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J Biol Chem, Vol. 274, Issue 38, 26815-26821, September 17, 1999


A Rho Exchange Factor Mediates Thrombin and Galpha 12-induced Cytoskeletal Responses*

Mousumi MajumdarDagger , Tammy M. SeasholtzDagger , Carolan BuckmasterDagger , Deniz Toksoz§, and Joan Heller BrownDagger

From the Dagger  Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636 and the § Department of Physiology, Tufts University, Boston, Massachusetts 02111

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thrombin induces astrocytoma cell rounding through a Rho-dependent pathway (Majumdar, M., Seasholtz, T. M., Goldstein, D., de Lanerolle, P., and Brown, J. H. (1998) J. Biol. Chem. 273, 10099-10106). The involvement of the G12 family of G proteins and the role of specific Rho exchange factors in transducing signals from the thrombin receptor to Rho-dependent cytoskeletal responses was examined. Microinjection of cDNAs for activated Galpha 12 or Galpha 13 induced cell rounding, and antibodies to Galpha 12 or Galpha 13 blocked the response to thrombin. In contrast, activation or inhibition of Galpha q function had relatively little effect. The cytoskeletal response to Galpha 12 was inhibited by microinjection of C3 exoenzyme, indicating Rho dependence. Two Rho-specific guanine nucleotide exchange factors (GEFs), oncogenic lbc and p115, increased the percentage of rounded cells 4-5-fold, and this was inhibited by C3. Mutant GEFs lacking the Dbl homology (DH) domain required for exchange factor activity failed to induce cell rounding. However, the DH mutants of lbc and p115 were efficacious inhibitors of rounding induced by thrombin or Galpha 12. The effects of lbc were dependent on an intact pleckstrin homology domain, which may be required for appropriate targeting of the Rho-GEF. These findings identify the Galpha 12 protein family as transducers of thrombin signaling to the cytoskeleton and provide the first evidence that a Rho-GEF transduces signals between G protein-coupled receptors and Rho-mediated cytoskeletal responses.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The small G protein Rho is one of a family of low molecular weight GTPases that act as molecular switches to regulate cellular responses. Among its many functions, Rho is involved in controlling the actin cytoskeleton, thereby regulating cell shape and polarity, cytokinesis, cell motility, and contraction (1, 2). Ligands for G protein-coupled receptors, including lysophosphatidic acid (LPA),1 bombesin, thrombin, and endothelin, have been shown to induce actin cytoskeletal rearrangements in a variety of cell types (3-7). These GPCR agonist-induced morphological changes are mimicked by microinjection of activated Rho and are abolished by C3 exoenzyme pretreatment (3-5), which specifically ribosylates and inhibits Rho function. In neuronal cell lines and PC-12 cells, LPA, thrombin, and prostaglandin E2 (all ligands for GPCRs) induce Rho-dependent retraction of cell processes and cell rounding (4, 6, 8). Our laboratory has also demonstrated a requirement for Rho in thrombin-stimulated rounding and retraction of processes in 1321N1 astrocytoma cells (9). Similar cytoskeletal rearrangements are believed to be crucial in growth cone guidance and cell migration and may be involved in development and plasticity of neuronal and glial cells.

Despite abundant evidence for the involvement of Rho family small G proteins in agonist-induced morphological responses, there is limited information regarding the molecular mechanisms linking agonist activation of GPCR to regulation of Rho-mediated events. The question of which heterotrimeric G proteins are responsible for coupling receptors to the downstream targets that transduce the effects of agonists on the actin cytoskeleton is of considerable interest. Notably, GPCR agonists that activate phospholipase C through Gq are not equally efficacious for inducing cytoskeletal changes. For example, thrombin but not carbachol causes cell rounding in astrocytoma cells (9), and LPA but not bradykinin causes process retraction in PC-12 cells (6). Several lines of evidence support the conclusion that thrombin-induced cell rounding is independent of phospholipase C activation, Ca2+ mobilization, or pertussis toxin-sensitive G protein activation (9, 10). Thus, heterotrimeric G proteins other than those of the Galpha q or Galpha i/o family are likely to be responsible for linking activation of selected GPCRs to Rho-mediated actin rearrangements.

The pertussis toxin-insensitive G protein Galpha 12 was first isolated as an oncogene (11) and subsequently shown to induce cellular transformation in fibroblasts (12, 13). Previous work from our laboratory used microinjection of antibodies to the C terminus of Galpha subunits to demonstrate that the thrombin receptor regulates DNA synthesis in 1321N1 cells through Galpha 12 (14). Other recently identified cellular functions that are regulated by G12 or its closely related family member G13 include stress fiber formation in fibroblasts (15, 16), SRE-mediated gene transcription (12, 17), activation of JNK (18-20), and activation of Na+/H+ exchange (21, 22). However, the direct effectors of Galpha 12/13 that mediate the aforementioned responses still remain to be identified.

