Pertussis toxin-insensitive activation of the heterotrimeric G-proteins Gi/Go by the NG108-15 G-protein activator.
A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergent-solubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG108-15 G-protein activator (NG-GPA)) increased [(35)S]guanosine 5'-O-(thiotriphosphate) ([(35)S]GTPgammaS) to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from Galpha(o). The NG-GPA also increased GTPgammaS binding to purified, recombinant Galpha(i2), Galpha(i3), and Galpha(o), but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTPgammaS binding to membrane-bound G-proteins and inhibited basal, forskolin- and hormone-stimulated adenylyl cyclase activity in DDT(1)-MF-2 cell membranes. In contrast to G-protein coupled receptor-mediated activation of heterotrimeric G-proteins in DDT(1)-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTPgammaS binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on G(i)/G(o) proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to G(i)/G(o) signaling systems.
Ribas
Catalina
C
Centro de Biologia Molecular, Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, Cantoblanco, 28049-Madrid, Spain.
Takesono
Aya
A
Sato
Motohiko
M
Hildebrandt
John D
JD
Lanier
Stephen M
SM
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D005910:Glioma; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009447:Neuroblastoma; D037342:Pertussis Toxin / Q000494:pharmacology*; D010761:Phosphorus Radioisotopes; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D010761:Phosphorus Radioisotopes; D009243:NAD; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D007545:Isoproterenol