A mutant substituting valine for glycine at position 49 of Gs alpha induces neuronal differentiation of PC12 cells without activation of adenylate cyclase.
A GTPase-deficient mutant of the alpha-subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylate cyclase, substituting valine for glycine 49 (G49V) was transiently expressed in COS7 cells. The basal level of cAMP as well as an agonist-dependent accumulation of cAMP was two-fold higher in transfectants of Gs alpha (G49V) than in those of the normal counterpart. A stable transformant of PC12 cells expressing Gs alpha (G49V) under the control of a metallothionein promoter was then established. Two independent clones showed neurite outgrowth when the mutated Gs alpha was expressed by adding Cd2+ to culture medium. However, the level of basal cAMP of the transformant of PC12 cells with Gs alpha (G49V) was lower than that of the parental cells. The response to an agonist of the adenosine A2-receptor was suppressed in transformants. Although a cAMP-responsive element (CRE) was slightly activated by transfection and transient expression of Gs alpha in PC12 cells, no activation but a suppression of CRE was observed with PC12 cells transiently expressing Gs alpha (G49V). These results suggest that the mutant Gs alpha (G49V) couples adenylate cyclase in a different way in PC12 cells and may transduce differentiation signals through a cAMP-independent pathway.
Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, Tokyo, Japan.
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AM
Kabir
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T
Kozasa
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Y
Kaziro
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Nakamura
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000172:deficiency / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005998:Glycine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology*; D009838:Oligodeoxyribonucleotides; D016716:PC12 Cells; D051381:Rats; D015398:Signal Transduction; D014162:Transfection; D014633:Valine
D009838:Oligodeoxyribonucleotides; D000242:Cyclic AMP; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D014633:Valine; D005998:Glycine