Receptor-dependent RhoA activation in G12/G13-deficient cells: genetic evidence for an involvement of Gq/G11.

The small GTPase RhoA is involved in the regulation of various cellular functions like the remodeling of the actin cytoskeleton and the induction of transcriptional activity. G-protein-coupled receptors (GPCRs), which are able to activate Gq/G11 and G12/G13 are major upstream regulators of RhoA activity, and G12/G13 have been shown to couple GPCRs to the activation of Rho by regulating the activity of a subfamily of RhoGEF proteins. However, the possible contribution of Gq/G11 to the regulation of RhoA activity via GPCRs is controversial. We have used a genetic approach to study the role of heterotrimeric G-proteins in the activation of RhoA via endogenous GPCRs. In pertussis toxin-treated Galpha12/Galpha13-deficient as well as in Galphaq/Galpha11-deficient mouse embryonic fibroblasts (MEFs), in which coupling of receptors is restricted to Gq/G11 and G12/G13, respectively, receptor activation results in Rho activation. Rho activation induced by receptor agonists via Gq/G11 occurs with lower potency than Rho activation via G12/G13. Activation of RhoA via Gq/G11 is not affected by the phospholipase-C blocker U73122 or the Ca2+-chelator BAPTA, but can be blocked by a dominant-negative mutant of the RhoGEF protein LARG. Our data clearly show that G12/G13 as well as Gq/G11 alone can couple GPCRs to the rapid activation of RhoA. Gq/G11-mediated RhoA activation occurs independently of phospholipase C-beta and appears to involve LARG.

The small GTPase RhoA plays a central role in the organization of the cellular actin cytoskeleton through its ability to stimulate the formation of actomyosin-based structures and to regulate their contractility (1). In addition to its role in the regulation of the actin cytoskeleton, RhoA has also been involved in various other cellular processes like the regulation of microtubule dynamics or transcriptional activity (1,2). Analogous to other regulatory guanine nucleotide-binding proteins Rho functions as a molecular switch by cycling between an inactive GDP-bound form and an active GTP-bound form. In the active state RhoA relays extracellular signals to a number of downstream effectors. These include protein kinases like Rho kinase or citron kinase, lipid kinases like phospholipase D, or phosphatidylinositol 4-phosphate 5-kinase as well as non-kinase proteins like rhothekin, rhophilin, or diaphanous (3). RhoA is activated through various receptors including those coupled to heterotrimeric G-proteins (4,5). Activation of RhoA through G-protein-coupled receptors (GPCRs) 1 is involved in a variety of physiological regulatory processes (6). One of the best described cellular paradigms for GPCR-mediated RhoA activation is the RhoA-dependent actin stress fiber formation in fibroblasts activated by various GPCR agonists like lysophosphatidic acid or thrombin. However, a GPCR/Rho-mediated regulation of actin-based structures has also been shown to occur in many other eukaryotic cells. For instance, in neuronal cells activation of Rho through lysophosphatidic acid or thrombin receptors leads to the formation of contractile actomyosin filaments thereby inducing neurite retraction and cell rounding (7,8). In vascular smooth muscle cells, the Rho-mediated pathway has been shown to contribute to the vasoconstrictor-induced actomyosin-based cell contraction (9,10), and the same pathway appears to be centrally involved in the platelet shape change response (11).
It is well established that G-proteins of the G 12 -family, G 12 and G 13 , can couple GPCRs to the activation of RhoA. Constitutively active mutants of G␣ 12 and G␣ 13 have been shown to induce actin stress fiber formation as well as other RhoA-dependent cellular effects (Ref. 12; for review see Ref. 6). Recent studies in reconstituted or co-transfected systems have demonstrated that a group of RhoGEF proteins, consisting of p115 RhoGEF, PDZ-RhoGEF, and LARG, interact with G␣ 12 and G␣ 13 through their RGS domains, thereby stimulating RhoA activity (13)(14)(15)(16). Receptors, which activate G 12 /G 13 also couple to G q and G 11 . It has been a controversial issue, whether G q /G 11 -mediated signaling contributes to the activation of RhoA via GPCRs. While various reports show Rho-dependent effects of constitutively active G␣ q mutants (Refs. 8 and 18; for review see Ref. 6) other studies demonstrated that active mutants of G␣ q are not able to induce Rho-mediated processes (12,17). The recent development of methods for the precipitation of the activated form of RhoA from cell lysates allowed to directly determine the effects of different G-protein ␣-subunits on Rho activity. It could be confirmed that constitutively active mutants of G␣ 12 /G␣ 13 can induce RhoA activation (19,20). However, again, conflicting data exist with regard to the potential role of G q /G 11 in GPCR-mediated RhoA activation. In NIH3T3 and HEK293T cells, expression of constitutively active G␣ qfamily members results in an increased level of active RhoA (21)(22)(23). In contrast, expression of mutant G␣ q in COS-7 cells does not induce activation of RhoA (24).
