From the Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received for publication, March 25, 2003
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ABSTRACT |
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INTRODUCTION |
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Semaphorins are a large family of secreted and transmembrane molecules that
play a central role in axon guidance in developing nervous system
(35).
In addition to the nervous system, they are widely expressed in embryonic and
adult tissues and are thought to mediate diverse processes such as cardiac and
skeletal development (6), tumor
growth and metastasis (7), and
the immune response (8,
9). The function of semaphorins
in the nervous system is mediated by Plexins, which can be classified into
four subfamilies: Plexin-A14, Plexin-B13, Plexin-C1, and
Plexin-D1 (10,
11). The most characterized
member of the semaphorins is semaphorin 3A (Sema3A), and a variety of
molecules have been shown to be involved in the intracellular signaling
pathway for the actions of Sema3A
(12,
13). Although most other
semaphorins appear to bind to and directly activate Plexins, Sema3A requires
receptor complexes consisting of Plexin-A1/2 and neuropilins
(10,
14). Plexin-B1 has been
identified as a receptor for semaphorin 4D (Sema4D, also known as CD100)
(10), and recent studies
indicate that Plexin-B1 directly interacts with PDZ-RhoGEF/leukemia-associated
Rho GEF (LARG) through its carboxyl-terminal PSD-95/Dlg/ZO-1 (PDZ)
domain-binding motif to induce RhoA activation and growth cone collapse in
response to Sema4D
(1519).
PDZ-RhoGEF was originally identified as a Rho-specific GEF with the PDZ domain
in its amino terminus that interacts with activated subunits of the
G12 family of heterotrimeric G proteins and can link G
protein-coupled receptors to RhoA activation
(20). In addition to RhoA
activation, Plexin-B1 directly interacts with Rac in a GTP-dependent manner
(21,
22), and this interaction
appears to inhibit Rac-dependent actions by sequestering active GTP-bound Rac
(23,
24). However, precise
mechanisms by which the activity of Plexin-B1 is regulated remain unclear.
Rnd GTPases, Rnd1, Rnd2, and Rnd3 (also known as RhoE), comprise a distinct branch of Rho family GTPases in that they have a low affinity for GDP and very low intrinsic GTPase activities (2527). In fibroblasts, transient expression of Rnd1 or Rnd3 leads to loss of stress fibers, retraction and rounding of the cell body, and production of extensively branching processes (27), and a part of these morphological effects of Rnd1 or Rnd3 is mediated by a novel Rnd GTPase-interacting protein, Socius (28). In Madin-Darby canine kidney epithelial cells, Rnd3 regulates cell migration speed and is involved in the alteration of the actin cytoskeleton associated with oncogenic transformation (26, 29). Among Rnd GTPases, Rnd1 and Rnd2 are abundantly expressed in the nervous system (27, 30), and Rnd2 induces neurite branching through its novel effector Rapostlin (31). Recently, Rnd1 has been shown to interact with Plexin-A1 and appears to be involved in the signaling pathway of Plexin-A1 (32, 33). However, the precise role of Rnd1 in the Plexin-mediated actions remains unknown. Here we provide evidence that Rnd1 directly interacts with the cytoplasmic region of Plexin-B1 and that this interaction potentiates RhoA activation by Plexin-B1 and induces contraction of COS-7 cells. We propose a role of Rnd1 in the Plexin-B1 signaling pathway leading to Rho activation and cytoskeletal rearrangements.
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EXPERIMENTAL PROCEDURES |
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Antibodies used were as follows: a mouse monoclonal anti-Myc antibody 9E10 (Upstate Biotechnology, Inc., Lake Placid, NY); a rabbit polyclonal anti-Myc antibody (MBL); a mouse monoclonal anti-HA antibody 12CA5 (Roche Applied Science); a mouse monoclonal anti-FLAG antibody M2 (Sigma); a mouse monoclonal anti-GFP antibody (Santa Cruz Biotechnology); horseradish peroxidase-conjugated secondary antibodies (DAKO); and Alexa 594-conjugated secondary antibodies (Molecular Probes, Inc., Eugene, OR).
Yeast Two-hybrid ScreeningA rat brain cDNA library fused to
the GAL4 activation domain of the pACT2 vector (Clontech) was screened using
pAS21/Rnd3S241 as a bait in the yeast strain Y190 according
to the manufacturer's instructions. Interaction between the bait and library
proteins activates transcription of the reporter gene HIS3 and
lacZ. From 107 transformants, 218 colonies grew on
selective medium lacking histidine and were also positive for
-galactosidase activity. One of these, clone 179, was found to encode
the carboxyl-terminal 438 amino acids of Plexin-B2.
