Abstract
The frizzled gene family of putative Wnt receptors encodes proteins that have a seven transmembrane-spanning 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 Galpha(o) or Galpha(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 Galpha(o) (a pertussis toxin substrate) and Galpha(q) for signaling by Frizzled-1, and an obligate role for the protein kinase C (likely mediated through stimulation of Galpha(q)) and mitogen-activated protein kinase network at the level of mitogen-activated protein kinase kinase.
MeSH terms
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3',5'-Cyclic-GMP Phosphodiesterases / antagonists & inhibitors
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Animals
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Clone Cells
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Culture Media, Conditioned
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Enzyme Inhibitors / pharmacology
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Fluorescent Antibody Technique, Indirect
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Frizzled Receptors
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GTP-Binding Proteins / antagonists & inhibitors
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GTP-Binding Proteins / metabolism*
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MAP Kinase Kinase Kinase 1*
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MAP Kinase Kinase Kinases / antagonists & inhibitors
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Mice
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Pertussis Toxin
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Phosphoinositide-3 Kinase Inhibitors
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Protein Kinase C / antagonists & inhibitors
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Protein Serine-Threonine Kinases*
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Proto-Oncogene Proteins / metabolism*
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Rats
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Receptors, G-Protein-Coupled
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Receptors, Neurotransmitter / metabolism*
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Signal Transduction*
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Teratocarcinoma / metabolism*
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Teratocarcinoma / pathology
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Tumor Cells, Cultured
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Virulence Factors, Bordetella / pharmacology
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Wnt Proteins
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Xenopus Proteins*
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Zebrafish Proteins*
Substances
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Culture Media, Conditioned
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Enzyme Inhibitors
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FZD1 protein, Xenopus
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Frizzled Receptors
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Fzd1 protein, mouse
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Phosphoinositide-3 Kinase Inhibitors
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Proto-Oncogene Proteins
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Receptors, G-Protein-Coupled
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Receptors, Neurotransmitter
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Virulence Factors, Bordetella
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Wnt Proteins
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Xenopus Proteins
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Zebrafish Proteins
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Fzd1 protein, rat
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Pertussis Toxin
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Protein Serine-Threonine Kinases
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Protein Kinase C
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MAP Kinase Kinase Kinase 1
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MAP Kinase Kinase Kinases
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Map3k1 protein, mouse
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3',5'-Cyclic-GMP Phosphodiesterases
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GTP-Binding Proteins