Modification of adipocyte membrane adenylyl cyclase activity by NAD: evidence against NAD-induced endogenous ADP-ribosylation of Gsalpha protein.
Treatment of saponin-permeabilized adipocytes with NAD enhanced adenylyl cyclase activity stimulated by GTgammaS, [Al/F(4)](-), isoproterenol, and forskolin in membrane fractions and potentiated isoproterenol-induced cAMP accumulation in whole cells. In parallel, when permeabilized adipocytes were incubated with [(32)P]NAD, there was significant incorporation of [(32)P]ADP-ribose in a 44-kDa acceptor membrane protein. This reaction was inhibited by l-arginine and was enhanced by the addition of GTPgammaS. Surprisingly, this 44-kDa protein could not be identified as Gs protein: (1) It was not recognized by Gsalpha specific antibody; (2) it did not comigrate with the major cholera toxin substrates in either 10% SDS-PAGE or two-dimensional electrophoresis; (3) a pretreatment of adipocytes with NAD did not decrease cholera toxin-mediated ADP-ribosylation of Gsalpha proteins on membrane fractions. Our results indicate that NAD did not induce endogenous ADP-ribosylation of Gsalpha in permeabilized rat adipocytes but nonetheless modified the adenylyl cyclase response.
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Unité de Recherche, Transduction Hormonale et Régulation Cellulaire, U 486 INSERM, Faculté de Pharmacie 5, rue J. B. Clément, Châtenay-Malabry, 92296, France.
Lambert
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Corrèze
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D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000187:drug effects*; D017667:Adipocytes / Q000201:enzymology*; D001120:Arginine / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D002463:Cell Membrane Permeability; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D009243:NAD / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects
D008565:Membrane Proteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002772:Cholera Toxin; D001120:Arginine; D000242:Cyclic AMP; D011065:Poly(ADP-ribose) Polymerases; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases