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J Biol Chem, Vol. 273, Issue 44, 28700-28707, October 30, 1998


Constitutively Active Galpha 12, Galpha 13, and Galpha q Induce Rho-dependent Neurite Retraction through Different Signaling Pathways*

Hironori KatohDagger , Junko AokiDagger , Yoshiaki YamaguchiDagger , Yoshimi Kitano§, Atsushi Ichikawa§, and Manabu NegishiDagger

From the Departments of Dagger  Molecular Neurobiology and § Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In neuronal cells, activation of a certain heterotrimeric G protein-coupled receptor causes neurite retraction and cell rounding via the small GTPase Rho. However, the specific heterotrimeric G proteins that mediate Rho-dependent neurite retraction and cell rounding have not yet been identified. Here we investigated the effects of expression of constitutively active Galpha subunits on the morphology of differentiated PC12 cells. Expression of GTPase-deficient Galpha 12, Galpha 13, and Galpha q, but not Galpha i2, caused neurite retraction and cell rounding in differentiated PC12 cells. These morphological changes induced by Galpha 12, Galpha 13, and Galpha q were completely inhibited by C3 exoenzyme, which specifically ADP-ribosylates and inactivates Rho. The tyrosine kinase inhibitor tyrphostin A25 blocked the neurite retraction and cell rounding induced by Galpha 13 and Galpha q. However, tyrphostin A25 failed to inhibit the Galpha 12-induced neuronal morphological changes. On the other hand, inhibition of protein kinase C or elimination of extracellular Ca2+ blocked the neurite retraction and cell rounding induced by Galpha q, whereas the morphological effects of Galpha 12 and Galpha 13 did not require activation of protein kinase C and extracellular Ca2+. These results demonstrate that activation of Galpha 12, Galpha 13, and Galpha q induces Rho-dependent morphological changes in PC12 cells through different signaling pathways.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The function of the nervous system depends on the highly specific pattern of connections formed between neurons during development. The specificity of these connections requires neurite extension toward the correct targets guided by the growth cone and remodeling of the initial pattern of connections in response to environmental signals (1). The Rho family of small GTPases (Rac, Cdc42, and Rho) has been demonstrated to play critical roles in the regulation of the cytoskeleton required for neurite extension and retraction. Studies on neuronal cell lines have shown that Rac and Cdc42 are required for the outgrowth of neurites, whereas Rho is required for their retraction (2-5). The downstream effectors involved in these GTPase-mediated neuronal morphological effects have been elucidated. The p21-activated kinase PAK1 was shown to act downstream of Rac and Cdc42 to induce neurite outgrowth (4). On the other hand, we recently revealed that the p160rhoA-binding kinase ROKalpha induces neurite retraction acting downstream of Rho (5). However, little is known about the signaling pathways upstream of these Rho family small GTPases in neuronal cells.

The activation of a certain heterotrimeric G protein-coupled receptor, such as the lysophosphatidic acid (LPA),1 sphingosine 1-phosphate, thrombin, and prostaglandin EP3 receptors, was shown to cause Rho-dependent neurite retraction in several neuronal cell lines (6-9). However, the heterotrimeric G proteins, which are coupled to those receptors for induction of neurite retraction, have not yet been identified. Previous studies demonstrated that pertussis toxin did not inhibit receptor-mediated neurite retraction (9, 10), indicating that this action is not mediated by Gi or Go. Furthermore, the activation of Gs by cholera toxin or an elevation of cAMP by forskolin failed to induce neurite retraction, but rather suppressed the receptor-mediated neurite retraction (9, 11), suggesting that Gs activation is not linked to induction of neurite retraction.

The G12 family of heterotrimeric G proteins, defined by Galpha 12 and Galpha 13, is the most recent family to be identified using a homology-based polymerase chain reaction (PCR) strategy (12). Although immediate downstream effectors have not yet been identified, studies with the constitutively active mutants of Galpha 12 and Galpha 13 have resulted in the identification of several novel functions regulated by these Galpha subunits, including transformation of fibroblasts (13, 14), activation of the c-Jun N-terminal kinase cascade (15-17), stimulation of stress fiber formation and focal adhesion assembly (18, 19), stimulation of the Na+-H+ exchanger (20-22), activation of phospholipase D (23), and induction of apoptosis (24). These studies also indicated that the Ras or Rho family small GTPases appear to be involved in the downstream responses regulated by Galpha 12 and Galpha 13.

