From the Department of Pharmacology, University of
California, San Diego, La Jolla, California 92093-0636, the
** Department of Microbiology, University of Illinois, Urbana, Illinois
61801, and the
Department of Physiology,
Tufts University School of Medicine, Boston, Massachusetts 02111
Received for publication, October 2, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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Recent reports have shown that several
heterotrimeric protein-coupled receptors that signal through
G Heterotrimeric G proteins are activated by exchange of GDP for GTP
in response to agonist occupation of G protein-coupled receptors
(GPCRs).1 In contrast, low molecular weight or small
G proteins are activated by guanine
nucleotide exchange factors (GEFs). Until fairly recently there was no
direct evidence or molecular mechanism for GPCR agonists activating
small G proteins. Pathways for activation of the small G protein Ras in
response to receptor tyrosine kinases and GPCRs have now been clearly
delineated (reviewed in Ref. 1). In addition, the observation that GPCR
agonists such as thrombin, bombesin, and lysophosphatidic acid (LPA)
activate cytoskeletal responses through pathways involving the small G
protein Rho (2) has now been explored mechanistically (3-9). Several
lines of evidence suggest that GPCR-stimulated,
Rho-dependent responses are mediated through activation of
the heterotrimeric G proteins G12 and G13 (reviewed in Refs. 10 and 11), which in turn associate with and may
activate Rho GEFs (12, 13).
The Rho family of small G proteins mediate numerous cytoskeletal
responses, including stress fiber formation, focal adhesion assembly,
and neurite retraction. In addition, many Rho family small G proteins
have been demonstrated to activate downstream kinases and to stimulate
gene transcription (14-17). Studies using the c-fos
promoter demonstrate that Rho can activate transcription through the
serum response element (SRE), independent of effects on the ternary
complex factor (16). The ternary complex factor-independent SRE
(SRE.L) has thus been frequently used as a readout for Rho-mediated transcriptional responses. The Rho effector which links Rho to the SRE
has not yet been identified; however several lines of evidence indicate
that Rho kinase is not required (18-20) and that protein kinase N (21)
or protein kinase C (PKC)-related serine/threonine kinase 2 (22) may
contribute significantly to this response.
Most of the GPCRs that have been shown to activate
Rho-dependent cytoskeletal rearrangements, gene expression,
or cellular contractility can couple to G It has been proposed that G The studies reported here were initiated to elucidate the mechanism by
which G Plasmids--
Plasmids encoding the G Cell Culture and Transfection--
COS-7 cells were maintained
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) in a 37 °C, humidified incubator with 10% CO2.
One day prior to transfection cells were set at 4 × 104 cells/ml. Cells were transfected overnight by calcium
phosphate precipitation, then washed and incubated in serum-free DMEM
supplemented with 1 mg/ml fatty acid-free bovine serum albumin,
penicillin, and streptomycin. 1321N1 cells were maintained in DMEM
supplemented with 5% fetal calf serum, penicillin (100 units/ml), and
streptomycin (100 µg/ml) in a 37 °C, humidified incubator with
10% CO2. For microinjection the cells were set onto 12-mm
round glass coverslips at a density of ~1.0 × 104
cells/slip. Where indicated, cells were incubated with recombinant Pasteurella multocida toxin (PMT) (37) for 24 h at
doses of 100-200 ng/ml serum-free medium. The Rho kinase
inhibitor Y-27632 was provided by Welfide Corp. (Hirakata-shi, Osaka,
Japan) and was used at 10 µM for 24 h before the
luciferase harvest. Phorbol 12-myristate 13-acetate (PMA) and
bisindoylmaleimide (GF109203X) were purchased from Calbiochem and used
at 100 mM and 3 µM, respectively, for 24 h prior to harvesting luciferase.
Coprecipitations--
24-48 h after transfection of COS-7 cells
with epitope-tagged Lbc, or p115RhoGEF constructs (or vector) and G Rho Activation Assay--
GST-Rhotekin RBD was produced in
Escherichia coli (DH5
4-24 h after transfection cells were rinsed with Tris-buffered saline
and lysed in buffer containing: 50 mM Tris HCl, pH 7.4, 10% glycerol, 0.1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 100 mM NaCl, 5 mM MgCl2, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin. The lysates were clarified by brief centrifugation and then
incubated with the Sepharose-bound GST-Rhotekin-RBD for 45 min at
4 °C. The beads and precipitated proteins were washed four times in cold lysis buffer and then the proteins were eluted by boiling in
Laemmli buffer and separated by SDS-PAGE. Proteins were transferred to
Immobilon-P membranes (Millipore) and immunoblotted with Rho-specific antibodies (Santa Cruz Biotechnology) followed by detection with horseradish peroxidase-conjugated secondary antibodies. Changes in Rho
were quantified using an LKB laser densitometer. The precipitated Rho
was normalized to the Rho present in whole cell lysate.
SRE.L-mediated Gene Expression--
COS-7 cells were set onto
six-well plates and transfected with the plasmids of interest or the
appropriate vectors along with the SRE.L-luciferase reporter plasmid.
24-48 h after transfection luciferase expression was assessed as
described previously (38). In selected experiments cells were
cotransfected with a plasmid encoding the GFP under a constitutive
promoter, to allow normalization of transfection efficiency and cell
viability. A portion of cell lysate was reserved, and fluorescent
emission from these samples was read in a fluorescence plate reader
(CTI Data model 7600).
Microinjection and Morphology--
1321N1 cells were set on
glass coverslips and microinjected as described in Majumdar et
al. (30). Injected cells were detected by expression of nuclear
GFP and actin morphology assessed by staining with rhodamine-conjugated
phalloidin (Molecular Probes).
Inositol Phosphate Accumulation--
After calcium phosphate
transfection, COS-7 cells were maintained in serum-free medium. 10 h after the transfection reagents were washed out, 1-2 µCi of
myo[3H]inositol/ml was added, and the cells were allowed
to label overnight. The cells were washed extensively to remove
unincorporated myo[3H]inositol and then incubated with 10 mM LiCl for 30 min. Reactions were terminated by the
addition of 10% trichloroacetic acid. The acidic lysates were
neutralized by ether extraction. Total [3H]inositol
(poly)phosphates were eluted by formate Dowex anion exchange
chromatography with 1 M NH4COOH, 0.1 M HCOOH.
