Differential Involvement of Galpha 12 and Galpha 13 in Receptor-mediated Stress Fiber Formation*

Antje GohlaDagger , Stefan OffermannsDagger , Thomas M. Wilkie§, and Günter SchultzDagger

From the Dagger  Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67-73, D-14195 Berlin, Germany and § Pharmacology Department, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G12 and G13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G12 or G13 in stress fiber formation induced through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and Galpha q/Galpha 11-deficient mice, we show that agonist-dependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A, endothelin ETA, and serotonin 5-HT2C receptors induced stress fiber formation in either the presence or absence of Galpha q/Galpha 11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1alpha receptors was dependent on Gq/G11 proteins. The activation of the Gq/G11-coupled endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber formation was dependent on Galpha 13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Galpha 12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Galpha q and Galpha 11 and that this involves either a Galpha 12 or a Galpha 13/EGF receptor-mediated pathway.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heterotrimeric guanine nucleotide-binding proteins (G-proteins)1 act as molecular switches that couple receptors for hormones, neurotransmitters, and other extracellular stimuli to effector systems such as enzymes or ion channels (1-4). G-proteins are composed of an alpha -, beta -, and gamma -subunit and are characterized by the identity of the alpha -subunit that binds and hydrolyzes GTP. On the basis of sequence and functional homologies, G-protein alpha -subunits can be classified into four families: (a) Galpha s, (b) Galpha i/o, (c) Galpha q, and (d) Galpha 12 (5).

The Galpha 12 subfamily consists of the ubiquitously expressed members Galpha 12 and Galpha 13, whose functions are still incompletely understood. They share a 67% sequence identity and are less than 45% homologous to other G-protein alpha -subunits (6). G12 and G13 are activated through various receptors, including receptors for thrombin, thromboxane A2, and lysophosphatidic acid (LPA) (7-9). Because no receptors selectively coupling to Galpha 12 or Galpha 13 have been found thus far, G12/G13-mediated signaling pathways have been studied using constitutively active mutants of Galpha 12 and Galpha 13. Both Galpha 12 and Galpha 13 can regulate the Na+/H+ antiporter (10-12), the Jun kinase/stress-activated protein kinase pathway (13), and the Rho-dependent formation of actin stress fibers (14). Whereas there are clear similarities in the effects induced by constitutively active forms of Galpha 12 and Galpha 13, differences in the involved signal transduction mechanisms have been reported (9, 11, 15). Functional differences between Galpha 12 and Galpha 13 are also suggested from studies on Galpha 13-deficient mice that die at midgestation due to an angiogenetic defect. Fibroblasts derived from Galpha 13-deficient mice express Galpha 12 but show severely impaired chemokinetic responses to thrombin (16). Effectors directly regulated by Galpha 12 and Galpha 13 remain to be discovered. Recently, it was shown that a guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, can be directly regulated by Galpha 13 (17, 18). Whereas Galpha 13 activates the p115 RhoGEF-catalyzed nucleotide exchange on Rho, Galpha 12 counteracts the Galpha 13-mediated stimulation. In addition, p115 RhoGEF functions as a GTPase-activating protein for both Galpha 12 and Galpha 13.

Studies conducted in plasma membrane preparations have shown that G12 and G13 are activated by receptors that also couple to Gq/G11 (7-9). The Gq family is constituted by four members (Gq, G11, G14, and G15/16) that couple receptors to beta -isoforms of phospholipase C (PLC) in a pertussis toxin (PTX)-insensitive manner, resulting in the generation of inositol phosphates and the mobilization of intracellular calcium (19-21). Whereas Galpha q and Galpha 11 are widely expressed and are primarily responsible for PTX-insensitive PLC-beta activation, the expression of Galpha 14 and Galpha 15/16 is restricted to a few tissues (22, 23). Studies on Galpha q/Galpha 11-double deficient mice and cells have confirmed that Galpha q and Galpha 11 are the primary mediators of PTX-insensitive PLC-beta activation and that their cellular functions are highly redundant (24, 25).

To analyze the role of Galpha q/Galpha 11, Galpha 12, and Galpha 13 in receptor-mediated rearrangements of the actin cytoskeleton, we studied the signaling processes induced through endogenous and heterologously expressed receptors in wild-type, Galpha q/Galpha 11-deficient and Galpha 13-deficient cells. Our data show that most, but not all, receptors tested mediate stress fiber formation in the absence of Galpha q and Galpha 11. Unexpectedly, we found that Galpha q/Galpha 11-independent stress fiber formation involved either Galpha 12 or Galpha 13. Moreover, the EGF receptor represents an essential intermediate in the Galpha 13-induced stress fiber formation but not the Galpha 12-induced stress fiber formation in fibroblasts.

