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Originally published In Press as doi:10.1074/jbc.M402943200 on June 8, 2004

J. Biol. Chem., Vol. 279, Issue 32, 33199-33205, August 6, 2004
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The Activity of the Plexin-A1 Receptor Is Regulated by Rac*

Laura J. Turner{ddagger}, Sarah Nicholls, and Alan Hall§

From the MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, Department of Biochemistry and Molecular Biology, Cancer Research UK, Oncogene and Signal Transduction Group, University College London, Gower Street, London, WC1E 6BT, United Kingdom

Received for publication, March 16, 2004 , and in revised form, June 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plexins constitute a large family of transmembrane proteins that act as receptors for the semaphorin family of ligands. They are best known for their role in growth cone guidance, although they also are widely expressed outside the nervous system. Plexins are thought to control axon guidance by modifying the growth cone cytoskeleton, and Rho GTPases have been strongly implicated in this response. However, the exact contribution of Rho proteins is unclear. Sema3A/Plexin-A1-induced growth cone collapse, for example, requires Rac activity, which is a surprising result given that this GTPase is usually associated with membrane protrusions. We show here that Sema3A-induced collapse of COS-7 cells expressing Plexin-A1 also requires Rac but not Rho activity and that the cytoplasmic tail of Plexin-A1 interacts directly with activated Rac. However, collapse induced by a constitutively activated version of Plexin-A1 does not require Rac. We propose a novel function for Rac, namely that it acts upstream of Plexin-A1 during semaphoring-induced collapse, to regulate the activity of the receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plexins are a family of transmembrane receptors characterized by the presence of a conserved intracellular domain of ~600 amino acids known as the sex and plexins domain (1). The sex and plexins domain is strikingly conserved across the plexin family (57-97% similarity) and consists of two blocks of high sequence conservation separated by a variable linker. Together with their co-receptors neuropilins, plexins act as functional receptors for members of the semaphorin family of growth cone guidance molecules (2-6). The prototypic semaphorin, Sema3A, has a potent inhibitory effect on sensory axons emanating from dorsal root ganglia (7). In addition, the binding of Sema3A to COS-7 cells expressing Plexin-A1 and neuropilin-1 (NP-1)1 has been shown to induce a morphological "collapse" response that may correlate with growth cone collapse (5).

To facilitate axon turning, growth cones are thought to translate sensory information into cytoskeletal rearrangements (8-13). Rho GTPases are important modulators of cytoskeletal assembly, and they have been strongly implicated in the regulation of axon growth, guidance, and motility (14). The best-characterized Rho family members, Rho, Rac and Cdc42, regulate the formation of contractile actin-myosin filaments (15), lamellipodia (16), and filopodia (17) respectively. Rho GTPases function as molecular switches, cycling between an "active" GTP-bound and an "inactive" GDP-bound conformation (18). Active GTPases interact directly with target proteins (also known as effectors) to trigger a downstream response (19), and one of the major criteria used to identify putative effectors is that the protein interacts preferentially with the active (GTP-bound) form of the GTPase.

In dorsal root ganglia and spinal motor neurons, Sema3A-induced collapse has been shown to require Rac activity (20-22). This is a surprising observation, since in other cell types and even in neuronal-like cell lines, Rac mediates membrane protrusion. This result has been observed by several groups, and in addition, plexins co-localize with Rac and F-actin in growth cones following stimulation with Sema3A (23). Sema3A-induced collapse has also been reported to require Cdc42 activity in motor, but not dorsal root ganglia, neurons (20, 21).

Interestingly, Rac·GTP interacts directly with a member of the B-family of plexins, Plexin-B1, through a partial CRIB motif (24-26), and in Drosophila, the binding of dPlex-B to dRac1 results in the inactivation of an important Rac effector, p21-activated kinase 1 (p65PAK-1) (27, 28). It has been proposed that stimulation of Plexin-B1 by its ligand, Sema4D, induces local sequestration of Rac, thereby inhibiting signaling to its effector pathways.

In this study, we show that Sema3A-induced collapse of COS-7 cells co-expressing Plexin-A1 and NP-1 requires both Rac and Cdc42 activity. We find that Rac·GTP associates directly with Plexin-A1 and that Rac is activated following Sema3A stimulation. Rho and Rho kinase (ROCK) are not required for this response, indicating that collapse is not a consequence of excessive contractile forces as has been shown for ephrin-A5-induced growth cone collapse mediated by the Eph family of tyrosine kinase receptors (29). Finally, a constitutively activated version of Plexin-A1 no longer requires Rac activity. Our results support a novel role for Rac, namely that it acts upstream of Plexin-A1 during semaphorin-induced collapse and is required for receptor activation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Mouse anti-vsv-G (clone P5D4) was from Roche Diagnostics Inc., mouse anti-Myc (clone 9E10) was prepared in the Hall laboratory, mouse anti-FLAG (clone M2) and rhodamine-phalloidin were from Sigma, mouse anti-Rac1 (clone 23A8) was from Upstate Biotechnology (Lake Placid, NY), rabbit anti-Myc was from Research Diagnostics (Flanders, NJ), goat anti-human IgG, Fc{gamma} fragment-specific (goat anti-hFc), and all of the secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), and the Rho kinase inhibitor Y-27632 was from Tocris Cookson, Inc. (Ellisville, MO). Recombinant Sema3A-Fc was purified by protein A-Sepharose affinity chromatography from the medium of a stable Chinese hamster ovary K1 cell line (a kind gift from Dr. B. Eickholt, Kings College, London, United Kingdom) expressing full-length chick Sema3A-Fc (30). For yeast interaction studies, the Saccharomyces cerevisiae Y190 (MAT{alpha}, gal4-542, gal80-538, his3, trp1-901, ade2-101, ura3-52, leu2-3,112, URA3::GAL1-lacZ, LYS2::GAL1-His3cyhr) yeast strain was used.