G G
Expression of constitutively active G G
To determine whether activation of endogenous G
The Rho dependence of G Activated G G
Although many Rho effectors have been identified, effectors mediating
Rho-dependent SRE.L activation have not been clearly delineated. To determine whether Rho kinase activity is required for
the SRE.L response to G Synergy between G
To further test this possibility we examined the response to
G
One downstream consequence of PLC activation is increased activity of
PKC. PKC has been shown to increase transcription from the activator
protein-1 (AP-1) response element. To determine whether stimulation of
the SRE.L by G Many Gq-coupled receptors have been suggested to
signal through Rho-dependent pathways, yet no signaling
pathway linking G Two assays demonstrated functional interactions between
G Induction of the SRE.L by Lbc was blocked by coexpression of C3
exoenzyme. A less expected observation was that C3 exoenzyme inhibited
the effect of G The other functional assay used to look for G In experiments designed to detect interactions of G We tested the possibility that the G There is evidence in other systems that Rho mediates responses to
G The physical interaction between G The finding that the activated form of G Since most GPCRs thought to induce Rho activation by coupling to
Gq can induce Rho-dependent responses,
but the pathways that mediate the interaction between G
q
and Rho have not yet been identified. In this report we present evidence that G
q expressed in COS-7 cells coprecipitates
with the Rho guanine nucleotide exchange factor (GEF) Lbc. Furthermore, G
q expression enhances Rho-dependent
responses. Coexpressed G
q and Lbc have a synergistic
effect on the Rho-dependent rounding of 1321N1 astrocytoma
cells. In addition, serum response factor-dependent gene
expression, as assessed by the SRE.L reporter gene, is synergistically activated by G
q and Rho GEFs. The synergistic effect of
G
q on this response is inhibited by C3 exoenzyme and
requires phospholipase C activation. Surprisingly, expression of
G
q, in contrast to that of G
12 and
G
13, does not increase the amount of activated Rho. We
also observe that G
q enhances SRE.L stimulation by
activated Rho, indicating that the effect of G
q occurs
downstream of Rho activation. Thus, G
q interacts
physically and/or functionally with Rho GEFs; however this does not
appear to lead to or result from increased activation of Rho. We
suggest that G
q-generated signals enhance responses
downstream of Rho activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q and thereby
activate phospholipase C (PLC) and PKC (9, 23, 24). Several reports
indicate that activation of G
q and its downstream
effectors is insufficient or unnecessary to induce Rho-mediated signals
(3, 9, 25-29). For example, LPA and thrombin induce Rho-mediated
responses in G
q/11-deficient cells (9, 23). On the other
hand, the
1-adrenergic, M1 muscarinic, and
metabotropic glutamate receptors were reported to require
G
q/11 to induce Rho-mediated changes in SRE.L-reporter gene expression (23, 24). In addition, some studies have shown that
expression of activated mutants of G
q leads to
Rho-mediated responses such as cytoskeletal rearrangements (30, 31),
transcriptional activation of the atrial natriuretic factor gene (24),
and SRE.L response element activation (23, 32). These studies suggest that stimulation of G
q, like G
12/13,
might result in Rho activation. There are, however, no published
studies demonstrating that G
q signaling activates Rho or
examining how G
q pathways regulate Rho-dependent responses.
12/13-coupled receptors
activate Rho via interaction of the G
subunit with Rho-specific GEFs such as p115RhoGEF (13, 30, 32; also see Refs. 10 or 11 for review).
Two Rho GEFs, p115RhoGEF and PDZ-RhoGEF, were shown to coprecipitate
with G
12/13 (12, 13, 33). Furthermore, the exchange
activity of purified p115RhoGEF was activated by G
13 (12), and downstream signaling to cytoskeletal and transcriptional responses was enhanced by coexpression of G
13 with
p115RhoGEF (32). In addition, catalytically inactive mutants of
p115RhoGEF (32) and PDZ-RhoGEF (13) have been shown to attenuate SRE.L responses due to G
12 and G
13, thrombin,
or LPA. Likewise, the catalytically inactive mutants of the Rho GEFs
lymphoid blast crisis (Lbc) and p115RhoGEF blocked the cytoskeletal
response induced by G
12 or thrombin (30). These findings
support a model in which the
subunits of G proteins interact with
Rho GEFs to activate Rho-mediated signaling pathways.
q signaling stimulates Rho-dependent
responses. Based on the findings of direct interaction of
G
12/13 with Rho GEFs, we hypothesized that
G
q might also interact with Rho GEFs. In this report we
demonstrate both a physical and a functional interaction between
G
q and the Rho GEF Lbc. Surprisingly, we report that
these interactions do not lead to or result from increased activation
of Rho. Rather, the interaction of G
q with
Rho-dependent pathways requires generation of PLC-mediated
second messengers and occurs downstream of Rho activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q,
G
12, and G
13 subunits were the generous
gifts of M. Simon and J. Exton. The G
sRC plasmid was provided by M. Farquar. The SRE.L-luciferase reporter plasmid was
provided by K. Kaibuchi. The plasmid encoding Green Lantern green
fluorescent protein (GFP), which was used to normalize transfections, was acquired from Life Technologies, Inc. The nuclear GFP plasmid used
in microinjection studies was a gift from G. Wahl. The cDNA plasmids for onco- and proto-Lbc are as described previously (34). Proto-Lbc was c-Myc-tagged by subcloning the proto-Lbc cDNA
into the pJ3M vector which contains a 5' c-Myc tag. The
resultant cDNA construct was subcloned into pMT3 for efficient
mammalian expression. The c-Myc-tagged p115RhoGEF was provided by M. Hart. L. Heasley kindly provided the PLC
CT (35), which we subcloned
into the pBJ mammalian expression vector using standard techniques.