Rat pheochromocytoma PC12 cells have served as a useful model system for studies of neuronal differentiation and morphology. When PC12 cells are exposed to nerve growth factor (NGF) for several days, they acquire many features of sympathetic neurons, such as an outgrowth of neurites. To investigate the role of the G12 family and other Galpha subunits in neuronal cell morphology, we microinjected expression plasmids encoding GTPase-deficient mutants of Galpha subunits into the nuclei of NGF-differentiated PC12 cells bearing neurites. We report here that expression of constitutively active mutants of Galpha 12, Galpha 13, and Galpha q caused Rho-dependent neurite retraction and cell rounding through different pathways.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- NGF (2.5 S) was purchased from Promega, and Clostridium botulinum C3 exoenzyme was obtained from Seikagaku Kogyo (Tokyo, Japan). Texas Red-coupled dextran was from Molecular Probes, Inc. Tyrphostin A25 and tyrphostin AG1478 were from Calbiochem. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was from Funakoshi Pharmaceuticals (Tokyo), and Ro31-8220 was from Nacalai Tesque (Kyoto, Japan). The sources of other materials are indicated below.

Construction of Expression Plasmids-- Wild-type mouse Galpha 12 and Galpha 13 were generous gifts from Dr. M. I. Simon (California Institute of Technology). Wild-type rat Galpha i2 was kindly provided by Dr. T. Katada (Tokyo University). The mammalian expression vector pEF-BOS was kindly provided by Dr. S. Nagata (Osaka University). Wild-type mouse Galpha q was generated by reverse transcription-PCR from mouse brain using primers 5'-GAGGCACTTCGGAAGAATGA-3' and 5'-AAGAACCAGTTTCTGGGAGG-3', and the PCR product was cloned into the pCR2.1 vector and sequenced completely. The cDNAs of the constitutively active mutants of Galpha 12 (Galpha 12 Q229L (Galpha 12QL)), Galpha 13 (Galpha 13 Q226L (Galpha 13QL)), Galpha q (Galpha q Q209L (Galpha qQL)), and Galpha i2 (Galpha i2 Q205L (Galpha i2QL)) were generated by PCR-mediated mutagenesis (25) and sequenced completely. The cDNA of RhoAV14 was obtained as described previously (5), and the cDNA of RhoAN19 was generated by PCR-mediated mutagenesis. The cDNAs of constitutively active mutants of Galpha subunits were inserted into the pcDNA3 expression vector (Invitrogen), and the cDNAs of RhoAV14 and RhoAN19 were inserted into pEF-BOS.

Cell Culture and Microinjection-- PC12 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 5% horse serum, 4 mM glutamine, 100 units/ml penicillin, and 0.2 mg/ml streptomycin under humidified conditions in 95% air and 5% CO2 at 37 °C. For microinjection, cells were seeded at a density 2 × 104 onto poly-D-lysine (Sigma)-coated 35-mm dishes, which were marked with a cross to facilitate the localization of injected cells. Microinjection was performed using an IMM-188 microinjection apparatus (Narishige, Tokyo). After cells had been differentiated in serum-free Dulbecco's modified Eagle's medium containing 50 ng/ml NGF for 3 or 4 days, 30 µg/ml concentrations of the indicated plasmids were microinjected into the nucleus with 1 mg/ml Texas Red-coupled dextran to visualize injected cells. C3 exoenzyme (100 µg/ml) was co-microinjected with expression plasmids. During microinjection, differentiated cells were maintained in Hepes-buffered Dulbecco's modified Eagle's medium (pH 7.4) at 37 °C. After microinjection, cells were replaced in NGF-containing serum-free Dulbecco's modified Eagle's medium and incubated for 3 h. To examine the effect of extracellular Ca2+, microinjected cells were replaced in Hepes-buffered saline containing 140 mM NaCl, 4.7 mM KCl, 5 mM MgCl2, 1.2 mM KH2PO4, 11 mM glucose, 2 mM EGTA, and 15 mM Hepes (pH 7.4). Cells were photographed at ×400 magnification under a phase-contrast microscope and by fluorescence of Texas Red-coupled dextran. For quantitative examinations, neurite-retracted cells were defined as the cells that almost completely retracted their neurites and caused rounding of the cell body within 3 h of the microinjection of plasmids. The percentages of neurite-retracted cells were calculated by counting at least 30 Texas Red-positive cells in the same field, and all data were obtained from triplicate experiments.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Expression of Constitutively Active Galpha Subunits in NGF-differentiated PC12 Cells-- To determine the role of the G12 family of heterotrimeric G proteins in neuronal cell morphology, we expressed constitutively active mutants of Galpha 12 and Galpha 13 in NGF-differentiated PC12 cells by nuclear microinjection of expression plasmids. Replacing a conserved glutamine with a leucine in the G3 region of the Galpha subunit, which corresponds to residue 229 in Galpha 12 and residue 226 in Galpha 13, has been shown to result in a GTPase-deficient, constitutively active form of the Galpha subunit (26, 27). As shown in Fig. 1, microinjection of expression plasmids (30 µg/ml) encoding constitutively active Galpha 12 (Galpha 12QL) and Galpha 13 (Galpha 13QL) into the nuclei of NGF-differentiated PC12 cells caused retraction of their extended neurites and rounding of the cell body within 3 h. Cells microinjected with the empty vector did not exhibit any morphological changes, indicating that there are no nonspecific effects due to nuclear microinjection itself. We also examined the effects of GTPase-deficient mutants of Galpha q (Galpha qQL) and Galpha i2 (Galpha i2QL) on differentiated PC12 cell morphology. As shown in Fig. 2, when expressed in differentiated PC12 cells, Galpha qQL mimicked Galpha 12QL and Galpha 13QL in induction of neurite retraction and rounding of the cell body. In contrast, expression of Galpha i2QL neither stimulated outgrowth nor caused retraction of neurites.


