
[
-
P]GTP
-labeled proteins were
separated by 15% SDS-PAGE after lysis of the cells. An autoradiogram of
the dried gel is shown here. The position of molecular mass markers is
indicated to the left of the
figure.
To evaluate the
specificity of the in situ labeling technique, competition
experiments were carried out using unoxidizable deoxyribonucleotides.
TRMP cells transfected with the human PDGF type- receptor gene
were used(33) . Cross-linking with
[
-
P]GTP
in permeabilized TRMP
cells was completely abolished in the presence of 100 µM cold dGTP or 100 µM cold dGDP, whereas addition of
cold dATP or dCTP did not affect labeling (Fig. 1B).
Thus, the observed 21-27-kDa GTP
-labeled bands were
the result of a specific affinity labeling, and only those proteins
that bound guanine nucleotides were labeled (Fig. 1B).
-
P]GTP
-labeling method,
Balb cells were stimulated with PDGF-BB for 2 min at 37 °C and then
permeabilized and labeled as described above (Fig. 2A).
Cell lysates were precleared up to three times with NRS coupled to
protein G-Sepharose (lane1). The precleared lysates
were independently immunoprecipitated with one of several different
specific anti-small G-protein antibodies. Lanes 3-5 show
the immunoprecipitation of GTP
-labeled RhoA, RhoB, and
Rap1A/B, precipitated with affinity-purified anti-RhoA, anti-RhoB, and
anti-Rap1A/B, respectively. The GTP
-labeled
p21
protein was immunoprecipitated from Balb
cells with mAb Y13-259 (lane6). Immunoprecipitated
[
-
P]GTP
-labeled proteins
migrated in a slightly retarded fashion during SDS-PAGE separation, due
to the cross-linking, presenting apparent molecular masses greater than
21 kDa.
-
P]GTP
. A, Balb
cells were stimulated with PDGF-BBh
for 2 min at 37 °C,
permeabilized and labeled with
[
-
P]GTP
as described in Fig. 1. The lysates were precleared with NRS-protein G-Sepharose (lane1) and immunoprecipitated with antibodies
specific for small G-proteins: affinity-purified anti-RhoA (lane3), affinity-purified anti-RhoB (lane4), affinity-purified anti-Rap1A/B (lane5), and anti-p21 mAb Y13-259 (lane6). Lane2 shows a small G-protein co-immunoprecipitated
with a specific anti-PDGF type-
receptor antibody. B,
anti-RhoA-specific immunoprecipitation of TRMP cell lysates labeled
with [
-
P]GTP
, without (lane1) or with (lanes 2-4) 100
µM competing cold deoxynucleotides. The immunoprecipitated
proteins were separated in a 15% SDS-PAGE after elution from the
Sepharose. An autoradiogram of the dried gel is shown here. The
position of molecular mass markers is indicated to the left of
the figure.
To further document the specificity of the labeling method
for known small G-proteins, the RhoA small G-protein was labeled in the
presence or absence of competing deoxynucleotides, and
immunoprecipitated by anti-RhoA antiserum. Labeling of RhoA in TRMP
cells (Fig. 2B, lane 1) was completely
abrogated by the addition of 100 µM cold dGDP (lane2) or 100 µM cold dGTP (lane3), whereas 100 µM cold dCTP (lane4) had no effect.
A Small G-protein Associates with the PDGF Type-
A specific rabbit anti-peptide serum directed against the
external region of the PDGF type-
Receptor
receptor co-precipitated a
23-27-kDa
[
-
P]GTP
-labeled protein along
with the PDGF type-
receptor from a labeled Balb cell lysate (Fig. 2A, lane2).
receptor/small G-protein interaction, the
amount of PDGF type-
receptor-associated G-proteins was compared
before and after stimulation with PDGF-BB. In cells stimulated for 2
and 5 min with PDGF-BB, there was a significant increase in the amount
of labeled G-proteins co-immunoprecipitated with the PDGF type-
receptor (Fig. 3A, compare lanes5 and 6 with lane4). In four independent
experiments, the binding of the small G-protein to the PDGF type-
receptor peaked at 2 min after PDGF-BB stimulation. Thus, there was a
rapid agonist-dependent induction of G-protein association to the PDGF
type-
receptor.
