From the Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4330
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ABSTRACT |
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Small GTP-binding proteins of the Rho family play
a critical role in signal transduction. However, there is still very
limited information on how they are activated by cell surface
receptors. Here, we used a consensus sequence for Dbl domains of Rho
guanine nucleotide exchange factors (GEFs) to search DNA data bases,
and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those
of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area
of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF
that was termed Lsc-homology (LH) domain. This
novel molecule, designated PDZ-RhoGEF, activated biological and
biochemical pathways specific for Rho, and activation of these pathways
required an intact DH and PH domain. However, the PDZ domain was
dispensable for these functions, and mutants lacking the LH domain were
more active, suggesting a negative regulatory role for the LH domain. A
search for additional molecules exhibiting an LH domain revealed a
limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This
prompted us to investigate whether PDZ-RhoGEF could interact with
representative members of each G protein family. We found that
PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the G The Ras superfamily of GTPases comprises approximately 50 members
that can be divided into several families, Ras, Rho, Sar, Rab, Arf, and
Ran, based on their primary sequence as well as on their cellular
activities (1-3). Whereas the Rab, Arf, and Sar groups participate in
the transport of proteins and vesicles among different intracellular
compartments, the Ran proteins function in nuclear transport, and Ras
plays a central role in cell proliferation and differentiation (2,
4). In the case of Ras, recent studies have revealed how it works at
the molecular level. This small GTP-binding protein exchanges GDP for
GTP upon activation of Ras-specific guanine nucleotide exchange factors
(GEFs)1 (5), and in the
GTP-bound state, Ras physically associates with Raf (6) thereby
recruiting this serine threonine kinase to the plasma membrane. This
causes the activation of Raf and initiates the activity of a sequential
cascade of kinases leading to the stimulation of mitogen-activated
protein kinases (MAPKs), p42MAPK and p44MAPK,
also known as extracellular signal-regulated kinases-2 and -1, respectively, which, in turn, control the activity of nuclear transcription factors that are critical for cell growth (7, 8).
The Rho family of GTP-binding proteins, which consists of the Rho, Rac,
and Cdc42 subfamilies, has been shown to regulate several aspects of
cytoskeleton function (4). For example, Rho participates in the
formation of actin stress fibers and mediates the redistribution of
cytoskeletal components (4, 9). Rac is involved in the regulation of
lamellipodia (pleat-shaped protrusions at the cell periphery) and
membrane ruffling (10); and Cdc42 regulates the formation of thin
finger-like cytoplasmic extensions known as filopodia (11). These
proteins play an important role in the regulation of cell morphology,
cell aggregation, tissue polarity, cytokinesis, cell motility, and also
in smooth muscle contraction (12-14). However, recent evidence
suggests that Rho proteins are also integral components of signaling
pathways leading to transcriptional control. For example, Rac and Cdc42
regulate the activity of the c-Jun amino-terminal kinase (JNK) thereby affecting the transcriptional activity of c-Jun (15), and Rho has been
recently shown to induce expression from the serum responsive element
(SRE) through the transcriptional activation of the serum response
factor (SRF) (16).
The functional activity of small GTP-binding proteins of the Ras
superfamily is tightly regulated in vivo by proteins that control their GDP/GTP bound state. Whereas GEFs promote the exchange of
GDP for GTP thus activating Ras-like proteins (2), GTPase-activating proteins increase the low intrinsic rate of GTP hydrolysis of small
GTPases (reviewed in Ref. 3) and are negative modulators. The
mechanisms of activation of GEFs for Ras by cell surface receptors have
been intensely investigated. For example, the biochemical route
connecting the epidermal growth factor-receptor tyrosine kinase to Ras
has been recently identified (5, 17), and includes the phosphorylation
of the receptor itself on tyrosine residues, thus creating docking
sites for adapter molecules such as Grb2 and Crk. These adapter
molecules, in turn, help recruit to the membrane Sos, a Ras-GEF,
thereby inducing the exchange of GDP for GTP on Ras. In contrast, the
signaling pathway regulating the activity of GEFs for Rho family
members is still poorly understood.
Many GEFs for Rho, Rac, and Cdc42, including dbl,
ost, lfc, lbc, vav,
ect2, tim, and net (reviewed in Refs.
18 and 19) were discovered by virtue of their ability to transform NIH
3T3 cells when overexpressed or when activated by truncations. All these proteins share a 250-amino acid stretch of significant sequence similarity with Dbl, termed Dbl homology (DH) domain, adjacent to a
pleckstrin-homology (PH) domain (18, 19). The DH domain was shown to be
responsible for nucleotide exchange activity toward GTPases of the Rho
family (20, 21). Of interest, one of these GEFs for Rho-like proteins,
the protein product of the vav proto-oncogene, proto-Vav,
also exhibits a Src homology (SH) 2 domain flanked by two SH3 domains
(22, 23), and we have recently shown that tyrosine phosphorylation of
proto-Vav by hematopoietic specific tyrosine kinases can activate its
GEF activity for Rac both in vitro and in vivo
(24, 25). However, no other GEF for Rho-like proteins has been found to
be regulated by tyrosine phosphorylation, nor to contain a
phosphotyrosine-binding domain such as an SH2 or PTB domains.
Furthermore, the vast majority of the known GEFs for small GTP-binding
proteins of the Rho family are expressed in a very restricted
tissue-specific manner (18, 19), and their mechanism of activation is
still largely unknown.
In this study, we explored the existence of novel GEFs for Rho-like
proteins possessing structural domains that might suggest a role in
signal transduction. Here, we report the identification of a novel,
ubiquitously expressed GEF for Rho-like proteins containing a PDZ
domain. This protein, termed PDZ-RhoGEF, was found to activate biochemical pathways specific for Rho, in a Rho-dependent
manner. Interestingly, PDZ-RhoGEF was found to be closely related to
the Drosophila DRhoGEF2, and recent genetic analysis
suggests that DRhoGEF2 acts downstream of the concertina
gene, a Drosophila G Expression Plasmids--
KIAA0380 (human PDZ-RhoGEF), kindly
provided by T. Nagase, Kazusa DNA Research Institute, Japan, was
subcloned into the pCEFL vector as an Asp718-NotI
fragment, thus generating the pCEFL-PDZ-RhoGEF expression plasmid.