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Angiotensin II, bradykinin, carbachol, endothelin-1, isoproterenol, lysophosphatidic acid, thrombin, and [Arg8]vasopressin were from Sigma. Endothelin-1(8-21), N-Suc-[Glu9,Ala11,15](IRL-1620), (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) hydrochloride, trans-azetidine-2,4-dicarboxylic acid, BQ-123, and BQ-788 were purchased from RBI.

Cell Culture-- Wild-type fibroblasts and fibroblasts lacking both Galpha q and Galpha 11 were derived from embryonic day 10.5 mouse embryos originating from intercrosses of Galpha q(-/+) and Galpha 11(-/+) mice. The generation of Galpha q and Galpha 11 mutant mice has been described previously (25). Fibroblasts lacking Galpha q/Galpha 11 or Galpha 13 were prepared and cultured as described previously (16, 26).

Expression Vectors-- Expression vectors carrying cDNAs of the human muscarinic M1 and M2 receptors, the murine serotonin 5-HT2C receptor, the human vasopressin V1A receptor, the human beta 2 adrenergic receptor, and beta -galactosidase have been described previously (27). Galpha 12G228A and Galpha 13G225A were generated by overlap extension polymerase chain reaction and cloned into pCIS (28), and mutations were verified by sequencing. The rat metabotropic glutamate receptor subtype 1alpha (mGluR1alpha ) cDNA was inserted in pcDNA-Amp (29), and the human angiotensin AT1A receptor cDNA was inserted in pcDNA3 (Invitrogen). Human endothelin ETA and ETB receptor cDNAs were cloned in pMEsf- (30), and the human bradykinin B2 receptor was carried by pcDNA1 (Invitrogen). RhoA G14V was in pcEXV (31). The dominant negative EGF receptor mutant EGFR-CD533 was carried by the pRK5 vector (32).

Microinjection-- For microinjection studies, cells were seeded at a density of approximately 103 cells/mm2 on glass coverslips imprinted with squares to facilitate the localization of injected cells and grown overnight. To obtain quiescent and serum-starved fibroblasts, cultures were rinsed in serum-free DMEM and incubated in DMEM supplemented with 25% Ham's F-12 medium, 0.2% NaHCO3, 10 mM Hepes, and 0.1% fetal bovine serum (modified DMEM) for 24 h, followed by a 48-h incubation in modified DMEM devoid of fetal bovine serum. Plasmids were injected into the nucleus together with Texas Red dextran (5 mg/ml; Molecular Probes) to visualize injected cells. Clostridium botulinum C3 exoenzyme, which was kindly provided by K. Aktories (Albert-Ludwigs-Universität, Freiburg, Germany), was co-microinjected with the cDNAs at a concentration of 100 µg/ml. To test the effects of dominant negative mutants of Galpha 12 (Galpha 12G228A; Galpha 12GA) and Galpha 13 (Galpha 13G225A; Galpha 13GA), plasmids encoding for receptors (0.1 µg/µl) were co-microinjected with Galpha 12GA or Galpha 13GA (0.5 µg/µl). As a control, receptor plasmids (0.1 µg/µl) supplemented with a vector encoding beta -galactosidase (0.5 µg/µl) to maintain the total amount of injected DNA constant were microinjected in an adjacent field on the same coverslip. The dominant negative EGF receptor mutant EGFR-CD533 (0.5 µg/µl) was co-expressed with various receptors (0.1 µg/µl) by nuclear microinjection. To test the specificity of dominant negative mutants of Galpha 12 and Galpha 13, RhoA G14V (0.1 µg/µl) was co-microinjected with Galpha 12GA or Galpha 13GA (0.5 µg/µl). About 150 cells/field were injected in each case, using a manual injection system (Eppendorf, Hamburg, Germany). To inhibit the EGF receptor tyrosine kinase activity pharmacologically, cells were incubated with the EGF receptor-selective tyrphostin AG1478 (1 µM) for 120 min before fixation. A 25-min treatment with the tyrosine phosphatase inhibitor vanadate (100 µM) was performed before fixation.

Intracellular Calcium Measurements-- For dye loading, cells were incubated with 2 µM fura-2 acetoxymethyl ester at 37 °C for 30 min in DMEM. Cells were then washed in 138 mM NaCl, 6 mM KCl, 1 mM MgCl2, 5.5 mM glucose, 1 mM CaCl2, and 20 mM Hepes (pH 7.4). Determinations of [Ca2+]i in single microinjected cells were performed in the same buffer, applying a digital imaging system and FUCAL 5.12 software (T.I.L.L. Photonics, München, Germany). Maximum and minimum fluorescence values were determined after the addition of 10 µM ionomycin (Calbiochem) and 10 mM CaCl2 or 5 mM EGTA.