Activation of Rho and other small GTPases requires the exchange of GDP for GTP, a process catalyzed by guanine nucleotide exchange factors (GEFs). Several GEFs including lbc, lfc, lsc, and p115 show specificity for Rho (23, 24). Very recent studies provide evidence that the Rho-specific GEFs can transduce signals from the G12 family of heterotrimeric G proteins to Rho. The p115 GEF was demonstrated to contain an RGS-like domain and act as a GTPase-activating protein for Galpha 12 and Galpha 13 (25, 26). Most importantly, Galpha 13 was demonstrated to enhance the Rho exchange activity of p115 (25). A related observation is that Galpha 13 can synergize with p115 to activate SRE-mediated gene transcription (17). Another Galpha 12 family binding protein, termed PDZ-RhoGEF or KIAA0380, also regulates G12/13 effects on SRE activation (27). Involvement of Rho exchange factors in GPCR- or G12/13-mediated effects on the actin cytoskeleton has not been demonstrated.

We hypothesized that the thrombin receptor mediates cell rounding through coupling to Galpha 12 and subsequent activation of a Rho exchange factor. To test this possibility we microinjected inhibitory antibody to Galpha 12, as well as an expression plasmid for activated Galpha 12. Our findings indicate that Galpha 12 is necessary and sufficient for thrombin-induced cell rounding. Furthermore, we demonstrate that thrombin- and Galpha 12-stimulated rounding are inhibited by mutants of the Rho-GEFs Lbc and p115. These data implicate GEFs as downstream mediators of G12/13-induced Rho-dependent cytoskeletal changes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Human 1321N1 astrocytoma cells were plated onto 100-mm plates at a density of 1.2 × 105 cells/ml and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) for 4 days. The cells were then triturated and set on 12-mm round glass coverslips (prewashed with acid and ethanol) at a density of ~1.0 × 104 cells/slip and allowed to grow in 5% fetal calf serum plus DMEM for 48 h before microinjection.

C3 Toxin Fusion Protein-- cDNA for the glutathione S-transferase-C3 fusion protein (kindly provided by Dr. J. Meinkoth, University of Pennsylvania) was used to transform JM109 Escherichia coli to produce the glutathione S-transferase-C3 fusion protein for purification. After transformation, the cells were lysed, and clarified extracts were incubated with GSH-Sepharose. After extensive washing, the C3 toxin protein was cleaved from the glutathione S-transferase by overnight incubation with thrombin. Thrombin was removed by incubation with para-aminobenzamidine-Sepharose, and the supernatant was concentrated in a Centricon-10 to a final concentration of 5 mg/ml protein. In experiments involving microinjection of the C3 toxin, the protein was first diluted to a concentration of 40 µg/ml in microinjection buffer (100 mM KCl, 5 mM NaPO4) before injection.

Microinjection-- 1321N1 cells were set on glass coverslips and grown as described above (see "Cell Culture"). Expression plasmids were prepared and purified by CsCl gradient and diluted to a concentration of 50 µg/ml in microinjection buffer. These diluted expression plasmids were microinjected into cell nuclei along with 100 µg/ml of a marker plasmid for nuclear green fluorescent protein (provided by the laboratory of Dr. Geoff Wahl (28)) using an Eppendorf microinjector and Zeiss Axiovert Microscope. After microinjection of expression plasmids, cells were incubated in DMEM with 5% fetal calf serum for 3 h, followed by 20 min in serum-free DMEM plus 0.1% bovine serum albumin with or without thrombin (0.5 units/ml), and then fixed and stained as described below. For microinjection of antibodies, the antibodies were concentrated to 10 mg/ml using a Centricon and then diluted to 5 mg/ml with IgG. Following microinjection, cells were incubated for 30 min in 5% DMEM plus fetal calf serum, after which they were switched to serum-free medium plus 0.1% bovine serum albumin with or without thrombin (0.5 units/ml) for 20 min. Cells were then fixed and stained for F-actin as described below. Microinjected cells were identified by staining for co-injected IgG or by directly staining for the injected antibody (from rabbit) using a secondary anti-rabbit antibody conjugated to fluorescein isothiocyanate.

Rounding (Reversal of Stellation) Assay and Immunofluorescence-- To detect actin morphology of microinjected cells, cells were fixed in 3.7% formaldehyde/phosphate-buffered saline, permeabilized with 0.3% Triton X-100/phosphate-buffered saline, and stained for 30 min with rhodamine-conjugated phalloidin (Molecular Probes, Inc., Eugene, OR). Microinjected cells were identified by detection of green fluorescent nuclei (expressing co-injected nuclear green fluorescent protein), and F-actin staining was visualized using a Zeiss-Axiophot microscope and a × 40 Neofluor objective lens. Approximately 100-150 injected cells were detected per slip, and these were scored for rounding based on absence of "stellate" morphology (i.e. lack of processes and rounded morphology).