To study the role of different G-proteins under more physiological conditions we used G␣ q /G␣ 11 -and G␣ 12 /G␣ 13 -double deficient embryonic fibroblasts (MEFs) to determine their role in the activation of Rho via endogenous receptors. Our data clearly show that G q /G 11 can couple GPCRs to the rapid activation of RhoA. This process occurs in a phospholipase C-␤independent manner and appears to involve the RhoGEF protein LARG.
Plasmids-HA-⌬DH/PH-LARG lacking the DH and the PH domain was generated by EcoNI and EcoRI digestion of full-length LARG and in-frame re-ligation using MungBean-nuclease and T 4 -DNA polymerase. Deletion was confirmed by sequencing (Li-cor 4200, Li-cor, Inc.). The modifying enzymes were purchased from New England Biolabs (Frankfurt, Germany), the ligase was obtained from Takara (Verviers, Belgium).
Cell Lines and Transfection-MEF cell lines were generated as described (25) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were transiently transfected using LipofectAMINE™ (Invitrogen, Karlsruhe, Germany) according to the manufacturer's protocol. Serum starvation and pertussis toxin treatment were done for 24 h.
Western Blot Analysis-Lysates of MEFs were analyzed by Western blotting after SDS-polyacrylamide gel electrophoresis and visualized by chemiluminescence detection using sheep anti-mouse (Amersham Biosciences) or goat anti-rabbit antibodies (Cell Signaling, Frankfurt, Germany) coupled to horseradish peroxidase and were visualized using ECL reagent (Amersham Biosciences). Monoclonal antibodies against RhoA (26C4) or c-Myc (9E10) and rabbit polyclonal antibodies against G␣ 13 (A-20) and G␣ q/11 (C-19) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, USA). Anti-G␣ 12 rabbit polyclonal antibody was described previously (26). Monoclonal HA antibody was obtained from Covance Research Products. Antibodies raised in rabbit against p115-RhoGEF, PDZ-RhoGEF, and LARG were described previously (26).
Rho Pull-down Assay-Activation of Rho was determined by a modified method described by Ren and Schwartz (29). MEF cells were seeded on 10-cm dishes and were either transfected with Myc-tagged RhoA and indicated plasmids or left untransfected for assessment of endogenous GTP-Rho. After serum starvation and pertussis toxin treatment for 24 h, cells were stimulated with different agonists and lysed in ice-cold lysis buffer (50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl 2 , 10 g/ml leupeptin, and aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Lysates were incubated for 45 min with glutathione-Sepharose beads (Amersham Biosciences) coupled with GST proteins fused to the Rho-binding domain (RBD) of rhotekin (29). Beads were washed four times with 600 l of ice-cold Tris buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM MgCl 2 , 10 g/ml leupeptin and aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride, and samples were collected by the addition of Laemmli buffer and subsequently analyzed using SDS-PAGE. Endogenous RhoA and transfected Myc-RhoA was detected using a mouse monoclonal antibody (26C4) and anti-Myc antibody (9E10), respectively (Santa Cruz Biotechnology).

RESULTS
Mouse embryonic fibroblast cell lines were generated from wild-type embryos as well as from embryos double deficient for G␣ q /G␣ 11 and G␣ 12 /G␣ 13 (30,31). Western blot analysis of lysates from these cell lines confirmed the absence of G␣ q /G␣ 11 and G␣ 12 /G␣ 13 in respective cells (Fig. 1A). We then determined, whether the different MEF cell lines express receptors for various ligands, which have been shown to induce the activation of G q /G 11 as well as G 12 /G 13 , like the lysophospholipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), bradykinin, or proteases like thrombin (Fig. 1B). By using RT-PCR, we found that wild type as well as G␣ q /G␣ 11and G␣ 12 /G␣ 13 -deficient MEFs express receptors for sphingosine 1-phosphate (S1P 1 , S1P 2 , and S1P 3 ), LPA (LPA 1 and LPA 2 ), bradykinin (B 2 ) as well as for thrombin (PAR-1). No expression was found for S1P 4 , S1P 5 , or LPA 3 receptors as well as PAR-2, Ϫ3 or Ϫ4 receptors. S1P 2 and S1P 3 receptors have been shown to couple to G 12 /G 13, G q /G 11 , as well as to G i -type G-proteins (32), and there is good evidence that LPA 1 and LPA 2

FIG. 2. Ca 2؉ transients and RhoA activation in wild type as well as G␣ 12 /G␣ 13 -and G␣ q /G␣ 11 -deficient cells.