pGEX-2T vector encoding a glutathione S-transferase (GST)
fusion protein containing amino acids 7-89 of Rhotekin, which is the
Rho-binding domain (RBD), was kindly provided by M. Schwartz (36).
cDNA plasmids, the cells were rinsed with phosphate-buffered saline
and then lysed in immunoprecipitation buffer containing: 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 5 mM CaCl2,
0.7% Triton X-100, 1 mM dithiothreitol, 1 mM
p-nitrophenyl phosphate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 7.5 µg/ml
aprotinin. Anti-FLAG M2 monoclonal (Sigma) or anti-c-Myc (Santa Cruz
Biotechnology) and protein G-Sepharose beads (Amersham Pharmacia
Biotechnology) were used to precipitate the epitope-tagged Lbc or
p115RhoGEF and associated proteins. The precipitates were washed four
times and then boiled in Laemmli sample buffer to elute, and the
resultant samples were separated by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE). Proteins were
electrophoretically transferred to Immobilon-P membranes (Millipore),
then immunoblotted with G
-specific antibodies (anti-G
q, G
12 and G
13 from
Santa Cruz Biotechnology; anti-G
q from Upstate Biotechnology).
strain) transformed with
pGEX-2T-Rhotekin RBD. Bacterial cultures were grown to
A600 = 0.6 and then induced with 0.1 mM isopropyl-
-D-thiogalactopyranoside overnight at room temperature. The bacteria were harvested in lysis
buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 10% glycerol)
containing protease inhibitors and were lysed by sonication (60 × 1 s on ice). After centrifugation (15 min @ 12,000 × g), the GST-Rhotekin-RBD was collected from the clarified
supernatant by rocking at 4 °C for 45 min with glutathione-Sepharose 4B (Amersham Pharmacia Biotech). The Sepharose was washed three times
with assay buffer, resuspended in fresh buffer, and aliquots snap-frozen in liquid N2 for future use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q Coimmunoprecipitates with Lbc but Not
p115RhoGEF--
To determine whether the G
q protein
associates with Rho GEFs, we cotransfected COS-7 cells with Myc-tagged
proto-Lbc or p115RhoGEF and constitutively active forms of either
G
q, G
12, or G
13. Analysis
of whole cell lysates by Western blotting with antibodies to specific
G
subunits confirmed that G
q, G
12, and
G
13 were overexpressed in the transfected cells (Fig.
1A). The Rho GEFs were
immunoprecipitated with anti-Myc antibodies and the precipitated proteins analyzed by Western blotting. Lbc or p115RhoGEF was present in
the immunoprecipitates from all samples transfected with the corresponding cDNA (Fig. 1B). Immunoblotting with
antibodies to specific G
proteins demonstrated that
G
q was present in Lbc precipitates (Fig. 1C).
In contrast, the levels of G
q detected in p115
precipitates were not significantly above that of cells transfected
with control vector in most experiments. Activated G
12
and G
13, were detected at similar levels in either Lbc
or p115RhoGEF immunoprecipitates. To further confirm the specificity of
these coprecipitations, we examined the ability of G
s to
coprecipitate with Lbc. Although the overexpressed G
s
was detected in transfected COS-7 cells, it did not coprecipitate
with Lbc (Fig. 2).
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Fig. 1.
Selective precipitation of
G q with Lbc. COS-7 cells were
transfected with empty vector, plasmid encoding Myc-tagged proto-Lbc,
or plasmid for Myc-tagged p115RhoGEF and with empty vector or plasmid
encoding the GTPase-deficient G
subunit of either G
q,
G
12, or G
13. A, whole cell
lysates from the transfected COS-7 cells were prepared and Western
blotted for the appropriate G
subunit, to ensure protein expression.
B, cell extracts were immunoprecipitated (IP)
with anti-Myc (9E10 mouse monoclonal) antibody and then Western blotted
with c-Myc antibody to visualize the epitope-tagged proto-Lbc and
p115RhoGEF. C, immunoprecipitates, as in B, were blotted
with antibodies against the appropriate G
subunit. Blots are
representative of two to three experiments, each performed in
duplicate.
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Fig. 2.
G s does
not coprecipitate with Lbc. COS-7 cells were transfected with
empty vector or plasmid encoding Myc-tagged proto-Lbc along with empty
vector or plasmid encoding the GTPase-deficient G
subunit of either
G
q or G
s. A, whole cell
lysates from the transfected COS-7 cells were prepared and Western
blotted for the appropriate G
subunit, to ensure protein expression.
B, cell extracts were immunoprecipitated (IP)
with anti-Myc (9E10 mouse monoclonal) antibody, which recognizes the
epitope-tagged proto-Lbc, and then Western blotted with antibodies
against the appropriate G
subunit. Blots are representative of two
experiments, each performed in duplicate.
q Does Not Activate Rho--
To examine effects of
G
q on Rho activation we expressed the G
q
subunit in COS-7 cells and used the RBD of a Rho effector (Rhotekin) to
affinity precipitate active Rho (36, 39). In initial studies we
compared the ability of the RBD of Rhotekin to precipitate
constitutively active RhoA versus wild-type RhoA expressed
in COS-7 cells. As shown in Fig.
3A, constitutively active RhoA
was efficiently affinity-precipitated, while wild-type RhoA was
not.
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Fig. 3.
G q does
not induce activation of RhoA. A, COS-7 cells were
transfected with hemagglutinin-tagged wild-type RhoA,
hemagglutinin-tagged L63RhoA or empty vector (pCMV5). Cell
lysates were prepared, and a portion of this whole cell lysate was
subjected to SDS-PAGE and Western blotting with anti-RhoA antibody to
measure total Rho (lower panel). The remaining portion of
the lysates was affinity-precipitated with the RBD of Rhotekin and
Western blotted with anti-RhoA. Only activated Rho binds to the
Rhotekin RBD (top panel). The doublet pattern is present
because the hemagglutinin-tagged RhoA migrates slower than the
endogenous RhoA. B, COS-7 cells were transfected with pCis
vector or various G
subunits. The cell extracts were analyzed as in
A. C, after transfection of COS-7 with G
q,
proto-Lbc or both active or total Rho was detected in cell
extracts as in A.
