The mechanism of aluminum-independent G-protein activation by fluoride and magnesium. 31P NMR spectroscopy and fluorescence kinetic studies.
With magnesium present, fluoride and aluminum ions activate heterotrimeric G-proteins by forming AlFx complexes that mimic the gamma phosphate of a GTP. We report compelling evidence for a newly proposed process of G-protein activation by fluoride and magnesium, without Al3+. With millimolar Mg2+ and F-, Gs and Gt activate adenylylcyclase and cGMP-phosphodiesterase, respectively. In 31P NMR, addition of magnesium to Gi1 alpha GDP or Gt alpha GDP solutions containing fluoride, but no Al3+, modifies the chemical shift of the GDP beta phosphorus, suggesting that magnesium interacts with the beta phosphate. Titration of this effect indicates that two Mg2+ are bound per G alpha. Biphasic activation kinetics, monitored by G alpha tryptophan fluorescence, suggests the rapid binding of one Mg2+ to G alpha GDP and the slow association of another Mg2+, in correlation with fluoride binding and G alpha activation. The deactivation rate upon fluoride dilution shows a second order dependence with respect to the residual F- concentration, suggesting the sequential release of at least three F-/G alpha. Thus, in millimolar Mg2+ and F-, and without Al3+, two Mg2+ and three F- bind sequentially to G alpha GDP and induce the switch to an active G alpha (GDP-MgF3)Mg state, which is structurally analogous to G alpha (GDP-AlFx)Mg and to G alpha (GTP)Mg.
Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Valbonne, France.
B
B
Antonny
M
M
Sukumar
J
J
Bigay
M
M
Chabre
T
T
Higashijima
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000535:Aluminum / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D015293:Transducin / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry
D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D014364:Tryptophan; D000535:Aluminum; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D019204:GTP-Binding Proteins; D015293:Transducin; D000262:Adenylyl Cyclases; D008274:Magnesium; D005459:Fluorides