For the -galactosidase filter assay, colonies of yeast transformants
were transferred onto Hybond-N filter papers (Amersham Biosciences) and
permeabilized in liquid nitrogen. Each filter was placed on a Whatman No. 2
filter paper that had been presoaked in Z buffer (60 mM
Na2HPO4, 40 mM NaH2PO4,
10 mM KCl, 1 mM MgSO4, 37.5 mM
-mercaptoethanol) containing 0.33 mg/ml
5-bromo-4-chloro-3-indoyl-
-D-galactopyranoside and was
incubated at 30 °C for 8 h.
Cell Culture and TransfectionCOS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 4 mM glutamine, 100 units/ml penicillin, and 0.2 mg/ml streptomycin under humidified conditions in 95% air and 5% CO2 at 37 °C. Transient transfections were carried out with LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions.
ImmunoblottingProteins were separated by 10 or 12.5% SDS-PAGE and were electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore Corp.). The membrane was blocked with 3% low fat milk in Tris-buffered saline and then incubated with primary antibodies. The primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and the ECL detection kit (Amersham Biosciences).
In Vitro Binding AssaysAll GST-fused proteins were purified from Escherichia coli as described previously (28). Nonfused Rnd1 was recovered by incubation with 10 units/ml thrombin (Sigma) for4hat4 °C, and then thrombin was removed by absorption to p-amino-benzamidine-agarose beads (Sigma). Protein concentration was determined by comparing with bovine serum albumin standards after SDS-PAGE and by staining with Coomassie Brilliant Blue.
For pull-down assays, COS-7 cells (7 x 105 cells) transfected with HA-tagged Rnd GTPases were rinsed once with phosphate-buffered saline (PBS) and lysed with the ice-cold cell lysis buffer (20 mM Tris-HCl, pH 7.4, 2 mM MgCl2,1mM dithiothreitol (DTT), 0.2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Cell lysates were then centrifuged for 10 min at 18,000 x g at 4 °C. The supernatants were incubated for 10 min at 4 °C with 10 µg of GST fusion proteins and subsequently incubated with glutathione-Sepharose beads for 1 h at 4 °C. After the beads were washed with the ice-cold cell lysis buffer, the bound proteins were eluted in Laemmli sample buffer and analyzed by SDS-PAGE and immunoblotting with anti-HA antibody.
To examine direct interaction between the cytoplasmic domains of Plexins
and Rnd GTPases, an overlay assay was performed according to the modified
method of Manser et al.
(36). E. coli cell
lysates expressing GST-fused cytoplasmic domains of Plexins were subjected to
SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was
soaked for 5 min in 6 M guanidinium hydrochloride dissolved in
buffer A (25 mM Hepes-NaOH, pH 7.0, 0.5 mM
MgCl2, 0.05% Triton X-100) at 4 °C, and the buffer was diluted
with an equal volume of buffer A and agitated for a further 5 min. This
process was repeated four times. The membrane was then agitated for 10 min
five times in buffer A; transferred to PBS containing 1% bovine serum albumin,
0.1% Triton X-100, 0.5 mM MgCl2, and 5 mM
DTT; and incubated in GAP buffer (25 mM Hepes-NaOH, pH 7.0, 5
mM MgCl2, 0.05% Triton X-100, 2.5 mM DTT, and
100 µM GTP) containing [-32P]GTP (6000
Ci/mmol; PerkinElmer Life Sciences)-loaded Rnd1. After washing with wash
buffer (25 mM Hepes-NaOH, pH 7.0, 5 mM MgCl2,
and 0.05% Triton-X-100), the membrane was dried, and bound radioactivity was
visualized with an FLA-3000 image analyzer (Fuji). Nonfused Rnd1 (1 µg)
were loaded with [
-32P]GTP by incubation in loading buffer
(20 mM Tris-HCl, pH 7.4, 25 mM NaCl, 0.1 mM
DTT, 0.5 µg/ml bovine serum albumin, and 2 mM MgCl2)
at 30 °C for 10 min.