12 or
G
13 in COS-7 cells led to increases in the amount of
RBD-precipitable, active Rho (Fig. 3B). In contrast
expression of constitutively active G
q did not increase
activation of Rho. The inability of G
q to activate Rho
could be due to a limiting concentration of its cognate exchange
factor, suggested by our previous experiments to be Lbc. We therefore
cotransfected Lbc with G
q and assessed the amount of
active Rho. Lbc led to Rho activation (Fig. 3C) as did other Rho GEFs (p115RhoGEF and PDZ-RhoGEF; data not shown). However G
q did not increase the ability of Lbc to activate Rho
(Fig. 3C). A range of concentrations of the
G
q and Lbc plasmids were tested, but at no
concentrations did G
q enhance Lbc-mediated Rho
activation (data not shown). In prior studies we have established that
[35S]GTP
S binding can be used to assess activation of
Rho (40). Expression of G
q alone or in combination with
Lbc failed to promote increased [35S]GTP
S binding to
Rho in lysates from COS-7 cells (data not shown).
q and RhoGEFs Synergistically Activate
Rho-dependent SRE.L-mediated Gene Transcription--
The
literature provides compelling evidence that G
q signals
through Rho-dependent pathways. A commonly used measure of
Rho-dependent signaling is stimulation of SRE.L-mediated
gene expression as originally described by Treisman's laboratory (16).
Both activated G
q and Lbc induced ~5-fold increases in
transcription of luciferase from an SRE.L-luciferase reporter gene in
COS-7 cells. Strikingly, when G
qRC and proto-Lbc were
coexpressed the SRE.L-luciferase was synergistically activated
(~25-fold). The data in Fig. 4 also demonstrate that the cooperative effect of G
q was
selective, since neither G
12 nor G
13
synergized with Lbc to induce SRE.L-luciferase expression. This
differential effect of G
q was maintained over a wide
range of doses of the G
subunit plasmids (from 0.5-2.5 µg/well;
data not shown) and thus was not dependent on high levels of G
subunit expression. Notably, G
q also synergized with
p115RhoGEF to activate the SRE.L response (Fig. 4) despite its minimal
ability to coimmunoprecipitate with this Rho GEF. Conversely,
G
12 and G
13 association with Lbc (Fig.
1C) did not lead to synergistic SRE.L activation (Fig.
4).
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Fig. 4.
G q
synergizes with Rho GEFs to induce Rho-dependent
SRE.L-mediated gene expression. COS-7 cells were transfected with
an SRE.L-luciferase reporter plasmid along with the empty pCis
vector or with pCis containing cDNA for the different G
subunits
as on the x axis. Cells were cotransfected with empty vector
(light gray), proto Lbc (medium gray), or
p115RhoGEF (black). Luciferase was quantified 24-48 h after
transfection. Data are averages ± S.E. of the fold over vector
control for three experiments, each performed in triplicate.
q could
induce transcriptional activation of the SRE.L reporter, we used a toxin that specifically activates G
q. PMT has been
demonstrated to cause transient activation of G
q and
downstream effectors in Xenopus oocytes (41). Furthermore,
this toxin has been used to investigate G
q-mediated
signaling pathways in mammalian cells (42, 43). Treatment of COS-7
cells with 100-200 ng/ml PMT for 24 h activated PLC as evidenced
by a 5-fold increase in total inositol phosphate formation. The ability
of PMT to stimulate SRE.L-mediated gene expression and to synergize
with Lbc (Fig. 5) confirmed that
endogenous G
q can also cooperate with Rho GEF-activated pathways.
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Fig. 5.
Activation of endogenous
G q synergizes with proto-Lbc on
SRE.L-mediated gene expression. COS-7 cells were transfected with
a control vector or proto-Lbc along with an SRE.L-luciferase reporter
plasmid. Following transfection cells were maintained in
serum-free medium (vehicle) or in serum-free medium
containing 100 ng/ml recombinant PMT for 24 h, then harvested and
assayed for luciferase activity. Data are representative of three
experiments, each performed in triplicate.
q effects on SRE.L-mediated gene
expression was examined by using C3 exoenzyme to inhibit Rho function. Expression of C3 exoenzyme (EFC3) completely inhibited the induction of
SRE.L-luciferase by G
q or Lbc. The synergy between
G
q and Lbc was also abolished, implicating Rho function
in mediating this synergistic response (Fig.
6). To confirm that the inhibitory effect
of C3 did not reflect its cytotoxicity, luciferase expression was
normalized to that of cotransfected GFP. These experiments demonstrated
that expression of C3 exoenzyme did not alter the cellular accumulation
of GFP (data not shown). In addition, the effect of another Rho family
small G protein, Cdc42, on SRE.L-luciferase expression was not
attenuated by C3. These data argue against nonspecific effects of C3
exoenzyme on cell viability or on luciferase expression.
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Fig. 6.
The synergistic SRE.L responses to
G q and Lbc in COS-7 cells are
Rho-dependent. COS-7 cells were transfected with an
SRE.L-luciferase reporter plasmid and the expression plasmids for the
proteins indicated on the x axis. Black bars
indicate cotransfection with an expression plasmid for C3 exoenzyme;
gray bars are vector-transfected controls. Luciferase was
quantified 24-48 h after transfection. Data are averages ± S.E.
of the fold increase over vector control for three experiments, each
performed in triplicate.
q and Lbc Cooperate to Induce
Cytoskeletal Changes--
We previously reported that 1321N1 human
astrocytoma cells show a marked cytoskeletal response to thrombin in
which the cell processes retract and the cell bodies become rounded. We
also showed that activated G
subunits or Rho GEFs could induce cell rounding (30). Although we reported that the effect of thrombin was
Rho- and G
12/13-dependent (30), we observed
a significant response to G
q. We used this preparation
to demonstrate interactions between G
q and Lbc in
another cell system and with a different functional readout. Cells were
microinjected with plasmid vectors containing cDNA for
G
qRC, proto-Lbc, or both and coinjected with cDNA
for nuclear GFP to identify injected cells. We established concentrations of G
qRC or proto-Lbc that alone induced
rounding in ~20% of the injected cells (versus 8% of
cells injected with pCis vector; Fig. 7).
