The G-protein G13 but Not G12 Mediates Signaling from Lysophosphatidic Acid Receptor via Epidermal Growth Factor Receptor to Rho*

Antje Gohla, Rainer Harhammer, and Günter SchultzDagger

From the Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67-73, D-14195 Berlin, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Lysophosphatidic acid (LPA) utilizes a G-protein-coupled receptor to activate the small GTP-binding protein Rho and to induce rapid remodeling of the actin cytoskeleton. We studied the signal transduction from LPA receptors to Rho activation. Analysis of the G-protein-coupling pattern of LPA receptors by labeling activated G-proteins with [alpha -32P]GTP azidoanilide revealed interaction with proteins of the Gq, Gi, and G12 subfamilies. We could show that in COS-7 cells, expression of GTPase-deficient mutants of Galpha 12 and Galpha 13 triggered Rho activation as measured by increased Rho-GTP levels. In Swiss 3T3 cells, incubation with LPA or microinjection of constitutively active mutants of Galpha 12 and Galpha 13 induced formation of actin stress fibers and assembly of focal adhesions in a Rho-dependent manner. Interestingly, the LPA-dependent cytoskeletal reorganization was suppressed by microinjected antibodies directed against Galpha 13, whereas Galpha 12-specific antibodies showed no inhibition. The tyrosine kinase inhibitor tyrphostin A 25 and the epidermal growth factor (EGF) receptor-specific tyrphostin AG 1478 completely blocked actin stress fiber formation caused by LPA or activated Galpha 13 but not the effects of activated Galpha 12. Also, expression of the dominant negative EGF receptor mutant EGFR-CD533 markedly prevented the LPA- and Galpha 13-induced actin polymerization. Coexpression of EGFR-CD533 and activated Galpha 13 in COS-7 cells resulted in decreased Rho-GTP levels compared with expression of activated Galpha 13 alone. These data indicate that in Swiss 3T3 cells, G13 but not G12 is involved in the LPA-induced activation of Rho. Moreover, our results suggest an involvement of the EGF receptor in this pathway.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Upon stimulation, a number of heterotrimeric G-protein-coupled receptors initiate cellular responses involving small GTP-binding proteins. For instance, the water-soluble phospholipid lysophosphatidic acid (LPA)1 binds to a G-protein-coupled heptahelical receptor (1, 2) and stimulates the activation of Ras and Rho proteins via different pathways (3, 4). In fibroblasts, LPA lowers cAMP levels in a pertussis toxin (PTX)-sensitive manner, suggesting the coupling of the LPA receptor to G-proteins of the Gi subfamily (5). LPA-induced activation of the Ras/Raf/mitogen-activated protein kinase cascade also occurs in a PTX-sensitive fashion (3, 6), and Gbeta gamma dimers of heterotrimeric G-proteins are thought to transduce this effect via intermediary protein-tyrosine kinases (7, 8). In contrast, in a large variety of cells, the stimulation of phospholipase C induced by LPA was shown to be PTX-insensitive (9), suggesting a coupling of the LPA receptor to members of the Gq subfamily. In addition to these pathways, LPA induces the Rho-dependent formation of focal adhesions and actin stress fibers in quiescent Swiss 3T3 fibroblasts (4, 10). A tyrosine kinase has been implicated in the LPA-induced Rho stimulation, since the tyrosine kinase inhibitor tyrphostin A 25 blocks the effects of LPA but not of microinjected Rho on actin polymerization (11). However, the G-proteins coupling the LPA receptor to Rho activation have not been specified. LPA-induced cytoskeletal reorganizations are PTX-insensitive and therefore presumably not transmitted via Gi subfamily members (12). Gq proteins are apparently not involved in this pathway, because neither the activation of protein kinase C nor mobilization of intracellular calcium or expression of a GTPase-deficient mutant of Galpha q induces the formation of actin stress fibers or focal adhesions (12-14). Interestingly, microinjection of constitutively active mutants of Galpha 12 and Galpha 13 into Swiss 3T3 cells triggers actin polymerization in a Rho-dependent manner (13).

In the present study, we determined the G-protein coupling pattern of the LPA receptor in membrane preparations and characterized the pathway leading from LPA receptor activation to stress fiber formation in intact cells. We report direct coupling of the LPA receptor to proteins of the G12, Gq, and Gi subfamilies. Furthermore, our data suggest that in intact cells G13 but not G12 mediates LPA-induced Rho activation involving the epidermal growth factor (EGF) receptor tyrosine kinase activity.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture-- Swiss 3T3 cells, kindly provided by Dr. Alan Hall (London), and COS-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. To obtain quiescent and serum-starved Swiss 3T3 cells, cultures were rinsed three times in serum-free DMEM and incubated in DMEM supplemented with 25% Ham's F-12, 0.2% NaHCO3, 10 mM Na-Hepes, and 0.1% fetal bovine serum (modified DMEM) for 24 h followed by an incubation in modified DMEM without fetal bovine serum for 18 h.