Fluorescence Microscopy-- Microinjected cells were stimulated with various agonists for 15 min at 37 °C, fixed in 4% paraformaldehyde for 20 min, and permeabilized in 0.2% Triton X-100 for 5 min. To visualize the cytoskeleton, cells were stained for polymerized actin by incubation with 0.5 µg/ml fluorescein isothiocyanate-phalloidin (Sigma) for 40 min. The coverslips were mounted on glass slides and examined using an inverted microscope (Zeiss Axiovert 100).

Quantification of Stress Fiber Formation-- For studies on endogenous receptors, the percentage of stress fiber-positive cells relates to the total number of investigated cells in randomly chosen visual fields. In the case of heterologously expressed receptors, microinjected cells were first identified by their Texas Red fluorescence, and then the fraction of stress fiber-positive cells among these cells was counted. Typically, the average receptor-dependent induction of actin stress fiber formation resulted in an increase of approximately 40% in fluorescence intensity compared with control cells when the average fluorescence intensity values of fluorescein isothiocyanate-labeled polymerized actin/cell were calculated using a digital imaging system and FUCAL 5.12 software. A minimum of 100 cells was investigated in each case. Experiments were performed in duplicate and repeated at least twice. In most cases, quantifications were performed by two independent observers in a blind manner, i.e. observers were not informed of the conditions used in the indicated experiment.

Assessment of cAMP Accumulation-- For the determination of cAMP levels, HEK 293 cells were seeded in 12-well plates at a density of 1 × 105 cells/well and grown overnight. Cells were transfected with cDNAs encoding the beta 2 adrenergic receptor and the M2 muscarinic receptor and either dominant negative Galpha 12 or Galpha 13 or the beta -galactosidase control, as described previously (27). The cells were subsequently labeled with [3H]adenine, and [3H]cAMP was isolated by double chromatography over Dowex and Alumina columns as described previously (33).

    RESULTS
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Stress fiber formation can be specifically induced by constitutively active mutants of Galpha 12 and Galpha 13 in a Rho-dependent manner (14). The G12- and G13-coupling receptors identified to date appear to activate Gq family members as well (7-9). Stress fiber formation induced through these receptors is accordingly accompanied by a PTX-insensitive stimulation of PLC-beta and a subsequent elevation of cytosolic [Ca2+]. To investigate whether Gq/G11-coupled receptors can, in general, activate G12/G13-mediated stress fiber formation and to test whether these cytoskeletal rearrangements are dependent on Gq/G11 signaling, we studied the agonist-induced stress fiber assembly through various Gq/11-coupled endogenous or heterologously expressed receptors in Galpha q/Galpha 11-deficient fibroblasts.

To assure a functional expression of the investigated receptors, we first measured the receptor-dependent calcium mobilization in wild-type fibroblasts. Fig. 1A (left part) shows that thrombin and LPA, acting through endogenous receptors, initiate a calcium mobilization in wild-type cells. The receptors for thrombin and LPA have been shown to couple to Gq/11, G12, G13, and Gi (8, 9). To test a possible involvement of Gi-type G-proteins in calcium mobilization induced through these receptors, Gi family members were inactivated by pretreating cells with PTX. The abrogation of Gi-mediated PLC activation slightly reduced the thrombin- and LPA-induced calcium mobilization (Fig. 1A, left part). Similarly, the agonist-dependent activation of ETA, ETB, AT1A, and B2 receptors that were heterologously expressed by intranuclear microinjection of respective cDNAs induced partially PTX-sensitive calcium responses. In contrast, elevation of [Ca2+]i mediated by the activation of heterologously expressed V1A, 5-HT2C, M1, and mGluR1alpha receptors was not affected by pretreatment of cells with PTX (Fig. 1A, right part). These data demonstrate that the tested receptors were functionally expressed at densities comparable to each other and to those of endogenous thrombin and LPA receptors.