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  		Fig. 1.   Neurite retraction and cell rounding induced by constitutively active Galpha 12 and Galpha 13. Expression plasmids (30 µg/ml) encoding Galpha 12QL, Galpha 13QL, or the empty vector were microinjected into the nuclei of NGF-differentiated PC12 cells. Cells were photographed before (left panels) and 3 h after (middle panels) microinjection under a phase-contrast microscope or by fluorescence of Texas Red-coupled dextran co-microinjected with the expression vectors (right panels). The arrows indicate injected cells. The results shown are representative of three independent experiments. The bar represents 50 µm.


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  		Fig. 2.   Neurite retraction and cell rounding induced by constitutively active Galpha q and RhoA. Expression plasmids (30 µg/ml) encoding Galpha qQL, Galpha i2QL, or RhoAV14 were microinjected into the nuclei of NGF-differentiated PC12 cells. Cells were photographed before (left panels) and 3 h after (middle panels) microinjection under the a phase-contrast microscope or by fluorescence of Texas Red-coupled dextran co-microinjected with the expression vectors (right panels). The arrows indicate injected cells. The results shown are representative of three independent experiments. The bar represents 50 µm.

Effect of C3 Exoenzyme on Constitutively Active Galpha Subunit-induced Neuronal Morphological Changes-- Previous studies have shown that the small GTPase Rho is required for neurite retraction in response to a certain G protein-coupled receptor agonist such as LPA (6-9). As shown in Fig. 2, microinjection of expression plasmids encoding a constitutively active form of RhoA, RhoAV14, caused the retraction of neurites and rounding of the cell body. These morphological changes induced by RhoAV14 were quite similar to those induced by Galpha 12QL, Galpha 13QL, and Galpha qQL (Figs. 1 and 2). Therefore, to examine whether the neuronal morphological changes induced by constitutively active forms of Galpha subunits were Rho-dependent, we co-microinjected the constitutively active Galpha -encoding plasmids into the cells with C3 exoenzyme from C. botulinum, which has been shown to catalyze ADP-ribosylation of Rho and the specifically suppress the action of Rho (28, 29). As shown in Figs. 3 and 4, co-microinjection of C3 exoenzyme (100 µg/ml) completely blocked both neurite retraction and cell rounding induced by Galpha 12QL, Galpha 13QL, and Galpha qQL. We also co-microinjected expression plasmids encoding the dominant-negative form of RhoA, RhoAN19, into the cells with the constitutively active mutants of Galpha subunits. Coexpression of RhoAN19 slightly blocked neurite retraction induced by Galpha subunits, but was less effective than co-injection of C3 exoenzyme (data not shown). RhoAN19 would not be an effective inhibitor for complete suppression of Galpha subunit-induced morphological changes due to coexpression of RhoAN19 and Galpha subunits or the requirement of a large amount of RhoAN19 for suppression.


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  		Fig. 3.   Effect of C3 exoenzyme on neuronal morphological changes induced by constitutively active Galpha subunits. C3 exoenzyme (100 µg/ml) was co-microinjected with expression plasmids (30 µg/ml) encoding Galpha 12QL, Galpha 13QL, or Galpha qQL into differentiated PC12 cells. Cells were photographed before (left panels) and 3 h after (middle panels) microinjection under a phase-contrast microscope or by fluorescence of Texas Red-coupled dextran co-microinjected with the expression vectors (right panels). The arrows indicate injected cells. The results shown are representative of three independent experiments. The bar represents 50 µm.