Membrane Preparations-- Membranes were prepared from subconfluent Swiss 3T3 cells. Cells were rinsed and scraped in a buffer containing 20 mM Tris/HCl, pH 8, 1 mM EDTA, 140 mM NaCl, 20 mM beta -mercaptoethanol, and 10 mM phenylmethylsulfonylfluoride. The pelleted cells were resuspended in the buffer described above devoid of NaCl but containing 20 µg/ml leupeptin. Cells were disrupted by forcing the suspension 10 times through a 26-gauge needle. Undisrupted cells were pelleted (750 × g, 2 min), and the supernatant was centrifuged at 50,000 × g for 30 min at 4 °C. Membranes were resuspended and stored at -70 °C until use. Protein concentrations were determined according to Lowry et al. (15).

SDS-PAGE and Immunoblotting-- For immunological detection of G-proteins, SDS-PAGE using 10% (w/v) polyacrylamide separating gels was employed. Proteins were transferred to nitrocellulose as described (16). The subtype specific antisera AS 232 and AS 233 (anti-alpha 12); AS 343, AS 342, and AS 272 (anti-alpha 13); AS 369 (anti-alpha q/11), and AS 266 (anti-alpha i) were characterized previously (17). Antisera against Galpha 12 were incubated with the peptides (10 µg/ml) used for their generation, as published elsewhere (17, 18). Detection of filter-bound proteins was carried out using the enhanced chemiluminescence (ECL) Western blotting system purchased from Amersham (19).

Photolabeling of Galpha Subunits-- Synthesis of [alpha -32P]GTP azidoanilide from [alpha -32P]GTP (NEN Life Science Products) was performed according to published protocols (20). For photolabeling of Galpha subunits, sedimented Swiss 3T3 membranes (200 µg of protein/tube) were preincubated in the absence or presence of 10 µM LPA at 30 °C, followed by the addition of [alpha -32P]GTP azidoanilide (3 µCi/tube; specific activity 3,000 Ci/mmol) at 30 °C for different times: 3 min for Galpha i, 10 min for Galpha q/11, and 30 min for Galpha 12 and Galpha 13 (18). Samples were irradiated for 15 s at 4 °C with a 254-nm UV lamp. Photolabeled membranes were pelleted and solubilized in 40 µl of 4% SDS (w/v) at room temperature, followed by the addition of precipitating buffer (280 µl) containing 10 mM Tris/HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% deoxycholate (w/v), 1% Nonidet P-40 (w/v), 1 mM dithiothreitol, 0.2 µM phenylmethylsulfonylfluoride, and 10 µg/ml aprotinin. Samples were centrifuged (13,000 × g) for 5 min at 4 °C to remove insoluble material. The antisera (20 µl) AS 369 for Galpha q/11, AS 233 for Galpha 12, AS 343 for Galpha 13, and AS 266 for Galpha i were added to the supernatants followed by constant rotation of the samples for 2 h at 4 °C. Protein A-Sepharose beads (7 µg) were added, and samples were incubated at 4 °C overnight. The immunoprecipitates were washed three times, boiled in SDS-sample buffer, and subjected to SDS-PAGE. Gels were subsequently dried and analyzed using autoradiography.

Transient Transfection of COS-7 Cells and Rho Activation Assay-- For transfection experiments, 1.2 × 105 COS-7 cells were plated in 10-cm diameter cell culture dishes, grown overnight, and subsequently serum-starved for 18 h. Cells were then washed with phosphate-buffered saline, and 12 µg of DNA mixed with 60 µl of LipofectAMINE (Life Technologies, Inc.) in 6 ml of Opti-MEM (Life Technologies, Inc.) were added. In cotransfection experiments with two different plasmids, 6 µg of each plasmid were added. The total amount of DNA was kept constant by supplementing with DNA from a vector encoding beta -galactosidase. Twelve h after transfection, COS-7 cell transfectants were labeled with [32P]orthophosphate (0.4 mCi/ml for 4 h) in phosphate-free Eagle's minimum essential medium. Cells were lysed on ice in 1 ml of 50 mM Hepes buffer, pH 7.4, containing 1% Triton X-100, 0.5% deoxycholate, 0.05% SDS, 150 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 1 mg/ml of bovine serum albumin, 10 mM benzamidine, leupeptin-aprotinin-soybean trypsin inhibitor (10 µg/ml), and 1 mM phenylmethylsulfonylfluoride. Nuclei and cellular debris were sedimented, and NaCl was added to lysates at a final concentration of 500 mM. After preclearing for 30 min with 8 µg of protein A-Sepharose, samples were incubated with 4 µg of rabbit polyclonal anti-Rho A (Santa Cruz Biotechnology) for 60 min at 4 °C. Immunocomplexes were collected with 15 µg of protein A-Sepharose for 90 min. The beads were washed eight times in 50 mM Hepes buffer, pH 7.4, 500 mM NaCl, 0.1% Triton X-100, and 0.005% SDS. Nucleotides were eluted in 5 mM EDTA, 2 mM dithiothreitol, 0.2% SDS, 0.5 mM GTP, and 0.5 mM GDP for 20 min at 68 °C and separated by thin-layer chromatography on polyethyleneiminecellulose plates (Merck) run in 1 M KH2PO4, pH 3.4. The positions of unlabeled GDP and GTP standards on the plates were visualized under 254-nm UV light. Quantitations of radiolabeled nucleotides were performed with phosphoimaging (Fuji BAS 1500).