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Fig. 1.   Intracellular calcium mobilization in response to activation of endogenous or heterologously expressed receptors. Wild-type fibroblasts (A) or Galpha q(-/-);Galpha 11(-/-) fibroblasts (B) were activated with 1 µM LPA or 1 unit/ml thrombin (left parts of A and B). For studies on heterologously expressed receptors, cells were first microinjected with expression plasmids carrying cDNAs of the indicated receptors (right parts of A and B) and then challenged with receptor agonists. To test the effect of PTX, cells were preincubated overnight in the absence (black-square) or presence of 100 ng/ml PTX (). The increases in free cytosolic [Ca2+]i over basal [Ca2+]i are shown (in nM; ordinate). Note the different scales in A and B. The ETA receptor was specifically activated with endothelin-1 (0.1 µM) in the presence of the ETB receptor antagonist BQ-788 (1 µM). The ETB receptor was specifically activated with the ETB receptor-specific agonist IRL-1620 (1 µM) in the presence the ETA receptor antagonist BQ-123 (10 µM). The V1A receptor was stimulated with AVP (1 µM), the AT1A receptor was activated with angiotensin II (1 µM), and bradykinin (1 µM) was used to activate the B2 receptor. The 5-HT2C receptor was stimulated with the serotonin receptor subtype-specific agonist DOI (1 µM), the M1 receptor was activated with carbachol (10 µM), and the mGluR1alpha receptor was activated with the metabotropic glutamate receptor agonist trans-azetidine-2,4-dicarboxylic acid (10 µM). Bars represent the mean values ± S.D. of calcium measurements in 30-50 individual cells in two independent experiments. C, typical calcium transients obtained after activation of heterologously expressed 5-HT2C and mGluR1alpha receptors in wild-type fibroblasts (upper panels) or Gq/11-deficient fibroblasts (lower panels). Cells were stimulated with 1 µM DOI to activate 5-HT2C receptors or with 10 µM trans-azetidine-2,4-dicarboxylic acid to activate mGluR1alpha receptors. The calcium responses of five individual cells each are shown. Arrows indicate the time of agonist addition.

To selectively study the agonist-dependent calcium mobilization induced by activation of the tested receptors in the absence of Gq- and G11-proteins, we used Galpha q/Galpha 11-double deficient embryonic fibroblasts. Activation of the heterologously expressed V1A, 5-HT2C, M1, and mGluR1alpha receptors did not elevate basal intracellular calcium levels, whereas stimulation of the endogenous receptors for thrombin and LPA and of the heterologous ETA, ETB, AT1A, and B2 receptors in Galpha q/Galpha 11-deficient cells induced a slight increase in the intracellular calcium concentration that was completely PTX-sensitive (Fig. 1B). Thus, the Galpha q/Galpha 11-deficient, PTX-treated fibroblasts did not respond to various receptor agonists with measurable, PLC-mediated elevation of [Ca2+]i.

The receptors for thrombin and LPA have previously been shown to mediate Rho-dependent stress fiber formation in Swiss 3T3 cells (34). To investigate whether the fibroblast cell lines derived from wild-type or Galpha q/Galpha 11-deficient mice provide a suitable assay system to study receptor-induced, Rho-dependent stress fiber formation, we first assessed the ability of thrombin and LPA to trigger stress fiber assembly in serum-starved, wild-type fibroblasts. Fig. 2A shows that both agonists clearly stimulated stress fiber formation in about 90% of the cells. Stress fiber assembly was insensitive to pretreatment with PTX but was completely blocked by C3 transferase, which specifically inactivates Rho by ADP-ribosylation (35). Similarly, agonist-dependent activation of the heterologously expressed ETA, V1A, B2, 5-HT2C, M1, and mGluR1alpha receptors resulted in a pronounced PTX-insensitive and C3 exoenzyme-sensitive stress fiber formation. We could not detect any actin polymerization in fibroblasts activated through the AT1A or ETB receptors, which clearly mediated calcium responses (Fig. 1A).


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Fig. 2.   Stimulation of actin stress fiber formation by activation of endogenous or heterologously expressed receptors. Wild-type fibroblasts (A) or Galpha q(-/-);Galpha 11(-/-) fibroblasts (B) were incubated with 1 µM LPA or 1 unit/ml thrombin (left parts of A and B). To study actin cytoskeletal rearrangements induced through heterologous receptors, the indicated receptors (right panel) were expressed by intranuclear microinjection of the corresponding cDNAs. Cells were subsequently challenged with the respective ligands as described in the legend of Fig. 1A for 15 min at 37 °C and fixed, and actin stress fibers were visualized with fluorescein isothiocyanate-phalloidin. To test the effect of PTX, cells were pretreated overnight in the absence (black-square) or presence of 100 ng/ml PTX (). To inactivate Rho, C3 toxin (100 ng/ml) was co-microinjected into the nucleus together with cDNAs encoding the various receptors (). For studies on endogenous receptors, the percentage of stress fiber-positive cells relates to the total number of investigated cells. In the case of heterologously expressed receptors, only microinjected cells detected by their Texas Red fluorescence were counted to determine the percentage of stress fiber-positive cells. The mean values of triplicates ± S.D. are shown. C, stress fiber assembly induced via 5-HT2C and mGluR1alpha receptors in wild-type (upper panels) or Galpha q(-/-);Galpha 11(-/-) fibroblasts (lower panels). Cells expressing 5-HT2C receptors or mGluR1alpha receptors were stimulated with DOI (1 µM) or trans-azetidine-2,4-dicarboxylic acid (10 µM), and actin stress fibers were visualized as described above. Typical examples of stress fibers in individual cells are shown.