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  		Fig. 4.   Quantification of effect of C3 exoenzyme on neurite retraction induced by constitutively active Galpha subunits. Differentiated PC12 cells were microinjected with expression plasmids (30 µg/ml) encoding Galpha 12QL, Galpha 13QL, Galpha qQL, Galpha i2QL, or the empty vector in the absence (-C3) or presence (+C3) of 100 µg/ml C3 exoenzyme. The percentages of neurite-retracted cells were determined 3 h after microinjection as described under "Experimental Procedures." Data are the means ± S.E. of triplicate experiments.

Effect of the Tyrosine Kinase Inhibitor Tyrphostin A25 on Neuronal Morphological Changes Induced by Constitutively Active Galpha Subunits-- A previous study showed that the tyrosine kinase inhibitor tyrphostin A25 inhibited stress fiber formation stimulated by LPA, but not by microinjection of constitutively active Rho into quiescent Swiss 3T3 cells, indicating that a tyrosine kinase is involved in the LPA-stimulated stress fiber formation acting upstream of Rho (30). Therefore, we examined the effect of tyrphostin A25 on the neuronal morphological changes induced by constitutively active Galpha subunits. As shown in Figs. 5 and 6, treatment of differentiated cells with tyrphostin A25 (150 µM) inhibited the Galpha 13QL- and Galpha qQL-induced neurite retraction and cell rounding. In contrast, the neurite retraction and cell rounding induced by Galpha 12QL were not influenced by this tyrosine kinase inhibitor. Thus, tyrphostin A25 specifically inhibited the signaling of Galpha 13QL and Galpha qQL. In addition, we examined the effect of another tyrosine kinase inhibitor, tyrphostin AG1478, on morphological changes induced by constitutively active Galpha subunits. Treatment of differentiated cells with tyrphostin AG1478 (10 µM) also specifically inhibited the Galpha 13QL- and Galpha qQL-induced neurite retraction and cell rounding, and Galpha 12QL-induced morphological changes were not inhibited by this inhibitor (data not shown).


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  		Fig. 5.   Effect of tyrphostin A25 on neuronal morphological changes induced by constitutively active Galpha subunits. After differentiated PC12 cells had been microinjected with expression plasmids (30 µg/ml) encoding Galpha 12QL, Galpha 13QL, or Galpha qQL, they were incubated with tyrphostin A25 (150 µM) for 3 h. Cells were photographed before (left panels) and 3 h after (middle panels) microinjection under a phase-contrast microscope or by fluorescence of Texas Red-coupled dextran co-microinjected with the expression vectors (right panels). The arrows indicate injected cells. The results shown are representative of three independent experiments. The bar represents 50 µm.


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  		Fig. 6.   Quantification of effect of tyrphostin A25 on neurite retraction induced by constitutively active Galpha subunits. After differentiated PC12 cells had been microinjected with expression plasmids (30 µg/ml) encoding Galpha 12QL, Galpha 13QL, or Galpha qQL, they were incubated with vehicle (-Tyr) or 150 µM tyrphostin A25 (+Tyr) for 3 h. The percentages of neurite-retracted cells were determined 3 h after microinjection as described under "Experimental Procedures." Data are the means ± S.E. of triplicate experiments.

Effects of Protein Kinase C Inhibition and Elimination of Extracellular Ca2+ on Neuronal Morphological Changes Induced by Constitutively Active Galpha Subunits-- A number of the cellular responses caused by activation of Galpha q have been shown to be mediated by activation of protein kinase C (PKC) or elevation of the intracellular Ca2+ concentration. Therefore, we examined whether activation of PKC was required for the neuronal morphological changes induced by constitutively active Galpha q and Galpha 12/Galpha 13. As shown in Figs. 7 and 9, down-regulation of endogenous PKC by a 24-h exposure to 1 µM TPA diminished the amount of neurite-retracted cells caused by Galpha qQL, whereas the Galpha 12QL- and Galpha 13QL-induced morphological changes were not significantly altered by PKC depletion of cells. Similar results were obtained by treatment of cells with the PKC inhibitor Ro31-8220 (300 nM) (Fig. 9). These results indicate that inhibition of PKC activity specifically interferes with the signaling pathway of Galpha q for neurite retraction and cell rounding.


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  		Fig. 7.   Effect of depletion of intracellular PKC on neuronal morphological changes induced by constitutively active Galpha subunits. Differentiated PC12 cells, which had been pretreated with TPA (1 µM) for 24 h, were microinjected with expression plasmids (30 µg/ml) encoding Galpha 12QL, Galpha 13QL, or Galpha qQL. Cells were photographed before (left panels) and 3 h after (middle panels) microinjection under a phase-contrast microscope or by fluorescence of Texas Red-coupled dextran co-microinjected with the expression vectors (right panels). The arrows indicate injected cells. The results shown are representative of three independent experiments. The bar represents 50 µm.

