Choleragen catalyzes ADP-ribosylation of the stimulatory G protein heterotrimer but not its free alpha-subunit.
The heterotrimeric (alpha beta gamma) stimulatory G protein (Gs) mediates activation of adenylylcyclase. Gs is inactive when GDP is bound to the guanine nucleotide binding site of the alpha-subunit (Gs alpha). Gs can be activated by fluoroaluminate or by binding GTP or GTP analogues, (e.g., GTP gamma S) in place of GDP. Magnesium ion is also required for the activation of Gs, and Gs alpha is a substrate for ADP-ribosylation catalyzed by choleragen (CT). Gs activation can also be accompanied by dissociation of Gs alpha from the G beta gamma-subunit complex. When dissociated Gs subunits were separated by chromatography, isolated Gs alpha could not be ADP-ribosylated by CT unless G beta was added back. RM/1 antiserum against Gs alpha was used to immunoprecipitate Gs, and the subunit composition of the immunoprecipitate was determined. When Gs was incubated with 2 mM MgCl2, the Gs heterotrimer was immunoprecipitated, and Gs alpha could be ADP-ribosylated by CT. Activation of Gs with GTP gamma S or fluoroaluminate in the presence of 2 mM MgCl2 did not cause Gs subunit dissociation nor did it affect the ability of Gs alpha to be ADP-ribosylated by CT. MgCl2 caused a dose-dependent decrease in the amount of G beta that coprecipitated with Gs alpha in the absence as well as the presence of GTP gamma S or fluoroaluminate. Gs subunit dissociation was accompanied by a corresponding decrease in CT-catalyzed ADP-ribosylation of Gs alpha regardless of whether or not GTP gamma S or fluoroaluminate was bound to Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Toyoshige
M
M
Membrane Biochemistry Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Okuya
S
S
Rebois
R V
RV
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism; D000535:Aluminum; D001665:Binding Sites; D002384:Catalysis; D002772:Cholera Toxin / Q000378:metabolism*; D005461:Fluorine; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015151:Immunoblotting; D015636:Magnesium Chloride; D011233:Precipitin Tests; D011485:Protein Binding; D014407:Tumor Cells, Cultured
D015636:Magnesium Chloride; D000246:Adenosine Diphosphate Ribose; C053629:fluoroaluminum; D005461:Fluorine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002772:Cholera Toxin; D000535:Aluminum; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases