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W2033221676 abstract "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 Gα subunits on the morphology of differentiated PC12 cells. Expression of GTPase-deficient Gα12, Gα13, and Gαq, but not Gαi2, caused neurite retraction and cell rounding in differentiated PC12 cells. These morphological changes induced by Gα12, Gα13, and Gαq 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 Gα13 and Gαq. However, tyrphostin A25 failed to inhibit the Gα12-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 Gαq, whereas the morphological effects of Gα12 and Gα13 did not require activation of protein kinase C and extracellular Ca2+. These results demonstrate that activation of Gα12, Gα13, and Gαq induces Rho-dependent morphological changes in PC12 cells through different signaling pathways. 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 Gα subunits on the morphology of differentiated PC12 cells. Expression of GTPase-deficient Gα12, Gα13, and Gαq, but not Gαi2, caused neurite retraction and cell rounding in differentiated PC12 cells. These morphological changes induced by Gα12, Gα13, and Gαq 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 Gα13 and Gαq. However, tyrphostin A25 failed to inhibit the Gα12-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 Gαq, whereas the morphological effects of Gα12 and Gα13 did not require activation of protein kinase C and extracellular Ca2+. These results demonstrate that activation of Gα12, Gα13, and Gαq induces Rho-dependent morphological changes in PC12 cells through different signaling pathways. lysophosphatidic acid polymerase chain reaction nerve growth factor 12-O-tetradecanoylphorbol-13-acetate protein kinase C. 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 (1Goodman C.S. Shatz C.J. Cell. 1993; 10 (suppl.): 77-98Abstract Full Text PDF Scopus (1012) Google Scholar). 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 (2Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Crossref PubMed Scopus (535) Google Scholar, 3Gebbink M.F.B.G. Kranenburg O. Poland M. Horck F.P.G. Houssa B. Moolenaar W.H. J. Cell Biol. 1997; 137: 1603-1613Crossref PubMed Scopus (139) Google Scholar, 4Daniels R.H. Hall P.S. Bokoch G.M. EMBO J. 1998; 17: 754-764Crossref PubMed Scopus (256) Google Scholar, 5Katoh H. Aoki J. Ichikawa A. Negishi M. J. Biol. Chem. 1998; 273: 2489-2492Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). 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 (4Daniels R.H. Hall P.S. Bokoch G.M. EMBO J. 1998; 17: 754-764Crossref PubMed Scopus (256) Google Scholar). On the other hand, we recently revealed that the p160rhoA-binding kinase ROKα induces neurite retraction acting downstream of Rho (5Katoh H. Aoki J. Ichikawa A. Negishi M. J. Biol. Chem. 1998; 273: 2489-2492Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). 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 (6Jalink K. Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (573) Google Scholar, 7Tigyi G. Fischer D.J. Sebök Á. Yang C. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 537-548Crossref PubMed Scopus (184) Google Scholar, 8Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Crossref PubMed Scopus (265) Google Scholar, 9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). 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 (9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 10Jalink K. Moolenaar W.H. J. Cell Biol. 1992; 118: 411-419Crossref PubMed Scopus (167) Google Scholar), 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 (9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 11Tigyi G. Fischer D.J. Sebök Á. Yang C. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 549-558Crossref PubMed Scopus (126) Google Scholar), suggesting that Gs activation is not linked to induction of neurite retraction.The G12 family of heterotrimeric G proteins, defined by Gα12 and Gα13, is the most recent family to be identified using a homology-based polymerase chain reaction (PCR) strategy (12Strathmann M.P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5582-5586Crossref PubMed Scopus (200) Google Scholar). Although immediate downstream effectors have not yet been identified, studies with the constitutively active mutants of Gα12 and Gα13 have resulted in the identification of several novel functions regulated by these Gα subunits, including transformation of fibroblasts (13Xu N. Bradley L. Ambudukar I. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6741-6745Crossref PubMed Scopus (174) Google Scholar, 14Voyno-Yasenetskya T.A. Pace A.M. Bourne H.R. Oncogene. 