Statistical Analysis-- Data were analyzed by analysis of variance. Postanalysis was performed using the Tukey test when p was <0.05. p values are given in the figure legends.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Microinjected Antibodies to Galpha 12 and Galpha 13 Inhibit Thrombin-induced 1321N1 Cell Rounding-- To determine whether Galpha 12 mediates the cytoskeletal effects of thrombin we microinjected a Galpha 12 C-terminal antibody into 1321N1 cells. The ability of thrombin to induce cell rounding was examined 30 min after antibody injection and compared with the response in cells microinjected with IgG. The Galpha 12 C-terminal antibody inhibited thrombin-induced rounding by nearly 80% (Fig. 1). Microinjection of a Galpha 13 antibody (CT100 from Dr. Melvin Simon) also dramatically decreased rounding. We also tested a commercially available C-terminal Galpha 13 antibody (Calbiochem) which gave a marked but less complete (~55%) inhibition (data not shown). The same concentration of a C-terminal Galpha q antibody caused only partial inhibition (~30%) of thrombin-induced rounding. To determine the specificity of the Galpha 12 and Galpha 13 antibodies, Western blot analysis was performed on lysates from COS cells transfected with plasmids encoding Galpha 12 or Galpha 13, as described previously (14). The Galpha 12 antibody recognized only Galpha 12; however, both Galpha 13 antibodies recognized Galpha 13 and, to a lesser extent, Galpha 12 (data not shown). Therefore, the ability of the Galpha 13 antibody to inhibit thrombin-stimulated rounding may be due in part to blockade of Galpha 12 function.


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  		Fig. 1.   Microinjection of antibodies to heterotrimeric G protein alpha  subunits inhibits thrombin-induced cell rounding. Cells were microinjected with antibodies against the C-terminal domains of the alpha  subunits for Gq, G12, and G13, at a final concentration of 5 mg/ml. Thirty minutes after injection, cells were changed to serum-free medium plus 0.1% bovine serum albumin with or without thrombin (0.5 units/ml) for 20 min and then fixed and stained for F-actin as described under "Experimental Procedures." Data are expressed as the mean ± S.E. from two separate experiments, each containing two or three coverslips. *, p < 0.05 compared with IgG plus thrombin; **, p < 0.001 compared with IgG plus thrombin.

Constitutively Activated Galpha 12 and Galpha 13 Induce Cell Rounding-- To determine which Galpha subunits could mimic the effects of thrombin, 1321N1 cells were microinjected with 50 µg/ml expression plasmids for the activated form of Galpha q (Galpha qRC), Galpha 12 (Galpha 12QL), or Galpha 13 (Galpha 13QL). The cells were fixed 3 h after injection and stained, and the percentage of round cells was determined. Approximately 10% of cells injected with control plasmid (pCIS) were rounded (Fig. 2A), while most showed a normal stellate, process-bearing shape like that seen in uninjected cells (Fig. 2B). Microinjection of constitutively activated Galpha 12 or Galpha 13 expression plasmids induced a loss of stellation and produced a round morphology in 40-50% of the injected cells. The morphology induced by microinjection of activated Galpha 12QL or Galpha 13QL was identical to that seen with thrombin stimulation of astrocytoma cells (9). Microinjection of the same concentration of constitutively activated Galpha q cDNA resulted in a significantly lower level of cell rounding (~20% of injected cells) than that seen with activated Galpha 12 or Galpha 13. Activated Galpha q was less efficacious than Galpha 12 even when the concentration of microinjected Galpha q was 50-fold above that of Galpha 12 (data not shown).


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  		Fig. 2.   Constitutively activated Galpha 12QL and Galpha 13QL stimulate rounding of 1321N1 cells. 1321N1 cell nuclei were microinjected with plasmids encoding for vector, Galpha qRC (50 µg/ml), Galpha 12QL (50 µg/ml), or Galpha 13QL (50 µg/ml) and cultured for 3 h, followed by 20 min in serum-free medium. Microinjected cells were identified and scored for rounding as described. A, data are expressed as the mean ± S.E. of values obtained from 3-6 coverslips examined in at least three separate experiments. *, p < 0.05 compared with control vector (pCIS); **, p < 0.001 compared with control vector (pCIS). B, photo showing representative cells co-injected with activated Galpha 12QL (50 µg/ml) expression plasmid and nuclear green fluorescent protein plasmid. Cells were stained for F-actin with rhodamine-phalloidin to examine cytoskeletal morphology (left panel) as described under "Experimental Procedures." Injected cells were visually identified by expression of co-injected nuclear green fluorescent protein plasmid (right panel).

Activated Rho, but Not Activated Ras or Activated Rac, Induces 1321N1 Cell Rounding-- A variety of cell-specific cytoskeletal responses have been shown to be induced by small G proteins. To directly examine the effect of Rho on astrocytoma cell morphology, we microinjected an expression plasmid for constitutively activated L63Rho into 1321N1 cell nuclei. The number of microinjected cells that rounded was increased 4-fold following injection of activated Rho (Fig. 3). Microinjection of another activated Rho mutant V12Rho was equally effective at inducing rounding (data not shown). The specificity of this response was assessed by comparison with responses to activated Rac (V12Rac) or Ras (V12Ras). Microinjection of cDNA for these small G proteins did not induce cell rounding (Fig. 3), or other detectable morphological changes (e.g. membrane ruffling or stress fiber formation) in cells examined at times between 1 and 3 h after injection. These data indicate that Rho is a specific mediator of 1321N1 cell rounding and that this Rho-mediated response is independent of the activation of Ras or Rac.