We then investigated whether stress fiber formation can be triggered in the absence of PLC-mediated calcium mobilization. Stress fiber assembly in response to the activation of M1 or mGluR1alpha receptors in Galpha q/Galpha 11-deficient fibroblasts was drastically reduced by 90-95% compared with wild-type cells, indicating that it largely depended on the presence of Gq/11-proteins (Fig. 2B). In contrast, stress fiber assembly triggered through the activation of the endogenous receptors for thrombin and LPA and heterologously expressed ETA, V1A, B2, and 5-HT2C receptors was induced as efficiently in Galpha q(-/-);Galpha 11(-/-) cells as in wild-type fibroblasts. In all cases, Galpha q/Galpha 11-independent stress fiber assembly was insensitive to PTX but was sensitive to C3 exoenzyme. We conclude that Gq/11- and Gi-proteins are dispensable for Rho-mediated actin reorganization induced through many, but not all, Gq/11-coupled receptors. Because activated forms of Galpha 12 and Galpha 13 are able to induce stress fiber assembly (14), these data suggest that Galpha q/Galpha 11- and Gi-independent, Rho-mediated stress fiber formation involves exclusively G12 family proteins.

By labeling activated G-proteins with [alpha -32P]GTP azidoanilide in plasma membrane preparations, we have previously demonstrated that LPA receptors activate Galpha 12 and Galpha 13, whereas LPA-induced stress fiber formation is selectively mediated via Galpha 13 and does not involve Galpha 12 (9). To test specific roles for Galpha 12 and Galpha 13 in the same cell, we have used dominant negative forms of Galpha 12 and Galpha 13 and Galpha 13-deficient fibroblasts. Receptors that were shown to mediate stress fiber formation in a Galpha q/Galpha 11-independent way (see Fig. 2B) were co-expressed with point mutants of Galpha 12 (Galpha 12GA) and Galpha 13 (Galpha 13GA). The analogous substitution of glycine for alanine in the nucleotide binding pocket of Galpha s has been shown to block Galpha s activation induced by receptor-mediated guanine nucleotide exchange. Therefore, this mutation is believed to create a dominant negative-acting protein (36, 37). Expression of Galpha 12GA and Galpha 13GA did not interfere with stress fiber formation induced by the constitutively active RhoA mutant RhoA G14V or by the incubation of the cells with orthovanadate (9), indicating that dominant negative forms of Galpha 12 or Galpha 13 did not unspecifically inhibit stress fiber formation (data not shown). In addition, the expression of dominant negative Galpha 12 or Galpha 13 did not interfere with the Gq/11-dependent calcium release (Table I), nor did dominant negative Galpha 12 or Galpha 13 interfere with signal transduction pathways dependent on Gi-type G-proteins. The latter is evident from the fact that Galpha 12GA and Galpha 13GA did not influence the carbachol-mediated inhibition of cAMP accumulation compared with control cells expressing beta -galactosidase (Table I). These data clearly demonstrate that dominant negative mutants of Galpha 12 and Galpha 13 do not interfere with Gq/11- and Gi-dependent signal transduction pathways.

                              
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Table I
Effect of dominant negative Galpha 12 and dominant negative Galpha 13 on calcium mobilization and inhibition of cAMP accumulation
Galpha 12GA and Galpha 13GA or the beta -galactosidase control (lacZ) were expressed in wild-type fibroblasts (left part) by microinjection of the corresponding cDNAs. Cells were then activated with 1 unit/ml thrombin or 1 µM LPA. The increases in free cytosolic [Ca2+]i over basal calcium are shown (in nM). The results represent the mean values ± S.D. of calcium measurements in 30-50 cells in two independent experiments. HEK 293 cells (right part) were transfected with M2 and beta 2 receptors together with Galpha 12GA, Galpha 13GA, or the beta -galactosidase control. Subsequently, cells were labeled with [3H]adenine and stimulated with 10 µM isoproterenol, and the [3H]cAMP accumulation was measured in the absence or presence of 100 µM carbachol (CCh). The mean values of triplicates ± S.D. are shown, as well as the inhibition of the isoproterenol-stimulated cAMP accumulation by carbachol (percentage).