When these concentrations of G
qRC and proto-Lbc were
coinjected their effect was more than additive, with nearly 50% of the
injected cells manifesting the rounded morphology (Fig. 7). In contrast
to what was observed with G
q, microinjected
G
12QL, at a concentration that elicited 20% rounding
alone, did not synergize with proto-Lbc (Fig. 7).
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Fig. 7.
Lbc selectively synergizes with
G q to induce astrocytoma cell
rounding. 1321N1 human astrocytoma cells were microinjected with
empty vector (pCis) or with G
subunits as indicated on the
x axis along with control vector or plasmid encoding
proto-Lbc. All cells were coinjected with plasmid for nuclear GFP to
allow identification of injected cells. Actin was visualized by
staining with rhodamine-conjugated phalloidin. Data shown are the
percent of microinjected cells that have a rounded appearance. Values
are the average ± S.E. from at least three experiments with a
minimum of 50 microinjected cells per condition per experiment.
q Synergizes with Rho--
Because
G
q synergized with Rho GEFs but did not elicit Rho
activation, we considered the possibility that G
q
cooperates downstream of Rho activation in effecting
Rho-dependent responses. Activated G
q,
G
12, G
13, or constitutively active RhoA
was expressed in COS-7 cells, and SRE.L-mediated gene expression was
assessed. Each of these G
subunits or activated RhoA induced
SRE.L-luciferase expression. Neither G
12 nor
G
13 synergized with cotransfected RhoA. In contrast,
G
q showed substantial synergy with activated RhoA (Fig.
8). These data indicate that the site at
which G
q synergizes with the Rho pathway to enhance
SRE.L-luciferase expression is at or downstream of Rho activation.
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Fig. 8.
G q
synergizes with Rho to induce SRE.L-mediated gene expression.
COS-7 cells were transfected with an SRE.L-luciferase reporter plasmid
and the expression plasmids for constitutively active G
subunits
along with control vector or plasmid encoding activated RhoA.
Luciferase was quantified 24 h after transfection. Values are the
average ± S.E. of the fold increase over vector control for three
experiments, each performed in triplicate.
q, we treated COS-7 cells with
the Rho kinase inhibitor Y-27632, which our laboratory has shown to inhibit DNA synthesis, cell migration, and morphological responses to
Rho (30, 40). We observed no attenuation of the
G
q-stimulated SRE.L response and no reduction in the
response to Lbc or in the synergistic response to coexpressed
G
q and Lbc.
q and Rho Pathways Requires
Activation of PLC, but Not PKC--
To determine whether the
activation state of G
q was critical for synergy with Rho
pathways, we compared the effects of wild-type G
q to
those of the constitutively active G
q. In COS-7 cells wild-type G
q did not significantly activate PLC (as
assessed by [3H]inositol phosphate formation), in
contrast to the marked stimulatory effect of G
qQL (Table
I). In parallel with this differential effect on PLC activity, the wild-type G
q did not induce
SRE.L-mediated gene expression and did not enhance the activation
elicited by Lbc (Fig. 9A).
These data suggested that activation of PLC was required for
G
q cooperation with the Rho pathway.
[3H]Inositol phosphate (IP) formation due to various mutants
of Gq and PLC
CT
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Fig. 9.
SRE.L activation is dependent on PLC
activation and independent of PKC. COS-7 cells were transfected
with the SRE.L-luciferase reporter plasmid, and luciferase was
determined as described. A, cells were transfected with
empty pCMV5 vector or one of the following G q
constructs: GTPase-deficient G
q
(G
qQL), wild-type G
q
(G
qWT), or GTPase-deficient G
q
that cannot activate PLC
(G
qQL/DNE) along
with control vector or proto-Lbc. B, COS-7 cells were
transfected with a GTPase-deficient G
q
(G
qRC) and/or Lbc along with control vector
plasmid encoding the C terminus of PLC
1
(PLC
CT). C, COS-7 cells were
transfected with a GTPase-deficient G
q
(G
qRC) and/or Lbc. After overnight
transfection, cells were treated with vehicle or with 3 µM GF109203X (to inhibit PKC) for 24 h prior to
harvest. Data are average ± S.E. of the fold increase over vector
control for two or three experiments performed in triplicate.
qDNE, an activated (Q209L) mutant of G
q
that also has three amino acids substituted to prevent coupling to PLC
(44). As previously demonstrated by Venkatakrishnan and Exton (44),
this mutant did not significantly stimulate inositol phosphate
formation (Table I). G
qDNE also failed to stimulate
SRE.L-mediated gene expression or synergize with Lbc (Fig.
9A), consistent with a requirement for PLC signaling
pathways. We also tested a cDNA that encodes the C terminus of
PLC
1 (PLC
CT), which has been suggested to interact with
G
q and thereby act as a competitive inhibitor blocking
activation of endogenous PLC activation (35). When the PLC
CT was
expressed along with G
qRC in COS-7 cells, inositol
phosphate accumulation was inhibited by approximately 50% (Table I).