-
P]GTP
-labeled protein
associated with PDGF type-
receptor. A, stimulation with PDGF-BB increases the association of the
small G-protein with the PDGF type-
receptor in a time-dependent
manner. Confluent Balb cells were serum-starved, and in vivo stimulated with 30 ng of PDGF-BBh
/ml for 2 min (lanes2 and 5) or 5 min (lanes3 and 6) at 37 °C (+), or left
untreated(-). Cells were permeabilized with LPC for 5 min on ice
and labeled with [
-
P]GTP after 30 min at 37
°C. Lysates were precleared three times with NRS. The (nonspecific)
proteins bound to the NRS-protein A beads after the final preclearing
were eluted and the proteins were separated by 15% SDS-PAGE (lanes
1-3). Precleared lysates were immunoprecipitated with a
specific anti-PDGF type-
-receptor antiserum, eluted, and separated
in the same gel (lanes 4-6). An autoradiogram of the
dried gel is shown here. The position of molecular mass markers are
indicated to the left of the figure. Co-precipitation of
labeled small G-proteins with the PDGF type-
receptor was
observed, and an increase in the amount of the
GTP
-labeled small G-binding protein associated with the
receptor at 2 min of stimulation with PDGF-BB (lane5) was apparent. This is a representative result from at
least four separate experiments. B, association of
the small G-protein with the PDGF type-
receptor requires a
functional PDGF type-
Receptor. Dog kidney epithelial cell lines
(TRMP), untransfected (WT, lane2),
transfected with human PDGF type-
receptor gene (lanes1 and 3), or transfected with a kinase-negative
mutant PDGF type-
receptor gene (L635R) (lane4)
were used. Cells were treated as described in Fig. 2. Lane1 is the eluate from the last preclearing by NRS-protein
A-Sepharose from the TRMP transfected with human PDGF type-
receptor gene. An autoradiogram of the dried gel is shown here. The
position of molecular size markers is indicated to the left of
the figure. A GTP
-labeled small G-protein
co-immunoprecipitated with the transfected human wild type PDGF
type-
receptor (lane3). In contrast, only
minimal amounts of labeled small G-proteins co-immunoprecipitate from
lysates of the parental cell line (lane2) or from
the cell line expressing the mutant kinase-negative receptor, L635R (lane4).
To determine whether the G-protein(s)/PDGF
type- receptor interaction was dependent on the expression of a
functionally-active PDGF type-
receptor, co-immmunoprecipitation
experiments were carried out in TRMP cells transfected with human wild
type or with a kinase-negative mutant PDGF type-
receptor gene. In
the mutant receptor, a single point mutation in the ATP-binding site
(L635R) inhibits the ligand-induced autophosphorylation of the PDGF
type-
receptor(33) . In cells stimulated with PDGF-BB,
much lower levels of a labeled small G-protein were associated with the
kinase-negative PDGF type-
receptor, as compared with the wild
type human PDGF type-
receptor (Fig. 3B, compare lane4 with lane3). Note that in
wild type TRMP cells, which lack PDGF type-
receptors (but express
low levels of PDGF type-
receptors),(
)
the anti-PDGF type-
receptor antibody co-immunoprecipitated
a similar amount of small G-protein as in the PDGF type-
receptor
kinase mutant-containing cells (Fig. 3B, compare lane2 with lane4). Taken
together, these results support the hypothesis that a
functionally-active PDGF type-
receptor is required for
ligand-stimulated G-protein binding to the receptor.
-labeling technique depends largely on the
ability to exchange radiolabeled nucleotide for endogenous nucleotide
under physiological conditions in semi-intact cells. This process
requires an extended incubation time with radiolabeled GTP prior to
chemical cross-linking. It was therefore necessary to determine the
functional status of the stimulated PDGF type-
receptor at the
time when the cross-linking occurred. The autophosphorylation
capability of the PDGF type-
receptor was assessed in cells that
were stimulated with PDGF-BB in vivo for 2 min, washed, and
kept permeabilized at 37 °C for 30 min before performing the
GTP
labeling. As shown in Fig. 4(panelA), the PDGF type-
receptor was
tyrosine-phosphorylated after PDGF-BB-stimulated cells were
GTP
-labeled, whereas in unstimulated cells the PDGF
type-
receptor remained largely unphosphorylated on tyrosine
residues. As expected, there was an increase in the amount of labeled
small G-protein associated with the PDGF type-
receptor upon
PDGF-BB stimulation (Fig. 4, panelB), which
closely correlated with the PDGF-BB-induced autophosphorylation of the
PDGF type-
receptor.
receptor in GTP
-labeled cells.
