(Received for publication, July 12, 1995)
From the
Rho, a member of the Ras superfamily of GTP-binding proteins,
regulates actin polymerization resulting in the formation of stress
fibers and the assembly of focal adhesions. In Swiss 3T3 cells,
heterotrimeric G protein-coupled receptors for lysophosphatidic acid
and gastrin releasing peptide stimulate Rho-dependent stress fiber and
focal adhesion formation. The specific heterotrimeric G protein
subunits mediating Rho-dependent stress fiber and focal adhesion
formation have not been defined previously. We have expressed
GTPase-deficient, constitutively activated G protein subunits and
mixtures of
and
subunits in Swiss 3T3 cells. Measurement of
actin polymerization and focal adhesion formation indicated that
GTPase-deficient
and
, but not the
activated forms of
or
stimulated
stress fiber and focal adhesion assembly. Combinations of
and
subunits were unable to stimulate stress fiber or focal adhesion
formation. G
- and
-mediated stress
fiber and focal adhesion assembly was inhibited by botulinum C3
exoenzyme, which ADP-ribosylates and inactivates Rho, indicating that
and
, but not other G protein
subunits or
complexes, regulate Rho-dependent
responses. The results define the integration of G
and
G
with the regulation of the actin cytoskeleton.
The heterotrimeric G protein-coupled receptors for
lysophosphatidic acid (LPA), ()gastrin releasing peptide
(GRP or bombesin), and thrombin are capable of stimulating the growth
of specific cell types(1, 2, 3) . LPA and
thrombin receptors have been shown to stimulate Ras GTP loading which
is required for the mitogenic response to these
ligands(4, 5) . Ras GTP loading in response to LPA and
thrombin activation of their receptors requires the heterotrimeric G
proteins G
or
G
(5, 6, 7) . GRP receptors are
mitogenic in Swiss 3T3 cells, lung epithelia, and small cell lung
carcinoma cells(2, 9) ; GRP receptors activate G
and phospholipase C activity but stimulate little or no GTP
loading of Ras(10) . In addition to the stimulation of
mitogenic responses, the LPA, GRP, and thrombin receptors regulate the
polymerization of actin to produce stress fibers and the assembly of
focal adhesions(11) . The stimulation of stress fiber and focal
adhesion assembly has been demonstrated to be regulated by
Rho(12, 13) .
The polymerization of actin and the
actin cytoskeleton is important for cell shape and regulatory responses
including chemotaxis and mitogenesis. In quiescent Swiss 3T3 cells, the
formation of stress fibers generally parallels the assembly of focal
adhesions. Focal adhesions are oligomeric protein complexes that
include p125, paxillin, talin,
-actinin, vinculin,
and other proteins(14, 15) . Focal adhesions link
actin stress fibers to integrins at the inner surface of the plasma
membrane. How Rho regulates the assembly of focal adhesions and the
formation of stress fibers is currently ill-defined. Similarly, the
heterotrimeric G proteins that couple LPA, GRP, and thrombin receptors
to Rho activation have not been defined. Pertussis toxin does not
inhibit receptor-stimulated actin polymerization or focal adhesion
assembly, indicating that neither G
nor G
mediates these responses(16) . Similarly, calcium
ionophores and phorbol esters do not stimulate Rho-dependent responses
suggesting that G
activation of phospholipase C is not
involved(16) .
To define which heterotrimeric G protein
subunits were involved in stimulating Rho-dependent actin
polymerization to produce stress fibers and focal adhesion assembly, we
microinjected expression plasmids encoding GTPase-deficient mutant
heterotrimeric G protein subunits into the nuclei of
serum-starved, quiescent Swiss 3T3 cells. For each G protein
subunit, the conserved glutamine (Q) adjacent to the G3 sequence of the
GDP/GTP binding domain of the polypeptide was mutated to a leucine (L).
The Gln
Leu mutation functionally inhibits the GTPase activity
of the polypeptide resulting in a constitutively activated G protein
subunit(17, 18) . This mutation corresponds to
residue 229 in
and 226 in
. In
addition, different combinations of
and
subunits were
microinjected. Each expression plasmid used in the experiments was
characterized previously for functional expression (19, 20, 21) . Following microinjection,
cells were stained with rhodamine-phalloidin to identify stress fibers
or anti-vinculin antibody for the identification of focal adhesions.
Microinjected cells were marked by coinjection of G protein subunit
expression plasmids with a plasmid encoding
-galactosidase.