Microinjection-- For microinjection studies, quiescent, serum-starved Swiss 3T3 cells (approximately 103 cells/mm2) plated on glass coverslips marked with squares to facilitate the localization of injected cells were used. The cDNAs of Galpha 12 (Q229L; Galpha 12QL), Galpha 13 (Q226L; Galpha 13QL), and Galpha q (R183C; Galpha qRC), carried by the cytomegalovirus promotor-containing vector pCis, Galpha 11 (Q209L; Galpha 11QL) cloned in pZeo, and empty vector controls, were applied for microinjection. Plasmids were microinjected into the nucleus alone or with Texas red-coupled dextran (5 mg/ml, Molecular Probes) to visualize injected cells. Clostridium botulinum C3 toxin was co-microinjected with the cDNAs described above at a final concentration of 100 µg/ml. After microinjection, the cells were returned to the incubator for a further 90 min until fixation. As indicated, cells were treated with tyrphostin A 25 (150 µM), tyrphostin A 1 (150 µM), or genistein (30 µg/ml) for 60 min. Tyrphostin AG 1478 (1 µM) or tyrphostin AG 1296 (10 µM) were added 120 min before fixation. To study cytoskeletal effects of extracellular factors, cells were incubated with LPA (0.3 µM), thrombin (40 ng/ml), EGF (10 ng/ml), or bombesin (10 nM) for 10 min. The phosphatase inhibitor orthovanadate (100 µM) was added to serum-starved cells 25 min before fixation. Antibodies against Galpha subunits were diluted and microinjected into the cytosol of cells followed by an incubation period of 60 min. The dominant negative EGF receptor mutant EGFR-CD533 carried by the pRK5 vector was expressed in Swiss 3T3 cells by nuclear microinjection. About 100 cells/field were injected in each case using a manual injection system (Eppendorf). Needles were pulled from capillaries with a horizontal micropipette puller (Sutter).

Fluorescence Microscopy-- Microinjected or growth factor-treated cells were fixed in 4% paraformaldehyde for 20 min and permeabilized in 0.2% Triton X-100 for 5 min. For localization of actin filaments, cells were stained with 0.5 µg/ml fluorescein isothiocyanate (FITC)-phalloidin (Sigma) for 40 min. Vinculin was detected after blocking with phosphate-buffered saline supplemented with 5% fetal bovine serum for 30 min, followed by incubation with a monoclonal anti-vinculin antibody (VIN-11-5; Sigma) for 60 min. Subsequently, cells were labeled with a FITC-conjugated goat anti-mouse antibody (Sigma) for 45 min. Antibody incubations were performed at room temperature. The coverslips were mounted on glass slides and examined on an inverted microscope (Zeiss Axiovert 100).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

To characterize the G-protein coupling pattern of the LPA receptor in Swiss 3T3 fibroblasts, we first determined the expression of different PTX-insensitive Galpha subunits in these cells by performing immunoblot experiments with subtype-specific antibodies. Immunoblotting of membrane proteins with an antibody specific for Galpha q/11 (AS 369) identified the expression of the corresponding 42-kDa proteins (Fig. 1, lane 1). Antisera raised against a C-terminal peptide of Galpha 12 (AS 232, AS 233) recognized a protein with an apparent molecular mass of 43 kDa (Fig. 1, lanes 2 and 3). In addition, these antisera cross-reacted with several unknown proteins of higher and lower molecular masses that had not been detected in rodent and nonrodent brain membranes (17). To assure the specificity of AS 232 and AS 233, which have been extensively characterized (17, 18), we performed peptide competition experiments. Upon preincubation with the peptide used for their generation, the detection of Galpha 12 was abolished (data not shown). Immunoblotting of membranes with an antiserum specific for Galpha 13 (AS 343) recognized a protein of approximately 43 kDa (Fig. 1, lane 4). These experiments indicate the expression of both Galpha 12 and Galpha 13 in Swiss 3T3 cell membranes.