Next we examined the role of Ca2+ signaling in the neuronal morphological changes induced by activated Galpha subunits. Differentiated PC12 cells were incubated in a Ca2+-free medium in the presence of 2 mM EGTA during expression of Galpha subunits. Under these conditions, expression of Galpha qQL failed to induce neurite retraction and cell rounding. In contrast, the neuronal morphological changes induced by Galpha 12QL and Galpha 13QL normally occurred in a Ca2+-free medium with EGTA (Figs. 8 and 9). These results indicate that Ca2+ influx is required for the Galpha qQL-induced neuronal morphological changes.


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  		Fig. 8.   Effect of elimination of extracellular Ca2+ on neuronal morphological changes induced by constitutively active Galpha subunits. After differentiated PC12 cells had been microinjected with expression plasmids (30 µg/ml) encoding Galpha 12QL, Galpha 13QL, or Galpha qQL, they were incubated in Ca2+-free medium containing EGTA (2 mM) for 3 h. Cells were photographed before (left panels) and 3 h after (middle panels) microinjection under a phase-contrast microscope or by fluorescence of Texas Red-coupled dextran co-microinjected with the expression vectors (right panels). The arrows indicate injected cells. The results shown are representative of three independent experiments. The bar represents 50 µm.


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  		Fig. 9.   Quantification of effects of inhibition of PKC and elimination of extracellular Ca2+ on neurite retraction induced by constitutively active Galpha subunits. After differentiated PC12 cells had been microinjected with expression plasmids (30 µg/ml) encoding Galpha 12QL, Galpha 13QL, or Galpha qQL, they were incubated in the absence (Control) or presence of 300 nM Ro31-8220 (+Ro31-8220) or in Ca2+-free medium containing 2 mM EGTA (Ca2+-free) for 3 h. The expression plasmids were microinjected into differentiated cells that had been treated with 1 µM TPA for 24 h before microinjection to induce down-regulation of endogenous PKC (PKC-depleted). The percentages of neurite-retracted cells were determined 3 h after microinjection as described under "Experimental Procedures." Data are the means ± S.E. of triplicate experiments.


    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Activation of a certain G protein-coupled receptor has been reported to induce Rho-dependent neurite retraction and cell rounding in neuronal cell lines (6-9). Here we have demonstrated that constitutively active forms of Galpha 12, Galpha 13, and Galpha q, but not Galpha i2, can trigger neurite retraction and cell rounding in NGF-differentiated PC12 cells. These morphological changes were similar to those induced by a constitutively active form of RhoA, RhoAV14 (Fig. 2); and C3 exoenzyme, which specifically ADP-ribosylates and inactivates Rho (28, 29), completely inhibited both neurite retraction and cell rounding induced by Galpha 12QL, Galpha 13QL, and Galpha qQL (Figs. 3 and 4), indicating that activation of Galpha 12, Galpha 13, and Galpha q induces neurite retraction and cell rounding through the Rho-dependent signaling pathway in differentiated PC12 cells.

Galpha 12 and Galpha 13, the members of the G12 class of heterotrimeric G proteins, show 67% amino acid identity to each other and often cause similar responses in various cell types, including transformation of fibroblasts, activation of the c-Jun N-terminal kinase cascade, and stimulation of stress fiber formation and focal adhesion assembly (31). We have also shown that both Galpha 12 and Galpha 13 can trigger Rho-dependent neurite retraction and cell rounding. These findings suggest that Galpha 12 and Galpha 13 may interact with a common effector. In this study, however, the tyrosine kinase inhibitor tyrphostin A25 blocked the Galpha 13QL-induced neurite retraction and cell rounding, whereas the Galpha 12QL-induced effects were not influenced by this tyrosine kinase inhibitor (Figs. 5 and 6), indicating that a tyrphostin-sensitive tyrosine kinase is involved in the signaling of Galpha 13, but not in that of Galpha 12. This finding strongly suggests that Galpha 12 and Galpha 13 interact with different effectors to regulate neuronal cell morphology. The differences in the sensitivity to tyrphostin between Galpha 12 and Galpha 13 were also shown in the signaling of Galpha 12- and Galpha 13-stimulated stress fiber formation and focal adhesion assembly in Swiss 3T3 fibroblasts (19). Furthermore, it was previously reported that Galpha 12 and Galpha 13 stimulate Na+-H+ exchangers through different mechanisms in COS-7 cells (32, 33). Therefore, it is likely that Galpha 12 and Galpha 13 activate different pathways to regulate their cellular functions.

Activation of Galpha q can stimulate the phospholipase C-beta family, which results in stimulation of PKC activity and elevation of the intracellular Ca2+ concentration. In this study, depletion of endogenous PKC by both TPA and the PKC inhibitor Ro31-8220 specifically diminished the amount of neurite-retracted cells induced by Galpha qQL, whereas the Galpha 12QL- and Galpha 13QL-induced morphological changes were not influenced (Figs. 7 and 9). In addition, elimination of extracellular Ca2+ also inhibited the effects of Galpha qQL, but not those of Galpha 12QL and Galpha 13QL (Figs. 8 and 9). It has been shown that inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, products of phospholipase C activation pathways, activate Ca2+-permeable channels in plasma membranes (34, 35). Recently, Galpha q was reported to activate inositol 1,4,5-trisphosphate-operated Ca2+-permeable channels (36). The requirement of extracellular Ca2+ for the Galpha q-induced morphological changes could be interpreted by this Galpha q-mediated Ca2+-permeable channel activation. Therefore, both PKC activation and Ca2+ influx are essential elements in the signaling of Galpha q upstream of Rho. We also examined the involvement of phospholipase C in the Galpha qQL signaling using the phospholipase C inhibitor U-73122, but this compound was cytotoxic for differentiated PC12 cells, and treatment with U-73122 alone caused cell detachment.2 Interestingly, both PKC activity and Ca2+ influx as a result of phospholipase C activation appeared to be required for LPA-induced neurite retraction in NGF-differentiated PC12 cells (7). Therefore, it is likely that a Gq-coupled LPA receptor stimulates phospholipase C activity, and the resultant activation of PKC and Ca2+ influx induces Rho-dependent neurite retraction in PC12 cells. In contrast, prostaglandin EP3 receptor-induced neurite retraction was mediated through a PKC-independent pathway (9), indicating that G12 or G13 mediates the action of the EP3 receptor.

Interestingly, the Galpha qQL-induced neurite retraction and cell rounding were also blocked by treatment of cells with the tyrosine kinase inhibitor tyrphostin A25 (Figs. 5 and 6), indicating that a tyrphostin-sensitive tyrosine kinase is involved in the signaling from Galpha q to Rho. This study did not show whether this tyrphostin-sensitive tyrosine kinase acts upstream or downstream of Ca2+ and PKC in the signaling from Galpha q to Rho. Since activation of Galpha q can directly stimulate phospholipase C, which results in stimulation of PKC activity and elevation of the intracellular Ca2+ concentration, this tyrphostin-sensitive tyrosine kinase may act downstream of Ca2+ and PKC. Recently, the novel nonreceptor tyrosine kinase PYK2 has been shown to mediate Gq-coupled receptor-stimulated activation of the mitogen-activated protein kinase cascade in PC12 cells, and the activity of this tyrosine kinase appears to be regulated by elevation of the intracellular Ca2+ concentration as well as by PKC activation (38). Therefore, PYK2 can be speculated to be a candidate for the tyrosine kinase, which links the signal of Galpha q to Rho for the induction of neurite retraction and cell rounding. A tyrphostin-sensitive tyrosine kinase was of course involved in the signaling of Galpha 13 to Rho. However, in contrast to Galpha q, Galpha 13 did not require either PKC activation or Ca2+ influx. Two potential explanations for the difference between the signaling pathways of Galpha 13 and Galpha q can be presented. One explanation is that an identical tyrosine kinase mediates the signals of Galpha 13 and Galpha q to Rho, but the pathways of both alpha  subunits to activate the tyrosine kinase are different; Galpha q activates the kinase through PKC and Ca2+ influx, whereas Galpha 13 activates the kinase independent of PKC. The other explanation is that different tyrosine kinases are involved in the pathways of both alpha  subunits. We have summarized these possible pathways of signal transduction of three alpha  subunits for Rho-dependent neurite retraction and cell rounding (Fig. 10).


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  		Fig. 10.   Model for signal transduction pathways from Galpha subunits to Rho activation leading to neurite retraction and cell rounding in differentiated PC12 cells. Expression of constitutively active mutants of Galpha 12, Galpha 13, and Galpha q induces neurite retraction and cell rounding through different signaling pathways, which, however, converge at activation of Rho.

Expression of a GTPase-deficient form of Galpha q in undifferentiated PC12 cells was recently shown to induce neurite outgrowth during 2-3 weeks using the retrovirus-mediated infection procedure (39). In contrast, our results showed that expression of Galpha qQL in NGF-differentiated PC12 cells triggered neurite retraction within 3 h after microinjection. Therefore, these opposite effects of Galpha q on the regulation of neurites may be due to different conditions of cells in differentiation or to a different time scale for examination of morphological effects.