1994; 9: 2559-2565PubMed Google Scholar), activation of the c-Jun N-terminal kinase cascade (15Prasad M.V.V.S.V. Dermott J.M. Heasley L.E. Johnson G.L. Dhanasekaran N. J. Biol. Chem. 1995; 270: 18655-18659Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 16Collins L.R. Minden A. Karin M. Brown J.H. J. Biol. Chem. 1996; 271: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 17Voyno-Yasenetskya T.A. Faure M.P. Ahn N.G. Bourne H.R. J. Biol. Chem. 1996; 271: 21081-21087Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), stimulation of stress fiber formation and focal adhesion assembly (18Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 19Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar), stimulation of the Na+-H+ exchanger (20Voyno-Yasenetskya T.A. Conklin B.R. Gilbert R.L. Hooley R. Bourne H.R. Barber D.L. J. Biol. Chem. 1994; 269: 4721-4724Abstract Full Text PDF PubMed Google Scholar, 21Hooley R., Yu, C.-Y. Symons M. Barber D.L. J. Biol. Chem. 1996; 271: 6152-6158Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 22Lin X. Voyno-Yasenetskya T.A. Hooley R. Lin C.-Y. Orlowski J. Barber D.L. J. Biol. Chem. 1996; 271: 22604-22610Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), activation of phospholipase D (23Plonk S.G. Park S.-K. Exton J.H. J. Biol. Chem. 1998; 273: 4823-4826Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and induction of apoptosis (24Althoefer H. Eversole-Cire P. Simon M.I. J. Biol. Chem. 1997; 272: 24380-24386Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). These studies also indicated that the Ras or Rho family small GTPases appear to be involved in the downstream responses regulated by Gα12and Gα13.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 Gα subunits in neuronal cell morphology, we microinjected expression plasmids encoding GTPase-deficient mutants of Gα subunits into the nuclei of NGF-differentiated PC12 cells bearing neurites. We report here that expression of constitutively active mutants of Gα12, Gα13, and Gαq caused Rho-dependent neurite retraction and cell rounding through different pathways.DISCUSSIONActivation of a certain G protein-coupled receptor has been reported to induce Rho-dependent neurite retraction and cell rounding in neuronal cell lines (6Jalink K. Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (573) Google Scholar, 7Tigyi G. Fischer D.J. Sebök Á. Yang C. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 537-548Crossref PubMed Scopus (184) Google Scholar, 8Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Crossref PubMed Scopus (265) Google Scholar, 9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Here we have demonstrated that constitutively active forms of Gα12, Gα13, and Gαq, but not Gαi2, 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 (28Aktories K. Weller U. Chhatwal G.S. FEBS Lett. 1987; 212: 109-113Crossref PubMed Scopus (169) Google Scholar, 29Sekine A. Fujiwara M. Narumiya S. J. Biol. Chem. 1989; 264: 8602-8605Abstract Full Text PDF PubMed Google Scholar), completely inhibited both neurite retraction and cell rounding induced by Gα12QL, Gα13QL, and GαqQL (Figs. 3 and 4), indicating that activation of Gα12, Gα13, and Gαq induces neurite retraction and cell rounding through the Rho-dependent signaling pathway in differentiated PC12 cells.Gα12 and Gα13, 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 (31Dhanasekaran N. Dermott J.M. Cell. Signalling. 1996; 8: 235-245Crossref PubMed Scopus (127) Google Scholar). We have also shown that both Gα12 and Gα13 can trigger Rho-dependent neurite retraction and cell rounding. These findings suggest that Gα12 and Gα13 may interact with a common effector. In this study, however, the tyrosine kinase inhibitor tyrphostin A25 blocked the Gα13QL-induced neurite retraction and cell rounding, whereas the Gα12QL-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 Gα13, but not in that of Gα12. This finding strongly suggests that Gα12 and Gα13interact with different effectors to regulate neuronal cell morphology. The differences in the sensitivity to tyrphostin between Gα12 and Gα13 were also shown in the signaling of Gα12- and Gα13-stimulated stress fiber formation and focal adhesion assembly in Swiss 3T3 fibroblasts (19Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Furthermore, it was previously reported that Gα12 and Gα13 stimulate Na+-H+ exchangers through different mechanisms in COS-7 cells (32Dhanasekaran N. Prasad M.V.V.S.V. Wadsworth S.J. Dermott J.M. van Rossum G. J. Biol. Chem. 1994; 269: 11802-11806Abstract Full Text PDF PubMed Google Scholar, 33Wadsworth S.J. Gebauer G. van Rossum G. Dhanasekaran N. J. Biol. Chem. 1997; 272: 28829-28832Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Therefore, it is likely that Gα12 and Gα13 activate different pathways to regulate their cellular functions.Activation of Gαq can stimulate the phospholipase C-β 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 GαqQL, whereas the Gα12QL- and Gα13QL-induced morphological changes were not influenced (Figs. 7 and 9). In addition, elimination of extracellular Ca2+ also inhibited the effects of GαqQL, but not those of Gα12QL and Gα13QL (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 (34Kuno M. Gardner P. Nature. 