G-protein-coupled receptors activate signal-transducing G-proteins, which consist of an α subunit and a βγ dimer. Membrane extraction with 5–7M urea has been used to uncouple receptors from endogenous G-proteins to permit reconstitution with purified G-proteins. We show that αi subunits are inactivated with 5M urea whereas the βγ dimer requires at least 7M urea for its inactivation. There is no significant loss of receptors. Surprisingly, Western-blot analysis indicates that the urea-denatured αi subunit remains mostly membrane-bound and that β is only partially removed. After 7M urea treatment, both αi1 and βγ subunits are required to restore high-affinity agonist binding and receptor-catalysed guanosine 5′-[γ-thio]triphosphate binding. We demonstrate the generality of this approach for four Gi-coupled receptors (α2A-adrenergic, adenosine A1, 5-hydroxytryptamine1A and µ-opioid) expressed in insect cells and two mammalian cell lines. Thus a selectivity of urea for G-protein α versus βγ subunits is established in both concentration and mechanism.

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