Serum-starved Balb cells were stimulated in vivo with PDGF-BB (lane3), or left untreated (lanes1 and 2), permeabilized with LPC, and labeled with
[
-
P]GTP, as described in Fig. 1A. Precleared lysates were immunoprecipitated
with an anti-PDGF type-
receptor antibody (lanes2 and 3), as described in Fig. 3. Lane1 shows the third preclearing with NRS. The eluted
samples were separated on a 5-15% gradient SDS-PAGE gel. The gel
was transferred to a nitrocellulose filter. PanelA,
the upper part of the nitrocellulose filter was immunoblotted with 4G10
anti-phosphotyrosine antibody (Wb:
-pTyr). The PDGF
type-
receptor was phosphorylated on tyrosine upon ligand binding. PanelB, the lower part of the nitrocellulose filter
was exposed to XAR5 Kodak film. An increase in the amount of the
labeled G-protein associated with the PDGF type-
receptor was
observed after PDGF-BB stimulation. Molecular mass markers are shown at right.
Identification of the G-protein Associated with the PDGF
Type-
Receptor
Analysis by Two-dimensional Protein
Separation
Members of the family of Ras-like small G-proteins
possess a high degree of homology (40, 41) . A
combination of the [-
P]GTP
labeling technique and two-dimensional gel electrophoresis has
been used to identify small G-proteins in different
tissues(29) . This technique was used here to identify the
small G-protein associated with the PDGF type-
receptor.
Serum-starved Balb cells that had been stimulated for 2 min with
PDGF-BB were permeabilized and GTP
-labeled as described.
The electrophoretic mobilities of the immunoprecipitated
GTP
-labeled proteins indicated that they migrated at a pI
range between 5 and 5.6, which appears to be a slightly more acidic
range than for native forms of these proteins (Fig. 5). This
finding has also been observed by Huber and Peter(29) . A
comparison of relative migration patterns (Fig. 5, panels
A-D), indicated that the PDGF type-
receptor-associated
small G-protein had a two-dimensional profile (panelA) strikingly similar to that of the Rho family of small
G-proteins (panelC). Only some spots of
immunoprecipitated Rap1A/B (panelB) migrated at
similar positions as those of the small G-protein co-immunoprecipitated
by the anti-PDGF receptor antibody. In contrast, two-dimensional
electrophoretic profile of the p21
protein (panelD) was significantly different from that of
the PDGF type-
receptor-associated small G-protein. These results
suggest that the PDGF receptor-associated small G-protein is closely
related to the Rho family of proteins.
-labeled small G-proteins immunoprecipitating with the
PDGF type-
receptor. Balb cells were treated as described in Fig. 1. The cells were stimulated in vivo with
PDGF-BBh
(30 ng/ml) for 2 min at 37 °C.
Permeabilization and GTP labeling were carried out as described.
Postnuclear lysis proteins were precleared three times with NRS
conjugated to protein G-Sepharose before the antibody-specific
immunoprecipitations. Aliquots of the same lysates were
immunoprecipitated with: anti-PDGF type-
receptor (A),
anti-Rap1A/B (B), anti-RhoB (C), and anti-p21 Y13-259 (D). Immunoprecipitated proteins were subjected to
two-dimensional IEF/followed by a 15% SDS-PAGE. Autoradiograms of the
dried gels are shown here. The positions of molecular mass markers are
indicated to the left of the figures. The dottedcircles in the figure represent the migration of the
ovalbumin two-dimensional-IEF marker (45 kDa and a pI of 5.1). The pH
gradient after electrophoresis was linear from 4.5 to
6.5.
Proteolytic Cleavage Analysis
To further identify
the small G-protein associated with the PDGF type- receptor,
[
-
P]GTP
-labeled proteins
co-immunoprecipitating with the receptor were digested with S.
aureus endoproteinase Glu-C (V8 protease), and the resulting
peptides were separated by a high resolution Tris-Tricine SDS-PAGE
system and then transferred to an Immobilon-PVDF membrane, as described
above (Fig. 6A). Three
[
P]GTP
-labeled, well separated,
and PDGF type-
receptor-associated peptides were detected (Fig. 6A, lane2). For comparison, a
number of known, affinity-purified small
[
-
P]GTP
-labeled G-proteins
were also subjected to V8 protease digestion, and the digestion
products analyzed. The digestion patterns of GTP
-labeled
RhoA (lane4), and of PDGF-type-
receptor-associated small G-protein (lane2) were
almost identical. The digestion pattern of RhoB protein (lane5) was also very similar to RhoA (lane4). Upon V8 protease digestion, labeled Rap1A/B protein (lane3) was converted to three peptides with similar
mobilities to the ones observed in the digestion of the PDGF type-
receptor-associated G-protein, plus an extra lower band that was unique
and was not present in digests of any of the other proteins. V8
protease digestion of the p21
protein (lane6) gave a fragment with a quite distinct mobility.
