An arginine residue is the site of receptor-stimulated, cholera toxin-catalysed ADP-ribosylation of pertussis toxin-sensitive G-proteins

Abstract

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 α2-C10 adrenergic receptor. As well as incorporation of radioactivity into 45,000 and 42,000 M r polypeptides which represent forms of G sα, there was weak labelling of a 40,000 M r polypeptide(s). Addition of the α2 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 G s. We have previously demonstrated that the 40,000 M r polypeptide(s) labelled in such a manner represents a combination of the α subunits of G i2 and G i3 [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 G i2 and G i3. However, cholera toxin-catalysed [ 32P]ADP-ribosylation of either the α subunits of the G i-subtypes or of forms of G s was unaffected by such treatment. By contrast, prolonged, but not short-term, exposure to neutral hydroxylamine removed radiolabel incorporated by cholera toxin from the G i-subtypes and from G s but did not remove [ 32P]ADP-ribose incorporated by pertussis toxin from the G i-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 G i2α and Arg 178 in G i3α.