Selective inactivation of guanine-nucleotide-binding regulatory protein (G-protein) alpha and betagamma subunits by urea.
G-protein-coupled receptors activate signal-transducing G-proteins, which consist of an alpha subunit and a betagamma 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(i) subunits are inactivated with 5 M urea whereas the betagamma 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(i) subunit remains mostly membrane-bound and that beta is only partially removed. After 7 M urea treatment, both alpha(i1) and betagamma 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 betagamma subunits is established in both concentration and mechanism.
Lim
W K
WK
Department of Pharmacology, The University of Michigan, Ann Arbor, MI 48109-0632, U.S.A.
Neubig
R R
RR
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D021122:Protein Subunits; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D014508:Urea / Q000008:administration & dosage / Q000494:pharmacology*
D021122:Protein Subunits; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014508:Urea; D020962:Heterotrimeric GTP-Binding Proteins