For microinjection, Swiss 3T3 cells were plated at
approximately 10% confluency on acid-washed glass coverslips in
Dulbecco's modified Eagle's medium (DMEM) with 5% bovine
calf serum (BCS) and 5% newborn calf serum (NCS). The next day, cells
were rinsed three times and placed in 0.1% BCS/DMEM. Twenty-four h
later, cells were rinsed three times in DMEM in the absence of serum
and incubated for an additional 18 h before microinjection. Injections
were performed with an Eppendorf automated microinjection system with
needles pulled from glass capillaries on a vertical pipette puller
(Kopf, Tujunga, CA). The cDNAs used for expression were inserted in
either pCMV5 or pCDNA3 and have been characterized for functional
expression. All plasmids were prepared by cesium chloride gradient
centrifugation and used at 100 ng/µl for microinjection into the
nuclei of Swiss 3T3 cells. The botulinum C3 exoenzyme pGEX2T vector was
a gift from Drs. S. Dillon and L. Feig, Tufts Medical School, Boston,
MA. The C3 fusion protein was induced, cleaved with thrombin, and
purified as described (22) . The pCMV5RhoAQL plasmid was a gift
from Dr. Sim Winitz, Scripps Research Foundation, La Jolla, CA. Two- to
three-h postnuclear injection cells were fixed in 3% paraformaldehyde
for 10 min. Cells were rinsed in phosphate-buffered saline and
permeabilized using 0.2% Triton X-100 for 5 min. The fixed and
permeabilized cells were then incubated with DMEM/5% BCS/5% NCS for 15
min. -Galactosidase was stained using a rabbit
anti-
-galactosidase antibody (Cappel) and a secondary
FITC-conjugated donkey anti-rabbit antibody (Pierce).
Rhodamine-phalloidin (Molecular Probes) was used to label F-actin.
Focal adhesions were stained using a mouse monoclonal anti-vinculin
antibody (Sigma) and a secondary FITC-conjugated sheep anti-mouse
(Cappel) antibody. When cells were stained for vinculin, a
rhodamine-conjugated goat anti-rabbit antibody (Cappel) was used for
detection of
-galactosidase. Coverslips were mounted on slides and
examined with a Nikon Diaphot TMD microscope with eipfluorescence.
Images of cells were captured using the IPLAB Spectrum digital image
analysis program (Signal Analytics Co., Vienna, VA). All experiments
were done at least 3 times with similar results.
Fig. 1shows that constitutively activated RhoA having
glutamine 63 mutated to leucine (RhoQL), which corresponds to the Q61L
mutation in p21, induces stress fiber formation
in Swiss 3T3 cells. Strikingly, the Gln
Leu mutant
subunits for G
(
QL) and G
(
QL), when expressed in Swiss 3T3 cells,
mimicked activated RhoQL in stimulating the formation of stress fibers.
The activated forms of
and
were
capable of stimulating stress fiber formation in quiescent Swiss 3T3
cells. Activated, GTPase-deficient forms of
(
QL) or
(
QL) were
unable to induce stress fiber formation (Fig. 2). Expression of
QL appears to disorder stress fibers and cause a loss
of cortical actin along the cytoplasmic surface of the plasma membrane.
Expression of
or
also did not induce stress fiber
formation in Swiss 3T3 cells. Similarly, neither
nor
had any effect on the actin network when injected into Swiss 3T3
cells (not shown). In addition, treatment of quiescent Swiss 3T3 cells
with forskolin to stimulate cAMP synthesis, phorbol esters to activate
protein kinase C, and ionomycin to elevate intracellular calcium does
not induce stress fiber formation(16) . Our results demonstrate
that
and
, but not other G protein
subunits or second messengers, stimulate stress fiber formation.
Figure 1:
Activated, GTPase-deficient mutants of
RhoA, , and
stimulate stress fiber
formation. Quiescent Swiss 3T3 cells were nuclear microinjected with
pCMV
gal in the presence of either pCMVRhoQL,
pCDNA3
QL, or pCDNA3
QL.
Microinjected cells were detected by indirect immunofluorescent
staining of
-galactosidase. Immunostaining of cells injected with
QL and
QL with specific C-terminal
peptide antisera showed expression of both constructs in injected cells
(not shown). Stress fibers were monitored using rhodamine-phalloidin.
Uninjected cells in the field that are negative for
-galactosidase
staining show few stress fibers, whereas cells expressing activated
Rho,
, or
show similar increased
levels of stress fibers.
Figure 2:
Stress fiber formation is specific for
G and G
. Swiss 3T3 cells were
nuclear microinjected with pCMV
gal in the presence of expression
plasmids encoding inserts for
QL,
QL,
QL,
and
, or
and
cDNAs.
Each expression plasmid was characterized to demonstrate functional
expression of the specific G protein
subunit in transfection
experiments (not shown). Microinjected cells were detected by staining
for
-galactosidase.
Expression of RhoQL in quiescent Swiss 3T3 cells also stimulates
focal adhesion assembly (Fig. 3). Microinjection and expression
of QL and
QL mimicked RhoQL in
stimulating focal adhesion assembly, as measured by the localization of
vinculin, at the leading edge of cells. Thus, activated forms of
and
regulate the polymerization
of actin and the assembly of focal adhesions similar to that observed
with RhoQL.
Figure 3:
Activated and
stimulate focal adhesion assembly. Swiss 3T3 cells
were nuclear microinjected with expression plasmids encoding RhoQL,
QL, or
QL in the presence of
pCMV
gal. Cells were then fixed and stained with an anti-vinculin
antibody to detect focal adhesions. Injected cells were detected by
staining for
-galactosidase.