DNA Constructs and Cloning Procedures—vsv-Plexin-A1 in pBKCMV was provided by Dr. A. Puschel (Westfalische Wilhelms Universitat, Munster, Germany) (6). Myc-Plexin-A1{Delta}SEM and Myc-Plexin-A1{Delta}ECT in pSecTag2 were provided by Dr. S. Strittmatter (Johns Hopkins, Baltimore, MD) (31). NP-1 in pMT21 was provided by Dr. M. Tessier-Lavigne (Howard Hughes Institute, Stanford University, Stanford, CA) (2). GTPase constructs in either pRK5Myc or pRK5FLAG were used.

Plexin-A1(cyt) (amino acids 1274-1894) was amplified by PCR from vsv-Plexin-A1 cDNA using the primers 1 (5'-T TTG AAT TCA CGT ACC CTC AAG CGG CTG-3') and 2 (5'-TT TGC GGC CGC TCA GCT GCT CAG GGC CAT TG-3') and subcloned into hemagglutinin-pYTH9 using the restriction sites EcoR1 and NotI. Plexin-A1(frag) (amino acids 1476-1623) was amplified by PCR from vsv-Plexin-A1 cDNA using the primers 3 (5'-GA TGA ATT CTA GAC GCC ATC ACA GGG GAG-3') and 4 (5'-TAT CTC GAG CTA GCT CTC GTA TCT GCT GAG-3') and subcloned into pGEX-4T2 using the restriction sites EcoR1 and XhoI. Plexin-A1 PH1a (amino acids 1274-1623) was obtained by PCR using the primers 5 (5'-CG GGA TCC CGT ACC CTC AAG CGG CTG-3') and 6 (5'-CG GAA TTC TCA GCT CTC GTA TCT GCT GAG-3') and sub-cloned into pRK5FLAG using the restriction sites BamH1 and EcoR1. Plexin-A1 PH2a (amino acids 1624-1894) was obtained by PCR using the primers 7 (5'-TC GGA TCC ATG CTG CGT ACA GCC AGT AG-3') and 8 (5'-GG GAA TTC TCA GCT GCT CAG GGC CAT TG-3') and subcloned into pRK5Myc using the restriction sites BamH1 and EcoR1. Plexin-A1{Delta}RBD (amino acids 1274-1595) was amplified by PCR using the primers 5 and 9 (5'-CG GAA TTC TCA CGA TGA CCC ATC CGT CAC-3') and subcloned into pRK5FLAG using the restriction sites BamH1 and EcoR1. Plexin-A1(cyt) (amino acids 1274-1894) was amplified by PCR using the primers 5 and 8 and subcloned into pRK5FLAG and pRK5Myc using the restriction sites BamH1 and EcoR1. Plexin-B1 PH1a (amino acids 1520-1870) was amplified by PCR using the primers 10 (5'-GGC GGA TCC AGG GAC TAT AAG AAG GTT C-3') and 11 (5'-TA TCT AGA TCA GGT CCG CTC TCC AGG GAC-3') and subcloned into pRK5FLAG using the restriction sites BamH1 and XbaI. Plexin-B1 PH1a{Delta}RBD (amino acids 1520-1846) was amplified by PCR using the primers 10 and 12 (5'-GC TCT AGA GGT TGC TCC ATC TGG GAC C-3') and subcloned into pRK5FLAG using the restriction sites BamH1 and XbaI. Plexin-B1 PH2a (amino acids 1871-2135) was amplified by PCR using the primers 13 (5'-TA GGA TCC CCA ATG CTG GAG GAT GTA G-3') and 14 (5'-GC TCT AGA CTA TAG ATC TGT GAC CTT G-3') and subcloned into pRK5Myc using the restriction sites BamH1 and XbaI. vsv-Plexin-A1-GGA (amino acids 1598-1600, LVP to GGA) was prepared by PCR-mediated site-directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. vsv-Plexin-A1 was used as a template, and the primers 15 (5'-GGG TCA TCG GTG GCA GGA GGA GCC AAG CAG ACA TCG GCC-3') and 16 (5'-GGC CGA TGT CTG CTT GGC TCC TCC TGC CAC CGA TGA CCC-3') were used. Plexin-A1 PH1a-GGA (amino acids 1274-1623) was amplified by PCR using the primers 5 and 6 and subcloned into pRK5FLAG using the restriction sites BamH1 and EcoR1. All of the constructs prepared were sequenced fully.

Cell Culture and Transfections—COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% complete fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). The Chinese hamster ovary K1 cell line was cultured in DME-Ham's F-10 medium plus L-glutamate (Invitrogen) supplemented with 10% {gamma}-globulin-free fetal calf serum (Ultrapure FCS, Sigma) and 100 units/ml penicillin and 100 µg/ml streptomycin. Cell lines were grown at 37 °C in 10% CO2 and passaged at 80-90% confluency. Cells were seeded in 6-well plates (1.5 x 105 cells/well) containing glass coverslips 16-24 h prior to transfection (1.5 µg of DNA/transfection) using the GenejuiceTM reagent (Novagen). For collapse assays, Sema3A-Fc (2 µg/ml in serum-free Dulbecco's modified Eagle's medium) was used to stimulate transfected cells for 30 min at 37 °C. Cells were washed once in phosphate-buffered saline (PBS) and then fixed in cold 4% paraformaldehyde for 20 min at room temperature.

Immunofluorescence—Fixed cells were quenched in 2.7 mg/ml NH4Cl/PBS for 10 min, permeabilized if required in 0.1% Triton X-100/PBS for 5 min, and blocked in 2% bovine serum albumin (BSA)/PBS for 20 min. For immunostaining, cells were incubated with primary antibodies diluted in 2% BSA/PBS for 1 h. Fluorescent conjugated secondary antibodies or rhodamine-phalloidin diluted in 2% BSA/PBS were applied to cells for 30 min at room temperature. Coverslips were mounted in Mowiol mountant (Calbiochem), and images were captured using a Bio-Rad MRC 1000 confocal microscope.