1987; 326: 301-304Crossref PubMed Scopus (469) Google Scholar, 35Irvine R.F. Moor R.M. Biochem. J. 1986; 240: 917-920Crossref PubMed Scopus (473) Google Scholar). Recently, Gαq was reported to activate inositol 1,4,5-trisphosphate-operated Ca2+-permeable channels (36Mochizuki-Oda N. Nakajima Y. Nakanishi S. Ito S. J. Biol. Chem. 1994; 269: 9651-9658Abstract Full Text PDF PubMed Google Scholar). The requirement of extracellular Ca2+ for the Gαq-induced morphological changes could be interpreted by this Gαq-mediated Ca2+-permeable channel activation. Therefore, both PKC activation and Ca2+ influx are essential elements in the signaling of Gαq upstream of Rho. We also examined the involvement of phospholipase C in the GαqQL 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. 2H. Katoh, J. Aoki, Y. Yamaguchi, Y. Kitano, A. Ichikawa, and M. Negishi, unpublished observation. 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 (7Tigyi G. Fischer D.J. Sebök Á. Yang C. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 537-548Crossref PubMed Scopus (184) Google Scholar). 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 (9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), indicating that G12 or G13 mediates the action of the EP3 receptor.Interestingly, the GαqQL-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 Gαq 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 Gαq to Rho. Since activation of Gαq 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 (38Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). Therefore, PYK2 can be speculated to be a candidate for the tyrosine kinase, which links the signal of Gαq to Rho for the induction of neurite retraction and cell rounding. A tyrphostin-sensitive tyrosine kinase was of course involved in the signaling of Gα13 to Rho. However, in contrast to Gαq, Gα13 did not require either PKC activation or Ca2+ influx. Two potential explanations for the difference between the signaling pathways of Gα13 and Gαq can be presented. One explanation is that an identical tyrosine kinase mediates the signals of Gα13 and Gαq to Rho, but the pathways of both α subunits to activate the tyrosine kinase are different; Gαq activates the kinase through PKC and Ca2+influx, whereas Gα13 activates the kinase independent of PKC. The other explanation is that different tyrosine kinases are involved in the pathways of both α subunits. We have summarized these possible pathways of signal transduction of three α subunits for Rho-dependent neurite retraction and cell rounding (Fig. 10).Expression of a GTPase-deficient form of Gαq in undifferentiated PC12 cells was recently shown to induce neurite outgrowth during 2–3 weeks using the retrovirus-mediated infection procedure (39Heasley L.E. Storey B. Fanger G.R. Butterfield L. Zamarripa J. Blumberg D. Maue R.A. Mol. Cell. Biol. 1996; 16: 648-656Crossref PubMed Google Scholar). In contrast, our results showed that expression of GαqQL in NGF-differentiated PC12 cells triggered neurite retraction within 3 h after microinjection. Therefore, these opposite effects of Gαq 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 Gα12, Gα13, and Gαq 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 (40Machesky L.M. Hall A. Trends Cell Biol. 1996; 6: 304-310Abstract Full Text PDF PubMed Scopus (253) Google Scholar). 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 activityin vitro, including Lbc, Lfc, and Lsc (3Gebbink M.F.B.G. Kranenburg O. Poland M. Horck F.P.G. Houssa B. Moolenaar W.H. J. Cell Biol. 1997; 137: 1603-1613Crossref PubMed Scopus (139) Google Scholar, 41Zheng Y. Olson M.F. Hall A. Cerione R.A. Toksoz D. J. Biol. Chem. 1995; 270: 9031-9034Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 42Glaven J.A. Whitehead I.P. Nomanbhoy T. Kay R. Cerione R.A. J. Biol. Chem. 1996; 271: 27374-27381Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 43Hart M.J. Sharma S. elMasry N. Qiu R.-G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In addition, they appear to be expressed in the same cell type (42Glaven J.A. Whitehead I.P. Nomanbhoy T. Kay R. Cerione R.A. J. Biol. Chem. 1996; 271: 27374-27381Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). 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 Gα 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 (37Threadgill R. Bobb K. Ghosh A. Neuron. 1997; 19: 625-634Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar,44Jin Z. Strittmatter S.M. J. Neurosci. 1997; 17: 6256-6263Crossref PubMed Google Scholar). In embryonic chick dorsal root ganglion, inhibition of Rho with C3 exoenzyme stimulated the outgrowth of neurites (44Jin Z. Strittmatter S.M. J. Neurosci. 1997; 17: 6256-6263Crossref PubMed Google Scholar), suggesting that activation of Rho suppresses neurite outgrowth in primary neurons. Therefore, Gα12, Gα13, and Gαq may play a negative regulator for neurite outgrowth through activation of Rho in primary neurons.In conclusion, we have here shown that activation of Gα12, Gα13, and Gαq 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. 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 (1Goodman C.S. Shatz C.J. Cell. 1993; 10 (suppl.): 77-98Abstract Full Text PDF Scopus (1012) Google Scholar). 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 (2Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Crossref PubMed Scopus (535) Google Scholar, 3Gebbink M.F.B.G. Kranenburg O. Poland M. Horck F.P.G. Houssa B. Moolenaar W.H. J. Cell Biol. 1997; 137: 1603-1613Crossref PubMed Scopus (139) Google Scholar, 4Daniels R.H. Hall P.S. Bokoch G.M. EMBO J. 1998; 17: 754-764Crossref PubMed Scopus (256) Google Scholar, 5Katoh H. Aoki J. Ichikawa A. Negishi M. J. Biol. Chem. 1998; 273: 2489-2492Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). 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 (4Daniels R.H. Hall P.S. Bokoch G.M. EMBO J. 1998; 17: 754-764Crossref PubMed Scopus (256) Google Scholar). On the other hand, we recently revealed that the p160rhoA-binding kinase ROKα induces neurite retraction acting downstream of Rho (5Katoh H. Aoki J. Ichikawa A. Negishi M. J. Biol. Chem. 1998; 273: 2489-2492Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). 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 (6Jalink K. Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (573) Google Scholar, 7Tigyi G. Fischer D.J. Sebök Á. Yang C. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 537-548Crossref PubMed Scopus (184) Google Scholar, 8Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Crossref PubMed Scopus (265) Google Scholar, 9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). 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 (9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 10Jalink K. Moolenaar W.H. J. Cell Biol. 1992; 118: 411-419Crossref PubMed Scopus (167) Google Scholar), 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 (9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 11Tigyi G. Fischer D.J. Sebök Á. Yang C. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 549-558Crossref PubMed Scopus (126) Google Scholar), suggesting that Gs activation is not linked to induction of neurite retraction. The G12 family of heterotrimeric G proteins, defined by Gα12 and Gα13, is the most recent family to be identified using a homology-based polymerase chain reaction (PCR) strategy (12Strathmann M.P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5582-5586Crossref PubMed Scopus (200) Google Scholar). Although immediate downstream effectors have not yet been identified, studies with the constitutively active mutants of Gα12 and Gα13 have resulted in the identification of several novel functions regulated by these Gα subunits, including transformation of fibroblasts (13Xu N. Bradley L. Ambudukar I. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6741-6745Crossref PubMed Scopus (174) Google Scholar, 14Voyno-Yasenetskya T.A. Pace A.M. Bourne H.R. Oncogene. 1994; 9: 2559-2565PubMed Google Scholar), activation of the c-Jun N-terminal kinase cascade (15Prasad M.V.V.S.V. Dermott J.M. Heasley L.E. Johnson G.L. Dhanasekaran N. J. Biol. Chem. 1995; 270: 18655-18659Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 16Collins L.R. Minden A. Karin M. Brown J.H. J. Biol. Chem. 1996; 271: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 17Voyno-Yasenetskya T.A. Faure M.P. Ahn N.G. Bourne H.R. J. Biol. Chem. 1996; 271: 21081-21087Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), stimulation of stress fiber formation and focal adhesion assembly (18Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 19Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar), stimulation of the Na+-H+ exchanger (20Voyno-Yasenetskya T.A. Conklin B.R. Gilbert R.L. Hooley R. Bourne H.R. Barber D.L. J. Biol. Chem. 1994; 269: 4721-4724Abstract Full Text PDF PubMed Google Scholar, 21Hooley R., Yu, C.-Y. Symons M. Barber D.L. J. Biol. Chem. 1996; 271: 6152-6158Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 22Lin X. Voyno-Yasenetskya T.A. Hooley R. Lin C.-Y. Orlowski J. Barber D.L. J. Biol. Chem. 1996; 271: 22604-22610Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), activation of phospholipase D (23Plonk S.G. Park S.