To demonstrate that the action of QL
and
QL were Rho-dependent, the cells were injected
with recombinant, purified botulinum C3 exoenzyme. Botulinum C3
exoenzyme has been shown to catalyze the ADP-ribosylation of Asn-41 in
the Rho polypeptide resulting in the inhibition of Rho
activity(23, 24) . Fig. 4shows that
microinjection of botulinum C3 exoenzyme inhibits LPA-stimulated stress
fiber formation. To demonstrate that the botulinum C3 exoenzyme was
selectively inhibiting Rho-dependent effects on the actin cytoskeleton,
cells were also stimulated with platelet-derived growth factor (PDGF).
PDGF has been shown to stimulate Rac1-dependent actin polymerization
that is associated with membrane ruffling(25, 26) ;
PDGF-stimulated membrane ruffling is unaffected by microinjection of
botulinum C3 exoenzyme (Fig. 4, middle panel).
Microinjection of botulinum C3 exoenzyme inhibited both
QL- and
QL-stimulated stress fiber
formation (Fig. 5) similar to the inhibition of LPA-stimulated
actin polymerization (Fig. 4). As predicted, botulinum C3
exoenzyme also inhibited LPA- and
QL-stimulated focal
adhesion formation (Fig. 6). In other experiments, the botulinum
C3 exoenzyme also inhibited
QL-stimulated focal
adhesion assembly (not shown).
Figure 4:
Botulinum C3 exoenzyme inhibition of
Rho-dependent actin polymerization. Cells were microinjected with
pCMVgal and purified botulinum C3 exoenzyme (100 ng/µl). After
2-3 h, cells were stimulated for 10 min with either LPA (200
ng/ml), PDGF (3 ng/ml), or buffer only (Control). Cells were fixed and
microinjected cells detected by staining for
-galactosidase.
LPA-stimulated stress fiber formation is observed in uninjected cells,
whereas stress fiber formation was inhibited by botulinum C3 exoenzyme
in the injected cells. PDGF-stimulated membrane ruffling was unaffected
in the botulinum C3 exoenzyme injected
cells.
Figure 5:
Botulinum C3 exoenzyme inhibits activated
- and
-stimulated stress fiber
formation. Swiss 3T3 cells were microinjected with pCMV
gal and
QL- or
QL-encoded expression
plasmids in the presence or absence of botulinum C3 exoenzyme (100
ng/µl). Microinjected cells were detected by staining for
-galactosidase.
Figure 6:
Botulinum C3 exoenzyme inhibits LPA- and
QL-stimulated focal adhesion assembly. Swiss 3T3
cells were microinjected with pCMV
gal in the presence or absence
of pCDNA3
QL and botulinum C3 exoenzyme (100
ng/µl). The indicated panel of cells was stimulated for 10 min with
LPA (200 ng/ml). Injected cells were detected by staining for
-galactosidase and focal adhesions monitored by staining for
vinculin. Uninjected cells in the upper panel responded to LPA
with the assembly of focal adhesions while focal adhesions are absent
in the botulinum C3 exoenzyme injected cell. Botulinum C3 exoenzyme
also inhibited focal adhesion assembly in response to
QL. Similar results were obtained for
QL (not shown).
These studies clearly demonstrate
that and
regulate Rho-dependent
actin polymerization resulting in stress fiber formation and the
assembly of focal adhesions. G
and G
have
been shown previously to interact with the thrombin
receptor(27) , and G
was shown to couple to the
bradykinin receptor(28) , two receptors that stimulate actin
polymerization responses in Swiss 3T3 cells. Both G
and
subunits are expressed in Swiss 3T3 cells
as determined by immunoblotting (not shown). Our results suggest that
and
behave similarly in their
ability to stimulate Rho-dependent stress fiber formation and focal
adhesion assembly. This finding indicates that
and
probably interact with a common effector regulating
Rho activation. Whether this effector is a dbl- or lbc-like Rho
exchange factor (29, 30) or a Rho GDI or GDS protein
characterized by Takai and co-workers (31, 32) is
presently unclear. Nonetheless, the results clearly demonstrate that
and
integrate heterotrimeric G
protein-coupled receptors with the regulation of Rho. Thus, it is
becoming increasingly apparent that specific G protein subunits
differentially regulate the activation of low molecular weight
GTP-binding proteins of the Ras and Rho
families(33, 34) . The Ras and Rho superfamily of low
molecular weight GTP-binding proteins are key regulators of major
phenotypic responses of cells including growth, apoptosis, chemotaxis,
and cell shape. The ability of seven transmembrane receptors to couple
to specific G proteins determines the ability of receptor agonists to
regulate the responses controlled by different low molecular weight
GTP-binding proteins. G
- and G
-coupled
receptors will function to regulate specific actin cytoskeleton
responses. Activated Rho exchange factors have been shown to alter the
growth and to transform NIH3T3 cells(29) . It is probable that
the ability of
and
to alter the
growth and to transform specific cell types is related in part to the
regulation of Rho and the downstream functions controlled by Rho. It
will be interesting to determine if regulatory events such as
activation of Na
/H
antiporter
activity by
and
(35, 36) involves Rho-dependent pathways as
well.