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  		Fig. 3.   Activated Rho, but not activated Rac or activated Ras, stimulates 1321N1 cell rounding. 1321N1 cells were microinjected with expression plasmids for vector, constitutively activated Rho (L63Rho), activated Rac (V12Rac), or activated Ras (V12Ras). Microinjected cells were identified and scored for rounding as described under "Experimental Procedures." The results are expressed as the means ± S.E. from either two experiments, each containing one to two coverslips (for V12Rac and V12 Ras) or five experiments, each containing two or three coverslips (for vector and L63Rho). **, p < 0.001 compared with control vector (pCMV5).

Rho Dependence of Thrombin- and Galpha 12-induced Cell Rounding-- The C3 exoenzyme from Clostridium botulinum ADP-ribosylates and specifically inhibits Rho function. To demonstrate that Galpha 12 stimulates cell rounding through a Rho-dependent mechanism, we coinjected C3 exoenzyme protein along with activated Galpha 12QL plasmid. Co-injection of C3 exoenzyme blocked the ability of Galpha 12 to induce rounding. Microinjection of C3 exoenzyme also inhibited thrombin-induced rounding (Fig. 4), confirming the inhibitory effect of extracellularly applied C3 exoenzyme on the response to thrombin in these cells (9). Cell rounding induced by activated Rho was also inhibited by C3 exoenzyme injection (Fig. 4), consistent with the concept that ADP-ribosylation of Rho blocks its ability to couple to downstream effectors. One likely effector of thrombin and Rho-mediated cell rounding is the Rho-dependent kinase, Rho kinase, which phosphorylates and inhibits myosin phosphatase, leading to an increase in phosphorylated myosin light chain (29). Recent studies from our laboratory demonstrate that a 10 µM concentration of the Rho kinase inhibitor Y27632 (30) fully blocks thrombin-stimulated myosin phosphorylation and cell rounding in 1321N1 cells.2


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  		Fig. 4.   C3 exoenzyme inhibits cell rounding induced by thrombin, activated Galpha 12QL, or activated L63Rho. Cells were co-injected with C3 protein (40 µg/ml) and expression plasmids for pCIS (vector), Galpha 12QL (50 µg/ml), or L63Rho (50 µg/ml). Microinjected cells were identified and scored for morphology as described. Data are expressed as the means ± S.E. from two separate experiments each containing two coverslips. **, p < 0.001 as compared with pCIS plus thrombin without C3 exoenzyme, Galpha 12QL without C3, or L63Rho without C3.

Activation of Rho-dependent Cell Rounding by a Rho-GEF-- To determine whether Rho-specific guanine nucleotide exchange factors, which serve as activators of Rho, could effect similar changes in 1321N1 cell shape, we microinjected expression plasmids for the Rho-specific GEF, lbc (see Fig. 5A). A truncated, activated form of lbc (onco-lbc), which lacks the region C-terminal to but includes the pleckstrin homology (PH) domain was found to be a highly efficacious activator of cell rounding (Fig. 6A). As evidence that this response was mediated through the ability of lbc to activate Rho, we demonstrated that onco-lbc-induced cell rounding was inhibited by C3 co-injection (Fig. 6A). Microinjection of the full-length proto-lbc construct had a significant but lesser effect on cell morphology. Expression plasmids for another Rho-specific exchange factor, p115 (Fig. 5B), were also microinjected. Both the full-length form (p115) and an N-terminal, truncated cDNA (Delta N-p115), which lacks the region that contains RGS sequence homology (26), induced cell rounding (Fig. 6B). The response to p115 was shown to be Rho-dependent based on inhibition by C3 exoenzyme co-injection (Fig. 6B).


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  		Fig. 5.   Domains and mutants of the Rho exchange factors lbc and p115GEF. A, onco-lbc contains the DH and PH domains but has amino acids 424-893 from the C terminus of proto-lbc (which is the full-length, wild type form) deleted. lbc no PH is onco-lbc minus residues 324-415 in the PH domain, while lbc no DH is onco-lbc with residues 76-295 in the DH domain deleted. B, p115GEF is the full-length p115GEF containing the DH and PH domains, while Delta N-p115 is the p115GEF construct with the first 249 amino acids in the N terminus deleted. p115 no DH is p115 with amino acids 466-547 in the DH domain deleted.


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  		Fig. 6.   Microinjection of lbc and p115GEF induce C3-sensitive 1321N1 cell rounding. A, cells were microinjected with expression plasmids for SRalpha (vector), proto-lbc (wild type) (50 µg/ml), onco-lbc (50 µg/ml), or co-injected with C3 protein (40 µg/ml) and onco-lbc plasmid. Microinjected cells were identified and scored for morphology as described under "Experimental Procedures." Data are expressed as the mean ± S.E. from 3-6 separate experiments each with one or two coverslips; **, p < 0.001 compared with vector (SRalpha ); *, p < 0.05 compared with vector (SRalpha ). B, cells were microinjected with expression plasmids for SRalpha (vector), Delta N-p115 (50 µg/ml), or p115 (50 µg/ml) or co-injected with C3 protein (40 µg/ml) and p115 (50 µg/ml) expression plasmid and assayed for rounding after staining as described above. Data are expressed as the mean ± S.E. from two or three experiments each with one or two coverslips. **, p < 0.001 compared with vector (SRalpha ).