Then, the coding sequence for the AU1 hexapeptide (DTYRYI) was cloned
in-frame with the open reading frame of PDZ-RhoGEF, immediately
upstream of the termination codon, thus generating a carboxyl-terminal
AU1-tagged PDZ-RhoGEF. cDNAs encoding deletion mutants of
PDZ-RhoGEF, as indicated in the corresponding figures, were generated
by restriction enzyme digestion or polymerase chain reaction
amplification using pCEFL-PDZ-RhoGEF-AU1 as a template. Sequences of
mutagenic oligonucleotides will be made available upon request.
Plasmids expressing epitope-tagged MAPK and JNK, pcDNA3 HA-MAPK and
pcDNA3 HA-JNK, respectively, as well as expression plasmids for
constitutively activated forms of Ras, RhoA, G Northern Blot Analysis--
Human multiple tissue Northern
blots, each lane containing 2 µg of poly(A)+ RNA, were
purchased from CLONTECH. Total RNA was isolated
from several cell lines by RNeasy kit (QIAGEN) according to the
manufacturer's instructions, then separated by electrophoresis on a
2% denaturing formaldehyde-agarose gel (20 µg of RNA/lane), and
transferred to HybondTM-N nylon membrane (Amersham Life
Science). The cDNA probe used for analysis of the PDZ-RhoGEF
mRNA was prepared using as a template a 1832-base pair
SacI-XbaI fragment derived from pCEFL-PDZ-RhoGEF, containing both 3'-translated and -untranslated regions of PDZ-RhoGEF cDNA. Human Cell Lines and Transfection--
Human kidney 293T cells and
COS-7 cells were maintained in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) supplemented with 10% fetal bovine serum.
NIH 3T3 fibroblasts were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% bovine calf serum. 293T cells were
transfected by the calcium-phosphate precipitation technique to examine
the expression of PDZ-RhoGEF and its mutants or using LipofectAMINE
PlusTM reagent (Life Technologies, Inc.) according to the
manufacturer's protocol for the co-immunoprecipitation study.
Transfection into NIH 3T3 cells were carried out by the
calcium-phosphate precipitation technique, and COS-7 cells were
transfected by the DEAE-dextran method. In each experiment, total
amount of DNA was adjusted to 3-10 µg/plate with vector alone
without insert. For the transfection into 293T cells, tissue culture
plates were treated with phosphate-buffered saline containing 20 µg/ml poly-D-lysine for 10 min before seeding the cells,
to prevent them from detaching from the plates.
Kinase Assays--
MAPK activity in cells transfected with an
epitope-tagged MAPK (HA-ERK2, referred in here as HA-MAPK) was
determined as described previously (15), using myelin basic protein
(Sigma) as a substrate. JNK assays in cells transfected with an
epitope-tagged JNK (HA-JNK) was also determined as described previously
(15), using purified, bacterially expressed GST-ATF2(96) fusion protein
as a substrate. Samples were separated by SDS-gel electrophoresis on
12% acrylamide gels. Autoradiography was performed with the aid of an
intensifying screen. Parallel lysates of cells transfected with the
HA-MAPK or HA-JNK expression plasmids were processed for Western blot analysis using an antibody against the HA epitope.
Reporter Gene Assays--
NIH 3T3 cells were transfected with
different expression plasmids together with 1.0 µg of pCMV- Immunoprecipitation and Western Blot Analysis--
To confirm
the expression of PDZ-RhoGEF and its mutants, 293T cells were
transfected with vector or expression vector for each PDZ-RhoGEF-AU1
DNA construct. Then, cells were cultured for 48 h, washed twice
with phosphate-buffered saline, and lysed at 4 °C in a buffer
containing 25 mM HEPES, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 20 mM
Lysates and anti-AU1 and anti-HA immunoprecipitates were analyzed by
Western blotting after SDS-polyacrylamide gel electrophoresis, transferred to ImmobilonTM-P Transfer Membranes (Amersham
Life Science) and immunoblotted with the corresponding rabbit antisera
or mouse monoclonal antibody as indicated in each figure. Immuno
complexes were visualized by enhanced chemiluminescence detection
(Amersham Life Science) using goat anti-rabbit or anti-mouse coupled to
horseradish peroxidase as a secondary antibody (Cappel). In this study,
we used mouse monoclonal antibodies anti-HA epitope (12CA5, BABCO) and
anti-AU1 epitope (BABCO). Polyclonal antiserum to Identification of a Novel PDZ Domain Containing Guanine Nucleotide
Exchange Factor for Rho Proteins--
We set out to identify candidate
GEFs for small GTP-binding proteins of the Rho family which contain
domains previously implicated in signal transduction. To that end, we
searched available DNA data bases using a consensus amino acid sequence
derived from the DH domain of known GEFs for Rho-related GTPases (2).
This search revealed the existence of a number of yet
uncharacterized proteins exhibiting DH-like domains (data not
shown). Subsequent analysis of their DNA sequences and their expected
translational products suggested that many of them encode putative GEFs
for Rho-like proteins. One of them, KIAA0380, accession AB002378, was
of particular interest and was further characterized. The corresponding
plasmid DNA was obtained from Dr. T. Nagase, Kazusa DNA Research
Institute, Japan, and its nucleotide sequence confirmed. Of interest,
its open reading frame encodes a protein of 1522 amino acids,
possessing areas of high homology to other signaling molecules (Fig.
1A). As depicted in Fig.
1B, this molecule contains a tandem of DH and PH domains,
being the DH domain closely related to those of p115-RhoGEF (30) (53%
identity, 74% similarity) and Lsc (31) (53% identity, 72%
similarity), and to the recently identified DRhoGEF2 (32, 33) (39%
identity, 64% similarity). Furthermore, this molecule displays 35% of
identity and 45% homology when analyzed for global homology with
p115-RhoGEF/Lsc. However, the DH and PH domains were more distantly
related to the DH domain of Dbl (20) and PH domain of pleckstrin (34),
respectively. This molecule also contains an NH2-terminal
region exhibiting extensive homology to a recently identified
structural domain termed PDZ (35), which is involved in protein-protein
interactions, and is also present in DRhoGEF2. As discussed above, the
DH domain is believed to be responsible for the nucleotide exchange
activity of GEFs, and both p115-RhoGEF and Lsc have been shown to
behave as Rho-specific guanine nucleotide releasing factors (30, 36). Thus, the newly identified molecule, which was tentatively named PDZ-RhoGEF, might represent a novel exchange factor for Rho.