ImmunoprecipitationCOS-7 cells (7 x 105 cells) cotransfected with Myc-tagged Plexin-B1 and HA-tagged Rnd1 were lysed with ice-cold cell lysis buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Nonidet P-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). After centrifugation, the supernatants were incubated with anti-Myc polyclonal antibody for 1 h and then with protein A-Sepharose (Amersham Biosciences) for 1 h. The beads were washed with the lysis buffer, and bound proteins were analyzed by SDS-PAGE and immunoblotting.
Immunofluorescence MicroscopyCOS-7 cells (2 x 104 cells) were seeded onto round 13-mm glass coverslips, and then they were transfected with expression vectors encoding Myc-tagged Plexin-B1 and GFP-Rnd1. Sixteen hours after transfection, cells on coverslips were fixed with 4% paraformaldehyde in PBS for 15 min. After residual formaldehyde had been quenched with 50 mM NH4Cl in PBS for 10 min, cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and incubated with 10% fetal bovine serum in PBS for 30 min to block nonspecific antibody binding. Then cells were incubated with anti-Myc antibody in PBS for 1 h, followed by the incubation with Alexa 594-conjugated secondary antibody in PBS for 1 h. Cells on coverslips were mounted in 90% glycerol containing 0.1% p-phenylenediamine dihydrochloride in PBS.
For the COS-7 cell contraction assay, a soluble form of Sema4D expressed as a fusion protein with the Fc fragment of human IgG1 (9) was harvested from the medium of transiently transfected COS-7 cells. Stimulation with Sema4D was performed by incubation of the cells with Sema4D-Fc-containing medium for 5 min at 37 °C. A specific inhibitor for Rho-associated kinase (Rho-kinase) Y-27632 (10 µM; a generous gift from Welfide Corp.) was added immediately after transfection. The size of transfected cells was determined from digital images acquired at x 20 magnification by using a Leica DC350F digital camera system equipped with a Nikon Eclipse E800 microscope and an Image-Pro Plus image analysis software (Media Cybernetics).
Measurement of RhoA ActivityMeasurement of RhoA activity was performed as described previously (37, 38). Briefly, COS-7 cells (7 x 105 cells) were transfected with an expression vector encoding HA-tagged wild-type RhoA together with Myc-tagged Plexin-B1 and GFP-tagged Rnd1 and were serum-starved for 16 h. Then they were stimulated with Sema4D-Fc for 5 min at 37 °C, and the cells were washed with the ice-cold Tris-buffered saline (20 mM Tris-HCl, pH 7.4, 300 mM NaCl). After centrifugation, the cells were lysed with the cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 30 mM MgCl2, 0.2% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Cell lysates were then centrifuged for 5 min at 10,000 x g at 4 °C, and the supernatants were incubated with 16 µg of GST-fused Rho-binding domain of mouse Rhotekin pre-bound to glutathione-Sepharose beads for 60 min at 4 °C. The beads were washed with the lysis buffer, and bound proteins were analyzed by SDS-PAGE and immunoblotting.
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RESULTS |
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The in vitro interaction studies were performed with the cytoplasmic domain of Plexin-B1. Therefore, we next examined whether Rnd1 interacts with the full length of Plexin-B1 in vivo in the presence or the absence of Sema4D, a ligand for Plexin-B1. Myc-tagged full-length Plexin-B1 and HA-tagged Rnd1 were expressed in COS-7 cells, and the cell lysate was immunoprecipitated with anti-Myc antibody. Stimulation of the cells with Sema4D was performed by the incubation with the medium from cells expressing the extracellular region of Sema4D fused with the Fc fragment of human IgG1 (9). As shown in Fig. 2, HA-tagged Rnd1 was coimmunoprecipitated with Myc-tagged Plexin-B1, and this interaction was not affected by the addition of Sema4D, indicating that Rnd1 shows a constitutive, ligand-independent interaction with Plexin-B1 in vivo. On the other hand, we could not detect the interaction between Rnd1 and the full length of Plexin-B1-GGA or between Plexin-B1 and Rnd1A45 by immunoprecipitation studies (Fig. 2). These results were consistent with those of in vitro binding studies.