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Fig. 1.   Immunological detection of G-protein alpha  subunits subunits expressed in Swiss 3T3 cells. Membrane proteins prepared from Swiss 3T3 cells (150 µg of protein/lane) were acetone-precipitated and separated by SDS-PAGE using separating gels containing 10% acrylamide. After blotting, nitrocellulose strips were incubated with anti-alpha q/11 (AS 369, lane 1), anti-alpha 12 (AS 232, lane 2; AS 233, lane 3), and anti-alpha 13 (AS 343, lane 4) antisera. All antisera were diluted 1:150. The ECL system was used for detection of filter-bound antibodies. The position of a 43-kDa marker protein is indicated.

In previous studies, interactions of the LPA receptor with G-proteins of the Gi and Gq subfamilies were postulated mainly from experiments showing the existence of PTX-sensitive as well as PTX-insensitive cellular effects of LPA (9). To directly show the LPA-mediated activation of G-proteins, membranes of subconfluent Swiss 3T3 cells were photolabeled with [alpha -32P]GTP azidoanilide in the presence or absence of LPA. G-protein Galpha subunits were subsequently immunoprecipitated with anti-alpha q/11 (AS 369), anti-alpha 12 (AS 233), anti-alpha 13 (AS 343), or anti-alpha i (AS 266) antisera and visualized by autoradiography. Fig. 2 shows that stimulation of membranes with 10 µM LPA induced an incorporation of [alpha -32P]GTP azidoanilide into Galpha subunits of Gq/11, G12, G13, and Gi proteins. For studying [alpha -32P]GTP azidoanilide incorporation into Galpha 12 and Galpha 13, the experiments were performed with prolonged incubation times of 30 min, whereas an agonist-induced activation of Galpha i was only observed with short incubation times, reflecting the substantial basal guanine nucleotide turnover of Gi proteins. No increased incorporation of [alpha -32P]GTP azidoanilide into Galpha 12 or Galpha 13 was found using lower concentrations of LPA (e.g. 1 µM).


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Fig. 2.   Coupling of the LPA receptor to G-proteins in Swiss 3T3 cell membranes. Cell membranes (200 µg of protein/tube) were photolabeled with [alpha -32P]GTP azidoanilide (3 µCi/tube) in the absence (-) or presence (+) of LPA (10 µM) as described under "Experimental Procedures." Membranes were solubilized and immunoprecipitated with anti-alpha q/11 (AS 369), anti-alpha 12 (AS 233), anti-alpha 13 (AS 343), and anti-alpha i (AS 266) antisera and protein A-Sepharose beads. Precipitated proteins were subjected to SDS-PAGE (separating gels with 10% acrylamide). Gels were dried, and labeled proteins were visualized by autoradiography. The position of a 43-kDa marker protein is indicated. Results are representative of three independent experiments.

Expression of constitutively active mutants of G12 subfamily members has been reported to induce a Rho-dependent formation of actin stress fibers in Swiss 3T3 cells (13). To further characterize the functional role of G12 and G13 in LPA-induced and Rho-mediated regulation of the actin cytoskeleton, we first expressed constitutively active mutants of Galpha 12 (Galpha 12 Q229L; Galpha 12QL) and Galpha 13 (Galpha 13 Q226L; Galpha 13QL) in Swiss 3T3 fibroblasts by microinjecting expression plasmids into quiescent cells. Both Galpha subunits stimulated the formation of actin stress fibers, whereas GTPase-deficient forms of Galpha q (Galpha q R183C; Galpha q RC) and Galpha 11 (Galpha 11 Q209L; Galpha 11 QL) were unable to induce stress fiber formation (Fig. 3). The expression of Galpha 12QL and Galpha 13QL was also accompanied by the formation of vinculin-containing spots with a characteristically elongated, arrowhead shape typical of Rho-induced assembly of focal adhesions (22) that were not observed upon microinjection of plasmids encoding Galpha qRC and Galpha 11QL (Fig. 3). The dependence of Galpha 12QL- and Galpha 13QL-mediated cytoskeletal effects on stimulation of Rho activity could be demonstrated employing purified recombinant C3 exoenzyme from C. botulinum (13). C3 exoenzyme has been shown to inhibit the isoforms of Rho but no other members of the Rho family (21, 22). Co-microinjection of C3 exoenzyme (100 µg/ml) abolished the actin stress fiber formation induced by Galpha 12QL and Galpha 13QL (Fig. 4). Likewise, focal adhesion assembly caused by both activated Galpha subunits was inhibited by C3 exoenzyme (data not shown). These results confirm that G12 and G13 are able to trigger Rho-dependent effects in Swiss 3T3 cells, whereas cytoskeletal remodeling typical of an activation of Cdc42 or Rac, i.e. formation of filopodia or membrane ruffles (23, 24), was not observed. Interestingly, the phosphotyrosine phosphatase inhibitor orthovanadate (100 µM), which has previously been shown to stimulate Rho in Swiss 3T3 fibroblasts (11), also induced a selective formation of actin stress fibers without membrane ruffling or formation of filopodia (not shown). To test G12 subfamily-induced Rho activation biochemically, we transiently overexpressed Galpha 12QL and Galpha 13QL in COS-7 cells and analyzed the incorporation of radioactive nucleotides in Rho A. Expression of activated Galpha 12 and Galpha 13, but not of the beta -galactosidase control, caused an accumulation of Rho-bound radioactive GTP. The ratio of bound GTP to total nucleotides bound to Rho [(GTP/GTP + GDP) × 100] was 22 ± 0.57% for the lacZ control, 28.75 ± 0.92% for Galpha 12QL, and 35.7 ± 4.1% for Galpha 13QL (Fig. 5). These data demonstrate that expression of Galpha 12QL and Galpha 13QL leads to an activation of Rho.