Yeast Two-hybrid Interaction Assays—A cDNA-encoding Plexin-A1(cyt) fused to the sequence encoding the GAL4 DNA-binding domain of the pYTH9 vector was stably integrated into the genome of the yeast strain Y190. Plexin-A1(cyt)::Y190 cells were transformed with cDNA encoding wild-type dRac, dRho, or dCdc42 fused to the sequence encoding the GAL4 activation domain in the pACTII vector. Putative interactions were assessed by growth on 3-AT-selective medium (33).

Slot Blot in Vitro GTPase Binding Assays—A nitrocellulose membrane was equilibrated in buffer A (50 mM Tris, 50 mM NaCl, and 5 mM MgCl2) plus 0.1 mM DTT and placed into a slot blot apparatus (Bio-Rad). GST alone, GST-PAK/CRIB, or GST-Plexin-A1(frag) (10 µg/protein, prepared according to Sander et al. (34)) was made up to 50 µl of total volume using binding buffer (1x 20 mM Tris-HCl (from a 1 M stock, pH 7.6) and 0.1 mM DTT) containing 1 mM MgCl2. These proteins were applied to the membrane and washed 3x in buffer A plus 0.1 mM DTT and then blocked in 1 M glycine, 5% marvel, 5% fetal calf serum, and 1% ovalbumin for 2 h at room temperature. Recombinant GTPases (35) (5 µg/reaction, diluted with binding buffer containing 1 mM MgCl2) were loaded with GTP by incubation for 10 min at 30 °C in 0.84x binding buffer containing 5 mM EDTA, 0.5 mg/ml BSA, and 1 µl of [{gamma}-32P]GTP (10 µCi/µl, 6000 Ci/mmol, PerkinElmer Life Sciences) and then placed on ice. The reactions were stopped by the addition of MgCl2 to 10 mM. To reduce background exposure levels, DTT was added to a final concentration of 0.1 mM. After blocking, the membrane was washed twice with buffer A containing 0.1 mM DTT and then incubated in buffer A containing 1 mM GTP, 1 mg/ml BSA, 0.1 mM DTT, and [{gamma}-32P]GTP-loaded GTPase and agitated gently for 10 min at 4 °C. The membrane was washed 3x in TBS-T (20 mM Tris-HCl (from a 1 M stock, pH 7.5), 150 mM NaCl, and 0.1% v/v Tween 20), and bound radiolabeled GTPases were visualized using autoradiography.

Immunoprecipitations—Cells were transfected 24 h prior to immunoprecipitation. Cells were washed in ice-cold buffer A containing 0.1 mM sodium orthovanadate and lysed at 4 °C in radioimmune precipitation buffer (1% v/v Nonidet P-40, 10 mM Tris-HCl (from a 1 M stock, pH 7.5), 140 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, and one complete protease inhibitor tablet) per well. Post-nuclear supernatants were prepared by centrifugation, and aliquots were removed to assess total protein levels. Lysates were incubated for 2 h at 4 °C with specific antibodies, and protein G-Sepharose and immunoprecipitates were harvested by centrifugation and washed three times in ice-cold radioimmune precipitation buffer. Immunoprecipitated proteins were eluted using SDS-sample buffer and analyzed by 10 or 15% SDS-PAGE and Western blot analysis.

GTPase Pull-down Assays—COS-7 cells were serum-starved 10 h post-transfection. After an additional 14 h, cells were stimulated for 0, 10, 20, 30, or 60 min with fresh medium, either alone or supplemented with 2 µg/ml Sema3A-Fc. GTP-bound Rac was affinity-purified using GST-PAK/CRIB coupled to glutathione-agarose beads. Proteins were eluted using SDS-sample buffer, and Rac·GTP levels were assessed by 15% SDS-PAGE and Western blot analysis using mouse anti-Rac1 (36).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sema3A-Fc-induced Collapse Requires Rac and Cdc42 but Not Rho or p160ROCK—A COS-7 collapse assay was used to investigate the involvement of Rho GTPases in Sema3A-Fc-mediated collapse. Cells were co-transfected with vsv-Plexin-A1 and NP-1 with or without a dominant negative GTPase mutant (Myc-N17Cdc42, Myc-N17Rac1, or Myc-N19RhoA). After 18 h, cells were stimulated with 2 µg/ml Sema3A-Fc and labeled using a goat anti-hFc antibody and either a mouse anti-Myc antibody or phalloidin. In each experiment, 100 cells positive for both Myc and Sema3A-Fc staining were counted and classified as either collapsed or not collapsed.

Dominant negative Cdc42 (Myc-N17Cdc42) completely inhibited Sema3A-Fc-induced collapse as only 30% of expressing cells collapsed in comparison to 70% of control cells (see Fig. 1, a-d and j). Dominant negative Rac (Myc-N17Rac1) also inhibited collapse with 45% of expressing cells collapsed in comparison to 70% of control cells (see Fig. 1, a, b, e, f, and j). This may be an underestimate of the true inhibition of collapse, because cells expressing dominant negative Rac showed a higher background of spontaneous collapse (40%) compared with control cells (20%). Western blot analysis was used to confirm that the inhibitory effects produced by GTPase mutants were not the result of changes in the expression levels of vsv-Plexin-A1 or NP-1 (Fig. 1i). Furthermore, the inhibitory effects observed were not a consequence of decreased surface expression of vsv-Plexin-A1 and NP-1, because Sema3A-Fc binds as efficiently with or without the dominant negative constructs (data not shown).