Interestingly, Galpha 12GA blocked thrombin-induced stress fiber formation by about 80% in Galpha q(-/-);Galpha 11(-/-) cells compared with the control vector, whereas Galpha 13GA had no inhibitory effect. In contrast, LPA-dependent actin reorganization was not affected by Galpha 12GA, whereas Galpha 13GA reduced the LPA-stimulated stress fiber assembly by about 70% (Fig. 3A). To further explore a specific role for Galpha 12 and Galpha 13 in stress fiber assembly, we co-expressed Galpha 12GA and Galpha 13GA with receptors shown to mediate stress fiber formation in a Gq/G11-independent way (see Fig. 2B). In Galpha q/Galpha 11-deficient cells, stress fiber formation induced through the stimulation of ETA and V1A receptors was strongly reduced by Galpha 12GA, whereas Galpha 13GA had no effect. Using the same experimental design, Galpha 13GA but not Galpha 12GA blocked stress fiber assembly induced through the B2 and 5-HT2C receptors. Thus, Galpha 12 and Galpha 13 appear to differentially couple receptors to Rho-dependent stress fiber formation. Stress fiber assembly induced by the activation of the M1 and mGluR1alpha receptors was dependent on Gq/11-mediated PLC-beta stimulation and calcium mobilization (see Fig. 2B). However, stress fiber formation induced through the M1 and mGluR1alpha receptors in wild-type fibroblasts was inhibited by 60-90% after expression of Galpha 13GA, whereas the expression of Galpha 12GA was without effect (data not shown).


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Fig. 3.   Effects of Galpha 12G228A and Galpha 13G225A on agonist-induced stress fiber formation in Galpha q(-/-);Galpha 11(-/-) fibroblasts. A, to test the effects of dominant negative mutants of Galpha 12 and Galpha 13, Galpha 12GA, Galpha 13GA, or the beta -galactosidase control were first expressed in Galpha q/Galpha 11-deficient cells by intranuclear microinjection of the corresponding cDNAs. Fibroblasts were then activated with 1 µM LPA or 1 unit/ml thrombin (left part). To assess the effects of Galpha 12GA and Galpha 13GA on stress fiber formation induced through heterologously expressed receptors, expression plasmids encoding the indicated receptors were co-microinjected into the nucleus together with Galpha 12GA and Galpha 13GA as described under "Experimental Procedures" (right part). Cells were stimulated with the respective ligands as described in the legend of Fig. 1A and stained for polymerized actin, and the percentage of stress fiber-positive cells was calculated as described in the legend of Fig. 2. The mean values of triplicates ± S.D. are shown. B, typical effects of Galpha 12GA and Galpha 13GA on stress fiber formation initiated via ETA receptors (left panel) and 5-HT2C receptors (right panel) in Galpha q(-/-);Galpha 11(-/-) fibroblasts. ETA and 5-HT2C receptors were co-expressed together with beta -galactosidase (lacZ), Galpha 12GA, or Galpha 13GA. Cells were activated with endothelin-1 (0.1 µM)/BQ-788 (1 µM) and DOI (1 µM) or left unstimulated (control), and actin stress fibers were visualized as described above.

To further prove a selective involvement of either Galpha 12 or Galpha 13 in receptor-mediated stress fiber formation, we used Galpha 13(-/-) fibroblasts. Fig. 4A demonstrates that the activation of the LPA, B2, and 5-HT2C receptors, which mediate stress fiber formation in a Galpha 13GA-sensitive manner, failed to induce stress fibers in Galpha 13(-/-) cells. In contrast, stimulation of the thrombin, ETA, and V1A receptors, which trigger stress fiber assembly in a Galpha 12GA-sensitive manner, occurred in almost all of the Galpha 13-deficient cells. This clearly confirms that both Galpha 12 and Galpha 13 are capable of mediating receptor-induced stress fiber accumulation. However, different receptors selectively utilize Galpha 12 or Galpha 13 for the stimulation of Rho activation and the subsequent formation of actin stress fibers.