The Rho-GEF Mutants Lacking Dbl Homology (DH) Domains Are Inactive but Inhibit Thrombin- and Galpha 12-stimulated Cell Rounding-- Mutants of lbc or p115 lacking DH domains cannot catalyze GDP/GTP exchange (24, 31). To investigate a role for lbc and/or p115 in the signaling pathway utilized by thrombin and Galpha 12, we examined the possibility that DH domain mutants of the Rho-GEFs would act to inhibit downstream signaling. Mutants of lbc and p115 lacking their DH domains were first examined for their ability to induce cell rounding (Fig. 7, A and B). In contrast to the marked stimulatory effects of onco-lbc or p115 shown in Fig. 6, A or B, the comparable mutants lacking DH domains ("lbc no DH" or "p115 no DH"; see Fig. 5) were ineffective (Fig. 7, A and B). Of greatest interest, expression of lbc no DH or p115 no DH inhibited cell rounding induced by thrombin by more than 80% (Fig. 7, A and B). Additionally, injection of the mutants lbc no DH or p115 no DH caused nearly complete inhibition of the induction of cell rounding by co-injected Galpha 12 (Fig. 7, A and B). As evidence that inhibition by the no DH mutant occurred upstream of Rho, lbc no DH failed to significantly inhibit cell rounding induced by activated (L63)Rho. To control for the specificity of the inhibition, we further determined that lbc no DH did not block cell rounding induced by colchicine (data not shown), which presumably elicits cytoskeletal responses through its direct effect on microtubules. An lbc mutant lacking the PH domain had no significant effect on cell morphology but, unlike the no DH mutant, did not act as an inhibitor of thrombin signaling (Table I).


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  		Fig. 7.   Microinjection of lbc no DH or p115 no DH inhibits thrombin- or Galpha 12-induced cell rounding. A, 1321N1 cells were microinjected with expression plasmids for vector (SRalpha ) or lbc no DH (50 µg/ml). In some experiments, cells were microinjected with L63Rho or activated Galpha 12QL (50 µg/ml) along with the SRalpha vector or lbc no DH. Microinjected cells were identified and scored for rounding as described previously. Data are expressed as the mean ± S.E. from three separate experiments containing two or three coverslips in each. **, p < 0.001 compared with thrombin control (SRalpha plus thrombin) or Galpha a12 control (SRalpha plus Galpha a12QL). B, cells were microinjected with expression plasmids for vector (SRalpha ) or p115 no DH (50 µg/ml) and Galpha 12QL (50 µg/ml). To test inhibition of rounding induced by activated Galpha 12QL, cells were injected with Galpha 12QL (50 µg/ml) and p115 no DH (50 µg/ml), and microinjected cells were stained, identified, and scored for rounding as described previously. **, p < 0.001 compared with thrombin control (SRalpha plus thrombin) or Galpha 12 control (SRalpha plus Galpha 12QL).

                              
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Table I
Effects of lbc no PH on 1321N1 cell rounding


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The thrombin receptor has been suggested to couple to Galpha 12 and Galpha 13 based on its ability to increase GTP binding to these PTX-insensitive heterotrimeric G proteins in platelets and a reconstituted baculovirus system (32, 33). Our laboratory has also shown Galpha 12 to be required for thrombin-stimulated mitogenesis, Ras activation, and AP-1-mediated gene transcription (14, 20, 34). However, because these transcriptional and mitogenic responses result from convergence of multiple pathways and are measured at long times following thrombin stimulation, it has been difficult to identify the effectors immediately downstream of thrombin and Galpha 12 signaling in these pathways. The study described here presents evidence that thrombin-induced cytoskeletal rearrangement in 1321N1 astrocytoma cells is also mediated through receptor coupling to the heterotrimeric G protein G12. Analysis of this rapid and robust response has allowed further dissection of the molecular events by which Galpha 12 signaling occurs (Fig. 8).


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  		Fig. 8.   Schematic of proposed signaling pathway leading to 1321N1 astrocytoma cell rounding (reversal of stellation). The thrombin receptor is shown to couple to Galpha 12, which in turn leads to Rho-GEF activation catalyzing exchange of GTP for GDP on Rho. The Rho effector, Rho kinase, is shown to mediate thrombin-induced rounding, since this response is fully inhibited by a µM concentration of the Rho kinase inhibitor Y27632 (M. Majumdar, unpublished observations). Previous studies indicate that increased myosin light chain phosphorylation (MLC-P) correlates with thrombin-stimulated cell rounding (9).