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  		FIG. 1.
Sema3A-Fc-induced collapse requires Rac and Cdc42 but not Rho or p160ROCK. Cells were transfected with the plasmids indicated and stimulated 18 h post-transfection with 2 µg/ml Sema3A-Fc for 30 min. In experiments using the p160ROCK inhibitor Y-27632, cells were stimulated 24 h post-transfection. a and b, cells expressing vsv-Plexin-A1 and NP-1. c and d, cells expressing vsv-Plexin-A1, NP-1, and Myc-N17Cdc42. e and f, cells expressing vsv-Plexin-A1, NP-1, and Myc-N17Rac1. g and h, cells expressing vsv-Plexin-A1, NP-1, and Myc-N19RhoA. a, TRITC-conjugated phalloidin. b, d, f, and h, goat anti-hFc. c, e, and g, mouse anti-Myc, clone 9E10. i, Western blot showing the expression levels of vsv-Plexin-A1, NP-1, and Myc-N17Cdc42 or Myc-N17Rac1 in each experiment. Lysates prepared from Sema3A-Fc-stimulated cells were divided into three and probed using mouse anti-vsv, goat anti-NP-1, and mouse anti-Myc antibodies. j, quantification of the effects of dominant negative GTPases and Y-27632 on the Sema3A-Fc-induced collapse. To inhibit Rho kinase, cells expressing vsv-Plexin-A1 and NP-1 were treated with 10 µM Y-27632 30 min prior to stimulation with Sema3A-Fc. 10 µM Y-27632 also was present in the culture medium containing Sema3A-Fc.

 
Dominant negative Rho (Myc-N19RhoA) did not inhibit Sema3A-Fc-induced collapse (see Fig. 1, a, b, g, h, and j). To confirm that collapse is not a consequence of actin/myosin-induced cell contraction, Y-27632, a small molecule inhibitor of the serine-threonine kinase p160ROCK, was added and this also had no effect on collapse (Fig. 1j).

Sema3A-Fc Induces Rac Activation—To determine whether Rac is activated in response to Sema3A-Fc, a GTPase pull-down assay was used. Cells were transfected with empty vector, NP-1, or vsv-Plexin-A1 and NP-1 and stimulated for 30 min with Sema3A-Fc, and Rac·GTP was precipitated from lysates using GST-PAK/CRIB bound to Sepharose beads. In COS-7 cells alone or cells expressing NP-1 only, the levels of Rac·GTP were almost undetectable following stimulation, whereas in cells expressing vsv-Plexin-A1 and NP-1, Sema3A-Fc induced a significant increase in the levels of active Rac (see Fig. 2). Additional experiments revealed modest activation 10 min after Sema3A-Fc stimulation, and maximum activation was seen at 30 min (see Fig. 2).



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  		FIG. 2.
Rac activation by Sema3A-Fc. Cells were transfected with vector only, NP-1, or vsv-Plexin-A1 and NP-1. After 10 h, cells were serum-starved. Stimulation with 2 µg/ml Sema3A-Fc was carried out 24 h post-transfection for 0, 10, 20, 30, or 60 min. Lysates were prepared, and aliquots were saved to assess total GTPase levels. GST-PAK/CRIB beads were used to pull down Rac·GTP. Equal amounts of beads and total lysates were assessed by Western blot analysis. Blots were probed using mouse anti-Rac1 and mouse anti-vsv antibodies.

 
Rac Acts Upstream of Plexin-A1 to Regulate the Activity of the Receptor—Takahashi and Strittmatter (31) demonstrate that mutants of Plexin-A1 lacking either the Sema domain (Plexin-A1{Delta}SEM) or the whole extracellular region (Plexin-A1{Delta}ECT) are constitutively active when introduced into COS cells (31). To test whether this response is still Rac-dependent, COS cells were transfected with Myc-Plexin-A1{Delta}SEM or Myc-Plexin-A1{Delta}ECT, either alone or in combination with green fluorescent protein or dominant negative Rac (FLAG-N17Rac1). When expressed alone, Myc-Plexin-A1{Delta}ECT and Myc-Plexin-A1{Delta}SEM induced collapse in 70 and 65% of cells, respectively (see Fig. 3, a, b, and d). However, dominant negative Rac was unable to inhibit collapse induced by constitutively active Plexin-A1 mutants (see Fig. 3, c and d). We conclude that Rac does not participate directly in the pathway leading from Plexin-A1 to the collapse response but it is required for Sema3A-induced collapse.



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  		FIG. 3.
Dominant negative Rac does not inhibit collapse induced by constitutively active Plexin-A1. Cells were transfected with Myc-Plexin-A1{Delta}ECT or Myc-Plexin-A1{Delta}SE, either alone or together with FLAG-N17Rac1. Collapse was assessed 18 h post-transfection. a, cells expressing Myc-Plexin-A1{Delta}ECT. b and c, cells expressing Myc-Plexin-A1{Delta}ECT and FLAG-N17Rac1. a and c, rabbit anti-Myc. b, mouse anti-FLAG. d, quantification of the effects of FLAG-N17Rac1 on collapse induced by Myc-Plexin-A1{Delta}ECT or Myc-Plexin-A1{Delta}SEM.