The data presented here demonstrated that constitutively active mutants of Galpha 12, Galpha 13, and Galpha q can induce neurite retraction and cell rounding through different signaling pathways, which, however, finally converge at activation of Rho (Fig. 10). Rho, like other small GTPases, functions as a molecular switch; it is active in its GTP-bound state and inactive in its GDP-bound state. Upstream activation of the cycle is mediated by guanine nucleotide exchange factors, which promote the exchange of GDP for GTP (40). A number of putative guanine nucleotide exchange factors for Rho and other Rho family GTPases have been identified, and some of these demonstrate Rho-specific guanine nucleotide exchange factor activity in vitro, including Lbc, Lfc, and Lsc (3, 41-43). In addition, they appear to be expressed in the same cell type (42). Therefore, one possibility for the existence of multiple guanine nucleotide exchange factors for Rho in the same cell type may be related to the existence of different signaling pathways from Galpha subunits to Rho activation.

Recent studies have shown the involvement of the Rho family of small GTPases in the regulation of neurite outgrowth in primary neurons (37, 44). In embryonic chick dorsal root ganglion, inhibition of Rho with C3 exoenzyme stimulated the outgrowth of neurites (44), suggesting that activation of Rho suppresses neurite outgrowth in primary neurons. Therefore, Galpha 12, Galpha 13, and Galpha q may play a negative regulator for neurite outgrowth through activation of Rho in primary neurons.

In conclusion, we have here shown that activation of Galpha 12, Galpha 13, and Galpha q can trigger Rho-dependent neurite retraction and cell rounding in differentiated PC12 cells through different signaling pathways. This study will contribute to the understanding of the signal transduction between heterotrimeric G protein-coupled receptors and Rho in neuronal cells.

    FOOTNOTES

* This work was supported in part by Grants-in-aid for Scientific Research 10155210, 10470482, and 09273105 from the Ministry of Education, Science, and Culture of Japan and by a grant from the Asahi Glass Research Foundation.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.: 81-75-753-4547; Fax: 81-75-753-4557; E-mail: mnegishi{at}pharm.kyoto-u.ac.jp.

The abbreviations used are: LPA, lysophosphatidic acid; PCR, polymerase chain reaction; NGF, nerve growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C.