-K. Exton J.H. J. Biol. Chem. 1998; 273: 4823-4826Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and induction of apoptosis (24Althoefer H. Eversole-Cire P. Simon M.I. J. Biol. Chem. 1997; 272: 24380-24386Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). These studies also indicated that the Ras or Rho family small GTPases appear to be involved in the downstream responses regulated by Gα12and Gα13. 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 Gα subunits in neuronal cell morphology, we microinjected expression plasmids encoding GTPase-deficient mutants of Gα subunits into the nuclei of NGF-differentiated PC12 cells bearing neurites. We report here that expression of constitutively active mutants of Gα12, Gα13, and Gαq caused Rho-dependent neurite retraction and cell rounding through different pathways. DISCUSSIONActivation of a certain G protein-coupled receptor has been reported to induce Rho-dependent neurite retraction and cell rounding in neuronal cell lines (6Jalink K. Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (573) Google Scholar, 7Tigyi G. Fischer D.J. Sebök Á. Yang C. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 537-548Crossref PubMed Scopus (184) Google Scholar, 8Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Crossref PubMed Scopus (265) Google Scholar, 9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Here we have demonstrated that constitutively active forms of Gα12, Gα13, and Gαq, but not Gαi2, 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 (28Aktories K. Weller U. Chhatwal G.S. FEBS Lett. 1987; 212: 109-113Crossref PubMed Scopus (169) Google Scholar, 29Sekine A. Fujiwara M. Narumiya S. J. Biol. Chem. 1989; 264: 8602-8605Abstract Full Text PDF PubMed Google Scholar), completely inhibited both neurite retraction and cell rounding induced by Gα12QL, Gα13QL, and GαqQL (Figs. 3 and 4), indicating that activation of Gα12, Gα13, and Gαq induces neurite retraction and cell rounding through the Rho-dependent signaling pathway in differentiated PC12 cells.Gα12 and Gα13, 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 (31Dhanasekaran N. Dermott J.M. Cell. Signalling. 1996; 8: 235-245Crossref PubMed Scopus (127) Google Scholar). We have also shown that both Gα12 and Gα13 can trigger Rho-dependent neurite retraction and cell rounding. These findings suggest that Gα12 and Gα13 may interact with a common effector. In this study, however, the tyrosine kinase inhibitor tyrphostin A25 blocked the Gα13QL-induced neurite retraction and cell rounding, whereas the Gα12QL-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 Gα13, but not in that of Gα12. This finding strongly suggests that Gα12 and Gα13interact with different effectors to regulate neuronal cell morphology. The differences in the sensitivity to tyrphostin between Gα12 and Gα13 were also shown in the signaling of Gα12- and Gα13-stimulated stress fiber formation and focal adhesion assembly in Swiss 3T3 fibroblasts (19Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Furthermore, it was previously reported that Gα12 and Gα13 stimulate Na+-H+ exchangers through different mechanisms in COS-7 cells (32Dhanasekaran N. Prasad M.V.V.S.V. Wadsworth S.J. Dermott J.M. van Rossum G. J. Biol. Chem. 1994; 269: 11802-11806Abstract Full Text PDF PubMed Google Scholar, 33Wadsworth S.J. Gebauer G. van Rossum G. Dhanasekaran N. J. Biol. Chem. 1997; 272: 28829-28832Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Therefore, it is likely that Gα12 and Gα13 activate different pathways to regulate their cellular functions.Activation of Gαq can stimulate the phospholipase C-β 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 GαqQL, whereas the Gα12QL- and Gα13QL-induced morphological changes were not influenced (Figs. 7 and 9). In addition, elimination of extracellular Ca2+ also inhibited the effects of GαqQL, but not those of Gα12QL and Gα13QL (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 (34Kuno M. Gardner P. Nature. 1987; 326: 301-304Crossref PubMed Scopus (469) Google Scholar, 35Irvine R.F. Moor R.M. Biochem. J. 1986; 240: 917-920Crossref PubMed Scopus (473) Google Scholar). Recently, Gαq was reported to activate inositol 1,4,5-trisphosphate-operated Ca2+-permeable channels (36Mochizuki-Oda N. Nakajima Y. Nakanishi S. Ito S. J. Biol. Chem. 1994; 269: 9651-9658Abstract Full Text PDF PubMed Google Scholar). The requirement of extracellular Ca2+ for the Gαq-induced morphological changes could be interpreted by this Gαq-mediated Ca2+-permeable channel activation. Therefore, both PKC activation and Ca2+ influx are essential elements in the signaling of Gαq upstream of Rho. We also examined the involvement of phospholipase C in the GαqQL 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. 2H. Katoh, J. Aoki, Y. Yamaguchi, Y. Kitano, A. Ichikawa, and M. Negishi, unpublished observation. 