PDZ-RhoGEF exhibits additional structural features, a proline-rich
region (amino acids 149 to 160) and another area (amino acid 290-486)
showing a high degree of homology (~34% identity and ~53%
similarity) to Lsc, p115-RhoGEF, and DRhoGEF2 (Fig. 1B), that was termed Lsc homology (LH) domain. Thus
PDZ-RhoGEF exhibits a number of characteristics that suggest a function
in signal transduction: a DH-PH domain highly related to those of Rho
GEFs, a PDZ domain, and an area of homology to three other GEFs.
PDZ-RhoGEF Is Widely Expressed--
The majority of the known GEFs
for small GTP-binding proteins of the Rho family are expressed in a
restricted tissue-specific manner. To investigate the pattern of
expression of PDZ-RhoGEF, we performed Northern blot analysis of RNAs
from a broad range of human tissues using a non-conserved region
including both 3'-translated and -untranslated sequences, nucleotides
3959-5790, of the PDZ-RhoGEF cDNA as a probe. As shown in Fig.
1C, a prominent RNA transcript of approximately 7 kilobases
was readily detected in many human tissues, albeit to a different
extent. PDZ-RhoGEF is highly expressed in the brain, testis, heart,
ovary, and placenta, and to lower levels in kidney, pancreas, spleen,
prostate, colon, skeletal muscle, lung, and liver. Whereas no
expression was detected in the thymus and small intestine, two RNA
species were expressed at comparable levels in peripheral blood
leukocytes. The nature of the smaller transcripts detected in
leukocytes, as well as that of the additional minor RNA species
detected in placenta, spleen, brain, and heart is still unknown, and
under current investigation. Similarly, transcripts for PDZ-RhoGEF were
detectable in frequently used mammalian cell lines, such as HeLa, 293T,
COS-7, and normal human keratinocytes, but limited expression was
observed in NIH 3T3 cells (data not shown). Thus, we can conclude that
PDZ-RhoGEF is expressed in a large variety of human tissues, rather
than in a tissue-restricted manner.
Expression of Wild-type and Truncated Mutants of
PDZ-RhoGEF--
To begin exploring the biochemical specificity of
PDZ-RhoGEF and the relative contribution of each structural domain, we
engineered expression plasmids for epitope-tagged forms of wild-type
and truncated PDZ-RhoGEF mutants. Initially, PDZ-RhoGEF was subcloned in an expression vector, pCEFL (37), and then the coding sequence for
the hexapeptide DTYRYI was cloned in-frame with the open reading frame
of PDZ-RhoGEF immediately upstream of the termination codon, thus
generating a carboxyl-terminal AU1-tagged PDZ-RhoGEF. As shown in
Fig. 2A, when transfected into
293T cells, wild-type PDZ-RhoGEF was readily detectable with an
anti-AU1 epitope-specific antibody. Similarly, each sequential
NH2-terminal deletion mutant, PDZ-RhoGEF Fails to Induce MAPK Cascades--
Whereas Ras controls
the MAPK cascade, recent data suggest that Rac and Cdc42 can regulate
the activity of the JNK pathway (15). Thus, as an approach to
investigate whether PDZ-RhoGEF activates small GTPases of the Ras, Rac,
or Cdc42 class, we investigated the ability of PDZ-RhoGEF and its
deletion mutants to activate MAPK and JNK in COS-7 cells. As shown in
Fig. 2B, none of the PDZ-RhoGEF expression plasmids enhanced
the activity of co-transfected HA-MAPK or HA-JNK, although,
12-O-tetradecanoylphorbol-13-acetate addition and activated
Ras potently stimulated MAPK, and anisomycin and activated Dbl strongly
activated JNK when used as controls under identical experimental
conditions. Thus, these data strongly suggest that PDZ-RhoGEF cannot
activate Ras and Rac/Cdc42 regulated pathways when expressed in COS-7 cells.
PDZ-RhoGEF Activates SRE in a Rho-dependent
Manner--
Recently, Rho proteins have been shown to signal to the
SRE through a pathway affecting the transcriptional activity of SRF, independent of any MAPK described to date (16). Thus, we next asked
whether PDZ-RhoGEF could induce expression from a reporter plasmid
containing a mutated SRE which eliminated the ternary complex
factor-binding site and which was shown to be potently activated by Rho
(16). As shown in Fig. 2C, an activated form of Rho potently
induced expression from this reporter system when used as a control
and, under identical experimental conditions, PDZ-RhoGEF caused a
remarkable, nearly 15-fold, elevation in CAT activity. Furthermore,
whereas deletion of the PDZ and the proline-rich region did not have
any demonstrable effect, deletion of the entire NH2-terminal regulatory domain enhanced the ability of
PDZ-RhoGEF to stimulate SRF-dependent transcription
when expressed at comparable levels. In contrast, as was expected, the
deletion mutant lacking also the DH domain failed to elevate CAT
expression. Thus, PDZ-RhoGEF potently stimulates
SRF-dependent transcription, and that can be enhanced upon
deletion of a putative negative regulatory region located between the
proline-rich region and the catalytically active DH domain.
As PDZ-RhoGEF induces the expression from an SRE-regulated reporter
plasmid, we next explored whether Rho mediates this effect. As an
approach, we took advantage of the finding that the botulinum toxin C3
ADP-ribosylates Rho thus preventing its activation (38, 39). As shown
in Fig. 3, cotransfection with a C3 toxin
expression plasmid abolished the stimulation of CAT activity by the
activated, NH2-terminal truncated form of PDZ-RhoGEF.
Similar results were obtained using wild-type PDZ-RhoGEF (data not
shown). To confirm a role for Rho, we then cotransfected PDZ-RhoGEF and
C3 with an expression plasmid for a Rho mutant that is insensitive to
the effects of C3, RhoI41 (38, 39). As shown in Fig. 3, RhoI41 restored
the ability of PDZ-RhoGEF to stimulate SRE-dependent expression. Taken together, we can conclude that PDZ-RhoGEF potently induces SRE, and that transcriptional stimulation requires a functional Rho protein. In line with these results, we have also found that PDZ-RhoGEF can potently induce the formation of actin stress fibers when expressed in Madin-Darby canine kidney cells, a typical
Rho-dependent effect (data not shown). Collectively, these
data indicate that PDZ-RhoGEF can effectively activate
Rho-dependent pathways.