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Interaction of Rnd1 with Plexin-B1 Induces COS-7 Cell Contraction in Response to Sema4DTo investigate the role of the interaction of Rnd1 with Plexin-B1, COS-7 cells were transfected with Myc-tagged full-length Plexin-B1 together with GFP or GFP-tagged Rnd1, and morphological changes in transfected cells were analyzed by the fluorescence of GFP after the addition of medium from mock or soluble Sema4D-transfected cells. Previous studies have demonstrated that class 3 semaphorins, such as Sema3A, induce the contraction of COS-7 cells when cells are cotransfected with Plexin-A1 and neuropilin, functional receptors for class 3 semaphorins, and that this morphological assay is utilized as a useful model system to study the properties of semaphorin receptors (14, 33, 39, 40). Stimulation with Sema4D had no detectable morphological change in COS-7 cells expressing GFP-Rnd1 alone or co-expressing GFP and Myc-Plexin-B1. In contrast, cells coexpressing GFP-Rnd1 and Myc-Plexin-B1 exhibit a dramatic contraction of the cell body after stimulation with Sema4D (Fig. 3A). On the other hand, coexpression of Rnd1 and Plexin-B1-GGA, a mutant of Plexin-B1 that had no ability to interact with Rnd1, or coexpression of Plexin-B1 and Rnd1A45, a Rnd1 mutant that failed to bind to Plexin-B1, had no morphological effect before and after stimulation with Sema4D (Fig. 3A). Quantitative analysis of COS-7 cell contraction was obtained by counting Plexin-B1 and Rnd1 double positive cells with an area less than 350 µm2 (Fig. 3B). These results suggest that the interaction of Rnd1 with Plexin-B1 is required for the Sema4D-induced COS-7 cell contraction. We also found that the COS-7 cell contraction in response to Sema4D was observed in the cells coexpressing Plexin-B1 and Rnd3. However, stimulation with Sema4D had no detectable morphological effect in the Plexin-B1/Rnd2-expressing cells (data not shown). Although the active form of Rac directly interacts with Plexin-B1 (21, 22), coexpression of Plexin-B1 with constitutively active form of Rac, Rac1L61, did not induce the COS-7 cell contraction in response to Sema4D (Fig. 3), this result being consistent with a previous report (24). Thus, the ability to induce the Plexin-B1-mediated COS-7 cell contraction is specific to Rnd1 or Rnd3. The majority of the contracted cells expressing Plexin-B1 and Rnd1 after stimulation with Sema4D took again a spread morphology when the cells were incubated in Sema4D-free medium for an additional 12 h (data not shown), indicating that this morphological effect is reversible and is not part of a cell death response but reflects a cytoskeletal event, as observed in the Sema3A-induced morphological change (39). In these experiments, immunostaining with anti-Myc antibody showed that expression levels and subcellular localizations of various constructs of Plexin-B1 were similar in the presence and the absence of GFP-Rnd1 (data not shown).
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Plexin-B1- and Rnd1-mediated COS-7 Cell Contraction Involves
PDZ-RhoGEF, RhoA, and Rho-kinaseSeveral lines of evidence indicate
that activation of RhoA is involved in the downstream signaling pathway of
Plexin-B1
(1518,
22). In hippocampal neurons,
Plexin-B1-mediated growth cone collapse in response to Sema4D was inhibited by
C3 exoenzyme, a Rho inhibitor, or by Y-27632, an inhibitor for its downstream
effector Rho-kinase (15).
Therefore, we next examined the involvement of RhoA and Rho-kinase in the
Plexin-B1 and Rnd1-mediated COS-7 cell contraction. The Sema4D-induced
contraction in the cells expressing Myc-tagged Plexin-B1 and GFP-tagged Rnd1
was suppressed by coexpression with HA-tagged dominant-negative RhoA
(RhoAN19) or by pretreatment of the cells with Y-27632
(Fig. 4). Although dominant
negative Rac blocked the Plexin-B1-mediated Rho-dependent stress fiber
formation in Swiss 3T3 fibroblasts
(22), coexpression of
HA-tagged dominant negative Rac1 (Rac1N17) had no effect on the
Sema4D-induced contraction in Plexin-B1/Rnd1-expressing cells
(Fig. 4). Recent studies have
shown that activation of RhoA by Plexin-B1 is mediated by PDZ-RhoGEF or LARG,
members of Rho-specific GEFs. PDZ-RhoGEF contains a PDZ domain in its
amino-terminal region, and the interaction between the PDZ domain of
PDZ-RhoGEF and the PDZ domain-binding motif at the carboxyl terminus of
Plexin-B1 is required for the RhoA activation by Plexin-B1
(1518).