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 (7Tigyi G. Fischer D.J. Sebök Á. Yang C. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 537-548Crossref PubMed Scopus (184) Google Scholar). 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 (9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), indicating that G12 or G13 mediates the action of the EP3 receptor.Interestingly, the GαqQL-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 Gαq 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 Gαq to Rho. Since activation of Gαq 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 (38Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). Therefore, PYK2 can be speculated to be a candidate for the tyrosine kinase, which links the signal of Gαq to Rho for the induction of neurite retraction and cell rounding. A tyrphostin-sensitive tyrosine kinase was of course involved in the signaling of Gα13 to Rho. However, in contrast to Gαq, Gα13 did not require either PKC activation or Ca2+ influx. Two potential explanations for the difference between the signaling pathways of Gα13 and Gαq can be presented. One explanation is that an identical tyrosine kinase mediates the signals of Gα13 and Gαq to Rho, but the pathways of both α subunits to activate the tyrosine kinase are different; Gαq activates the kinase through PKC and Ca2+influx, whereas Gα13 activates the kinase independent of PKC. The other explanation is that different tyrosine kinases are involved in the pathways of both α subunits. We have summarized these possible pathways of signal transduction of three α subunits for Rho-dependent neurite retraction and cell rounding (Fig. 10).Expression of a GTPase-deficient form of Gαq in undifferentiated PC12 cells was recently shown to induce neurite outgrowth during 2–3 weeks using the retrovirus-mediated infection procedure (39Heasley L.E. Storey B. Fanger G.R. Butterfield L. Zamarripa J. Blumberg D. Maue R.A. Mol. Cell. Biol. 1996; 16: 648-656Crossref PubMed Google Scholar). In contrast, our results showed that expression of GαqQL in NGF-differentiated PC12 cells triggered neurite retraction within 3 h after microinjection. Therefore, these opposite effects of Gαq 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 Gα12, Gα13, and Gαq 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 (40Machesky L.M. Hall A. Trends Cell Biol. 1996; 6: 304-310Abstract Full Text PDF PubMed Scopus (253) Google Scholar). 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 activityin vitro, including Lbc, Lfc, and Lsc (3Gebbink M.F.B.G. Kranenburg O. Poland M. Horck F.P.G. Houssa B. Moolenaar W.H. J. Cell Biol. 1997; 137: 1603-1613Crossref PubMed Scopus (139) Google Scholar, 41Zheng Y. Olson M.F. Hall A. Cerione R.A. Toksoz D. J. Biol. Chem. 1995; 270: 9031-9034Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 42Glaven J.A. Whitehead I.P. Nomanbhoy T. Kay R. Cerione R.A. J. Biol. Chem. 1996; 271: 27374-27381Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 43Hart M.J. Sharma S. elMasry N. Qiu R.-G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In addition, they appear to be expressed in the same cell type (42Glaven J.A. Whitehead I.P. Nomanbhoy T. Kay R. Cerione R.A. J. Biol. Chem. 1996; 271: 27374-27381Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). 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 Gα 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 (37Threadgill R. Bobb K. Ghosh A. Neuron. 1997; 19: 625-634Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar,44Jin Z. Strittmatter S.M. J. Neurosci. 1997; 17: 6256-6263Crossref PubMed Google Scholar). In embryonic chick dorsal root ganglion, inhibition of Rho with C3 exoenzyme stimulated the outgrowth of neurites (44Jin Z. Strittmatter S.M. J. Neurosci. 1997; 17: 6256-6263Crossref PubMed Google Scholar), suggesting that activation of Rho suppresses neurite outgrowth in primary neurons. Therefore, Gα12, Gα13, and Gαq may play a negative regulator for neurite outgrowth through activation of Rho in primary neurons.In conclusion, we have here shown that activation of Gα12, Gα13, and Gαq 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. Activation of a certain G protein-coupled receptor has been reported to induce Rho-dependent neurite retraction and cell rounding in neuronal cell lines (6Jalink K. Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (573) Google Scholar, 7Tigyi G. Fischer D.J. Sebök Á. Yang C. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 537-548Crossref PubMed Scopus (184) Google Scholar, 8Postma F.R. Jalink K. Hengeveld T. Moolenaar W.H. EMBO J. 1996; 15: 2388-2395Crossref PubMed Scopus (265) Google Scholar, 9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Here we have demonstrated that constitutively active forms of Gα12, Gα13, and Gαq, but not Gαi2, 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 (28Aktories K. Weller U. Chhatwal G.S. FEBS Lett. 1987; 212: 109-113Crossref PubMed Scopus (169) Google Scholar, 29Sekine A. Fujiwara M. Narumiya S. J. Biol. Chem. 1989; 264: 8602-8605Abstract Full Text PDF PubMed Google Scholar), completely inhibited both neurite retraction and cell rounding induced by Gα12QL, Gα13QL, and GαqQL (Figs. 3 and 4), indicating that activation of Gα12, Gα13, and Gαq induces neurite retraction and cell rounding through the Rho-dependent signaling pathway in differentiated PC12 cells. Gα12 and Gα13, 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 (31Dhanasekaran N. Dermott J.M. Cell. Signalling. 1996; 8: 235-245Crossref PubMed Scopus (127) Google Scholar). We have also shown that both Gα12 and Gα13 can trigger Rho-dependent neurite retraction and cell rounding. These findings suggest that Gα12 and Gα13 may interact with a common effector. In this study, however, the tyrosine kinase inhibitor tyrphostin A25 blocked the Gα13QL-induced neurite retraction and cell rounding, whereas the Gα12QL-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 Gα13, but not in that of Gα12. This finding strongly suggests that Gα12 and Gα13interact with different effectors to regulate neuronal cell morphology. The differences in the sensitivity to tyrphostin between Gα12 and Gα13 were also shown in the signaling of Gα12- and Gα13-stimulated stress fiber formation and focal adhesion assembly in Swiss 3T3 fibroblasts (19Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Furthermore, it was previously reported that Gα12 and Gα13 stimulate Na+-H+ exchangers through different mechanisms in COS-7 cells (32Dhanasekaran N. Prasad M.V.V.S.V. Wadsworth S.J. Dermott J.M. van Rossum G. J. Biol. Chem. 1994; 269: 11802-11806Abstract Full Text PDF PubMed Google Scholar, 33Wadsworth S.J. Gebauer G. van Rossum G. Dhanasekaran N. J. Biol. Chem. 1997; 272: 28829-28832Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Therefore, it is likely that Gα12 and Gα13 activate different pathways to regulate their cellular functions. Activation of Gαq can stimulate the phospholipase C-β 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 GαqQL, whereas the Gα12QL- and Gα13QL-induced morphological changes were not influenced (Figs. 7 and 9). In addition, elimination of extracellular Ca2+ also inhibited the effects of GαqQL, but not those of Gα12QL and Gα13QL (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 (34Kuno M. Gardner P. Nature. 1987; 326: 301-304Crossref PubMed Scopus (469) Google Scholar, 35Irvine R.F. Moor R.M. Biochem. J. 1986; 240: 917-920Crossref PubMed Scopus (473) Google Scholar). Recently, Gαq was reported to activate inositol 1,4,5-trisphosphate-operated Ca2+-permeable channels (36Mochizuki-Oda N. Nakajima Y. Nakanishi S. Ito S. J. Biol. Chem. 1994; 269: 9651-9658Abstract Full Text PDF PubMed Google Scholar). The requirement of extracellular Ca2+ for the Gαq-induced morphological changes could be interpreted by this Gαq-mediated Ca2+-permeable channel activation. Therefore, both PKC activation and Ca2+ influx are essential elements in the signaling of Gαq upstream of Rho. We also examined the involvement of phospholipase C in the GαqQL 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. 2H. Katoh, J. Aoki, Y. Yamaguchi, Y. Kitano, A. Ichikawa, and M. Negishi, unpublished observation. 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 (7Tigyi G. Fischer D.J. Sebök Á. Yang C. Dyer D.L. Miledi R. J. Neurochem. 1996; 66: 537-548Crossref PubMed Scopus (184) Google Scholar). 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 (9Katoh H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 29780-29784Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), indicating that G12 or G13 mediates the action of the EP3 receptor. Interestingly, the GαqQL-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 Gαq 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 Gαq to Rho. Since activation of Gαq 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 (38Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). Therefore, PYK2 can be speculated to be a candidate for the tyrosine kinase, which links the signal of Gαq to Rho for the induction of neurite retraction and cell rounding. A tyrphostin-sensitive tyrosine kinase was of course involved in the signaling of Gα13 to Rho. However, in contrast to Gαq, Gα13 did not require either PKC activation or Ca2+ influx. Two potential explanations for the difference between the signaling pathways of Gα13 and Gαq can be presented. One explanation is that an identical tyrosine kinase mediates the signals of Gα13 and Gαq to Rho, but the pathways of both α subunits to activate the tyrosine kinase are different; Gαq activates the kinase through PKC and