 
Since Plexin-B1 has been shown to directly interact with Rac, we examined whether Rac might also interact directly with Plexin-A1. First, DNA encoding the 620 amino acid cytoplasmic tail of Plexin-A1 was subcloned into the yeast two-hybrid bait vector, pYTH9 (see Fig. 4a). The construct produced, hemagglutinin-Plexin-A1(cyt), was subsequently integrated into the genome of the yeast strain Y190, and Western blot analysis confirmed that the protein was expressed. Y190::Plexin-A1(cyt) yeast were then transformed with wild-type Rac, Rho, or Cdc42, and as shown in Fig. 4b, an interaction with Rac was clearly observed. To confirm this observation, a fragment of the cytoplasmic tail of Plexin-A1 was subcloned into the Escherichia coli expression vector, pGEX-4T2 (see Fig. 4a). The fragment chosen corresponded to the 149 amino acid stretch of Plexin-B1 previously defined as the minimal region sufficient for interaction with Rac (26). The GST fusion protein (GST-Plexin-A1(frag)) was used in a slot blot in vitro GTPase binding assay using [{gamma}-32P]GTP-bound Rac. GST-PAK/CRIB was used as a positive control. As shown in Fig. 4, c and d, Rac·GTP interacts with the cytoplasmic tail of Plexin-A1. vsv-Plexin-A1-GGA Is unable to Mediate Collapse but Still Activates Rac following Sema3A-Fc Stimulation—Plexin-A1 does not contain an obvious CRIB motif (24, 37), but a triple substitution mutant (vsv-Plexin-A1-GGA) was constructed analogous to a Plexin-B1 mutant (Plexin-B1-GGA) previously shown to no longer interact with Rac (see Fig. 5a) (24). Cells expressing NP-1 together with either vsv-Plexin-A1 or vsv-Plexin-A1-GGA were stimulated with Sema3A-Fc. In cells expressing NP-1 and vsv-Plexin-A1-GGA, only 7% of cells with bound Sema3A-Fc collapsed compared with 85% of cells expressing NP-1 and vsv-Plexin-A1 (see Fig. 5, b-d). This effect was not a consequence of vsv-Plexin-A1-GGA failing to reach the cell surface as unpermeabilized cells displayed a strong staining of vsv-Plexin-A1-GGA and efficient interaction with Sema3A-Fc (data not shown).



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  		FIG. 4.
Rac·GTP interacts with the cytoplasmic tail of Plexin-A1. a, schematic showing the regions of Plexin-A1 used in the interaction assays. b, Y190::Plexin-A1(cyt) was transformed with wild-type dRac1, dRhoA, or dCdc42, and the ability of the clones to grow on 3-AT-selective plates was assessed. Plexin-A1 interacted with dRac1 but not dCdc42. c, slot blot assays were used to assess the ability of the GST-Plexin-A1(frag) fusion protein to associate with recombinant Rac1·GTP. GST-PAK/CRIB was used as a positive control for Rac1 interaction, and GST alone was used as a negative control. d, quantitation of the slot blot assay in c.

 



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  		FIG. 5.
Rac activation by Sema3A-Fc occurs independently of the collapse response. a, schematic showing the vsv-Plexin-A1-GGA mutant prepared by site-directed mutagenesis. b-d, cells expressing vsv-Plexin-A1-GGA and NP-1 were stimulated 24 h post-transfection for 30 min with 2 µg/ml Sema3A-Fc. Unpermeabilized cells were stained using mouse anti-vsv and goat anti-hFc antibodies. b, mouse anti-vsv. c, goat anti-hFc. d, quantification of the effect of vsv-Plexin-A1-GGA on collapse. e, Sema3A-Fc induces Rac activation in cells expressing vsv-Plexin-A1-GGA and NP-1. Pull-down assays were performed as in Fig. 2.

 
To determine whether or not vsv-Plexin-A1-GGA was still capable of mediating Sema3A-Fc-induced Rac activation, pull-down assays were performed. Cells were transfected with NP-1, either alone or in combination with vsv-Plexin-A1 or vsv-Plexin-A1-GGA. These experiments demonstrated that Sema3A-Fc was able to induce Rac activation in cells expressing NP-1 and vsv-Plexin-A1-GGA (see Fig. 5e).

Potential Autoinhibitory Interactions within the Cytoplasmic Tail of Plexin-A1—The discovery that Plexin-B1 and Plexin-A1 interact with Rac·GTP raises the possibility that these receptors may be targets of Rac. A characteristic feature of many Rho GTPase effector proteins, e.g. WASP or p65PAK, is the presence of an autoinhibitory interaction (38-42) between sequences located in the N- and C-terminal parts of the protein, which is relieved upon GTPase binding.

The possibility that a similar interaction might occur within the cytoplasmic tail of Plexin-A1 was explored using immunoprecipitation experiments. Fragments of Plexin-A1 and Plexin-B1 were subcloned into the mammalian expression vectors, pRK5FLAG or pRK5Myc (see Fig. 6a), and cells were transfected with various combinations. Immunoprecipitations were performed using mouse anti-Myc or mouse anti-FLAG antibodies, and precipitated proteins were assessed by Western blot analysis. Blots were probed with mouse anti-Myc and mouse anti-FLAG antibodies. A strong interaction could be seen between the N-terminal (PH1a) and C-terminal (PH2a) cytoplasmic sequences of Plexin-A1 (see Fig. 6, b and c). Similar interactions could be seen using the corresponding fragments of Plexin-B1 (Fig. 6, b and c). This finding demonstrates the potential for an intramolecular interaction within the cytoplasmic tail of Plexin family members or an intermolecular interaction between homodimerized receptors.



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  		FIG. 6.
Intramolecular/intermolecular interaction within the cytoplasmic tail of Plexin-A1 involve the Rac-binding region. a, schematic showing the fragments of Plexin-A1 and Plexin-B1 used in immunoprecipitation studies. Cells were transfected with the indicated plexin fragments. Lysates were prepared, and aliquots were saved to assess total protein levels. Immunoprecipitations were performed using mouse anti-Myc or mouse anti-FLAG antibodies as indicated, and precipitated proteins were assessed by Western blot analysis. Blots were probed with mouse anti-Myc and mouse anti-FLAG antibodies. b, Myc-Plexin-A1 PH2a precipitates FLAG-Plexin-A1 PH1a, and Myc-Plexin-B1 PH2a precipitates FLAG-Plexin-B1 PH1a. c, FLAG-Plexin-A1 PH1a precipitates Myc-Plexin-A1 PH2a, and FLAG-Plexin-B1 PH1a precipitates Myc-Plexin-B1 PH2a. d, Myc-Plexin-A1 PH2a precipitates FLAG-Plexin-A1 PH1a-GGA. e, Myc-Plexin-A1 PH2a is unable to precipitate FLAG-Plexin-A1 PH1a{Delta}RBD. f, Myc-Plexin-B1 PH2a precipitates FLAG-Plexin-B1 PH1a{Delta}RBD weakly compared with FLAG-Plexin-B1 PH1a. g, Plexin-A1 cytoplasmic regions can dimerize.

 
The GGA triple point mutation prevents Plexin-A1 from mediating Sema3A-Fc-induced collapse and inhibits Rac binding to Plexin-B1 (24). To determine whether this affects the proposed intra/intermolecular interactions, the PH2a segment of Plexin-A1 was co-expressed with a PH1a-GGA segment. As seen in Fig. 6d, PH1a-GGA still interacted with Myc-Plexin-A1 PH2a, although the level of this interaction was reduced. The Rac-binding region of Plexin-B1 has been mapped to the very end of the fragment corresponding to PH1a shown in Fig. 6a. The fragments lacking this region (PH1a{Delta}RBD) were made from Plexin-A1 and Plexin-B1 (28 and 24 amino acid deletions, respectively) and co-expressed with fragment PH2a. As seen in Fig. 6e, the PH2a fragment of Plexin-A1 was unable to associate with PH1a{Delta}RBD. A similar result was found with the corresponding Plexin-B1 fragments (Fig. 6f). In addition, to examine whether the cytoplasmic regions of Plexin-A1 can interact through an intermolecular interaction, the cytoplasmic tail was subcloned into pRK5FLAG and pRK5Myc (see Fig. 6a). As seen in Fig. 6g, after co-transfection, FLAG-Plexin-A1(cyt) can be efficiently co-precipitated with Myc-Plexin-A1(cyt).

Finally, to determine whether or not active GTPases influence the proposed intra/intermolecular interactions, the PH1a and PH2a segments of Plexin-A1 were co-expressed with either constitutively activated Cdc42 (FLAG-L61Cdc42) or Rac (FLAG-L61Rac1). The presence of FLAG-L61Cdc42 had no affect on the interaction between Plexin-A1 PH1a and PH2a, whereas the addition of FLAG-L61Rac1 significantly reduced the amount of FLAG-Plexin-A1 PH1a precipitated (see Fig. 7).



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  		FIG. 7.
Activated Rac, but not Cdc42, inhibits the intra/intermolecular interaction within the cytoplasmic tail of Plexin-A1. Cells were transfected with the indicated plexin fragments, either alone or in combination with constitutively activated GTPases (FLAGL61Cdc42 or FLAG-L61Rac1). Lysates were prepared, and aliquots were saved to assess total protein levels. Immunoprecipitations were performed using a mouse anti-Myc antibody, and precipitated proteins were assessed by Western blot analysis. Blots were probed with mouse anti-Myc and mouse anti-FLAG antibodies. Myc-Plexin-A1 PH2a precipitates FLAG-Plexin-A1 PH1a in the presence of FLAG-L61Cdc42, but the amount precipitated is significantly reduced in the presence of FLAG-L61Rac1.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It has been reported previously that Rac plays a role in signaling downstream of Sema3A in the growth cone collapse assay of primary neurons (20-22). Here we show that Rac activity is required during Sema3A-induced collapse of COS-7 cells expressing the Plexin-A1 and NP-1 co-receptors and furthermore that Rac is activated following Sema3A stimulation. Intriguingly, however, collapse induced by constitutively active Plexin-A1 does not require Rac activity. Therefore, it appears that Rac is not required for receptor-induced collapse per se but is required for a ligand-induced collapse. One possible way to account for this is that Plexin-A1 is a Rac target and that Rac is required for receptor activation. This explanation fits well with our observations that (i) the cytoplasmic tail of Plexin-A1 interacts directly with Rac·GTP, (ii) the cytoplasmic region of Plexin-A1 can form intramolecular or intermolecular interactions through sequences located in the N- and C-terminal parts of the tail that overlap with the region required for Rac binding, and (iii) constitutively activated Rac can inhibit this intra/intermolecular interaction. Many of the characterized Rho GTPase targets such as p65PAK, WASp, and mDia are known to form autoinhibitory intramolecular interactions, which can be relieved upon binding of an active GTPase. Therefore, we propose that the binding of Rac·GTP relieves an autoinhibitory conformation of the cytoplasmic tail of Plexin-A1.

The requirement for Rac but not Rho or p160ROCK downstream of Sema3A suggests that the collapse we observe in COS cells is not a consequence of excessive actin/myosin contractile forces driven by Rho. Others have reached the same conclusion using neurons (43). To date, many of the ideas surrounding neuronal morphogenesis such as growth cone collapse and neurite extension have assumed that Rho acts antagonistically to Rac/Cdc42, the former producing contraction/retraction responses and the latter producing protrusive/extension processes (14, 45, 46). Thus lysophosphatidic acid-induced neurite retraction is mediated by Rho (47) as is ephrin-A5-induced growth cone collapse (29). The observation that Sema3A-induced collapse requires active Rac suggests that this simple model may be an oversimplification. Indeed, Fan et al. (48) have provided in vivo evidence that Rac is required in slit-mediated midline repulsion in Drosophila, whereas Jurney et al. (49) and Marston et al. (50) demonstrate the involvement of Rac in ephrin-A2- and ephrin-B2-induced repulsive responses, respectively. It appears that growth cone guidance may be a more complicated process than a simple Rho/Rac antagonism at the level of the actin cytoskeleton.

The activation of Rac by Sema3A that we describe here is in contrast to reports by others that stimulation of another plexin family member, Plexin-B1, by its ligand Sema4D does not result in Rac activation (51). In addition, others (24, 25, 32) have failed to detect an association of Rac with Plexin-A1. The reasons for these discrepancies are not known. Through the use of the Plexin-A1-GGA mutant, we do know that Rac activation is not a consequence of the collapse-induced response. As for the direct interaction between Plexin-A1 and Rac, this is clearly much more difficult to detect than the interaction of Rac with some of its better-characterized targets such as PAK. However, in our hands it is comparable to the interaction seen between Rac and Plexin-B1. Sequence analysis demonstrates that unlike Plexin-B1, Plexin-A1 does not contain a recognizable CRIB motif. However, three critical amino acids (LVP) within the CRIB region of Plexin-B1 are conserved at a similar location in Plexin-A1. These residues have previously been shown to be required for the association between Plexin-B1 and Rac (24). Mutation of these residues in Plexin-A1 (Plexin-A1-GGA) produces a receptor that is unable to mediate Sema3AFc-induced collapse, although it can still activate Rac. Since this GGA mutant is still capable of participating in intramolecular interactions within the cytoplasmic tail, this would be consistent with this mutant existing in an inactive conformation that is no longer responsive to activation by Rac. However, the situation may be yet more complicated since other GTPases have been reported to interact with Plexin-B1 but not with the GGA mutant, including Rnd1 (44) and RhoD (32). Whether or not all of these putative GTPase interactions play a role in vivo downstream of Plexin-A1 will require further analysis.


   FOOTNOTES
 
* The work was supported generously by a program grant from Cancer Research UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of an MRC Ph.D. fellowship. Back

§ To whom correspondence should be addressed. Tel.: 44-20-7679-7909; Fax: 44-20-7679-7804; E-mail: alan.hall{at}ucl.ac.uk.

1 The abbreviations used are: NP-1, neuropilin-1; Sema3A, semaphorin 3A; CRIB, Cdc42/Rac-interactive binding; PAK1, p21-activated kinase 1; ROCK, Rho kinase/p160ROCK/ROK; goat anti-hFc, goat anti-human IgG Fc{gamma} fragment-specific; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RBD, Rac-binding domain; Sema4D, semaphorin 4D; TRITC, tetramethylrhodamine isothiocyanate; d, Drosophila. Back