To determine whether PDZ-RhoGEF is involved in the Plexin-B1 and Rnd1-mediated
COS-7 cell contraction, we coexpressed GFP-tagged Rnd1 and Myc-tagged
Plexin-B1 lacking the carboxyl-terminal PDZ domain-binding motif
(Plexin-B1-C) in COS-7 cells. Plexin-B1-
C could interact with
Rnd1 in vivo, as shown by immunoprecipitation studies
(Fig. 2). However,
morphological changes were not detected in the cells coexpressing Rnd1 and
Plexin-B1-
C after Sema4D stimulation
(Fig. 4). Overexpression of the
PDZ domain of PDZ-RhoGEF has been shown to inhibit the Plexin-B1-mediated
cytoskeletal effect, and the PDZ domain of PDZ-RhoGEF therefore serves as the
dominant negative form probably by dissociating endogenous PDZ-RhoGEF or LARG
from Plexin-B1 (18). We showed
that expression of the FLAG-tagged PDZ domain of PDZ-RhoGEF also suppressed
the Sema4D-induced contraction in the Plexin-B1/Rnd1-expressing cells
(Fig. 4). These results
indicate that the PDZ-RhoGEF/RhoA/Rho-kinase pathway participates in the
Plexin-B1 and Rnd1-mediated COS-7 cell contraction. In these experiments,
expressions of wild-type Plexin-B1 and Plexin-B1-
C were almost the same
levels, and more than 80% of Plexin-B1 and Rnd1 double positive cells also
expressed RhoAN19, Rac1N17, or the PDZ domain of
PDZ-RhoGEF (data not shown).
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Interaction of Rnd1 with Plexin-B1 Potentiates Sema4D-induced RhoA
ActivationWe have shown that RhoA and PDZ-RhoGEF are involved in
the Plexin-B1 and Rnd1-mediated cell contraction. This result led us to
examine whether the interaction of Rnd1 with Plexin-B1 affects the
PDZ-RhoGEF-mediated RhoA activation by Plexin-B1 in COS-7 cells. To measure
the level of active GTP-bound RhoA in the cells, we used GST-fused Rho-binding
domain of Rhotekin to precipitate GTP-bound active RhoA from the cell lysates
(37). We could not detect
endogenous RhoA in COS-7 cells with anti-RhoA antibody. Therefore, we
cotransfected HA-tagged wild-type RhoA with Myc-tagged Plexin-B1 and
GFP-tagged Rnd1 in COS-7 cells, and precipitated GTP-bound RhoA was detected
with anti-HA antibody. When COS-7 cells were cotransfected with Plexin-B1 and
HA-tagged wild-type RhoA, a slight increase in RhoA activity was observed
after stimulation with Sema4D. In contrast, coexpression of Rnd1 with
Plexin-B1 dramatically increased the GTP loading of RhoA in the cells in
response to Sema4D (Fig. 5). On
the other hand, the potentiation of RhoA activity was not observed in the
cells coexpressing Rnd1 and Plexin-B1-GGA or coexpressing Rnd1 and
Plexin-B1-C. These results suggest that the interaction of Rnd1 with
Plexin-B1 potentiates PDZ-RhoGEF-mediated RhoA activation by Plexin-B1 in
vivo. Thus, the abilities of Plexin-B1 mutants to enhance the RhoA
activity correlated with their abilities to induce the cell contraction.
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Rnd1 Promotes the Interaction between Plexin-B1 and PDZ-RhoGEFFinally, we examined the possibility that Rnd1 regulates the interaction between Plexin-B1 and PDZ-RhoGEF. Myc-tagged Plexin-B1 and FLAG-tagged PDZ-RhoGEF were expressed in COS-7 cells, and the cell lysate was immunoprecipitated with anti-Myc antibody. Plexin-B1 was coimmunoprecipitated with PDZ-RhoGEF, and this interaction was significantly enhanced by coexpression with Rnd1 (Fig. 6). Deletion of the carboxyl-terminal PDZ domain-binding motif of Plexin-B1 completely abolished the interaction between Plexin-B1 and PDZ-RhoGEF in the presence of Rnd1, indicating that the increased interaction of Plexin-B1 with PDZ-RhoGEF is also mediated through its carboxyl-terminal PDZ domain-binding motif. On the other hand, Plexin-B1-GGA also interacted with PDZ-RhoGEF, but coexpression of Rnd1 did not promote this interaction (Fig. 6). These results suggest that the interaction of Rnd1 with the cytoplasmic domain of Plexin-B1 promotes the interaction between PDZ-RhoGEF and the carboxyl terminus of Plexin-B1. We also tested whether active Rac regulates the interaction of Plexin-B1 with PDZ-RhoGEF and the Plexin-B1-mediated RhoA activation, but both of them were not influenced by expression of Rac1L61 (data not shown).
