Pertussis toxin-catalyzed ADP-ribosylation of G(o) 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, L353A/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).
Avigan
J
J
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Murtagh
J J
JJ
Stevens
L A
LA
Angus
C W
CW
Moss
J
J
Vaughan
M
M
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D037342:Pertussis Toxin; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012160:Retina / Q000378:metabolism; D013379:Substrate Specificity; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000378:metabolism / Q000494:pharmacology*
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D015293:Transducin