PDZ-RhoGEF Associates Physically with Heterotrimeric G Protein
As an approach, we co-expressed in 293T cells the AU1-tagged
full-length PDZ-RhoGEF together with PDZ-RhoGEF Binds Heterotrimeric G Proteins through a Novel
Structural Domain Designated LH--
To investigate further the
identity of the structural domains of PDZ-RhoGEF involved in binding to
G
To further study the role of the LH domain in signaling, we
investigated the ability of each deletion mutant to activate SRE, a
Rho-dependent event. As shown in Fig. 6C, the PH
and DH domains are strictly required to induce SRE, and as described
above, deletion of the PDZ and PDZ + proline-rich domains did not
affect the ability of PDZ-RhoGEF to stimulate SRE. However, deletion of
the entire NH2-terminal regulatory region or the LH domain
enhanced the activity of PDZ-RhoGEF. Thus, these data strongly suggest
that the NH2-terminal regulatory region exerts a negative
regulatory activity on the catalytic DH/PH domains, and that the LH
domain may be responsible for this inhibitory activity.
A DH and PH Deletion Mutant of PDZ-RhoGEF Prevents Signaling from
G Although small GTP-binding proteins of the Rho family play a
critical role in a variety of cellular functions, including the organization of the actin cytoskeleton and the activity of biochemical routes regulating gene expression and cell growth, how these GTPases are activated by cell surface receptors is still largely unknown. Thus,
we investigated whether novel exchange factors for Rho-related GTPases
might exist, exhibiting functional domains suggestive of a role in
signal transduction. For this, we took advantage of the observation
that all known GEFs for Rho proteins exhibit a DH domain, a 250-amino
acid stretch of significant sequence similarity to Dbl, a transforming
protein that was originally isolated from a diffuse B cell lymphoma
(46). Using a consensus sequence for DH domains of Rho-exchange factors
(2) to search DNA data bases, we identified a yet uncharacterized
molecule exhibiting a putative DH domain. Detailed analysis of its
primary sequence revealed that this molecule contained additional areas
of similarity with known modular domains, including a PH domain, a PDZ
domain, a proline-rich region, and an area of homology to p115-RhoGEF, DRhoGEF2 and Lsc, that was not found in any other GEF described so far,
and that was termed LH domain. As many of these protein regions are
likely candidates to participate in signal transmission, we decided to
investigate further this novel putative exchange factor.
When the DH domain of this new molecule was compared with those of
other DH containing proteins, we found that it was highly related to
that of two Rho-specific GEFs, p115-RhoGEF (30) and Lsc (31), and to a
recently described Drosophila Rho GEF, DRhoGEF2 (33).
However, PDZ-RhoGEF was more distantly related to the DH domain of
exchange factors activating Rac1 and/or Cdc42 such as Tiam1 (47), Vav
(48), Ost (49), and Dbl (46), and to those acting on Ras, including Sos
(50) and Ras-GRF (51). Consistent with this observation, expression of
epitope-tagged forms of this novel GEF did not elevate the activity of
co-transfected HA-tagged forms of MAPK and JNK, but potently stimulated
the transcriptional activity of SRF, as judged by the use of a reporter
plasmid under the control of a mutated SRE (16). Furthermore,
experiments with the use of botulinum C3 exoenzyme, which
ADP-ribosylates and inactivates Rho, and an inactivation resistant form
of Rho, RhoI41, indicated that the enhanced expression from the
SRE-driven plasmid was dependent on the availability of a functional
Rho. In addition, we have recently observed that expression of this molecule in Madin-Darby canine kidney cells potently induces the formation of actin stress fibers, to an extent comparable to that caused by expressing activated forms of
Rho.2 Thus, taken together,
the primary sequence similarity with Rho GEFs and these biochemical
profiles strongly suggest that this novel DH-containing molecule, that
was designated PDZ-RhoGEF, can stimulate in vivo
Rho-specific pathways.
In addition to the DH domain, a PH domain is present in all GEFs for
Rho-related proteins described so far, located adjacent to the carboxyl
end of the DH domain (18). PH domains are found in a wide variety of
signaling molecules (52) and have been implicated in both
protein-protein and protein-lipid interactions (53). Although a DH
domain is necessary and sufficient for the exchange activity on Rho
proteins in vitro (20), the integrity of the PH domain is
required for the activity in vivo of this family of exchange
factors (31) most likely by facilitating membrane translocation (31).
Consistent with those observations, deletion of the DH domain, PH
domain, or DH/PH domains abolished the biochemical activity of
PDZ-RhoGEF. Similarly, PDZ-RhoGEF readily induced the appearance of
foci of transformation when expressed in NIH 3T3 cells, and this
activity required the presence of an intact DH and PH domain (data not
shown), supporting a critical role for the DH and PH domains for the
functional activity of PDZ-RhoGEF.
The most striking feature of this novel exchange factor was the
presence of a PDZ domain, a protein-protein interaction domain originally identified as an area of homology between the product of the
Drosophila dlg tumor suppresor gene and the synaptic protein PSD-95 (54), currently found in more than 60 distinct gene products (35). These domains can either bind specific recognition sequences such
as the (S/T)XV motif at the carboxyl termini of certain
proteins, or they can form hetero- or homodimers, suggesting that this
modular protein-binding domain can participate in the formation of
macromolecular complexes (35). Thus, the PDZ domain was expected to
contribute to PDZ-RhoGEF function. Surprisingly, however, when this
domain was deleted, we did not observe any demonstrable effect on the ability of PDZ-RhoGEF to induce Rho-dependent pathways.
Similarly, deletion of the PDZ domain was shown not to affect the
biological activities of the Rac1 exchange factor Tiam1 (55). Although, based on these results, we cannot rule out the possibility that the PDZ
domain facilitates the interaction of PDZ-RhoGEF and other exchange
factors with yet to be identified signaling molecules (see below), we
therefore decided to focus our efforts on other putative functional
domains. One such interesting domain is a stretch of 197 amino acids
located upstream from the DH domain, that was termed LH domain for
Lsc homology domain, and that is also found in
Lsc, p115-RhoGEF, and DRhoGEF2 but not in any other GEF. This suggests
that the LH domain represents a distinctive feature of this subgroup of
exchange factors, which might bear functional relevance. Indeed,
deletion of the LH domain enhanced the ability of PDZ-RhoGEF to
stimulate SRE, to an extent comparable to that of a truncation mutant
lacking the entire NH2-terminal regulatory region.
Similarly, we found that NH2-terminal truncated or
LH-deleted forms of PDZ-RhoGEF exhibit enhanced focus forming activity
in NIH 3T3 cells when compared with the wild-type
form.3 Thus, PDZ-RhoGEF, like
other GEFs, appears to be negatively regulated by inhibitory sequences
within the non-catalytic region (18), and this inhibitory function most
likely resides in the LH domain.