Ca2+influx, whereas Gα13 activates the kinase independent of PKC. The other explanation is that different tyrosine kinases are involved in the pathways of both α subunits. We have summarized these possible pathways of signal transduction of three α subunits for Rho-dependent neurite retraction and cell rounding (Fig. 10). Expression of a GTPase-deficient form of Gαq in undifferentiated PC12 cells was recently shown to induce neurite outgrowth during 2–3 weeks using the retrovirus-mediated infection procedure (39Heasley L.E. Storey B. Fanger G.R. Butterfield L. Zamarripa J. Blumberg D. Maue R.A. Mol. Cell. Biol. 1996; 16: 648-656Crossref PubMed Google Scholar). In contrast, our results showed that expression of GαqQL in NGF-differentiated PC12 cells triggered neurite retraction within 3 h after microinjection. Therefore, these opposite effects of Gαq 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 Gα12, Gα13, and Gαq 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 (40Machesky L.M. Hall A. Trends Cell Biol. 1996; 6: 304-310Abstract Full Text PDF PubMed Scopus (253) Google Scholar). 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 activityin vitro, including Lbc, Lfc, and Lsc (3Gebbink M.F.B.G. Kranenburg O. Poland M. Horck F.P.G. Houssa B. Moolenaar W.H. J. Cell Biol. 1997; 137: 1603-1613Crossref PubMed Scopus (139) Google Scholar, 41Zheng Y. Olson M.F. Hall A. Cerione R.A. Toksoz D. J. Biol. Chem. 1995; 270: 9031-9034Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 42Glaven J.A. Whitehead I.P. Nomanbhoy T. Kay R. Cerione R.A. J. Biol. Chem. 1996; 271: 27374-27381Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 43Hart M.J. Sharma S. elMasry N. Qiu R.-G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In addition, they appear to be expressed in the same cell type (42Glaven J.A. Whitehead I.P. Nomanbhoy T. Kay R. Cerione R.A. J. Biol. Chem. 1996; 271: 27374-27381Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). 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 Gα 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 (37Threadgill R. Bobb K. Ghosh A. Neuron. 1997; 19: 625-634Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar,44Jin Z. Strittmatter S.M. J. Neurosci. 1997; 17: 6256-6263Crossref PubMed Google Scholar). In embryonic chick dorsal root ganglion, inhibition of Rho with C3 exoenzyme stimulated the outgrowth of neurites (44Jin Z. Strittmatter S.M. J. Neurosci. 1997; 17: 6256-6263Crossref PubMed Google Scholar), suggesting that activation of Rho suppresses neurite outgrowth in primary neurons. Therefore, Gα12, Gα13, and Gαq may play a negative regulator for neurite outgrowth through activation of Rho in primary neurons. In conclusion, we have here shown that activation of Gα12, Gα13, and Gαq 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." @default.
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W2033221676 title "Constitutively Active Gα12, Gα13, and Gαq Induce Rho-dependent Neurite Retraction through Different Signaling Pathways" @default.
W2033221676 cites W1512107284 @default.
W2033221676 cites W1513979636 @default.
W2033221676 cites W1515601041 @default.
W2033221676 cites W1530463005 @default.
W2033221676 cites W1543811682 @default.
W2033221676 cites W1545267219 @default.
W2033221676 cites W1595029695 @default.
W2033221676 cites W1950300271 @default.
W2033221676 cites W1964570827 @default.
W2033221676 cites W1965685644 @default.
W2033221676 cites W1973237558 @default.
W2033221676 cites W1974836258 @default.
W2033221676 cites W1985074372 @default.
W2033221676 cites W1990540386 @default.
W2033221676 cites W1998414644 @default.
W2033221676 cites W1999788892 @default.
W2033221676 cites W2001013796 @default.
W2033221676 cites W2005339591 @default.
W2033221676 cites W2022164476 @default.
W2033221676 cites W2030051082 @default.
W2033221676 cites W2037669016 @default.
W2033221676 cites W2038351958 @default.
W2033221676 cites W2047454594 @default.
W2033221676 cites W2061126410 @default.
W2033221676 cites W2064483656 @default.
W2033221676 cites W2067741165 @default.
W2033221676 cites W2074603949 @default.
W2033221676 cites W2077537799 @default.
W2033221676 cites W2080953730 @default.
W2033221676 cites W2085287004 @default.
W2033221676 cites W2087277315 @default.
W2033221676 cites W2088274904 @default.
W2033221676 cites W2108680081 @default.
W2033221676 cites W2117406052 @default.
W2033221676 cites W2121666423 @default.
W2033221676 cites W2130196728 @default.
W2033221676 cites W2150647809 @default.
W2033221676 cites W2151584690 @default.
W2033221676 cites W2157441732 @default.
W2033221676 cites W2164923114 @default.
W2033221676 cites W2170467038 @default.
W2033221676 cites W2187930412 @default.
W2033221676 cites W2214371436 @default.
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