Activation of Rat Frizzled-1 Promotes Wnt Signaling and Differentiation of Mouse F9 Teratocarcinoma Cells via Pathways That Require Gαq and Gαo Function

Abstract

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven transmembranespanning motif characteristic of G-protein-linked receptors, although no loss-of-function studies have demonstrated a requirement for G-proteins for Wnt signaling by the gene product of frizzled-1. Medium conditioned by mouse F9 teratocarcinoma stem cells stably transfected to express either Xenopus Wnt-5a or Wnt-8 was used to test primitive endoderm formation of F9 stem cells. F9 stem cells expressing the rat Frizzled-1 receptors demonstrated endoderm formation in response to conditioned medium containing Wnt-8 but not to medium containing Wnt-5a. Primitive endoderm formation stimulated by Wnt-8 acting on the rat Frizzled-1 receptor was blocked by treatment with pertussis toxin by depletion of either G␣ o or G␣ q via antisense oligodeoxynucleotides, as well as by inhibitors of protein kinase C (bisindoylmaleimide) and of mitogen-activated protein kinase kinase (PD98059). Our results demonstrate the requirement for G-protein subunits G␣ o (a pertussis toxin substrate) and G␣ q for signaling by Frizzled-1, and an obligate role for the protein kinase C (likely mediated through stimulation of G␣ q ) and mitogen-activated protein kinase network at the level of mitogen-activated protein kinase kinase.Wnts are a class of vertebrate genes encoding secreted signaling proteins, which appear to modulate diverse processes in developing vertebrate embryos and in some adult tissues (1-4). The actions of Wnts are thought to be mediated by the function of members of the frizzled gene family of prospective heptihelical receptors (5-10). In the absence of a Wnt signal, active glycogen-synthase kinase 3 (zeste white 3/shaggy in Drosophila) phosphorylates ␤-catenin at an amino-terminal site (11), targeting it for ubiquitination and degradation through a proteosome pathway that also involves axin and the product of the adenomatous polyposis coli gene (12-21). Signaling by Wnt-1 via Frizzled homologues activates the function of Dishevelled, which represses the activity of glycogen-synthase kinase-3 (3, 22), promoting elevation of intracellular ␤-catenin levels and accumulation of ␤-catenin in nuclei (11, 23). Nuclear ␤-catenin interacts with members of the lymphoid-enhancer factor/T-cell factor (LEF/TCF) classes of architectural high mobility group (HMG) box transcription factors (24 -27) to regulate expression of genes involved in vertebrate development (24, 28 -32).Some Wnts can work through a pathway distinct from the glycogen-synthase kinase 3-mediated one described above (2,(33)(34)(35), depending upon what receptors are present. Xwnt-5a, unlike Wnt-1 and Xwnt-8, does not induce a duplication of the axis in Xenopus when ectopically expressed, but instead causes morphogenetic defects (33, 34). Whereas Xwnt-1, -8, -8b, and -3a are functionally equivalent in axis induction assays, Xwnt5a, -4, and -11 are functionally equivalent in this second, distinct Wnt signaling activity (36,37). This classification of Wnt ligand resembles the cl...

From individual Wnt pathways towards a Wnt signalling network

“…A graph representation including the major components of this pathway is indicated in figure 1c. (Liu et al 1999a), whereas triggering Frizzled-2 signalling through the corresponding inducible Frizzled receptor requires the action of Ga o and Ga t (Liu et al 1999b). Further evidence for an involvement of G-proteins in canonical Wnt signalling was provided by the observation that overexpression of RGS4, a regulator of G-protein signalling, in early Xenopus embryos interferes with Wnt-8-induced secondary axis formation (Wu et al 2000).…”

Dual Regulation of Glycogen Synthase Kinase-3β by the α1A-Adrenergic Receptor

“…In the Wg/Wnt signaling pathway in Drosophila and vertebrates, the extracellular Wg/ Wnt signal is transduced from the membrane receptor Frizzled through Dsh/Dvl to inactivate GSK-3 (3,40,47,48). Frizzled-1 has been reported to signal through G␣ o and G␣ q (49), and Wg-dependent inhibition of GSK-3 requires a PMA-sensitive isoform of PKC (40). Conflicting results have been obtained as to whether the inhibition of GSK-3 involves phosphorylation of the protein or the activation of Akt (40,47,48,50), but it is well accepted that GSK-3 binding to axin and other proteins is critical for its regulation by the Wg/Wnt signaling pathway (3,4).…”

Inhibition by Wnt-1 or Wnt-3a of nerve growth factor-induced differentiation of PC12 cells is reversed by bisindolylmaleimide-I but not by several other PKC inhibitors

“…This pathway signals via a pertussis toxin-sensitive G-protein coupled system (Liu et al, 1999) and thus can be inhibited by pertussis toxin. As is shown in Figure 7, pertussis toxin did not affect the inhibition by Wnt-1 of NGF-induced neurite outgrowth from either twnt3-C10 or twnt-4 cells.…”