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

Abstract

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.

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

“…To address this issue, disulfide cross-linking studies were also carried out with receptor-containing membranes that had been pretreated with high concentration (5 M) of the chaotropic agent, urea. Previous studies have shown that this strategy leads to the almost complete inactivation or removal of heterotrimeric G proteins, while leaving uncoupled receptors fully functional (35,36 Fig. 8A), in good agreement with previous studies using a similar approach to uncouple heterologously expressed 5-HT 2C receptors (35).…”

“…8). Consistent with previous reports (35,36), urea treatment greatly reduced the activity of heterotrimeric G proteins (Fig. 8A), probably due to denaturation of heterotrimeric G proteins and other nonintegral membrane proteins (36).…”

“…Consistent with previous reports (35,36), urea treatment greatly reduced the activity of heterotrimeric G proteins (Fig. 8A), probably due to denaturation of heterotrimeric G proteins and other nonintegral membrane proteins (36). In contrast, urea treatment had essentially no effect on the extent of agonist-dependent disulfide cross-linking displayed by the Y254C/A489C, Y254C/Q490C, Y254C/T491C, and Y254C/L492C mutant receptors (Fig.…”

“…Binding reactions were terminated by rapid filtration on presoaked GF/B filters followed by several washes with cold phosphate buffer (pH 7.4). The bound radioactivity was determined by liquid scintillation spectrometry.Urea Treatment of Cell Membranes-To inactivate G proteins coupled to heterologously expressed mutant M 3 muscarinic receptors, we treated membranes prepared from receptor-expressing COS-7 cells with a high concentration of urea (35,36), using a procedure similar to that described by Lim and Neubig (36). Briefly, membranes were prepared from transfected COS-7 cells grown in a 100-mm dish as described above and centrifuged at 8,000 ϫ g (4°C) for 30 min.…”

C5a Receptor Oligomerization

“…An identical cross-linking pattern was obtained, indicating that the presence of peripheral membrane proteins does not influence the ability of C5a receptors to cross-link. Treatment of membranes with urea has been shown to uncouple GPCRs from G proteins yet maintain receptor activity (35,36), thus allowing for functional reconstitution with purified G proteins. Also, 5 M urea treatment can remove InaD, a PDZ domaincontaining scaffold protein, from photoreceptor membranes (37).…”