Expression of PLC
CT led to a dose-dependent inhibition of G
q-induced SRE.L activation (not shown), with the
highest dose almost completely abolishing SRE.L activation by
G
qRC (Fig. 9B). The synergistic SRE.L
response due to G
q and Lbc was also markedly inhibited
by expression of PLC
CT. Expression of the PLC
CT had no effect on
SRE.L activation by Lbc alone. Our findings using
G
qQL/DNE and the PLC
CT further support the hypothesis that activation of PLC is required for the synergistic interaction between G
q and Rho signaling pathways.
q could result from PKC activation, we
treated COS-7 cells with PMA. Although PMA induced transcription from
an AP-1-luciferase reporter, it did not activate the SRE.L or enhance
the ability of Lbc to induce SRE.L-mediated transcription (data not
shown). In addition, the PKC inhibitor GF109203X failed to affect the
SRE.L responses to G
q and/or Lbc (Fig. 9C),
whereas it blocked the PMA-induced activation of
AP-1-dependent transcription (data not shown). These
observations suggest that activation of PKC is not necessary or
sufficient for G
q to induce transcription from the
SRE.L.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q and Rho has been identified. In fact
there is conflicting data on the issue of whether and how
G
q might activate Rho (reviewed in Ref. 11). Therefore,
our initial goal was to determine whether G
q activates
Rho and if this occurs via interaction with Rho GEFs, as has been
suggested for G
12 and G
13. In this report we demonstrate that G
q can associate with Lbc, a Rho
GEF, and that G
q-generated signals cooperate with Rho to
enhance two distinct Rho-mediated responses. Although these functional
and physical interactions suggest that G
q signals
through a Rho GEF, our data indicate that the interaction between these
pathways occurs distal to the activation of Rho.
q and Lbc on Rho-dependent pathways. The
first was activation of an SRE.L-luciferase reporter gene, a response
that a number of laboratories have utilized as a readout of
Rho-dependent signaling (16, 23, 29, 32). In COS-7 cells
both G
q and Lbc activated this reporter.
G
q synergized with either proto-Lbc or p115RhoGEF to
activate the SRE.L-luciferase, while G
12 and
G
13 did not synergize with these Rho GEFs.
q and abolished the synergistic response elicited by G
q and Lbc coexpression. Nonspecific effects
of the C3 toxin on luciferase expression or cell viability were ruled out, since C3 had no effect on constitutive GFP expression or SRE.L-luciferase expression induced by Cdc42. Thus the effect of C3
indicates that Rho activity is indeed required for G
q
signaling to the SRE.L.
q and Rho
interactions was the morphological response of 1321N1 cells. Since cell
rounding is rapid and robust, appearing in less than 3 h after
microinjection (4, 30), it represents an early consequence of Rho
activation. In our previous studies we demonstrated that proto-Lbc
induced C3-sensitive rounding (30). Our current findings reveal that
G
q synergizes with proto-Lbc to stimulate this response. As observed for SRE.L activation, G
12 did not enhance
Lbc-induced rounding. Importantly, the selective enhancement of the
cytoskeletal response to Lbc by G
q indicates that the
synergy between G
q and Lbc is not an artifact of using
the SRE.L reporter gene assay as a readout.
q
with Rho GEFs, we asked whether G
q expressed in COS-7
cells associates with coexpressed Rho GEFs. Two Rho GEFs, Lbc and
p115RhoGEF, were tested, and their interactions with G
q,
G
12, G
13, and G
s were compared. The finding that emerged from these studies was that G
q coprecipitated specifically with Lbc. This was
independent of the epitope tag on Lbc and was seen with both the
full-length proto form and a truncated, onco, form of Lbc (data not
shown). Lbc did not coprecipitate G
s, confirming that
there is G
subunit specificity, even in the transfected COS-7
cell system. Consistent with previously published findings,
G
q did not associate with p115RhoGEF, although
coprecipitation of G
12 or G
13 with
p115RhoGEF was demonstrable (12, 33). Strikingly, synergy on the SRE.L followed the opposite pattern, that is G
q synergized
with p115RhoGEF, while G
12 and G
13 did
not. The discrepancy between these findings and those reported by Mao
et al. (32) may reflect cell type-specific differences.
Nonetheless, our data demonstrate that functional cooperation between
Rho GEFs and G
subunits is not correlated with the physical
interaction observed in the coprecipitation studies.
q-Lbc interaction
enhances the exchange activity of Lbc, which should result in enhanced activation of Rho in COS-7 cells. Rho activation was observed in cells
expressing Lbc or other Rho GEFs, as demonstrated by an increase in the
fraction of Rho that bound to the RBD of Rhotekin. Furthermore, the
ability of expressed G
subunits to activate Rho in COS-7 cells was
confirmed by the stimulatory effect of both G
12 and
G
13 on Rho activation. However Rho activation was not
observed in cells expressing G
q nor did we observe a
synergistic effect of G
q on Lbc induced Rho activation.
It is of course possible that the rhotekin-RBD assay is not sensitive
enough to detect a modest Rho activation by G
q. However,
since Rho activation by G
12 and G
13 was
detectable, while activation by G
q was not, at the least
significantly less Rho is activated by G
q. Furthermore, the Rho activation assay was carried out under the same conditions in
which both physical and functional interactions of G
q
and Lbc were seen. Thus, it appears that Rho activation neither results from nor explains G
q synergy with Lbc.
q. In published studies inhibition of Rho with C3
exoenzyme blocked G
q-stimulated SRE activation (23) and
neurite retraction (31). In addition, depletion of G
q by
antisense (45) or G
q/11-knockout (9) prevented
Rho-dependent actin reorganization in smooth muscle cells
and fibroblasts. While these observations indicate that
G
q and Rho pathways are interdependent, they do not
directly demonstrate that Rho is activated by G
q
expression. Preliminary data from Hall's laboratory suggests that
G
q expression increases the amount of activated Rho
(46). Furthermore agonists that activate Gq-coupled
receptors, have been shown to stimulate Rho redistribution (47, 48).
While the relationship between Rho activation and its cellular
distribution is complex, it is generally believed that inactive Rho is
sequestered in the cytosol and translocates to the membrane or
cytoskeleton upon stimulation (reviewed in Ref. 11). Interestingly, our
preliminary experiments suggest that G
q increases the
amount of Rho in the particulate fraction of COS-7 cell extracts. This
altered Rho localization may be distinct from its activation but could
contribute to enhancement of Rho-dependent signaling.
13 and p115RhoGEF has
been shown to increase the guanine nucleotide exchange activity of p115RhoGEF in vitro (12). Notably other G protein-Rho GEF
interactions have not, to our knowledge, been directly shown to
increase Rho activation. Indeed, G
12 associates with
p115RhoGEF but does not stimulate its exchange activity (12).