A, in Fura-2/AM-loaded MEF cells, the intracellular free Ca 2ϩ concentration was recorded as described under "Materials and Methods," and the effects of LPA (5 M; left panels) and thrombin (1 unit/ml; right panels) were determined. The time of addition of the stimuli is indicated by arrows. The boldface lines represent the means of 20 cells in a representative experiment. The intra-assay variability is given by vertical lines representing the S.D. over 20 cells in the experiment. B, serum-starved MEFs (WT, wild-type; G␣ q/11 -defic., G␣ q/11 -deficient; G␣ 12/13 -defic., G␣ 12/13 -deficient) were pertussis toxin-treated for 24 h, and the cells were stimulated via endogenous receptors by thrombin (1 unit/ml), LPA (5 M) and bradykinin (10 M), and activated RhoA was precipitated as described in under "Materials and Methods." Shown are Western blots using an anti-RhoA antibody on the GST-RBD precipitates as well as on corresponding lysates. C, to verify the complete ADP-ribosylation of G i after pretreatment of cells with pertussis toxin, untreated, and PTX-treated (100 ng/ml; for 24 h) G␣ 12 /G␣ 13 -deficient cells were lysed, and lysates were analyzed by Western blotting with an antibody against G i . Complete ADP-ribosylation after PTX treatment is indicated by the decreased mobility of G i -type G-proteins in urea-containing SDS-polyacrylamide gels. Data shown are representative of at least three independently performed experiments.
receptors have a very similar G-protein coupling pattern (33). Similarly, the thrombin receptor PAR-1 has also been shown to couple to all three G-protein families (11, 34 -36). Notably, the pattern of expressed receptors did not differ between all three cell lines indicating that they are well suited for a comparative analysis of receptor-mediated effects.
In further experiments we used LPA, bradykinin and thrombin to stimulate cells through their endogenous receptors. To study the role of G q /G 11 and G 12 /G 13 in GPCR-induced Rho activation, we restricted coupling of receptors to these two G-protein families by pretreating cells with pertussis toxin (PTX), which uncouples receptors from G i -type G-proteins. Fig.  2C shows that PTX treatment completely ADP-ribosylated ␣-subunits of G i in G␣ 12 /G␣ 13 -deficient cells under the used experimental conditions. As expected, none of the stimuli was able to induce Ca 2ϩ transients in G␣ q/ G␣ 11 -deficient cells whereas Ca 2ϩ transients were induced in wild-type and G␣ 12/ G␣ 13 -deficient cells ( Fig. 2A and data not shown). In pertussis toxin-treated G␣ q/ G␣ 11 -deficient cells, in which coupling of receptors is restricted to G 12 /G 13 , thrombin, and LPA resulted in an activation of RhoA (Fig. 2B). This confirms that G 12 /G 13 are able to mediate receptor-dependent RhoA activation independently of any signaling via G q /G 11 . Bradykinin, which induced Rho activation in PTX-treated wild-type cells had no effect on Rho activity in G␣ q /G␣ 11 -deficient cells, suggesting that the bradykinin B 2 receptor is not coupled to G 12 /G 13 , but may induce Rho activation through G q /G 11 (Fig. 2B). To test the potential ability of G q /G 11 to mediate receptor-dependent Rho activation more directly, we tested the effect of thrombin, LPA, and bradykinin on RhoA activation in PTX-pretreated G␣ 12 / G␣ 13 -deficient cells, in which coupling of receptors is restricted to G q /G 11. All three receptor agonists were still able to activate RhoA through G q /G 11 (Fig. 2B).