Several observations support the involvement of Galpha 12 in thrombin-mediated cytoskeletal rearrangement. First, microinjection of the constitutively activated subunit of Galpha 12 alone, in the absence of agonist, is sufficient to elicit a morphological response (i.e. cell rounding and retraction of processes) identical to that seen with thrombin treatment. This finding is consistent with a recent study, published while this manuscript was in preparation, which found retraction of neurites in PC-12 cells to be induced by activated Galpha 12 or Galpha 13 (35). More compelling evidence supporting the involvement of Galpha 12 in thrombin receptor-induced 1321N1 cell rounding is the observation that the response is fully inhibited by microinjection of C-terminal inhibitory antibodies to Galpha 12. The finding that thrombin-induced rounding could also be blocked by microinjection of an antibody to the C terminus of Galpha 13 suggested that the G12 and G13 pathways might both be required for thrombin-induced rounding. However, while the Galpha 12 antibody was specific for Galpha 12, both Galpha 13 antibodies tested reacted with Galpha 12 as well as Galpha 13. Thus, the inhibitory effect of the Galpha 13 antibodies may result from blockade of Galpha 12 signaling. Gohla et al. (16) examined the ability of antibodies to the C-terminal of Galpha 12 and Galpha 13 to block LPA-induced stress fiber formation and reported inhibition only with antibodies to Galpha 13. More recent work from the same group has shown that antibodies to Galpha 12, but not Galpha 13, were capable of inhibiting thrombin-stimulated stress fiber formation (42). These findings suggest that the LPA receptor preferentially couples to Galpha 13, while the thrombin receptor couples to Galpha 12 to induce cytoskeletal responses.

Our findings further implicate a Rho guanine nucleotide exchange factor in the thrombin receptor/Galpha 12/13-induced cytoskeletal response. Specifically, we demonstrate that two distinct Rho exchange factors, lbc and p115, both stimulate cell rounding and retraction of cell processes. The response to these Rho-GEFs, as well as the response to thrombin and Galpha 12, is C3-sensitive, demonstrating that Rho mediates the effects of all of these stimuli. Further experiments show that the guanine nucleotide exchange activity, which is localized to the DH domain, is required for this Rho-GEF induced cytoskeletal response, since neither the lbc nor p115 mutants which lack exchange activity stimulate rounding. Microinjection of a mutant lbc, which lacks the PH domain was also unable to induce cell rounding or process retraction, demonstrating a requirement for the PH domain in these morphological responses. Surprisingly, this same mutant was able to induce stress fibers in fibroblasts (31). However, the PH domain has been shown to be required for transformation (24, 31, 36-38), has a critical role in targeting GEFs to their site of action (31, 39) and appears to be important for optimal guanine nucleotide exchange activity (38, 40).

Most critical to the conclusion that Rho-GEFs transduce signals from thrombin/Galpha 12 to Rho is the finding that microinjection of either lbc or p115 mutants lacking the DH exchange factor domain (lbc no DH or p115 no DH) can inhibit thrombin- and Galpha 12-stimulated rounding. Importantly, the lbc no DH mutant was unable to significantly inhibit L63Rho-induced rounding, indicating that the inhibition occurs upstream of Rho. In addition, the lbc no DH mutant was unable to block colchicine-induced 1321N1 cell rounding. These observations mitigate against nonspecific inhibitory effects of the mutant Rho-GEFs on the actin cytoskeleton.

The finding that lbc lacking its PH domain, while inactive, did not inhibit thrombin-induced rounding in 1321N1 cells suggests that the lbc PH domain could be the region required to compete with the endogenous Rho exchange factor that is the direct target of thrombin and Galpha 12. Whether this exchange factor is lbc, p115, or another as yet undetermined Rho-GEF is currently unknown. It is of interest that the PH domains in p115 and lbc show a greater degree of homology to each other (~27%) than to the comparable PH domain in the Rac specific exchange factor Dbl (24) or to PH domains in other proteins. Nonetheless we cannot exclude the possibility that the PH domains in the Rho-GEFs compete for Galpha 12 binding to other proteins that do not have exchange factor activity. For example Galpha 12 has been shown to interact with the PH domain in the Bruton's tyrosine kinase (43), a member of the Tec/Bmx family of non-receptor tyrosine kinases recently suggested to mediate Rho-GEF activation by Galpha 12 family proteins (44). Continued studies to determine which exchange factors bind to the Galpha 12 subunit and whether the PH domain competes for this binding are being carried out in our laboratory.

The p115 GEF contains not only PH and DH domains but also an RGS domain. This domain binds to the alpha  subunits of G12 and G13, for which p115 functions as a GTPase-activating protein (25, 26). Another Rho-specific GEF (termed PDZ-RhoGEF, cloned as KIAA0380) was also found to interact directly with Galpha 12 and Galpha 13 through an Lsc homology domain, with RGS domain homology (27). Inhibition of thrombin- and Galpha 12-induced cell rounding by p115 no DH could therefore be due to an inhibitory interaction of either the PH or the RGS domain with Galpha 12. While lbc has not yet been shown to directly interact with Galpha 12/13 or to contain a well defined RGS domain, some RGS homology was detectable within the PH domain.3 Thus, direct interactions of lbc as well as p115 with G protein alpha  subunits are possible.

Several recent studies of heterotrimeric G protein signaling indicate that tyrosine kinases act upstream of Rho activation. Notably, these studies reported that a tyrphostin A25-sensitive tyrosine kinase was required for the effects of LPA and Galpha 13 on Rho-dependent cytoskeletal responses (16, 35, 41) but that the response to Galpha 12 was not blocked by this tyrosine kinase inhibitor (16, 35). Our preliminary data also suggest that the thrombin/Galpha 12-induced morphological response is not inhibited by tyrphostin A25 or PP2, which inhibits src-like tyrosine kinases. However, studies using more selective inhibitors of Src family or other nonreceptor tyrosine kinases are required to rule out involvement of tyrosine kinases in the pathway by which thrombin and Galpha 12 activate Rho-GEFs and elicit cell rounding.

