Rho GTPase-dependent transformation by G protein-coupled receptors.

G protein coupled receptors (GPCRs) constitute the largest family of cell surface receptors, with more than 1000 members, and are responsible for converting a diverse array of extracellular stimuli into intracellular signaling events. Most members of the family have defined roles in intermediary metabolism and generally perform these functions in well-differentiated cells. However, there is an increasing awareness that some GPCRs can also regulate proliferative signaling pathways and that chronic stimulation or mutational activation of receptors can lead to oncogenic transformation. Activating mutations in GPCRs are associated with several types of human tumors and some receptors exhibit potent oncogenic activity due to agonist overexpression. Additionally, expression screening analyses for novel oncogenes identified GPCRs whose expression causes the oncogenic transformation of NIH3T3 mouse fibroblasts. These include Mas, G2A, and the PAR-1 thrombin receptor. In this review we summarize the signaling and transforming properties of these GPCR oncoproteins. What has emerged from these studies is the delineation of a GTPase cascade where transforming GPCRs cause aberrant growth regulation via activation of Rho family small GTPases.

Expression cloning of Xenopus Os4, an evolutionarily conserved gene, which induces mesoderm and dorsal axis.

Multiple factors, including members of the FGF, TGF beta, and Wnt family of proteins, are important mediators in the regulation of dorsal-ventral pattern formation during vertebrate development. By using an expression cloning approach to identify novel factors that could regulate dorsal-ventral patterning in the Xenopus embryo, we isolated the Xenopus homologue of the human Os4 gene by virtue of its ability to induce a secondary dorsal axis. While Os4 homologues have been identified in a variety of species, and human Os4 is overexpressed in human tumors, the biological function of Os4 is unknown. To explore the mechanism by which Xenopus Os4 (XOs4) induces a secondary dorsal axis, we used Xenopus explant and whole-embryo assays. The secondary axis induced by XOs4 is distinct from that induced by activation of Wnt or FGF pathways but similar to that induced by inhibition of BMP signaling or activation of an Activin pathway. However, XOs4 did not inhibit BMP signaling in dissociated animal cap explants, indicating that XOs4 does not inhibit BMP signaling. Similar to activation of an Activin-like pathway, expression of XOs4 induces molecular markers for mesoderm in animal cap explants, although expression of gastrula-stage mesodermal markers was very weak and substantially delayed. Yet, XOs4 does not require activity of the Activin signal-transduction pathway for mesoderm induction as dominant-negative components of the Activin/Nodal/Vg1 pathway did not prevent XOs4-mediated induction of mesodermal derivatives. Finally, like Activin/Nodal/Vg1 pathways, XOs4 requires FGF signaling for expression of mesoderm markers. Results presented in this study demonstrate that XOs4 can induce mesoderm and dorsalize ventral mesoderm resulting in ectopic dorsal axis formation, suggesting a role for this large evolutionarily conserved gene family in early development.

G2A is an oncogenic G protein-coupled receptor.

G2A is a heptahelical cell surface protein that has recently been described as a potential tumor suppressor, based on its ability to counteract transformation of pre-B cells and fibroblasts by Bcr-Abl, an oncogenic tyrosine kinase. We have isolated cDNAs encoding G2A in the course of screening libraries for clones that cause oncogenic transformation of NIH3T3 fibroblasts. When expressed at high levels in NIH3T3 cells by retroviral transduction, G2A induced a full range of phenotypes characteristic of oncogenic transformation, including loss of contact inhibition, anchorage-independent survival and proliferation, reduced dependence on serum, and tumorigenicity in mice. When expressed by transfection, G2A greatly enhanced the ability of a weakly oncogenic form of Raf-1 to transform NIH3T3 cells. These results demonstrate that G2A is potently oncogenic both on its own and in cooperation with another oncogene. Expression of G2A in fibroblasts and endothelial cells resulted in changes in cell morphology and cytoskeleton structure that were equivalent to those induced by the G protein subunit Galpha13. Transformation of NIH3T3 cells via G2A expression was completely suppressed by co-expression of LscRGS, a GTPase activating protein that suppresses signaling by Galpha12 and Galpha13. Hyperactivity of Galpha12 or Galpha13 has previously been shown to result in activation of Rho GTPases. G2A expression resulted in activation of Rho, and transformation via G2A was suppressed by a dominant negative form of RhoA. These results indicate that G2A may be directly coupled to Galpha13, and that it is the activation of this Rho-activating Galpha protein which is responsible for the ability of G2A to transform fibroblasts.