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Fig. 3.   Stimulation of actin stress fiber formation and focal adhesion assembly by constitutively active Galpha subunits. Expression plasmids (100 ng/µl) containing the cDNAs of Galpha 12 (Q229L), Galpha 13 (Q226L), Galpha q (R183C) or Galpha 11 (Q209L), or the empty vector (control) were microinjected into the nucleus of serum-starved, subconfluent Swiss 3T3 fibroblasts. Cells were fixed after 90 min with paraformaldehyde and permeabilized with Triton X-100. Actin stress fibers were revealed using FITC-labeled phalloidin (left panels), and focal adhesions were localized with a mouse anti-vinculin antibody followed by a FITC-conjugated goat anti-mouse antibody (right panels). Shown are cells from one of four independent experiments.


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Fig. 4.   Botulinum C3 exoenzyme abolishes the cytoskeletal effects induced by constitutively active Galpha 12, Galpha 13, and LPA. Serum-starved Swiss 3T3 cells were stimulated with LPA (200 ng/ml) or microinjected with expression plasmids (100 ng/µl) encoding Galpha 12 (Q229L) or Galpha 13 (Q226L). Cells in the right panel were co-microinjected with botulinum C3 exoenzyme (100 µg/ml). Actin stress fibers were visualized by indirect immunofluorescence as described under "Experimental Procedures." Cells from one of three independent experiments are shown.


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Fig. 5.   Rho-activation due to overexpression of constitutively active Galpha 12 and Galpha 13. COS-7 cells were transiently transfected with cDNAs encoding activated Galpha 12 and Galpha 13. After metabolic labeling with [32P]orthophosphate, cells were lysed and RhoA was immunoprecipitated. A, radioactive nucleotides bound to Rho were eluted and resolved by TLC. The position of GDP and GTP standards is indicated. B, ratio of GTP to total labeled nucleotides complexed to Rho [(GTP/GTP + GDP) × 100]. Data represent means ± S.D. (n = 2). Error bars not shown are masked by the symbols.

To characterize the function of individual G12 subfamily members for LPA-induced activation of Rho in intact cells, we microinjected antibodies directed against the carboxyl termini of Galpha 12 and Galpha 13. Actin stress fiber formation caused by LPA at a concentration of 0.3 µM was suppressed by application of Galpha 13-specific antiserum AS 343 (dilution of 1:50), whereas microinjection of the corresponding preimmune serum failed to influence LPA-induced cytoskeletal effects. Inhibition of LPA-induced stress fiber formation was also obtained using other Galpha 13-specific antisera, i.e. AS 342 and AS 272 (not shown). Surprisingly, the Galpha 12-specific antisera (AS 232, AS 233) did not inhibit the LPA-induced actin polymerization using dilutions of 1:1 to 1:50 (Fig. 6). The application of antibodies against Galpha q/11 (AS 369) showed no effects on cytoskeletal reorganisation caused by LPA. These results suggest that in intact Swiss 3T3 cells, LPA-induced Rho stimulation may be mediated by G13 but not by G12.


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Fig. 6.   Suppression of LPA-induced cytoskeletal effects by antibodies against Galpha 13. Formation of actin stress fibers is shown in serum-starved Swiss 3T3 cells stimulated with 0.3 µM LPA (control). Cells were microinjected with anti-alpha 13-antiserum AS 343, corresponding preimmune serum (AS 343-P), anti-alpha 12-antiserum AS 232, anti-alpha 12-antiserum AS 233, or anti-alpha q/11-antiserum AS 369, each diluted 1:50. Actin stress fibers were visualized by fluorescently tagged phalloidin, as described under "Experimental Procedures." Cells from one of three independent experiments are shown.