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Fig. 4.   Stimulation of actin stress fiber formation in Galpha 13(-/-) cells. A, Galpha 13-deficient fibroblasts were challenged with 1 µM LPA or 1 unit/ml thrombin (left panel). The control represents the percentage of residual stress fibers in untreated, serum-starved fibroblasts (control). Stress fiber accumulation mediated by heterologously expressed receptors was investigated after nuclear microinjection of cDNAs corresponding to the indicated receptors (right panel). Cells were then activated with the respective ligands and stained for polymerized actin, and the percentage of stress fiber-positive cells was determined as described in the legends of Figs. 1A and 2. The mean values of duplicates ± S.D. are shown. B, stress fiber assembly induced through ETA and 5-HT2C receptors in wild-type (left panel) or Galpha 13-deficient fibroblasts (right panel). ETA or 5-HT2C receptors or beta -galactosidase were expressed by microinjection, and cells were activated with endothelin-1 (0.1 µM)/BQ-788 (1 µM) or DOI (1 µM) or left unstimulated (control). Actin stress fibers were visualized as described above.

We have previously demonstrated that the EGF receptor is an essential signaling intermediate in LPA-induced, G13-mediated stress fiber formation but not in the G12-induced stress fiber formation in Swiss 3T3 fibroblasts (9). To investigate a general involvement of the EGF receptor in G13-dependent rearrangements of the actin cytoskeleton, we first determined the effects of the EGF receptor-specific tyrosine kinase inhibitor tyrphostin AG1478 on the receptor-induced stimulation of stress fiber formation in wild-type fibroblasts. Fig. 5A shows that tyrphostin AG1478 blocked the G13-mediated stress fiber formation induced through activation of the LPA, B2, and 5-HT2C receptors. In contrast, the G12-mediated stress fiber formation triggered by stimulation of the thrombin, ETA, or V1A receptors was not reduced by inactivation of the EGF receptor with tyrphostin AG1478. Furthermore, a cytosolically truncated EGF receptor (EGFR-CD533) was expressed in fibroblasts. This EGF receptor mutant exerts a dominant negative function on EGF receptor signaling due to the formation of signaling-incompetent heterodimers with endogenous EGF receptors (32). In agreement with the effects observed after inhibition of the EGF receptor tyrosine kinase activity with tyrphostin AG1478, expression of EGFR-CD533 abolished the G13-mediated stress fiber formation induced through LPA, B2, and 5-HT2C receptors, whereas the G12-dependent stress fiber formation was not sensitive to an inhibition of EGF receptor function (Fig. 5B).


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Fig. 5.   Involvement of EGF receptors in stress fiber formation. To study the contribution of EGF receptors in stress fiber assembly, wild-type fibroblasts were incubated with the EGF receptor-selective tyrphostin AG1478 (1 µM; ) or with the DMSO control (black-square) for 120 min (A). In parallel experiments (B), the dominant negative EGF receptor mutant EGFR-CD533 () or the beta -galactosidase control (black-square) was expressed by nuclear microinjection of the corresponding cDNA. Cells were activated through the indicated endogenous or heterologously expressed receptors with the respective ligands, as described in the legend of Fig. 1A, and stress fibers were stained as described under "Experimental Procedures." The mean values ± S.D. of three independent experiments are shown.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The aim of the present study was to investigate the involvement of Gq/G11- and G12/G13-proteins in receptor-dependent stress fiber formation. Whereas constitutively active mutants of Galpha 12 and Galpha 13 are known to induce stress fiber assembly (14), it is not clear whether physiological activation of G12/G13 through receptors is alone sufficient to mediate actin reorganization. Our data demonstrate that activation of LPA, thrombin, ETA, V1A, B2, and 5-HT2C receptors efficiently triggers PTX-insensitive stress fiber formation in the absence of Gq/11-dependent calcium mobilization. We conclude that Gq/11- and Gi-proteins are dispensable for stress fiber formation evoked through many Gq/11-coupled receptors.

Stress fiber assembly induced by activation of M1 and mGluR1alpha receptors was dependent on Gq/11-mediated PLC-beta stimulation and calcium mobilization. However, using dominant negative mutants of Galpha 12 and Galpha 13, we could show that stress fiber formation induced through M1 and mGluR1alpha receptors in wild-type fibroblasts was inhibited in 60-90% of the cells after the expression of Galpha 13GA, whereas the expression of Galpha 12GA was without effect. In addition, the activation of M1 and mGluR1alpha receptors in Galpha 13-deficient fibroblasts failed to promote stress fiber assembly (data not shown). These results suggest that Gq/G11-dependent signaling can critically contribute to G13-mediated stress fiber formation induced through some receptors, although the expression of constitutively active Gq-proteins alone is not sufficient to form stress fibers (9, 14). Experiments in cardiomyocytes have indicated an involvement of Galpha q in receptor-induced and Rho-mediated actin reorganization (38).