Additionally, while an interaction of G
12 or
G
13 with PDZ-RhoGEF was shown by coprecipitation and
indirectly by assessing SRE.L activation, neither G
12
nor G
13 has, to our knowledge, been directly
demonstrated to enhance PDZ-RhoGEF exchange activity or
PDZ-RhoGEF-induced Rho activation (13). These data, together with the
findings reported here, suggest caution in interpreting physical
interactions between G
subunits and Rho GEFs as evidence that the
exchanger is regulated by the G
protein.
q is
required to enhance Rho-mediated signaling, along with experiments
using the DNE mutant of activated G
q and the inhibitory
PLC
CT construct, support the conclusion that activation of PLC
is
required for the synergistic interaction of G
q and Rho.
Thus PLC, the primary effector of G
q signaling, appears
to play a central role in a pathway that enhances Rho-mediated
responses. Although PKC is downstream of PLC in many pathways,
activation of PKC with PMA did not mimic the effects of
G
q on SRE.L-luciferase expression, and inhibition of PKC
did not reduce this response. Subsequent experiments will be needed to
identify the PLC-dependent signals and effectors that
interact with and enhance Rho-mediated responses.
12/13 also activate G
q and PLC, these
processes are likely to occur in concert. Interestingly, in 1321N1
cells, thrombin, which stimulates PLC and activates Rho, can induce
cell rounding. In contrast, cell rounding is not induced by carbachol,
which activates PLC (4, 49) but not Rho, or by LPA, which activates Rho
but only weakly stimulates PLC in these
cells.2 These findings are
consistent with the notion that signals generated by
G
q/PLC may be insufficient to induce Rho activation, but
required to enhance responses elicited through Rho signaling pathways.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants GM36927 (to J. H. B.) and AI38396 (to B. A. W.), National Institutes of Health NCI Grant CA62029 (to D. T.), and by an American Heart Association, New England affiliate, grant-in-aid (to D. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Markey Fellow. Work was performed in partial fulfillment of the Ph.D. degree in the Biomedical Sciences Graduate Program.
¶ Supported by an American Heart Association, Western States Affiliate, postdoctoral fellowship award.
Recipient of a summer fellowship from the American Society for
Pharmacology and Experimental Therapeutics.
§§ To whom correspondence should be addressed. Tel.: 858-534-2595; Fax: 858-534-4337; E-mail: jhbrown@ucsd.edu.
Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M008961200
2 S. A. Sagi, T. M. Seasholtz, D. Goldstein, and J. H. Brown, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
AP-1, activator protein-1;
C3, C.
botulinum C3 transferase;
DMEM, Dulbecco's modified Eagle's
medium;
GEF, guanine nucleotide exchange factor;
GFP, green fluorescent
protein;
GST, glutathione S-transferase;
Lbc, lymphoid blast
crisis;
LPA, lysophosphatidic acid;
PAGE, polyacrylamide gel
electrophoresis;
PKC, protein kinase C;
PLC, phospholipase C;
PMA, phorbol 12-myristate 13-acetate;
PMT, Pasteurella multocida
toxin;
RBD, Rho-binding domain;
SRE, serum response element;
GTPS, guanosine 5'-O-(thio)triphosphate.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | van Biesen, T., Luttrell, L. M., Hawes, B. E., and Lefkowitz, R. J. (1996) Endocr. Rev. 17, 698-714[Medline] [Order article via Infotrieve] |
2. | Ridley, A. J., and Hall, A. (1992) Cell 70, 389-399[Medline] [Order article via Infotrieve] |
3. |
Gohla, A.,
Harhammer, R.,
and Schultz, G.
(1998)
J. Biol. Chem.
273,
4653-4659 |
4. |
Majumdar, M.,
Seasholtz, T. M.,
Goldstein, D.,
de Lanerolle, P.,
and Brown, J. H.
(1998)
J. Biol. Chem.
273,
10099-10106 |
5. | Bishop, A. L., and Hall, A. (2000) Biochem. J. 348, 241-255[CrossRef][Medline] [Order article via Infotrieve] |
6. | Narumiya, S., Ishizaki, T., and Watanabe, N. (1997) FEBS Lett. 410, 68-72[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Amano, M.,
Chihara, K.,
Kimura, K.,
Fukata, Y.,
Nakamura, N.,
Matsuura, Y.,
and Kaibuchi, K.
(1997)
Science
275,
1308-1311 |
8. |
Essler, M.,
Amano, M.,
Kruse, H.-J.,
Kaibuchi, K.,
Weber, P. C.,
and Aepfelbacher, M.
(1998)
J. Biol. Chem.
273,
21867-21874 |
9. |
Gohla, A.,
Offermanns, S.,
Wilkie, T. M.,
and Schultz, G.
(1999)
J. Biol. Chem.
274,
17901-17907 |
10. |
Seasholtz, T. M.,
Majumdar, M.,
and Brown, J. H.
(1999)
Mol. Pharmacol.
55,
949-956 |
11. | Sah, V. P., Seasholtz, T. M., Sagi, S. A., and Brown, J. H. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 459-489[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Hart, M. J.,
Jiang, X.,
Kozasa, T.,
Roscoe, W.,
Singer, W. D.,
Gilman, A. G.,
Sternweis, P. C.,
and Bollag, G.
(1998)
Science
280,
2112-2114 |
13. |
Fukuhara, S.,
Murga, C.,
Zohar, M.,
Igishi, T.,
and Gutkind, J. S.
(1999)
J. Biol. Chem.
274,
5868-5879 |
14. | Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650[Abstract] |
15. | Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito, M., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) EMBO J. 15, 2208-2216[Abstract] |
16. | Hill, C. S., Wynne, J., and Treisman, R. (1995) Cell 81, 1159-1170[Medline] [Order article via Infotrieve] |
17. | Treisman, R., Alberts, A. S., and Sahai, E. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 643-651[Medline] [Order article via Infotrieve] |
18. |
Sahai, E.,
Alberts, A. S.,
and Treisman, R.
(1998)
EMBO J.
17,
1350-1361 |
19. |
Fujisawa, K.,
Madaule, P.,
Ishizaki, T.,
Watanabe, G.,
Bito, H.,
Saito, Y.,
Hall, A.,
and Narumiya, S.
