CHOLERAGEN CATALYZES ADP-RIBOSYLATION OF THE STIMULATORY G-PROTEIN HETEROTRIMER BUT NOT ITS FREE ALPHA-SUBUNIT

The heterotrimeric (alpha beta gamma) stimulatory G protein (G(S)) med iates activation of adenylylcyclase. G(S) is inactive when GDP is boun d to the guanine nucleotide binding site of the alpha-subunit (G(S) al pha). G(S) can be activated by fluoroaluminate or by binding GTP or GT P analogues, (e.g., GTP gamma S) in place of GDP. Magnesium ion is als o required for the activation of G(S), and G(S) alpha is a substrate f or ADP-ribosylation catalyzed by choleragen (CT). G(S) activation can also be accompanied by dissociation of G(S) alpha from the G beta gamm a-subunit complex. When dissociated G(S) subunits were separated by ch romatography, isolated G(S) alpha could not be ADP-ribosylated by CT u nless G beta was added back. RM/1 antiserum against G(S) alpha was use d to immunoprecipitate G(S), and the subunit composition of the immuno precipitate was determined. When G(S) was incubated with 2 mM MgCl2, t he G(S) heterotrimer was immunoprecipitated, and G(S) alpha could be A DP-ribosylated by CT. Activation of G(S) with GTP gamma S or fluoroalu minate in the presence of 2 mM MgCl2 did not cause G(s) subunit dissoc iation nor did it affect the ability of G(S) alpha to be ADP-ribosylat ed by CT. MgCl2 caused a dose-dependent decrease in the amount of G be ta that coprecipitated with G(S) alpha in the absence as well as the p resence of GTP gamma S or fluoroaluminate. G(S) subunit dissociation w as accompanied by a corresponding decrease in CT-catalyzed ADP-ribosyl ation of G(S) alpha regardless of whether or not GTP gamma S or fluoro aluminate was bound to G(S) alpha. If G(S) subunits were dissociated w ith 120 mM MgCl2 and fluoroaluminate, and the concentration of MgCl2 w as subsequently reduced to 2 mM, subunit reassociation occurred, and t he ability of G(S) alpha to be ADP-ribosylated by CT was restored.