Selective inactivation of guanine-nucleotide-binding regulatory protein (G-protein) α and βγ subunits by urea

G-protein-coupled receptors activate signal-transducing G-proteins, which consist of an alpha subunit and a beta gamma dimer. Membrane extraction with 5-7 M urea has been used to uncouple receptors from endogenous G-proteins to permit reconstitution with purified G-proteins. We show that alpha (1) subunits are inactivated with 5 M urea whereas the beta gamma dimer requires at least 7 M urea for its inactivation. There is no significant loss of receptors. Surprisingly, Western-blot analysis indicates that the urea-denatured alpha (1) subunit remains mostly membrane-bound and that beta is only partially removed. After 7 M urea treatment, both alpha (11) and beta gamma subunits are required to restore high-affinity agonist binding and receptor-catalysed guanosine 5'-[gamma -thio]triphosphate binding. We demonstrate the generality of this approach for four G(i)-coupled receptors (alpha (2A)-adrenergic, adenosine A1,5-hydroxytryptamine(1A) and mu -opioid) expressed in insect cells and two mammalian cell lines. Thus a selectivity of urea for G-protein alpha versus beta gamma subunits is established in both concentration and mechanism.