Because of the possibility that the LH domain might have a regulatory
function, we searched data bases for molecules displaying sequences
related to the LH domain. Surprisingly, we found that the catalytic
region of a recently described GTPase-activating proteins for
heterotrimeric G proteins, RGS14, exhibited a limited sequence
similarity to the LH domain. RGSs were initially identified as
homologues of Sst2 proteins, which are negative regulators of pheromone
signaling in yeast (56). This protein family, currently with 19 members, shares a 120-amino acid core, termed GH domain, which is
essential for accelerating the rate of GTP hydrolysis on G These findings may have important implications regarding the function
of G proteins of the G12 family, G However, the nature of the molecules linking G12 family, G
12 and
G
13, and that this interaction was mediated by the LH
domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF
mediates the activation of Rho by G
12 and
G
13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that
transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of
members of the G
12 family which, in turn, activate an
exchange factor acting on Rho.
INTRODUCTION
Top
Abstract
Introduction
References
12 homolog. Here, we
found that PDZ-RhoGEF physically associates in vivo with
activated
subunits of heterotrimeric G proteins of the
G12 family, G
12 and G
13.
Association was found to occur through a novel structural domain,
termed Lsc homology (LH) domain, located
between the PDZ and the DH domain, and also present in the
NH2-terminal, regulatory domain of the lsc
proto-oncogene product and its human homolog, p115-RhoGEF. This LH
domain is distantly related to the G protein-binding region of a family of proteins known as regulators of G protein
signaling (RGSs) (26). Together, our present findings
suggest the existence of a novel pathway by which the large family of G
protein-coupled receptors communicates to Rho through the activation of
G12/G13 and the physical association between
G
12 or G
13 with LH containing GEFs for
Rho, thereby stimulating Rho-dependent pathways.
EXPERIMENTAL PROCEDURES
q,
G
i2, G
s, G
12, and
G
13,
1 and
2 subunits of G
proteins and RhoI41, a RhoA mutant insensitive to the C3 toxin
activity, were described previously (15, 27). Reporter plasmids that express the chloramphenicol acetyltransferase (CAT) gene under the
control of the wild-type serum response element from the
c-fos promoter (SREwt), or a mutant lacking the ternary
complex factor-binding site (SREmutL) as well as an expression vector
for the C3 toxin were kindly provided by R. Treisman (16).
-actin cDNA (2.0 kilobase pairs) was also used
as a control probe. Probes were labeled using a Random Primer DNA labeling kit (Boehringer-Mannheim) with [
-32P]dCTP,
and RNA hybridization performed as described (28).
gal, a
plasmid expressing the enzyme
-galactosidase, and 1.0 µg of
pSREmutL, the reporter plasmid expressing the CAT gene. 293T cells were
transfected with expression vectors for PDZ-RhoGEF or its mutants
together with 0.5 µg of pCMV-
gal and 0.5 µg of pSREwt, the
reporter plasmid expressing a luciferase gene under the control of the
wild-type SRE. After overnight incubation, NIH 3T3 cells and 293T cells were washed twice with phosphate-buffered saline, and kept for approximately 24 h in Dulbecco's modified Eagle's medium
supplemented with 0.5% calf serum or 0.5% fetal bovine serum,
respectively. Cells were then lysed using reporter lysis buffer
(Promega). Additional DNAs were added to the transfection mixtures as
indicated in each figure. CAT activity was assayed in the cell extracts
by incubation at 37 °C for 10-16 h in the presence of 0.25 µCi of
[14C]chloramphenicol (100 mCi/mmol) (ICN) and 200 µg/ml
butyryl-CoA (Sigma) in 0.25 M Tris-HCl, pH 7.4. Labeled
butyrylated products were extracted using a mixture of Xylenes
(Aldrich) and counted as described (15). Luciferase activity
in cell extracts was measured using Luciferase assay system (Promega).
-Galactosidase activity present in each sample was assayed by a
colorimetric method, and used to normalize for transfection efficiency.
-glycerophosphate, 1 mM sodium vanadate, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/ml
leupeptin. For the co-immunoprecipitation study of PDZ-RhoGEF and
heterotrimeric G protein subunits, 293T cells were transfected with
vector or expression vector for PDZ-RhoGEF-AU1 or its mutants together
with vector or expression vectors carrying cDNAs for the
constitutively activated forms of G
s G
i2,
G
q, G
12, and G
13
(G
sQL, G
i2QL, G
q QL,
G
12QL, and G
13QL, respectively) as well
as plasmids expressing
1 and
2 cDNAs
or epitope-tagged G
12QL and G
13QL
(HA-G
12QL and HA-G
13QL, respectively).
After culture for 48 h, the cells were washed twice with
phosphate-buffered saline, then lysed at 4 °C in 900 µl of lysis
buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM MgCl2, 1 mM sodium vanadate, 1%
Triton X-100, 10 µM AlCl3, 10 mM
sodium fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride), and centrifuged at
15,000 rpm for 10 min at 4 °C. The epitope-tagged PDZ-RhoGEF and its
mutants or HA-G
12QL and HA-G
13QL were
immunoprecipitated from the cleared lysates by incubation for 1 h
at 4 °C with the specific antibody against AU1 or HA, respectively.
Immuno complexes were recovered with the aid of Gamma-bind Sepharose
beads (Pharmacia).
subunits of
heterotrimeric G proteins was kindly provided by William Simonds, and
anti-G
12/13 rabbit polyclonal serum was described before
(29). Rabbit polyclonal antisera to G
q and to
G
s were purchased from Santa Cruz Laboratories and to
G
i2 from Upstate Biotechnology.
RESULTS
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Fig. 1.
PDZ-RhoGEF contains several domains involved
in signal transduction and is widely expressed in many human tissues.
A, amino acid sequence of PDZ-RhoGEF. The expected
translational product of PDZ-RhoGEF, accession number AB002378, is
depicted using the 1-letter amino acid code. B, sequence comparison of
PDZ-RhoGEF with proteins possessing PDZ, LH, DH, and PH domains.
Accession numbers: DRhoGEF2, AF032870; Rhophilin, U43194; GRIP, U88572;
Lsc, U58203; p115-RhoGEF, U64105; proto-Dbl, P10911; pleckstrin,
P08567. C, Northern blot analysis. A radiolabeled probe
encompassing nucleotide sequence corresponding to both 3'-translated
and -untranslated regions of PDZ-RhoGEF cDNA (1832 base pairs) was
hybridized with poly(A)+ RNA blots prepared from the
indicated human tissues. The probe for actin mRNA was hybridized
with the same membrane as a control. The arrow indicates the
band of actin mRNA.