(1998)
J. Biol. Chem.
273,
18943-18949 |
20. | Sahai, E., Ishizaki, T., Narumiya, S., and Treisman, R. (1999) Curr. Biol. 9, 136-145[CrossRef][Medline] [Order article via Infotrieve] |
21. | Morissette, M. R., Sah, V. P., Glembotski, C. C., and Brown, J. H. (2000) Am. J. Physiol. 278, H1769-H1774 |
22. |
Quilliam, L. A.,
Lambert, Q. T.,
Mickelson-Young, L. A.,
Westwick, J. K.,
Sparks, A. B.,
Kay, B. K.,
Jenkins, N. A.,
Gilbert, D. J.,
Copeland, N. G.,
and Der, C. J.
(1996)
J. Biol. Chem.
271,
28772-28776 |
23. |
Mao, J.,
Yuan, H.,
Xie, W.,
Simon, M. I.,
and Wu, D.
(1998)
J. Biol. Chem.
273,
27118-27123 |
24. |
Sah, V. P.,
Hoshijima, M.,
Chien, K. R.,
and Brown, J. H.
(1996)
J. Biol. Chem.
271,
31185-31190 |
25. | Tigyi, G., Fischer, D. J., Sebok, A., Yang, C., Dyer, D. L., and Miledi, R. (1996) J. Neurochem. 66, 537-548[Medline] [Order article via Infotrieve] |
26. |
Buhl, A. M.,
Johnson, N. L.,
Dhanasekaran, N.,
and Johnson, G. L.
(1995)
J. Biol. Chem.
270,
24631-24634 |
27. | Vouret-Craviari, V., Van Obberghen-Schilling, E., Scimeca, J. C., Van Obberghen, E., and Pouyssegur, J. (1993) Biochem. J. 289, 209-214[Medline] [Order article via Infotrieve] |
28. | Jalink, K., and Moolenaar, W. H. (1992) J. Cell Biol. 118, 411-419[Abstract] |
29. |
Coso, O. A.,
Montaner, S.,
Fromm, C.,
Lacal, J. C.,
Prywes, R.,
Teramoto, H.,
and Gutkind, J. S.
(1997)
J. Biol. Chem.
272,
20691-20697 |
30. |
Majumdar, M.,
Seasholtz, T. M.,
Buckmaster, C.,
Toksoz, D.,
and Brown, J. H.
(1999)
J. Biol. Chem.
274,
26815-26821 |
31. |
Katoh, H.,
Aoki, J.,
Yamaguchi, Y.,
Kitano, Y.,
Ichikawa, A.,
and Negishi, M.
(1998)
J. Biol. Chem.
273,
28700-28707 |
32. |
Mao, J.,
Yuan, H.,
and Wu, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12973-12976 |
33. |
Kozasa, T.,
Jiang, X.,
Hart, M. J.,
Sternweis, P. M.,
Singer, W. D.,
Gilman, A. G.,
Bollag, G.,
and Sternweis, P. C.
(1998)
Science
280,
2109-2111 |
34. |
Sterpetti, P.,
Hack, A. A.,
Bashar, M. P.,
Park, B.,
Cheng, S.-D.,
Knoll, J. H. M.,
Urano, T.,
Feig, L. A.,
and Toksoz, D.
(1999)
Mol. Cell. Biol.
19,
1334-1345 |
35. | Beekman, A., Helfrich, B., Bunn, P. A., Jr., and Heasley, L. E. (1998) Cancer Res. 58, 910-913[Abstract] |
36. |
Ren, X.-D.,
Kiosses, W. B.,
and Schwartz, M. A.
(1999)
EMBO J.
18,
578-585 |
37. |
Wilson, B. A.,
Ponferrada, V. G.,
Vallance, J. E.,
and Ho, M.
(1999)
Infect. Immun.
67,
80-87 |
38. |
Collins, L. R.,
Minden, A.,
Karin, M.,
and Brown, J. H.
(1996)
J. Biol. Chem.
271,
17349-17353 |
39. |
Kranenburg, O.,
Poland, M.,
van Horck, F. P. G.,
Drechsel, D.,
Hall, A.,
and Moolenaar, W. H.
(1999)
Mol. Biol. Cell
10,
1851-1857 |
40. |
Seasholtz, T. M.,
Majumdar, M.,
Kaplan, D. D.,
and Brown, J. H.
(1999)
Circ. Res.
84,
1186-1193 |
41. |
Wilson, B. A.,
Zhu, X.,
Ho, M.,
and Lu, L.
(1997)
J. Biol. Chem.
272,
1268-1275 |
42. |
Seo, B.,
Choy, E. W.,
Maudsley, S.,
Miller, W. E.,
Wilson, B. A.,
and Luttrell, L. M.
(2000)
J. Biol. Chem.
275,
2239-2245 |
43. |
Sabri, A.,
Pak, E.,
Alcott, S. A.,
Wilson, B. A.,
and Steinberg, S. F.
(2000)
Circ. Res.
86,
1047-1053 |
44. |
Venkatakrishnan, G.,
and Exton, J. H.
(1996)
J. Biol. Chem.
271,
5066-5072 |
45. |
Hirshman, C. A.,
and Emala, C. W.
(1999)
Am. J. Physiol.
277,
L653-L661 |
46. | Kjøller, L., and Hall, A. (1999) Exp. Cell. Res. 253, 166-179[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Fleming, I. N.,
Elliot, C. M.,
and Exton, J. H.
(1996)
J. Biol. Chem.
271,
33067-33073 |
48. | Keller, J., Schmidt, M., Hussein, B., Rumenapp, U., and Jakobs, K. H. (1997) FEBS Lett. 403, 299-302[CrossRef][Medline] [Order article via Infotrieve] |
49. | Post, G. R., Collins, L. R., Kennedy, E. D., Moskowitz, S. A., Aragay, A. M., Goldstein, D., and Brown, J. H. (1996) Mol. Biol. Cell 7, 1679-1690[Abstract] |