2 H. Katoh, J. Aoki, Y. Yamaguchi, Y. Kitano, A. Ichikawa, and M. Negishi, unpublished observation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Goodman, C. S., and Shatz, C. J. (1993) Cell 10 (suppl.), 77-98
  2. Kozma, R., Sarner, S., Ahmed, S., and Lim, L. (1997) Mol. Cell. Biol. 17, 1201-1211[Abstract]
  3. Gebbink, M. F. B. G., Kranenburg, O., Poland, M., Horck, F. P. G., Houssa, B., and Moolenaar, W. H. (1997) J. Cell Biol. 137, 1603-1613[Abstract/Free Full Text]
  4. Daniels, R. H., Hall, P. S., and Bokoch, G. M. (1998) EMBO J. 17, 754-764[CrossRef][Medline] [Order article via Infotrieve]
  5. Katoh, H., Aoki, J., Ichikawa, A., and Negishi, M. (1998) J. Biol. Chem. 273, 2489-2492[Abstract/Free Full Text]
  6. Jalink, K., Corven, E. J., Hengeveld, T., Morii, N., Narumiya, S., and Moolenaar, W. H. (1994) J. Cell Biol. 126, 801-810[Abstract/Free Full Text]
  7. Tigyi, G., Fischer, D. J., Sebök, Á., Yang, C., Dyer, D. L., and Miledi, R. (1996) J. Neurochem. 66, 537-548[Medline] [Order article via Infotrieve]
  8. Postma, F. R., Jalink, K., Hengeveld, T., and Moolenaar, W. H. (1996) EMBO J. 15, 2388-2395[Medline] [Order article via Infotrieve]
  9. Katoh, H., Negishi, M., and Ichikawa, A. (1996) J. Biol. Chem. 271, 29780-29784[Abstract/Free Full Text]
  10. Jalink, K., and Moolenaar, W. H. (1992) J. Cell Biol. 118, 411-419[Abstract/Free Full Text]
  11. Tigyi, G., Fischer, D. J., Sebök, Á., Yang, C., Dyer, D. L., and Miledi, R. (1996) J. Neurochem. 66, 549-558[Medline] [Order article via Infotrieve]
  12. Strathmann, M. P., and Simon, M. I. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5582-5586[Abstract/Free Full Text]
  13. Xu, N., Bradley, L., Ambudukar, I., and Gutkind, J. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6741-6745[Abstract/Free Full Text]
  14. Voyno-Yasenetskya, T. A., Pace, A. M., and Bourne, H. R. (1994) Oncogene 9, 2559-2565[Medline] [Order article via Infotrieve]
  15. 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]
  16. Collins, L. R., Minden, A., Karin, M., and Brown, J. H. (1996) J. Biol. Chem. 271, 17349-17353[Abstract/Free Full Text]
  17. Voyno-Yasenetskya, T. A., Faure, M. P., Ahn, N. G., and Bourne, H. R. (1996) J. Biol. Chem. 271, 21081-21087[Abstract/Free Full Text]
  18. Buhl, A. M., Johnson, N. L., Dhanasekaran, N., and Johnson, G. L. (1995) J. Biol. Chem. 270, 24631-24634[Abstract/Free Full Text]
  19. Gohla, A., Harhammer, R., and Schultz, G. (1998) J. Biol. Chem. 273, 4653-4659[Abstract/Free Full Text]
  20. Voyno-Yasenetskya, T. A., Conklin, B. R., Gilbert, R. L., Hooley, R., Bourne, H. R., and Barber, D. L. (1994) J. Biol. Chem. 269, 4721-4724[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-Yasenetskya, T. A., Hooley, R., Lin, C.-Y., Orlowski, J., and Barber, D. L. (1996) J. Biol. Chem. 271, 22604-22610[Abstract/Free Full Text]
  23. Plonk, S. G., Park, S.-K., and Exton, J. H. (1998) J. Biol. Chem. 273, 4823-4826[Abstract/Free Full Text]
  24. Althoefer, H., Eversole-Cire, P., and Simon, M. I. (1997) J. Biol. Chem. 272, 24380-24386[Abstract/Free Full Text]
  25. Ito, W., Ishiguro, H., and Kurosawa, Y. (1991) Gene (Amst.) 102, 67-70[CrossRef][Medline] [Order article via Infotrieve]
  26. Masters, S. B., Miller, R. T., Chi, M.-H., Chang, F.-H., Beiderman, B., Lopez, N. G., and Bourne, H. R. (1989) J. Biol. Chem. 264, 15467-15474[Abstract/Free Full Text]
  27. Osawa, S., Dhanasekaran, N., Woon, C. W., and Johnson, G. L. (1990) Cell 63, 697-706[CrossRef][Medline] [Order article via Infotrieve]
  28. Aktories, K., Weller, U., and Chhatwal, G. S. (1987) FEBS Lett. 212, 109-113[CrossRef][Medline] [Order article via Infotrieve]
  29. Sekine, A., Fujiwara, M., and Narumiya, S. (1989) J. Biol. Chem. 264, 8602-8605[Abstract/Free Full Text]
  30. Nobes, C. D., Hawkins, P., Stephens, L., and Hall, A. (1995) J. Cell Sci. 108, 225-233[Abstract]
  31. Dhanasekaran, N., and Dermott, J. M. (1996) Cell. Signalling 8, 235-245[CrossRef][Medline] [Order article via Infotrieve]
  32. Dhanasekaran, N., Prasad, M. V. V. S. V., Wadsworth, S. J., Dermott, J. M., and van Rossum, G. (1994) J. Biol. Chem. 269, 11802-11806[Abstract/Free Full Text]
  33. Wadsworth, S. J., Gebauer, G., van Rossum, G., and Dhanasekaran, N. (1997) J. Biol. Chem. 272, 28829-28832[Abstract/Free Full Text]
  34. Kuno, M., and Gardner, P. (1987) Nature 326, 301-304[CrossRef][Medline] [Order article via Infotrieve]
  35. Irvine, R. F., and Moor, R. M. (1986) Biochem. J. 240, 917-920[Medline] [Order article via Infotrieve]
  36. Mochizuki-Oda, N., Nakajima, Y., Nakanishi, S., and Ito, S. (1994) J. Biol. Chem. 269, 9651-9658[Abstract/Free Full Text]
  37. Threadgill, R., Bobb, K., and Ghosh, A. (1997) Neuron 19, 625-634[CrossRef][Medline] [Order article via Infotrieve]
  38. Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376, 737-745[CrossRef][Medline] [Order article via Infotrieve]
  39. Heasley, L. E., Storey, B., Fanger, G. R., Butterfield, L., Zamarripa, J., Blumberg, D., and Maue, R. A. (1996) Mol. Cell. Biol. 16, 648-656[Abstract]
  40. Machesky, L. M., and Hall, A. (1996) Trends Cell Biol. 6, 304-310[CrossRef][Medline] [Order article via Infotrieve]
  41. Zheng, Y., Olson, M. F., Hall, A., Cerione, R. A., and Toksoz, D. (1995) J. Biol. Chem. 270, 9031-9034[Abstract/Free Full Text]
  42. 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]
  43. 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]
  44. Jin, Z., and Strittmatter, S. M. (1997) J. Neurosci. 17, 6256-6263[Abstract/Free Full Text]

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