Since the tyrosine kinase inhibitor tyrphostin A 25 has been shown to block stress fiber formation evoked by LPA, but not by microinjected Rho protein, an involvement of a tyrosine kinase acting upstream of Rho in the pathway of LPA-induced Rho activation appears to be likely (11). To further study the mechanism whereby G13 mediates the LPA-induced activation of Rho, we investigated effects of tyrosine kinase inhibitors on cytoskeletal changes induced by different stimuli. As demonstrated in Fig. 7A, tyrphostin A 25 (150 µM) completely inhibited stress fiber formation induced by LPA and Galpha 13QL (100 ng/µl of cDNA) but not the cytoskeletal effects of Galpha 12QL (100 ng/µl of cDNA), further confirming the notion that LPA signals via G13 to Rho. Focal adhesion assembly caused by LPA or Galpha 13QL was also blocked by incubation with tyrphostin A 25, whereas focal adhesions induced by Galpha 12QL were not influenced by this tyrosine kinase inhibitor (Fig. 7B). To exclude that these differences in tyrphostin sensitivity were results of different expression levels of Galpha 12QL and Galpha 13QL, we studied cytoskeletal effects caused by various concentrations of expression plasmids. Cytoskeletal effects of Galpha 13QL induced by injection of up to 1 µg/µl of cDNA were abolished by tyrphostin A 25. However, Galpha 12QL-induced submaximal stress fiber formation after microinjection of 10 ng/µl of cDNA was tyrphostin A 25-insensitive. Unlike tyrphostin A 25, the tyrosine kinase inhibitor genistein (30 µg/ml), which has previously been shown to act downstream of Rho (12), blocked the formation of actin stress fibers induced by LPA, Galpha 13QL, and Galpha 12QL (Fig. 7A). The biologically inactive tyrphostin A 1 (150 µM) showed no effects on cytoskeletal changes caused by LPA or the activated Galpha subunits (data not shown). Using more specific inhibitors of tyrosine kinases, we found that 1 µM EGF receptor-specific tyrosine kinase inhibitor tyrphostin AG 1478 (25) completely blocked both the LPA- and Galpha 13QL-induced formation of actin stress fibers (Fig. 8). Lower concentrations of tyrphostin AG 1478 (e.g. 250 or 500 nM) partially suppressed actin polymerization caused by Galpha 13QL. Cytoskeletal effects caused by Galpha 12QL were not influenced by this tyrphostin at concentrations of up to 20 µM. Tyrphostin AG 1296 (10 µM), a selective platelet-derived growth factor receptor tyrosine kinase inhibitor, failed to affect stress fiber formation induced by LPA and Galpha 13QL or by Galpha 12QL (Fig. 8). Actin stress fiber formation caused by orthovanadate was also inhibited by tyrphostin A 25 and tyrphostin AG 1478 but not by tyrphostin AG 1296 (data not shown).


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Fig. 7.   Effects of tyrosine kinase inhibitors on cytoskeletal reorganization caused by LPA, active Galpha 13, or Galpha 12. Serum-starved Swiss 3T3 cells were treated with 0.3 µM LPA for 10 min or microinjected with plasmids (100 ng/µl) encoding Galpha 12 (Q229L) or Galpha 13 (Q226L). Cells were incubated with control buffer (-), tyrphostin A 25 (150 µM), or genistein (30 µg/ml) for 60 min as indicated, fixed in paraformaldehyde, and permeabilized using Triton X-100. Actin filaments (A) or focal adhesions (B) were visualized with FITC-phalloidin and an anti-vinculin antibody, respectively, as described under "Experimental Procedures." Shown are cells from one of four independent experiments.


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Fig. 8.   Inhibition of G13-mediated Rho activation by tyrphostin AG 1478. Actin stress fiber formation is shown in Swiss 3T3 cells treated with LPA (0.3 µM) or injected with plasmids encoding constitutively active Galpha 12 or Galpha 13. As indicated, cells were incubated with tyrphostin AG 1478 (1 µM), tyrphostin AG 1296 (10 µM), or buffer (control). Actin cytoskeleton was visualized by staining of fixed cells with FITC-phalloidin. Cells from one of three independent experiments are shown.