Quantitative analysis of the radioactivity on the Immobilon filter is
shown in Fig. 6B. The analysis of these proteolytic
cleavage studies further demonstrated that the small G-protein
associated with the PDGF type-
receptor belongs to the Rho family
of small G-proteins.
P] GTP-
-labeled small G-proteins. A, [
-
P]GTP-labeled small
G-proteins from Balb cells were immunoprecipitated with anti-PDGF
type-
receptor antibody or with specific anti-small G-protein
antibodies, as described. Immunoprecipitates were digested with S.
aureus proteinase Glu-C V8. A small aliquot of uncleaved
immunoprecipitated PDGF receptor-associated small G-protein is shown in lane1. The V8-cleaved G proteins immunoprecipitating
with anti-PDGF receptor, anti-Rap1A/B, anti-RhoA, anti-RhoB, and
anti-Ras are shown in lanes 2-6, respectively. The
samples were separated in a high resolution Tris-Tricine gel (16.5% T,
3% C). The gel was transferred to an Immobilon-PVDF membrane. The
membrane was exposed for 1 day with an enhancing screen at -70
°C. The positions of low molecular mass markers are indicated to
the left of the figures. B, InstantImager analysis of
the membrane shown in panelA. The membrane was
counted for 18 h in an InstantImager. The figure shows a profile with
the quantitation of the radioactivity throughout each lane containing
the V8-digested peptides. The analysis was done by counting the
radioactivity through a rectangle comprising each whole lane of the
gel. The width of each rectangle was kept constant for all the lanes.
The profile is presented in counts, in arbitrary units. These
experiments were repeated at least four
times.
The Small G-protein Co-immunoprecipitated by the
Anti-PDGF Type-
The association of a Rho small G-protein with the
PDGF type- Receptor Antibody Is a Substrate for the Exoenzyme
C3 Transferase
receptor was confirmed by an alternative strategy. In
these experiments, purified recombinant C3 transferase was utilized to
ADP-ribosylate Rho proteins in Balb cell membranes. The exoenzyme C3
transferase from C. botulinum specifically ADP-ribosylates Rho
proteins on amino acid Asn-41, which is located in the putative
effector domain(42, 43) . The members of Rho subfamily
are the major substrates for the ADP-ribosylation by C. botulinum ADP-ribosyltransferase C3 exoenzyme. In contrast, the members of
the Rac subfamily are very poor substrates for the C3
transferase(44, 45, 46) .
P]ADP-ribosylated, demonstrating the presence
of Rho proteins in Balb cells (Fig. 7, panel A, lane2; and panel B, lane1). This was confirmed through direct immunoprecipitation
of RhoA and RhoB by specific antibodies (Fig. 7B, lanes3 and 4). As suggested by the amount
of radioactivity incorporated, RhoA was the predominant C3 substrate in
Balb membranes (lane3). An ADP-ribosylated
p21-protein was co-immunoprecipitated by the anti-PDGF type-
receptor antibody. Quantitative analysis indicated 3-fold more
radioactivity in anti-PDGF type-
receptor immunoprecipitates than
in the anti-p21
or NRS immunoprecipitates used
as negative controls (Fig. 7B, compare lane2 with lanes5 and 6).
Moreover, the amount of [
P]-ADP-ribosylated
protein in PDGF type-
receptor immunoprecipitates represented
10-25% of the [
P]ADP-ribosylated proteins
immunoprecipitated by the anti-RhoA and anti-RhoB antibodies,
respectively.
P]ADP-ribosylated protein in presence (+)
or in absence(-) of 6-7 µg of C3 transferase/ml. In panelB, membranes (100 µg of protein/reaction)
were prepared from serum-starved Balb cells that had been stimulated
for 2 min with PDGF-BB. The ribosylation reactions were stopped by
lysing the membranes with HTG buffer, as described under
``Materials and Methods.'' The precleared lysates were
independently immunoprecipitated with anti-PDGF type-
receptor or
with several specific anti-small G-protein antibodies: anti-PDGF
type-
receptor (lane2), anti-RhoA (lane3), anti-RhoB (lane4), anti-Ras (lane5), and NRS (lane6). Lane1 shows the ribosylated material in 20 µg of
membrane protein. An ADP-ribosylated small G-protein was
co-immunoprecipitated by the anti-PDGF type-
receptor antibody (lane2). ADP-ribosylated RhoA is the predominant C3
substrate in Balb cell membranes (lane3), followed
by RhoB (lane4), which migrated as a double band.
p21 protein was not ribosylated (lane5).