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DISCUSSION |
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Rac in its active GTP-bound state has been shown to interact directly with Plexin-B1 (21, 22). We showed that the Rnd1 binding region in Plexin-B1 overlaps with the region responsible for the interaction with Rac and that mutation of three amino acid residues in Plexin-B1 (L1849G, V1850G, and P1851A), which abolishes the interaction of Plexin-B1 with Rac, also suppressed the interaction with Rnd1, suggesting that Rnd1 and Rac use a similar or an identical binding site in Plexin-B1. However, coexpression of Plexin-B1 with constitutively active Rac1 in COS-7 cells had no effect on the RhoA activation by Plexin-B1 and did not induce the cell contraction even after stimulation with Sema4D. This may be due to the lack of ability of constitutively active Rac1 to facilitate the interaction of Plexin-B1 and PDZ-RhoGEF. These observations suggest that effects of the interaction of Plexin-B1 with Rnd1 and Rac on the Plexin-B1 signaling are largely different and that potentiation of PDZ-RhoGEF-mediated RhoA activation is an essential step for the induction of cell contraction by Plexin-B1.
It has been recently reported that Rnd1 also interacts with Plexin-A1 and induces COS-7 cell collapse when cotransfected with Plexin-A1 (32, 33). Considering the lack of the PDZ domain-binding motif at the carboxyl terminus of Plexin-A1 and inability of Plexin-A1 to associate with PDZ-RhoGEF or LARG (16), the intracellular signaling pathways of Plexin-A1 and Plexin-B1 for the morphological changes in COS-7 cells are likely to be different. In addition, the cell collapse induced by Rnd1 and full-length Plexin-A1 in COS-7 cells does not require ligand stimulation (33), whereas the cell contraction induced by Rnd1 and full-length Plexin-B1 requires Sema4D stimulation. At present, it is not known why there is a difference in ligand dependence between Plexin-A1 and Plexin-B1 for the induction of the morphological changes in COS-7 cells in the presence of Rnd1, but further elucidation of signal transductions of Plexin family receptors will help us to understand precise mechanisms by which Rnd1 regulates Plexin-mediated cellular functions.
Previous studies have shown that Rnd1 possesses an antagonistic effect on G
protein-coupled receptor-mediated RhoA activation signals, such as
lysophosphatidic acid-induced formation of actin stress fibers and focal
adhesions in fibroblasts (26,
27,
35,
41). In contrast, we have
demonstrated that Rnd1 promotes the Plexin-B1-mediated RhoA activation,
suggesting that Rnd1 oppositely regulates the RhoA activation induced by G
protein-coupled receptors and Plexin-B1. PDZ-RhoGEF, which interacts with
Plexin-B receptors through the PDZ domain, is also known to associate with
activated G12 or G
13 through the
regulators of G protein signaling-like domain (RGS domain, also known as Lsc
homology domain) and to mediate RhoA activation induced by certain G
protein-coupled receptors (20,
42). In the light of the
Rnd1-facilitated interaction of Plexin-B1 and PDZ-RhoGEF, Rnd1 may inhibit
RhoA activation by G protein-coupled receptors due to sequestration of
PDZ-RhoGEF to Plexin-B-like receptors. Thus, Rnd1 may act as a key regulator
of the signal transductions of both Plexin-B receptors and G protein-coupled
receptors.
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FOOTNOTES |
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These authors contributed equally to this work.
To whom correspondence should be addressed: Laboratory of Molecular
Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto
606-8502, Japan. Tel.: 81-75-753-4547; Fax: 81-75-753-7688; E-mail:
mnegishi{at}pharm.kyoto-u.ac.jp.
1 The abbreviations used are: GEF, guanine nucleotide exchange factor; LARG,
leukemia-associated Rho GEF; PDZ, PSD-95/Dlg/ZO-1; GST, glutathione
S-transferase; HA, hemagglutinin; GFP, green fluorescent protein;
PBS, phosphate-buffered saline; DTT, dithiothreitol; Rho-kinase,
Rho-associated kinase.
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REFERENCES |
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