The inhibitory effects observed with tyrphostins suggested a participation of the EGF receptor in Galpha 13QL-induced Rho activation. Therefore, we expressed an EGF receptor mutant lacking 533 C-terminal amino acids (EGFR-CD533) in Swiss 3T3 cells by microinjection. It has been shown that this mutant exerts a dominant negative function on EGF receptor signaling by formation of signaling-incompetent heterodimers with the wild type receptor (26). Fig. 9 shows that expression of the truncated receptor markedly suppressed actin stress fiber formation caused by LPA or by Galpha 13QL. In agreement with the tyrphostin data shown above, Galpha 12QL-induced actin polymerization was not inhibited by expression of EGFR-CD533. Furthermore, expression of the dominant negative EGF receptor mutant also abolished the cytoskeletal effects caused by orthovanadate (Fig. 9). To assess a role for the EGF receptor in Galpha 13-mediated Rho activation, we transiently coexpressed Galpha 13QL and EGFR-CD533 in COS-7 cells and determined the activation state of Rho by analyzing Rho-bound nucleotides. Fig. 10 demonstrates that the Galpha 13QL-induced Rho-GTP accumulation was attenuated by co-expression of the mutated EGF receptor. The ratio of bound GTP to total nucleotides bound to Rho [(GTP/GTP + GDP) × 100] was 17.8 ± 1.9% for the lacZ control, 25.8 ± 0.2% for the coexpression of Galpha 13QL with lacZ, and 22.5 ± 0.1% for the coexpression of Galpha 13QL with EGFR-CD533. This demonstrates that the dominant negative EGF receptor (EGFR-CD533) was able to inhibit Galpha 13-induced activation of Rho. The expression of EGFR-CD533 with lacZ did not affect basal Rho-GTP levels compared with lacZ (not shown). These data clearly suggest the EGF receptor as a critical component in the pathway leading from Galpha 13 to Rho activation.


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Fig. 9.   Suppression of cytoskeletal effects by expressing the dominant negative EGF receptor mutant EGFR-CD533. Cells were incubated with LPA (0.3 µM) or orthovanadate (100 µM) or microinjected with plasmids encoding Galpha 12QL or Galpha 13QL. In addition, fibroblasts were co-microinjected with plasmids encoding the dominant negative EGF receptor mutant (EGFR-CD533) or the empty vector (control). After fixation, cells were stained with FITC-phalloidin. Shown are cells from one of three independent experiments.


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Fig. 10.   Reduction of Galpha 13-induced Rho activation after expression of the dominant negative EGF receptor mutant EGFR-CD533. COS-7 cells were transfected with either lacZ alone or Galpha 13QL/lacZ or Galpha 13QL/EGFR-CD533. Cells were labeled with [32P]orthophosphate for 4 h, and RhoA was immunoprecipitated from the lysates. A, migration of Rho-associated radioactive nucleotides after separation by TLC in comparison to cold GDP/GTP standards. B, ratio of GTP to total labeled nucleotides complexed to Rho [(GTP/GTP + GDP) × 100]. Data represent means ± S.D. (n = 2). Error bars not shown are contained within the symbols.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The phospholipid LPA has been characterized as a multifunctional messenger acting on adenylyl cyclases, phospholipases, mitogen-activated protein kinases, and the actin cytoskeleton via different pathways (9). Previous studies demonstrated the existence of high affinity, guanine nucleotide-sensitive binding sites for LPA in membranes prepared from Swiss 3T3 cells and rat brains (27). Recently, the cloning of LPA receptors from neuronal cells and Xenopus oocytes has been described (1, 2). It has been suggested from experiments using PTX that some cellular effects of LPA are mediated by G-proteins belonging to Gi or Gq subfamilies (9). The signaling cascade coupling the LPA receptor to an activation of Rho and subsequent remodeling of the actin cytoskeleton leading to stress fiber formation has, however, remained unclear. Results from previous studies indicated that LPA-induced Rho signaling is PTX-insensitive and that constitutively activated Galpha q subunits are not able to stimulate Rho, excluding Gi and Gq proteins from this pathway (9, 13). On the other hand, expression of constitutively active mutants of Galpha 12 and Galpha 13 has been shown to trigger Rho-mediated stress fiber formation in Swiss 3T3 cells (13). Therefore, we studied the involvement of G12 subfamily proteins in the pathway from LPA receptor to stimulation of Rho/stress fiber formation. Activated G12 and G13 induced Rho-mediated stress fiber formation (see Fig. 3; Ref. 13). Furthermore, we were able to demonstrate activation of Rho by constitutively active mutants of Galpha 12 and Galpha 13 (see Fig. 5). In photolabeling experiments, both members of the G12 subfamily appeared to interact with the LPA receptor; however, in intact cells, only G13, but not G12, mediated signaling from LPA receptor to Rho. This was suggested by discriminating effects of microinjected subtype-specific antibodies, by differences in tyrphostin sensitivity, and by the different requirement of EGF receptor function in LPA and Galpha 13QL- versus Galpha 12QL-induced Rho activation (see below). These data led us to identify G13 as a mediator from LPA receptors to Rho. A likely explanation for the observed discrepancy between the photoaffinity assay and microinjection/Rho activation studies is that photolabeling studies had to be performed employing a high concentration of LPA (10 µM), whereas a significantly lower concentration of 0.3 µM LPA was sufficient to induce maximum Rho activation in live Swiss 3T3 cells. Moreover, our data suggest that selectivity in receptor signaling is often found to be higher in intact complex systems than in in vitro assays. Similar conclusions may be deducible from the specificity of Gbeta gamma dimers in signaling pathways, since studies with antisense oligonucleotides performed in intact cells revealed a higher selectivity than biochemical assays using purified components (28-31). The identification of G13 as signal transducer from LPA receptor to Rho may suggest that other agonists of G-protein-coupled receptors that induce effects on the actin cytoskeleton may also act via G13 and/or G12.