It has been reported that stimulation of a voltage-dependent calcium channel in rat portal vein myocytes through AT1A receptors involves G13 (39). In contrast to these studies, we could not detect any G12/G13-dependent actin polymerization in fibroblasts activated through AT1A receptors. This may suggest that G13 is indirectly involved in calcium-current regulation via AT1A receptors.

Stimulation of ETA receptors effectively triggered Galpha 12-dependent stress fiber formation. In contrast, the activation of ETB receptors failed to induce stress fiber assembly. These results suggest that ETA receptors activate G12, whereas ETB receptors do not couple to G12 family members, which evoke actin reorganization.

We have previously demonstrated that the LPA receptor induces stress fiber formation selectively via Galpha 13. By labeling activated G-proteins with [alpha -32P]GTP azidoanilide in plasma membrane preparations, it was shown that LPA activates both G12 and G13, but that this activation of G12 was apparently not linked to stress fiber assembly (9). To test a possible selective involvement of Galpha 12 and Galpha 13 in stress fiber formation through other receptors, we have used dominant negative forms of Galpha 12 and Galpha 13 and Galpha 13-deficient cells. Our data show that stress fiber formation induced through the thrombin, ETA, and V1A receptors was mediated by Galpha 12 and occurred in the absence of Galpha 13. In contrast, stress fiber accumulation induced through the LPA, B2, and 5-HT2C receptors was completely dependent on the presence of Galpha 13 and was not reduced by a dominant negative form of Galpha 12. This clearly shows that both Galpha 12 and Galpha 13 are capable of mediating receptor-induced stress fiber formation, although different receptors selectively recruit Galpha 12 or Galpha 13 for the regulation of actin stress fiber accumulation.

The EGF receptor has been identified as a critical component in LPA/G13-mediated stress fiber formation in Swiss 3T3 fibroblasts (9). Using the EGF receptor-selective tyrphostin AG1478 and the dominant negative EGF receptor mutant EGFR-CD533, we now show that receptors utilizing either G12 or G13 for stress fiber formation can be further distinguished by the involvement of EGF receptors in the process of actin polymerization. Whereas EGF receptors contributed to the LPA, B2, and 5-HT2C receptor-mediated, Galpha 13-dependent stress fiber formation in wild-type fibroblasts, the Galpha 12-dependent stress fiber assembly induced through thrombin, ETA, and V1A receptors occurred via a different, EGF receptor-independent pathway. These results implicate the EGF receptor as a signaling intermediate of general importance in Galpha 13-dependent stress fiber formation in fibroblasts.

Whereas no difference in the coupling of e.g. thrombin or LPA receptors to G12 and G13 has been observed in plasma membrane fractions, it is conceivable that receptors preferentially activate G12 or G13 under physiological conditions in intact cells. This could be caused by different affinities of receptors for G12 and G13 or by the subcellular localization of the involved downstream signaling components like EGF receptors. It is also possible that in the intact cell, G12 and G13 are specifically preassembled with individual receptors. Finally, regulators of G-protein signaling (RGS) proteins may selectively influence the Galpha 12- or Galpha 13-mediated signaling in a receptor-specific way. The recently described GEF for Rho, p115 RhoGEF, functions as an RGS protein for Galpha 12 and Galpha 13 in vitro, but its GEF-activity is only regulated by Galpha 13 (17, 18). However, it is not clear how p115 RhoGEF behaves in intact cells. In addition, other RGS proteins specific for Galpha 12 or Galpha 13 may exist.

In summary, we have taken advantage of a Galpha q/Galpha 11-deficient cell line and dominant negative mutants of Galpha 12 and Galpha 13 and Galpha 13-deficient cells to demonstrate that many Gq/G11-coupled receptors induce stress fiber formation in the absence of Galpha q and Galpha 11 and that this involves either a Galpha 12 or a Galpha 13/EGF receptor-dependent pathway.

    ACKNOWLEDGEMENTS

We thank U. Brandt and B. Klages for excellent technical assistance and A. Hall, F. Hess, and T. Masaki for expression plasmids.

    FOOTNOTES

* This work was supported by funds from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie and by National Institutes of Health Grant DK47890 (to T. M. W.).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. Tel.: 49-30-8445-1812; Fax: 49-30-8445-1818; E-mail: gschultz{at}zedat.fu-berlin.de.

    ABBREVIATIONS

The abbreviations used are: G-protein, heterotrimeric guanine nucleotide-binding protein; LPA, lysophosphatidic acid; PTX, pertussis toxin; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GEF, guanine nucleotide exchange factor; PLC, phospholipase C; DOI, 2,5-dimethoxy-4-iodoamphetamine; DMEM, Dulbecco's modified Eagle's medium.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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