TC21 and Ras share indistinguishable transforming and differentiating activities.

Constitutively activated mutants of the Ras-related protein TC21/R-Ras2 cause tumorigenic transformation of NIH3T3 cells. However, unlike Ras, TC21 fails to bind to and activate the Raf-1 serine-threonine kinase. Thus, whereas Ras transformation is critically dependent on Raf-1 TC21 activity is promoted by activation of Raf-independent signaling pathways. In the present study, we have further compared the functions of Ras and TC21. First we determined the basis for the inability of TC21 to activate Raf-1. Whereas Ras can interact with the two distinct Ras-binding sequences in NH2-terminus of Raf-1, designated RBS1 and Raf-Cys, TC21 could only bind Raf-Cys. Thus, the inability of TC21 to bind to RBS1 may prevent it from promoting the translocation of Raf-1 to the plasma membrane. Second, we found that TC21 is an activator of the JNK and p38, but not ERK, mitogen-activated protein kinase cascades and that TC21 transforming activity was dependent on Rac function. Thus, like Ras, TC21 may activate a Rac/JNK pathway. Third, we determined if TC21 could cause the same biological consequences as Ras in three distinct cell types. Like Ras, activated TC21 caused transformation of RIE-1 rat intestinal epithelial cells and terminal differentiation of PC12 pheochromocytoma cells. Finally, activated TC21 blocked serum starvation-induced differentiation of C2 myoblasts, whereas dominant negative TC21 greatly accelerated this differentiation process. Therefore, TC21 and Ras share indistinguishable biological activities in all cell types that we have evaluated. These results support the importance of Raf-independent pathways in mediating the actions of Ras and TC21.

Mas oncogene signaling and transformation require the small GTP-binding protein Rac.

The Mas oncogene encodes a novel G-protein-coupled receptor that was identified originally as a transforming protein when overexpressed in NIH 3T3 cells. The mechanism and signaling pathways that mediate Mas transformation have not been determined. We observed that the foci of transformed NIH 3T3 cells caused by Mas were similar to those caused by activated Rho and Rac proteins. Therefore, we determined if Mas signaling and transformation are mediated through activation of a specific Rho family protein. First, we observed that, like activated Rac1, Mas cooperated with activated Raf and caused synergistic transformation of NIH 3T3 cells. Second, both Mas- and Rac1-transformed NIH 3T3 cells retained actin stress fibers and showed enhanced membrane ruffling. Third, like Rac, Mas induced lamellipodium formation in porcine aortic endothelial cells. Fourth, Mas and Rac1 strongly activated the JNK and p38, but not ERK, mitogen-activated protein kinases. Fifth, Mas and Rac1 stimulated transcription from common DNA promoter elements: NF-kappaB, serum response factor (SRF), Jun/ATF-2, and the cyclin D1 promoter. Finally, Mas transformation and some of Mas signaling (SRF and cyclin D1 but not NF-kappaB activation) were blocked by dominant negative Rac1. Taken together, these observations suggest that Mas transformation is mediated in part by activation of Rac-dependent signaling pathways. Thus, Rho family proteins are common mediators of transformation by a diverse variety of oncogene proteins that include Ras, Dbl family, and G-protein-coupled oncogene proteins.

Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase.