-127,
-238,
-702,
and
-956, lacking progressively, the PDZ domain, the proline-rich
region, the LH domain, and the DH domain, as depicted in Fig.
2A, were also detected in transfected 293T cells.
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Fig. 2.
Activation of the SRE, but not MAPK cascades,
by PDZ-RhoGEF. A, structures and expression of
epitope-tagged wild type and NH2-terminal truncated mutants
of PDZ-RhoGEF. On the left panel, structure of the proteins
encoded by each expression plasmid: -127,
-238,
-702, and
-956 constructs code for amino acid residues 127 to 1522, 238 to
1522, 238 to 1522, 702 to 1522, and 956 to 1522 of PDZ-RhoGEF,
respectively. On the right panel, lysates from cells
transfected with the vector control or with expression plasmid carrying
epitope-tagged forms of PDZ-RhoGEF (PDZ-RhoGEF-AU1) and its mutants were
immunoprecipitated with anti-AU1 antibody and subjected to Western blot
analysis with the antibody to AU1. Bands were visualized by the
enhanced chemiluminescence technique using the appropriate horseradish
peroxidase-conjugated goat antiserum. B, effects of
wild-type and mutant PDZ-RhoGEF on MAPK and JNK activity. COS-7 cells
were transfected with pcDNA3-HA-MAPK (1 µg/plate) or
pcDNA3-HA-JNK1 (2 µg/plate) for MAPK and JNK assays,
respectively, together with pCEFL vector or expression vectors carrying
cDNAs for wild-type and NH2-terminal truncated mutants
of PDZ-RhoGEF, the mutationally activated Ras (Ras-V12) and onco-Dbl,
as indicated (PDZ-RhoGEF and its mutants: 3 µg/plate, Ras-V12 and
onco-Dbl: 1 µg/plate). Treatments of cells with 10 µg/ml anisomycin
for 20 min or 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate
(TPA) for 10 min were used as a control. Kinase reactions
were performed in anti-HA immunoprecipitates from the corresponding
cellular lysates as described under "Experimental Procedures." Data
represent the mean ± S.E. of three independent experiments,
expressed as fold increase with respect to vector-transfected cells
(vector). Autoradiograms correspond to representative
experiments. Western blot (WB) analysis was performed in the
corresponding cellular lysates and immunodetected with the antibody to
HA. C, effects of wild-type and mutants of PDZ-RhoGEF on the
activity of SRE. NIH 3T3 cells were cotransfected with pSREmutL (1 µg/plate) and pCMV-
-gal (1 µg/plate) plasmid DNAs together with
expression vectors carrying cDNAs for wild-type and
NH2-terminal truncated mutants of PDZ-RhoGEF (2 µg/plate)
or the constitutively activated mutants of Ras and RhoA (1.0 µg/plate), as indicated. Cells were processed as described under
"Experimental Procedures." The data represent CAT activity
normalized by the
-galactosidase activity present in each cellular
lysate, expressed as fold induction with respect to control cells, and
are the mean ± S.E. of triplicate samples from a typical
experiment. Similar results were obtained in three separate
experiments.
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Fig. 3.
Involvement of functional Rho proteins in the
SRE activation by PDZ-RhoGEF. NIH 3T3 cells were co-transfected
with pSREmutL (1 µg/plate) and pCMV- -gal (1 µg/plate) plasmid
DNAs and expression vectors for the activated mutant of PDZ-RhoGEF (2 µg/plate,
-702), the C3 toxin, and the C3-insensitive mutant of
RhoA (5 µg/plate, RhoI41), as indicated. The data represent CAT
activity normalized by the
-galactosidase activity present in each
cellular lysate, expressed as fold induction with respect to control
cells, and are the mean ± S.E. of triplicate samples from a
typical experiment. Similar results were obtained in three independent
experiments.
Subunits of the G12 Family--
A genetic screen for
components of the Rho signaling pathway in Drosophila led to
the identification of DRhoGEF2, the Drosophila homolog of
PDZ-RhoGEF, as an essential molecule for directing cell shape changes
associated with gastrulation during early embryo development (32, 33).
Of interest, two other molecules were previously identified as critical
for gastrulation, folded gastrulation, a secreted protein (40), and
concertina, a G protein
subunit related to
G
12 (41). Although a direct link between the folded gastrulation receptor, concertina and DRhoGEF2 is yet to be
established, these studies suggested that this Rho-GEF might act
downstream from heterotrimeric G proteins. Furthermore, a computer
assisted search for proteins sharing areas of homology to the LH domain of PDZ-RhoGEF revealed that this domain exhibits limited similarity to
a region within the putative catalytic domain of RGS14 (26), a newly
discovered RGS for G protein
subunits (Fig.
4). Together, these findings prompted us
to explore whether PDZ-RhoGEF could interact directly with a
heterotrimeric G proteins.
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Fig. 4.
Comparison of the amino acid sequences of
PDZ-RhoGEF, p115-RhoGEF, and RGS14. The sequences between residues
290 and 486 of PDZ-RhoGEF, residues 25 and 232 of p115-RhoGEF and
residues 57 and 235 of RGS14 were optimally aligned on the basis of
residue identity (black) and similarity
(gray).
1
2
dimers or activated forms of representative members of each G protein
subunit family, G
q, G
s,
G
i2, and G
12 and G
13 in
293T cells. In each case, we used G
proteins rendered constitutively
active by replacing a critical glutamine residue within the C3 region
that is essential for GTP hydrolysis for leucine (QL mutants) (1).
Detailed biochemical characterization of each G protein subunit has
been previously reported in our laboratory (29, 42-45). As shown in
Fig. 5A, the tagged PDZ-RhoGEF
and all transfected G protein
subunits and
heterodimers were
expressed, as judged by Western blot analysis using anti-epitope
monoclonal antibodies and G protein subtype-specific antisera.
Surprisingly, we found that when PDZ-RhoGEF was immunoprecipitated,
both members of the G
12 family, G
12 and
G
13, co-immunoprecipitated with this putative nucleotide exchange factor for Rho. No other G protein subunit was found to
co-immunoprecipitate with PDZ-RhoGEF, nor were G
12 and
G
13 detectable in immunoprecipitates from control
samples (Fig. 5A). To confirm these findings, we
co-expressed the AU1-tagged PDZ-RhoGEF with NH2-terminal
HA-tagged forms of G
12 and G
13, and
performed anti-AU1 and anti-HA Western blots on both anti-HA and
anti-AU1 immunoprecipitates. As shown in Fig. 5B, the
activated forms of G
13 efficiently co-immunoprecipitated
PDZ-RhoGEF. Similar results were obtained with G
12,
although the co-immunoprecipitation of PDZ-RhoGEF was less efficient
because the HA-tagged G
12 was poorly expressed (data not
shown). We concluded that both members of the G
12 family
physically associate in vivo with PDZ-RhoGEF, thus providing
a direct link between heterotrimeric G proteins and small GTP-binding
proteins of the Ras superfamily.