   ACKNOWLEDGMENTS
 
We thank Dr. B. Eickholt for the kind gift of the Sema3A-Fc-expressing Chinese hamster ovary K1 cell line and Drs. A. Puschel, S. Strittmatter, and M. Tessier-Lavigne for the gift of cDNAs.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Maestrini, E., Tamagnone, L., Longati, P., Cremona, O., Gulisano, M., Bione, S., Tamanini, F., Neel, B. G., Toniolo, D., and Comoglio, P. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 674-678[Abstract/Free Full Text]
  2. He, Z., and Tessier-Lavigne, M. (1997) Cell 90, 739-751[CrossRef][Medline] [Order article via Infotrieve]
  3. Tamagnone, L., Artigiani, S., Chen, H., He, Z., Ming, G. I., Song, H., Chedotal, A., Winberg, M. L., Goodman, C. S., Poo, M.-M., Tessier-Lavigne, M., and Comoglio, P. M. (1999) Cell 99, 71-80[CrossRef][Medline] [Order article via Infotrieve]
  4. Winberg, M. L., Noordermeer, J. N., Tamagnone, L., Comoglio, P. M., Spriggs, M. K., Tessier-Lavigne, M., and Goodman, C. (1998) Cell 95, 903-916[CrossRef][Medline] [Order article via Infotrieve]
  5. Takahashi, T., Fournier, A., Nakamura, F., Wang, L. H., Murakami, Y., Kalb, R. G., Fujisawa, H., and Strittmatter, S. M. (1999) Cell 99, 59-69[CrossRef][Medline] [Order article via Infotrieve]
  6. Rohm, B., Ottemeyer, A., Lohrum, M., and Puschel, A. W. (2000) Mech. Dev. 93, 95-104[CrossRef][Medline] [Order article via Infotrieve]
  7. Luo, Y., Raible, D., and Raper, J. A. (1993) Cell 75, 217-227[CrossRef][Medline] [Order article via Infotrieve]
  8. Bentley, D., and Toronian-Raymond, A. (1986) Nature 323, 712-715[CrossRef][Medline] [Order article via Infotrieve]
  9. Williamson, T., Gordon-Weeks, P. R., Schachner, M., and Taylor, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15221-15226[Abstract/Free Full Text]
  10. Tanaka, E., and Kirschner, M. W. (1995) J. Cell Biol. 128, 127-137[Abstract/Free Full Text]
  11. Zheng, J. Q., Wan, J. J., and Poo, M. M. (1996) J. Neurosci. 16, 1140-1149[Abstract/Free Full Text]
  12. Challacombe, J. F., Snow, D. M., and Letourneau, P. C. (1996) J. Cell Sci. 109, 2031-2040[Abstract]
  13. Challacombe, J. F., Snow, D. M., and Letourneau, P. C. (1997) J. Neurosci. 17, 3085-3095[Abstract/Free Full Text]
  14. Luo, L. (2000) Nat. Rev. Neurosci. 1, 173-180[CrossRef][Medline] [Order article via Infotrieve]
  15. Ridley, A. J., and Hall, A. (1992) Cell 70, 389-399[CrossRef][Medline] [Order article via Infotrieve]
  16. Ridley, A. J., Paterson, H. F., Johnston C. L., Diekmann, D., and Hall, A. (1992) Cell 70, 401-410[CrossRef][Medline] [Order article via Infotrieve]
  17. Nobes, C. D., and Hall, A. (1995) Cell 81, 53-62[CrossRef][Medline] [Order article via Infotrieve]
  18. Schmidt, A., and Hall, A. (2002) Genes Dev. 16, 1587-1609[Free Full Text]
  19. Bishop, A. L., and Hall, A. (2000) Biochem. J. 348, 241-255
  20. Jin, Z., and Strittmatter, S. M. (1997) J. Neurosci. 17, 6256-6263[Abstract/Free Full Text]
  21. Kuhn, T. B., Brown, M. D., Wilcox, C. L., Raper, J. A., and Bamburg J. R. (1999) J. Neurosci. 19, 1965-1975[Abstract/Free Full Text]
  22. Vastrik, I., Eickholt, B. J., Walsh, F. S., Ridley, A., and Doherty, P. (1999) Curr. Biol. 9, 991-998[CrossRef][Medline] [Order article via Infotrieve]
  23. Fournier, A. E., Nakamura, F., Kawamoto, S., Goshima, Y., Kalb, R. G., and Strittmatter, S. M. (2000) J. Cell Biol. 149, 411-422[Abstract/Free Full Text]
  24. Vikis, H. G., Li, W., He, Z., and Guan, K. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12457-12462[Abstract/Free Full Text]
  25. Rohm, B., Rahim, B., Kleiber, B., Hovatta, I., and Puschel, A. W. (2000) FEBS Lett. 486, 68-72[CrossRef][Medline] [Order article via Infotrieve]
  26. Driessens, M. H., Hu, H., Nobes, C. D., Self, A., Jordens, I., Goodman, C. S., and Hall, A. (2001) Curr. Biol. 11, 339-344[CrossRef][Medline] [Order article via Infotrieve]
  27. Hu, H., Marton, T. F., and Goodman, C. (2001) Neuron 32, 39-51[CrossRef][Medline] [Order article via Infotrieve]
  28. Vikis, H. G., Sequin, L., and Guan, K.-L. (2002) Genes Dev. 16, 836-845[Abstract/Free Full Text]
  29. Wahl, S., Barth, H., Ciossek, T., Aktories, K., and Mueller, B. K. (2000) J. Cell Biol. 149, 263-270[Abstract/Free Full Text]
  30. Eickholt, B. J., Morrow, R., Walsh, F. S., and Doherty, P. (1997) Mol. Cell. Neurosci. 9, 358-371[CrossRef][Medline] [Order article via Infotrieve]
  31. Takahashi, T., and Strittmatter, S. (2001) Neuron 29, 429-439[CrossRef][Medline] [Order article via Infotrieve]
  32. Zanata, S. M., Hovatta, I., Rohm, B., and Puschel, A. W. (2002) J. Neurosci. 22, 471-477[Abstract/Free Full Text]
  33. Aspenstrom, P., and Olson, M. F. (1995) Methods Enzymol. 256, 228-241[Medline] [Order article via Infotrieve]
  34. Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R.A., and Collard, J.G. (1999) J. Cell Biol. 147, 1009-1022[Abstract/Free Full Text]
  35. Self, A., and Hall, A. (1995) Methods Enzymol. 256, 3-10[Medline] [Order article via Infotrieve]
  36. Patel, J. C., Hall, A., and Caron, E. (2002) Mol. Biol. Cell 13, 1215-1226[Abstract/Free Full Text]
  37. Burbelo, P. D., Dreschsel, D., and Hall, A. (1995) J. Biol. Chem. 270, 29071-29074[Abstract/Free Full Text]
  38. Rohatgi, R., Ho, H. Y., Kirschner, M. W. (2000) J. Cell Biol. 150, 1299-1310[Abstract/Free Full Text]
  39. Prehoda, K. E., Scott, J. A., Mullins, R. D., and Lim, W. A. (2000) Science 290, 801-806[Abstract/Free Full Text]
  40. Zhao, Z. S., Manser, E., Chen, X. Q., Chong, C., Leung, T., and Lim, L. (1998) Mol. Cell. Biol. 18, 2153-2163[Abstract/Free Full Text]
  41. Tu, H., and Wigler, M. (1999) Mol. Cell. Biol. 19, 602-611[Abstract/Free Full Text]
  42. Zenke, F. T., King, C. C., Bohl, B. P., and Bokoch, G. M. (1999) J. Biol. Chem. 274, 32565-32573[Abstract/Free Full Text]
  43. Arimura, N., Inagaki, N., Chihara, K., Menager, C., Nakamura, N., Amano, M., Iwamatsu, A., Goshima, Y., and Kaibuchi, K. (2000) J. Biol. Chem. 275, 23973-23980[Abstract/Free Full Text]
  44. Oinuma, I., Katoh, H., Harada, A., and Negishi, M. (2003) J. Biol. Chem. 278, 25671-25677[Abstract/Free Full Text]
  45. Dickson, B. J. (2001) Curr. Opin. Neurobiol. 11, 103-110[CrossRef][Medline] [Order article via Infotrieve]
  46. Patel, B. N., and Van Vactor, D. L. (2002) Curr. Opin. Cell Biol. 14, 221-229[CrossRef][Medline] [Order article via Infotrieve]
  47. Jalink, K., van Corven, E. J., Hengeveld, T., Morii, N., Narumiya, S., and Moolenaar, W. H. (1994) J. Cell Biol. 126, 801-810[Abstract/Free Full Text]
  48. Fan, X., Labrador, J. P., Hing, H., and Bashaw, G. J. (2003) Neuron 40, 113-127[CrossRef][Medline] [Order article via Infotrieve]
  49. Jurney, W. M., Gallo, G., Letourneau, P. C., and McLoon, S. C. (2002) J. Neurosci. 22, 6019-6028[Abstract/Free Full Text]
  50. Marston, D. J., Dickinson, S., and Nobes, C. D. (2003) Nat. Cell Biol. 5, 879-888[CrossRef][Medline] [Order article via Infotrieve]
  51. Swiercz, J. M., Kuner, R., Behrens, J., and Offermans, S. (2002) Neuron 35, 51-63[CrossRef][Medline] [Order article via Infotrieve]

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