Previously, Nobes et al. (11) have indicated the existence of a protein-tyrosine kinase acting in the LPA pathway upstream of Rho. One major finding of the present study is that LPA- as well as Galpha 13QL-induced effects on the actin cytoskeleton can be blocked with tyrphostins, suggesting that a tyrosine kinase activity is required for the signaling from G13 to Rho. Although an inhibition of GTPase activity of transducin by tyrphostin A 25 has been reported (32), it appears unlikely that the suppression of Galpha 13QL-induced Rho activation by this inhibitor might be a result of nonspecific interactions between tyrphostin A 25 and the G-protein, since GTPase-deficient mutants of Galpha subunits were used. Furthermore, also the specific EGF receptor inhibitor tyrphostin AG 1478 blocked the effects. The implication of EGF receptor kinase activity in the LPA/Galpha 13QL-induced, Rho-dependent stress fiber formation was substantiated by the expression of a dominant negative EGF receptor mutant in Swiss 3T3 cells. In addition, Galpha 13-induced Rho-GTP accumulation was at least partially reversed after overexpression of the truncated EGF receptor mutant. Recently, several G-protein-coupled receptors have been reported to induce a ligand-independent activation of the EGF or platelet-derived growth factor receptor with subsequent activation of mitogen-activated protein kinase cascades, and an involvement of Gi-type G-proteins in this transactivation pathway has been suggested (33-35). However, neither active Galpha i nor Gbeta gamma dimers lead to a reorganization of the actin cytoskeleton (13). Our data suggest an involvement of the receptor tyrosine kinase EGF receptor in the pathway of G13-mediated actin stress fiber formation. The presumably complex mechanism whereby the EGF receptor influences signaling mediated by activated Galpha 13 has yet to be resolved. Using serum-starved Swiss 3T3 cells stimulated with LPA, we found neither a coimmunoprecipitation of Galpha 13 with the EGF receptor or with a nonreceptor tyrosine kinase of the Src family nor a tyrosine phosphorylation of Galpha 13, arguing against the possibility of a direct interaction between Galpha 13 and a tyrosine kinase.2 On the other hand, strong similarities were observed between cytoskeletal events induced by LPA or Galpha 13QL and those caused by orthovanadate, pointing to the possibility that activated Galpha 13 may cause a ligand-independent stimulation of the EGF receptor by interacting with a phosphotyrosine phosphatase. The identification of a putative receptor tyrosine kinase/phosphatase cycle controlling the communication between activated G13 and Rho is under current investigation. Taken together, our data indicate that G13 couples the LPA receptor to Rho activation in Swiss 3T3 cells. In addition, we provide evidence for an involvement of the EGF receptor in the pathway leading from G13 to Rho activation/stress fiber formation.

    ACKNOWLEDGEMENTS

We thank Nadine Albrecht for expert technical assistance. We are grateful to Dr. Klaus Aktories (Freiburg) for providing botulinum C3 exoenzyme, Dr. Alan Hall (London) for the donation of Swiss 3T3 fibroblasts, Dr. Melvin I. Simon (Pasadena) for providing cDNAs of wild type and constitutively active forms of Galpha q, Galpha 11, Galpha 12, and Galpha 13, Dr. Christoph Sachsenmaier (München/Seattle) and Andreas Herrlich (Berlin/Karlsruhe) for the construct encoding the EGF receptor mutant EGFR-CD533, and Dr. Karsten Spicher (Berlin/Los Angeles) for providing antisera against Galpha subunits. Furthermore, we thank Dr. Thomas Gudermann and Dr. Christian Harteneck for helpful suggestions and critical reading of the manuscript and Dr. Frank Kalkbrenner for help with the microinjection technique.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.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.

Dagger To whom all correspondence should be addressed. Tel.: 49-30-838 3190; Fax: 49-30-831 5954.

1 The abbreviations used are: LPA, lysophosphatidic acid; G-protein, heterotrimeric guanine nucleotide-binding protein; PTX, pertussis toxin; EGF, epidermal growth factor; EGFR, EGF receptor; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate.

2 A. Gohla and R. Harhammer, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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