These results, together with the data on V8 digestion
and two-dimensional IEF/SDS-PAGE analysis, confirmed that a small G
protein of the Rho family is associated with the PDGF-BB-stimulated
PDGF type- receptor in Balb fibroblasts.
receptor in response to PDGF-BB
stimulation. Two-dimensional electrophoresis and proteolytic cleavage
analysis indicated that the small G-protein associated with the PDGF
type-
receptor is a member of the Rho family of G-proteins. These
results were confirmed by demonstrating that the small G-protein
co-immunoprecipitated by the anti-PDGF receptor antibody was a
substrate for the ADP-ribosyltransferase C3 exoenzyme, which
specifically ADP-ribosylates Rho proteins(45) . Thus, the PDGF
type-
receptor may form a complex with small G-proteins upon
binding PDGF-BB, and the Rho G-protein is likely to be an important
component of the proteins making up the multimeric signaling complex of
the PDGF type-
receptor.
receptor is of sufficient affinity as to allow co-immunoprecipitation
with an unstimulated receptor. Thus, the association appears to differ
substantially from the chain of adaptor proteins involved in the
transmission of signals from the PDGF type-
receptor to
p21
. There was a significant increase in the
amount of GTP
-labeled Rho small G-protein associated with
the PDGF type-
receptor upon ligand binding, and this increase
correlated with PDGF type-
receptor tyrosine autophosphorylation.
This agonist-induced association also appeared to require a
``functional'' receptor. The Rho protein did not undergo
ligand-induced association with a mutant receptor protein that was
unable to bind ATP. This mutant was tyrosine kinase-negative, but this
result does not necessarily imply that receptor tyrosine kinase
activity is required for Rho association. In addition to being
kinase-deficient, this same mutant receptor does not undergo
conformation changes or dimerization in response to PDGF-BB binding,
and either of these two events, rather than tyrosine kinase activity,
may instead be required for Rho association.
receptor may reflect a change in the rate of
GTP-GDP exchange rate, with a resulting change in the ability of the
Rho to be cross-linked by the GTP
-labeling technique, as
well as a change in the amount of Rho protein bound. This alternative
possibility for PDGF-induced signaling through Rho is under
investigation.
receptor? Biochemical and genetic
data implicate p21
in growth factor-stimulated
pathways involving tyrosine and serine/threonine kinases. One of the
targets or mediators of p21
in these cascades
appears to be
Raf-1(63, 64, 65, 66, 67, 68, 69, 70, 71, 72) .
The biochemical targets of the Rho family of small G-proteins, however,
remain largely uncharacterized. There is now evidence that a
genistein-sensitive tyrosine kinase is required in the Rho-mediated
signal transduction pathways involved in thrombin-induced changes in
cytoskeleton(45) . It has been shown in Swiss-3T3 cells and in
human platelet cytosolic extracts that activation of
phosphatidylinositol 3-kinase is dependent on
Rho(73, 74) . In addition, in rat liver membranes,
RhoA protein has a role in the activation of the membrane-associated
phospholipase D(75) , although no evidence exists for a direct
interaction between phospholipase D and Rho proteins. Recently, others
have identified a serine/threonine kinase, STE 20, as a target of Cdc42
and Rac1(76) . This in vitro study demonstrated that
this kinase complexes specifically with the activated (or GTP-bound)
p21, inhibiting p21GTPase activity and leading to kinase
autophosphorylation and activation. The resulting autophosphorylated
kinase had a decreased activity for Cdc42/Rac1, freeing the p21 for
further stimulatory activities or for down-regulation by
GTPase-activating proteins. Thus, there may be as-yet-unexplored
regulatory interactions between the PDGF type-
receptor and the
Rho small G-protein family, analogous to the link between the PDGF
type-
receptor and p21
.
, human recombinant platelet-derived growth
factor-BB; mAb, monoclonal antibody; NRS, normal rabbit serum; LPC, L-
-lysophosphatidylcholine; PVDF, polyvinylidene
difluoride; GDS, guanine dissociation stimulator; Tricine, N-tris(hydroxymethyl)methylglycine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.