In GN4 rat liver epithelial cells, angiotensin II (Ang II) and other agonists which activate phospholipase C stimulate tyrosine kinase activity in a calcium-dependent, protein kinase C (PKC)-independent manner. Since Ang II also produces a proliferative response in these cells, we investigated downstream signaling elements traditionally linked to growth control by tyrosine kinases. First, Ang II, like epidermal growth factor (EGF), stimulated AP-1 binding activity in a PKC-independent manner. Because increases in AP-1 can reflect induction of c-Jun and c-Fos, we examined the activity of the mitogen-activated protein (MAP) kinase family members Erk-1 and -2 and the c-Jun N-terminal kinase (JNK), which are known to influence c-Jun and c-Fos transcription. Ang II stimulated MAP kinase (MAPK) activity but only approximately 50% as effectively as EGF; again, these effects were independent of PKC. Ang II also produced a 50- to 200-fold activation of JNK in a PKC-independent manner. Unlike its smaller effect on MAPK, Ang II was approximately four- to sixfold more potent in activating JNK than EGF was. Although others had reported a lack of calcium ionophore-stimulated JNK activity in lymphocytes and several other cell lines, we examined the role of calcium in GN4 cells. The following results suggest that JNK activation in rat liver epithelial cells is at least partially Ca(2+) dependent: (i) norepinephrine and vasopressin hormones that increase inositol 1,4,5-triphosphate stimulated JNK; (ii) both thapsigargin, a compound that produces an intracellular Ca(2+) signal, and Ca(2+) ionophores stimulated a dramatic increase in JNK activity (up to 200-fold); (iii) extracellular Ca(2+) chelation with ethylene glycol tetraacetic acid (EGTA) inhibited JNK activation by ionophore and intracellular chelation with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl-ester (BAPTA-AM) partially inhibited JNK activation by Ang II or thapsigargin; and (iv) JNK activation by Ang II was inhibited by pretreatment of cells with thapsigargin and EGTA, a procedure which depletes intracellular Ca(2+) stores. JNK activation following Ang II stimulation did not involve calmodulin; either W-7 nor calmidizolium, in concentrations sufficient to inhibit Ca(2+)/calmodulin-dependent kinase II, blocked JNK activation by Ang II. In contrast, genistein, in concentrations sufficient to inhibit Ca(2+)-dependent tyrosine phosphorylation, prevented Ang II and thapsigargin-induced JNK activation. In summary, in GN4 rat liver epithelial cells, Ang II stimulates JNK via a novel Ca(2+)-dependent pathway. The inhibition by genistein suggest that Ca(2+)-dependent tyrosine phosphorylation may modulate the JNK pathway in a cell type-specific manner, particularly in cells with a readily detectable Ca(2+)-regulated tyrosine kinase.

Differential effects of P2-purinoceptor antagonists on phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors.

1. Stimulation of P2Y-purinoceptors on turkey erythrocytes and many other cell types results in activation of phospholipase C. In contrast, we have observed recently that P2Y-purinoceptors on C6 rat glioma cells are not coupled to phospholipase C, but rather, inhibit adenylyl cyclase. 2. In this study we investigated the pharmacological selectivity of the P2-purinoceptor antagonists, suramin, reactive blue 2, and pyridoxal phosphate 6-azophenyl 2',4'-disulphonic acid (PPADS) for phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors. 3. In C6 glioma cells, suramin and reactive blue 2 competitively antagonized the inhibitory effect of 2MeSATP on adenylyl cyclase (pKB = 5.4 +/- 0.2 and 7.6 +/- 0.1, respectively), whereas PPADS at concentrations up to 100 microM had no effect. 4. In contrast, in the turkey erythrocyte preparation, PPADS at concentrations up to 30 microM was a competitive antagonist of P2Y-purinoceptor-stimulated phospholipase C activity (pKB = 5.9 +/- 0.1). Suramin and reactive blue 2 produced both a shift to the right of the concentration-effect of 2MeSATP for the activation of phospholipase C and a significant decrease in the maximal inositol phosphate response. 5. Turkey erythrocytes also express a phospholipase C-coupled beta-adrenoceptor. Concentrations of PPADS that competitively inhibited the P2Y-purinoceptor-mediated response had only minimal effects on the activation of phospholipase C by beta-adrenoceptors. In contrast, suramin and reactive blue 2 produced a non-competitive inhibition, characterized by decreases in the maximal response to isoprenaline with no change in the potency of this beta-adrenoceptor agonist. 6. The differential effect of PPADS on P2Y-purinoceptors of C6 glioma cells and turkey erythrocytes adds further support to the idea that different P2Y-purinoceptor subtypes mediate coupling to adenylylcyclic and phospholipase C.