The studies reported here are among the first to show that mutant GEFs can be used to inhibit signaling through Rho-dependent pathways and to demonstrate that a mutant Rho-GEF can block agonist or heterotrimeric G protein-stimulated cytoskeletal responses. Other recent studies demonstrate inhibition of agonist and Galpha 12/13 effects on SRE-mediated gene expression by no DH mutants of p115 and PDZ-RhoGEF (17, 27). We are currently investigating the mechanism and specificities of Galpha 12 signaling to Rho exchange factors and the question of how Rho is activated in response to GPCR agonists and heterotrimeric G proteins.

    ACKNOWLEDGEMENTS

We thank Dr. Matthew J. Hart for providing the p115-RhoGEF plasmid and mutants and for helpful discussions. We also thank Dr. James Feramisco for advice and discussions concerning the microinjection experiments and Dr. Geoff Wahl for permission to use the nuclear green fluorescent protein plasmid in these microinjection experiments.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM 36927 (to J. H. B.).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.

To whom correspondence should be addressed: Dept. of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636. Tel.: 619-534-2595; Fax: 619-534-4337; E-mail: jhbrown@ucsd.edu.

2 M. Majumdar, D. Goldstein, and J. Heller Brown, unpublished observations.

3 D. Toksoz, unpublished results.

    ABBREVIATIONS

The abbreviations used are: LPA, lysophosphatidic acid; GEF, guanine nucleotide exchange factor; DMEM, Dulbecco's modified Eagle's medium; PH, pleckstrin homology; SRE, serum response element; GPCR, G protein-coupled receptor; DH, Dbl homology domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Narumiya, S. (1996) J. Biochem. (Tokyo) 120, 215-228[Abstract/Free Full Text]
2. Van Aelst, L., and D'Souza-Schory, C. (1997) Genes Dev. 11, 2296-2322
3. Ridley, A. J., and Hall, A. (1992) Cell 70, 389-399[CrossRef][Medline] [Order article via Infotrieve]
4. 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]
5. Rankin, S., Morii, N., Narumiya, S., and Rozengurt, E. (1994) FEBS Lett. 354, 315-319[CrossRef][Medline] [Order article via Infotrieve]
6. Tigyi, G., Fischer, D. J., Sebok, A., Yang, C., Dyer, D. L., and Miledi, R. (1996) J. Neurochem. 66, 537-548[Medline] [Order article via Infotrieve]
7. Koyama, Y., and Baba, A. (1996) Glia 16, 342-350[CrossRef][Medline] [Order article via Infotrieve]
8. Katoh, H., Negishi, M., and Ichikawa, A. (1996) J. Biol. Chem. 271, 29780-29784[Abstract/Free Full Text]
9. Majumdar, M., Seasholtz, T. M., Goldstein, D., de Lanerolle, P., and Brown, J. H. (1998) J. Biol. Chem. 273, 10099-10106[Abstract/Free Full Text]
10. Jalink, K., and Moolenaar, W. H. (1992) J. Cell Biol. 118, 411-419[Abstract/Free Full Text]
11. Chan, A. M.-L., Fleming, T. P., McGovern, E. S., Chedid, M., Miki, T., and Aaronson, S. A. (1993) Mol. Cell. Biol. 13, 762-768[Abstract/Free Full Text]
12. Fromm, C., Coso, O. A., Montaner, S., Xu, N., and Gutkind, J. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 91, 10098-10103
13. Jiang, H., Wu, D., and Simon, M. I. (1993) FEBS Lett. 330, 319-322[CrossRef][Medline] [Order article via Infotrieve]
14. Aragay, A. M., Collins, L. R., Post, G. R., Watson, A. J., Feramisco, J. R., Brown, J. H., and Simon, M. I. (1995) J. Biol. Chem. 270, 20073-20077[Abstract/Free Full Text]
15. Buhl, A. M., Johnson, N. L., Dhanasekaran, N., and Johnson, G. L. (1995) J. Biol. Chem. 270, 24631-24634[Abstract/Free Full Text]
16. Gohla, A., Harhammer, R., and Schultz, G. (1998) J. Biol. Chem. 273, 4653-4659[Abstract/Free Full Text]
17. Mao, J., Yuan, H., and Wu, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12973-12976[Abstract/Free Full Text]
18. Prasad, M. V. V. S. V., Dermott, J. M., Heasley, L. E., Johnson, G. L., and Dhanasekaran, N. (1995) J. Biol. Chem. 270, 18655-18659[Abstract/Free Full Text]
