Activation of Rat Frizzled-1 Promotes Wnt Signaling and Differentiation of Mouse F9 Teratocarcinoma Cells via Pathways That Require Gαq and Gαo Function*
- From the ‡Department of Pharmacology, State University of New York, Stony Brook, New York 11794-8651,§Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8661, and ¶Howard Hughes Medical Institute and Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195
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 Gαo or Gαq 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 Gαo (a pertussis toxin substrate) and Gαq for signaling by Frizzled-1, and an obligate role for the protein kinase C (likely mediated through stimulation of Gαq) and mitogen-activated protein kinase network at the level of mitogen-activated protein kinase kinase.
Footnotes
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↵* 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.
All authors contributed equally to the work presented herein.
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↵‖ To whom correspondence should be addressed: Dept. of Pharmacology, State University of New York, Stony Brook, NY 11794-8651. Tel.: 516-444-7873; Fax: 516-444-7696; E-mail: craig@pharm.sunysb.edu.
- Abbreviations:
- Rfz-1
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rat frizzled 1
- ODN
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oligodeoxynucleotide
- tPA
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tissue plasminogen activator
- PE
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primitive endoderm
- GFP
-
green fluorescent protein
- EV
-
empty vector
- PCR
-
polymerase chain reaction
- PKC
-
protein kinase C
- MEK
-
mitogen-activated protein kinase kinase DOTAP,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate
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- Received July 20, 1999.
- Revision received August 4, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
