PERTUSSIS TOXIN-CATALYZED ADP-RIBOSYLATION OF GO-ALPHA WITH MUTATIONS AT THE CARBOXYL TERMINUS

The guanine nucleotide-binding protein G(o-alpha) has been implicated in the regulation of Ca2+ channels in neural tissues. Covalent modification of G(o-alpha) by pertussis toxin-catalyzed ADP-ribosylation of a cysteine (position 351) four amino acids from the carboxyl terminus decouples G(o-alpha) from receptor. To define the structural requirements for ADP-ribosylation, preparations of recombinant G(o-alpha) with mutations within the five amino acids at the carboxyl terminus were evaluated for their ability to serve as pertussis toxin substrates. As expected, the mutant in which cysteine 351 was replaced by glycine (C351G) was not a toxin substrate. Other inactive mutants were G352D and L353-DELTA/Y354-DELTA. Mutations that had no significant effect on toxin-catalyzed ADP-ribosylation included G350D, G350R, Y354-DELTA, and L353V/Y354-DELTA. Less active mutants were L353G/Y354-DELTA, L353-DELTA/Y354-DELTA, and L353G. ADP-ribosylation of the active mutants, like that of wild-type G(o-alpha), was enhanced by the beta-gamma subunits of bovine transducin. It appears that three of the four terminal amino acids critically influence pertussis toxin-catalyzed ADP-ribosylation of G(o-alpha).