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Fig. 5.
Direct interaction of PDZ-RhoGEF with
heterotrimeric G protein subunits of the
G12 family. A, 293T cells were
transfected with vector or expression vector (C) for
PDZ-RhoGEF-AU1 (P-AU1) together with vector or expression plasmids
carrying cDNAs for the constitutively activated mutants of
G
s, G
i2, G
q,
G
12, and G
13 as well as plasmids
expressing
1 and
2 cDNAs, as
indicated. Lysates, prepared as described under "Experimental
Procedures," were immunoprecipitated (IP) with anti-AU1
antibody and subjected to Western blot (WB) analysis using
anti-AU1 antibody and G protein subtype-specific antisera. In addition,
total cellular lysates (TCL) were subjected to Western blot
analysis with G protein subtype-specific antisera. B,
lysates from cells transfected with vector or expression plasmids for
PDZ-RhoGEF-AU1 together with vector or expression vectors carrying
cDNAs for an epitope-tagged and activated form of
G
13 (HA-G
13QL) were immunoprecipitated
with anti-AU1 and anti-HA antibody, and were subjected to Western blot
analysis with antibodies to HA and AU1, respectively. In addition,
total cellular lysates were used in Western blot analysis with anti-AU1
and anti-HA antibodies. Bands were visualized by the enhanced
chemiluminescence technique using the appropriate horseradish
peroxidase-conjugated goat antiserum. In this figure, G
,
AU1, and HA are used for abbreviations anti-G
antiserum, anti-AU1 antibody, and anti-HA antibody, respectively.
12 and G
13, we used the progressive
truncated forms of PDZ-RhoGEF described above, lacking the PDZ domain
(
-127), the PDZ domain, and the proline-rich domain (
-238), and
the entire NH2-terminal regulatory region (
-702). We
also engineered additional deletion mutants, lacking the LH domain
(
LH), the PH domain (
PH), and the DH/PH domain (
DH/PH), as
depicted in Fig. 6A. All
deletion mutants were efficiently expressed when transfected into 293T
cells (see above and Fig. 6A). However, when co-expressed
with an activated epitope-tagged form of G
12 and
G
13, we found that all PDZ-RhoGEF deletion mutants lacking the LH domain were unable to associate in vivo with
these G protein
subunits (Fig. 6B). These data indicate
that the ability of PDZ-RhoGEF to bind heterotrimeric G proteins of the
G
12 family is strictly dependent upon the structural
integrity of its LH domain.
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Fig. 6.
Interaction of PDZ-RhoGEF with heterotrimeric
G protein subunits through the LH domain.
A, structure and expression of deletion mutants of
PDZ-RhoGEF. Structure of the proteins encoded by the expression
plasmids used in this figure are shown (left and in Fig.
2A).
-LH,
-PH, and
-DH/PH constructs, lacking LH,
PH, and both DH and PH domains, respectively, codes for amino acid
sequences in which residues 171-484, 956-1054, and 738-1054 were
deleted from PDZ-RhoGEF, respectively. Lysates from cells transfected
with vector or with expression plasmid carrying an epitope-tagged
PDZ-RhoGEF (PDZ-RhoGEF-AU1) and its deletion mutants were
immunoprecipitated with anti-AU1 antibody and these immunoprecipitates
were subjected to Western blot analysis with the antibody against AU1.
B, 293T cells were transfected with vector or expression
plamids for G
12QL or G
13QL together with
vector or expression vectors carrying cDNAs for wild type and
deletion mutants of PDZ-RhoGEF, as indicated. Lysates, prepared as
described under "Experimental Procedures," were immunoprecipitated
with anti-AU1 antibody and subjected to Western blot (WB)
analysis using specific antiserum for heterotrimeric G proteins of the
G12 family. Total cellular lysates (TCL) were
also subjected to Western blot analysis using the same antiserum. In
A and B, bands were visualized by the enhanced
chemiluminescence technique using the appropriate horseradish
peroxidase-conjugated goat antiserum. C, effects of
wild-type and deletion mutants of PDZ-RhoGEF on the activity of the
SRE. NIH-3T3 cells were co-transfected with pSREmutL (1 µg/plate) and
pCMV-
-gal (1 µg/plate) plasmid DNAs together with expression
vectors carrying cDNAs for wild-type and deletion mutants of
PDZ-RhoGEF, as indicated. Cells were processed as described under
"Experimental Procedures." The data represent CAT activity
normalized by the
-galactosidase activity present in each cellular
lysate, expressed as fold induction with respect to control cells, and
are the mean ± S.E. of triplicate samples from a typical
experiment. Similar results were obtained in three independent
experiments.
12 and G
13 to Rho-dependent
Pathways--
The availability of biochemically inactive PDZ-RhoGEF
mutants prompted us to ask whether they can affect signaling from
G
12 and G
13 to SRE, a
Rho-dependent event (27). For these experiments, we chose
to use 293T cells as the transfection efficiency in these cells is
greater than that in NIH 3T3 cells, thus allowing us to control the
expression of each transfected DNA construct. As shown in Fig.
7, expression in 293T cells of PDZ-RhoGEF
lacking the DH/PH domains or the entire NH2-terminal region + DH domain (
-956) did not affect SRE activation by RhoQL, when used
as a control. In contrast, the PDZ-RhoGEF DH/PH deletion mutant
specifically blocked SRE activation by G
12 and
G
13. Similarly, this PDZ-RhoGEF mutant diminished the
activation of SRE when mediated by lysophosphatidic acid receptors
(data not shown). Thus, PDZ-RhoGEF
DH/PH behaves as a dominant
negative mutant, probably by preventing the coupling of
G
12 and G
13 to the endogenously expressed
PDZ-RhoGEF or to other related GEFs.
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Fig. 7.
Inhibition of
G 12- and
G
13-mediated SRE activation by the
DH and PH deletion mutant of PDZ-RhoGEF. 293T cells were
cotransfected with pSREwt (0.5 µg/plate) and pCMV-
-gal (0.5 µg/plate) plasmid DNAs together with expression vectors carrying
cDNAs for the constitutively activated mutants of
G
12, G
13, and RhoA,
-DH/PH,
-956,
and green fluorescent protein (GFP) (control), as indicated.
Cells were processed as described under "Experimental Procedures."
The data represent luciferase activity normalized by the
-galactosidase activity present in each cellular lysate, expressed
as a percentage relative to that observed in control, and are the
mean ± S.E. of triplicate samples from a typical experiment.
Similar results were obtained in three independent experiments.
DISCUSSION
proteins
(56). RGS14, together with RGS12, represent novel members of this
family, characterized for being substantially larger (~60 and ~140
kDa, respectively) than the majority of the other known RGSs (~25
kDa) (26). This data suggested that the LH domain might confer to
PDZ-RhoGEF the ability to interact with G protein
subunits. Indeed,
we found that PDZ-RhoGEF could form stable complexes in vivo
specifically with members of the G12 family of G protein
subunits, G
12 and G
13, and that this
interaction required the presence of an intact LH domain. Taken
together, we can conclude that PDZ-RhoGEF can interact physically with
a particular subset of G
proteins, thereby providing a direct link between hetereotrimeric G proteins and small GTP-binding proteins of
the Rho family.
12 and
G
13. These ubiquitously expressed G proteins were
discovered by M. Simon's group upon amplification of mouse brain
cDNA by polymerase chain reaction using degenerated
oligonucleotides corresponding to regions highly conserved among G
proteins (57). G
12 and G
13 exhibit 67%
amino acid identity with each other, but only 35-44% of amino acid
identity to
subunits of other classes, such as Gq,
Gi, and Gs (57). Furthermore, whereas members
of the G
q family of G proteins activate
phosphatidylinositol-specific phospholipases, the G
s
family stimulate adenylyl cyclases, and G
i inhibits
adenylyl cyclases and activate certain phosphodiesterases and promote
the opening of several ion channels (58-60), members of the
G
12 family of GTPases appear not to affect any of these
second messenger-generating systems (60). In this regard, the finding
that concertina (cta), a Drosophila
gene involved in embryogenesis (41), and that G
12 and
G
13 can behave as remarkably potent oncogenes (29, 42), provided early indications that this G protein class might be involved
in growth regulation, albeit through poorly defined mechanisms. Intense
investigation in many laboratories has recently generated a wealth of
information on how G
12 and G
13 may act
(see Ref. 61, for a recent review). In particular, one such study (62) demonstrated that activated G
12 and G
13,
but not G
i2 and G
q or different
combinations of
and
subunits, mimicked the effect of activated
RhoA on stress fibers and focal adhesion assembly, and we have recently
provided evidence that G
12 stimulates nuclear responses
and cellular transformation through Rho (27). Furthermore, several
studies have now provided evidence that members of the G12
family of G proteins link many G protein-coupled receptors, including
receptors for lysophosphatidic acid, thrombin, thromboxane A2, and acetylcholine to the activation of
Rho-dependent pathways and, in many cases, cell growth
control (27, 63-66).
12 and
G
13 to Rho remained largely unknown. In this regard, our
present results suggest that PDZ-RhoGEF or other LH-containing RhoGEF
might act downstream from these G protein
subunits in a biochemical
route leading to Rho activation (see Fig.
8). Moreover, while our study was in the
process of submission, it was reported that G
13 could enhance the in vitro Rho-GEF activity of p115-RhoGEF, a
distinct LH domain-containing exchange factor (67, 68). However, how the interaction between RhoGEFs and G
12 and/or
G
13 leads to Rho activation in vivo is still
unclear. For PDZ-RhoGEF, it is possible that binding of G12
proteins to its LH domain results in the translocation of PDZ-RhoGEF to
the membrane where it can act on Rho. Alternatively, binding of
G12 to the LH domain might de-repress the functional
activity of PDZ-RhoGEF by preventing its negative modulatory effect. In
addition, although the functional significance of the PDZ domain is
still unclear, it is noticeable that some receptors known to induce
G
12 and activate Rho exhibit a COOH-terminal PDZ-binding
motif, such as SVV in the case of both human and murine
lysophosphatidic acid receptors, EDG-2 and VZG-1, respectively (69,
70). Thus, it is also possible that certain G12-coupled
receptors might facilitate the recruitment of PDZ-RhoGEF by binding to
its PDZ domain and, simultaneously, activating G12
proteins. These, as well as other possibilities, are under current
investigation.
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Fig. 8.
Proposed mechanism whereby G-protein coupled
receptors stimulate Rho-dependent pathways. The
stimulation of certain G protein-coupled receptors activates
G 12 and G
13 which, in turn, interact with
PDZ-RhoGEF through its LH domain, thereby causing the activation of
PDZ-RhoGEF by a still unclear mechanism. Subsequently, the activated
PDZ-RhoGEF catalyzes the exchange of GDP for GTP on Rho through its DH
and PH domains, leading to the activation of Rho-dependent
pathways. Additional possibilities are also discussed in the
text.
We can conclude that our present findings support the existence of a
novel mechanism whereby the large family of G protein-coupled cell
surface receptors can stimulate Rho-dependent pathways
(Fig. 8). This pathway involves the activation of G12
and/or G
13, which, in turn, will interact directly with
Rho-exchange factors containing a
G
12/G
13-binding region, such as an LH
domain. This would result in the activation of Rho by a still unclear
mechanism, thereby stimulating the activity of
Rho-dependent pathways that, ultimately, would affect the
cytoskeletal structure, nuclear gene expression, and cellular growth.
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FOOTNOTES |
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* 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.
Supported by a postdoctoral fellowship from the Spanish Ministerio
de Educación y Cultura.
§ To whom correspondence should be addressed: Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, 30 Convent Dr., Bldg. 30, Rm. 212, Bethesda, MD 20892-4330. Tel.: 301-496-6259; Fax: 301-402-0823; E-mail: gutkind{at}nih.gov.
2 M. Zohar and J. S. Gutkind, unpublished data.
3 S. Fukuhara and J. S. Gutkind, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: GEF, guanine nucleotide exchange factor; LH domain, Lsc homology domain; MAPK, mitogen-activated protein kinase; JNK, c-Jun amino-terminal kinase; SRE, serum response element; SRF, serum response factor; GAP, GTPase-activating protein; DH domain, Dbl homology domain; PH domain, pleckstrin-homology domain; SH, Src homology; RGS, regulators of G protein signaling; CAT, chloramphenicol acetyltransferase.
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REFERENCES |
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