19. Voyno-Yasenetskaya, T. A., Faure, M., Ahn, N. G., and Bourne, H. R. (1996) J. Biol. Chem. 271, 21081-21087[Abstract/Free Full Text]
20. Collins, L. R., Minden, A., Karin, M., and Brown, J. H. (1996) J. Biol. Chem. 271, 17349-17353[Abstract/Free Full Text]
21. Hooley, R., Yu, C.-Y., Symons, M., and Barber, D. L. (1996) J. Biol. Chem. 271, 6152-6158[Abstract/Free Full Text]
22. Lin, X., Voyno-Yasenetskaya, T., Hooley, R., Lin, C.-Y., Orlowski, J., and Barber, D. L. (1996) J. Biol. Chem. 271, 22604-22610[Abstract/Free Full Text]
23. Glaven, J. A., Whitehead, I. P., Nomanbhoy, T., Kay, R., and Cerione, R. A. (1996) J. Biol. Chem. 271, 27374-27381[Abstract/Free Full Text]
24. Hart, M. J., Sharma, S., elMasry, N., Qiu, R.-G., McCabe, P., Polakis, P., and Bollag, G. (1996) J. Biol. Chem. 271, 25452-25458[Abstract/Free Full Text]
25. Hart, M. J., Jiang, X., Kozasa, T., Roscoe, W., Singer, W. D., Gilman, A. G., Sternweis, P. C., and Bollag, G. (1998) Science 280, 2112-2114[Abstract/Free Full Text]
26. Kozasa, T., Jiang, X., Hart, M. J., Sternweis, P. M., Singer, W. D., Gilman, A. G., Bollag, G., and Sternweis, P. C. (1998) Science 280, 2109-2111[Abstract/Free Full Text]
27. Fukuhara, S., Murga, C., Zohar, M., Igishi, T., and Gutkind, J. S. (1999) J. Biol. Chem. 274, 5868-5879[Abstract/Free Full Text]
28. Kanda, T., Sullivan, K. F., and Wahl, G. M. (1998) Curr. Biol. 8, 377-385[CrossRef][Medline] [Order article via Infotrieve]
29. Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 273, 245-248[Abstract]
30. Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T., Morishita, T., Tamakawa, H., Yamagami, K., Inui, J., Maekawa, M., and Narumiya, S. (1997) Nature 389, 990-994[CrossRef][Medline] [Order article via Infotrieve]
31. Olson, M. F., Sterpetti, P., Nagata, K., Toksoz, D., and Hall, A. (1997) Oncogene 15, 2827-2831[CrossRef][Medline] [Order article via Infotrieve]
32. Offermanns, S., Laugwitz, K.-L., Spicher, K., and Schultz, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 504-508[Abstract/Free Full Text]
33. Barr, A. J., Brass, L. F., and Manning, D. R. (1997) J. Biol. Chem. 272, 2223-2229[Abstract/Free Full Text]
34. Post, G. R., Collins, L. R., Kennedy, E. D., Moskowitz, S. A., Aragay, A. M., Goldstein, D., and Brown, J. H. (1996) Mol. Biol. Cell 7, 1679-1690[Abstract]
35. Katoh, H., Aoki, J., Yamaguchi, Y., Kitano, Y., Ichikawa, A., and Negishi, M. (1998) J. Biol. Chem. 273, 28700-28707[Abstract/Free Full Text]
36. Sterpetti, P., Hack, A. A., Bashar, M. P., Park, B., Cheng, S.-D., Knoll, J. H. M., Urano, T., Feig, L. A., and Toksoz, D. (1999) Mol. Cell. Biol. 19, 1334-1345[Abstract/Free Full Text]
37. Qian, X., Vass, W. C., Papageorge, A. G., Anborgh, P. H., and Lowy, D. R. (1998) Mol. Cell. Biol. 18, 771-778[Abstract/Free Full Text]
38. Soisson, S., Nimnual, A., Uy, M., Bar-Sagi, D., and Kuriyan, J. (1998) Cell 95, 259-268[CrossRef][Medline] [Order article via Infotrieve]
39. Whitehead, I., Kirk, H., Tognon, C., Trigo-Gonzalez, G., and Kay, R. (1995) J. Biol. Chem. 270, 18388-18395[Abstract/Free Full Text]
40. Liu, X., Wang, H., Eberstadt, M., Schnuchel, A., Olejniczak, E. T., Meadows, R. P., Schkeryantz, J. M., Janowick, D. A., Harlan, J. E., Harris, E. A., Staunton, D. E., and Fesik, S. W. (1998) Cell 95, 269-277[CrossRef][Medline] [Order article via Infotrieve]
41. Nobes, C. D., Hawkins, P., Stephens, L., and Hall, A. (1995) J. Cell Sci. 108, 225-233[Abstract]
42. Gohla, A., Offermanns, S., Wilkie, T. M., and Schultz, G. (1999) J. Biol. Chem. 274, 17901-17907[Abstract/Free Full Text]
43. Jiang, Y., Ma, W., Wan, Y., Kozasa, T., Hattori, S., and Huang, X.-Y. (1998) Nature 395, 808-813[CrossRef][Medline] [Order article via Infotrieve]
44. Mao, J., Xie, W., Yuan, H., Simon, M. I., Mano, H., and Wu, D. (1998) EMBO J. 17, 5638-5646[CrossRef][Medline] [Order article via Infotrieve]

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