Next we assessed whether G q /G 11 -mediated RhoA activation requires a functional phospholipase C-␤ or increases in intracellular free [Ca 2ϩ ]. Receptor-mediated Rho activation in PTX-treated G␣ 12 /G␣ 13 -deficient cells was not inhibited by the phospholipase C-␤ inhibitor U73122 or the Ca 2ϩ -chelator BAPTA, although both agents completely blocked receptormediated induction of Ca 2ϩ transients in the same cells (Fig.  3, A and B). These data indicate that GPCRs can mediate RhoA activation via G q /G 11 in a phospholipase C-␤-independent manner. To further characterize G 12 /G 13 -and G q /G 11 -mediated RhoA activation through GPCRs, we determined concentration-response relationships for thrombin-induced RhoA activation in PTX-treated wild-type, G␣ q /G␣ 11 -, and G␣ 12 /G␣ 13deficient cells (Fig. 4, A and B). Thrombin-induced RhoA activation in PTX-treated wild-type cells and in G␣ q/ G␣ 11deficient cells with half-maximal and maximal concentrations of 10 Ϫ4 -10 Ϫ3 and 10 Ϫ2 units/ml, respectively. However, when G-protein coupling was restricted to G q /G 11 in PTXtreated G␣ 12/ G␣ 13 -deficient cells, thrombin was considerably less potent, and maximal RhoA activation was observed only at thrombin concentrations of 1 unit/ml. While both G 12 /G 13 and G q /G 11 can mediate RhoA activation, receptor-dependent activation through G q /G 11 requires agonist concentrations about two orders of magnitude higher than activation through G 12 /G 13 .
The fact that RhoA activation via G q /G 11 occurred independently of phospholipase C-␤ prompted us to consider the involvement of RGS domain-containing RhoGEF proteins, which have been suggested to directly link G 12 /G 13 to Rho activation. Of the three members of this subgroup of RhoGEF proteins, p115 RhoGEF, PDZ-RhoGEF, and LARG, we found only LARG to be expressed in MEFs (Fig. 5A). This is consistent with the much wider expression pattern of LARG compared with p115RhoGEF and PDZ-RhoGEF, which are mainly found in hematopoietic and neuronal cells, respectively (26,37,38). To test the potential role of LARG in G q /G 11 -mediated Rho activation, we constructed a mutant of LARG lacking the DH and PH domains required for its RhoGEF activity (Fig. 5B). Expression of (⌬DH/PH)-LARG in PTX-treated G␣ 12 /G␣ 13 -deficient cells efficiently blocked rapid actin stress fiber formation induced by LPA (Fig. 5C). Furthermore, expression of ⌬DH/PH-LARG also dramatically reduced the cellular F-actin content in the presence of 10% fetal calf serum (Fig. 5C). Similarly, ⌬DH/ PH-LARG was found to block activation of Myc-tagged RhoA in response to thrombin and LPA in PTX-treated G␣ 12 /G␣ 13 -deficient cells (Fig. 5D). These data suggest that LARG is critically involved in the phospholipase C-␤-independent RhoA activation via G q /G 11.

FIG. 5. Involvement of LARG in
RhoA activation in G␣ 12 /G␣ 13 -deficient MEFs. A, expression of RhoGEF proteins in G␣ 12 /G␣ 13 -deficient cells. Shown is the Western blot analysis using polyclonal antibodies against LARG, PDZ-RhoGEF, and p115-RhoGEF on total mouse brain cholate extracts (BCE; for PDZ-RhoGEF and LARG) or platelet extracts (Plat.; for p115RhoGEF) as well as on lysates of G␣ 12 /G␣ 13 -deficient MEFs (MEF). B, model of the HA-tagged ⌬DH/ PH-LARG mutant. C, PTX-treated G␣ 12 / G␣ 13 -deficient MEFs transiently transfected with HA-tagged ⌬DH/PH-LARG were either kept in 10% fetal calf serum (unstarved) or were maintained in 0.5% fatty acid-free bovine serum albumin overnight (starved). After a short incubation in the absence or presence of LPA (1 M), cells were fixed and stained with BODIPY-phallacidin and anti-HA antibodies to visualize F-actin or the epitope tagged ⌬DH/PH-LARG, respectively. D, effect of ⌬DH/PH-LARG on RhoA activation in G␣ 12 /G␣ 13 -deficient MEFs. Cells were transfected with c-Myc-tagged RhoA, either without (two left lanes) or with the HA-tagged ⌬DH/PH mutant of LARG (two right lanes). After serum starvation and pertussis toxin treatment thrombin (1 unit/ml), LPA (5 M), and bradykinin (10 M) were added together (T/L/B) to the cells, and activated RhoA was precipitated as described. Precipitates (GST-RBD pulldown) and a defined part of the lysates were immunoblotted with monoclonal c-Myc antibody (anti-c-Myc) to detect activated and total RhoA in transfected cells. The expression of the HA-tagged ⌬DH/PH mutant of LARG was controlled by an immunoblot of lysates using HA-monoclonal-antibody (anti-HA). Data shown are representative of at least three independently performed experiments.