An arginine residue is the site of receptor-stimulated, cholera toxin-catalysed ADP-ribosylation of pertussis toxin-sensitive G-proteins.
Cholera toxin-catalysed [32P]ADP-ribosylations were performed in the absence of guanine nucleotides on membranes of a clone (1C) of Rat 1 fibroblasts which express high levels of the alpha 2-C10 adrenergic receptor. As well as incorporation of radioactivity into 45,000 and 42,000 M(r) polypeptides which represent forms of Gs alpha, there was weak labelling of a 40,000 M(r) polypeptide(s). Addition of the alpha 2 adrenergic agonist UK14304 to such assays enhanced markedly the incorporation of radioactivity into the 40,000 M(r) polypeptide(s) but did not alter labelling of the forms of Gs. We have previously demonstrated that the 40,000 M(r) polypeptide(s) labelled in such a manner represents a combination of the alpha subunits of Gi2 and Gi3 [Milligan et al. (1991) J. biol. Chem. 266, 6447-6455]. Mercuric acetate treatment of membranes prelabelled with [32P]ADP-ribose by exposure to pertussis toxin and [32P]NAD removed totally the radiolabel from both Gi2 and Gi3. However, cholera toxin-catalysed [32P]ADP-ribosylation of either the alpha subunits of the Gi-subtypes or of forms of Gs was unaffected by such treatment. By contrast, prolonged, but not short-term, exposure to neutral hydroxylamine removed radiolabel incorporated by cholera toxin from the Gi-subtypes and from Gs but did not remove [32P]ADP-ribose incorporated by pertussis toxin from the Gi-subtypes. It is concluded that ADP-ribosylation of pertussis toxin-sensitive G-proteins by cholera toxin, which can be induced by exposure of membranes to agonists for receptors which interact with that G-protein, is at an arginine residue. It is suggested that this residue may be Arg 179 in Gi2 alpha and Arg 178 in Gi3 alpha.
Milligan
G
G
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Mitchell
F M
FM
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001120:Arginine; D001792:Blood Platelets / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D037342:Pertussis Toxin; D010761:Phosphorus Radioisotopes; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D010761:Phosphorus Radioisotopes; D011942:Receptors, Adrenergic, alpha; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D001120:Arginine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins