A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. II. Purification, characterization, and reconstitution.
In the previous paper, we reported the identification of a 74-kDa G-protein that co-purifies with the alpha 1-adrenergic receptor following ternary complex formation. We report here on the purification and characterization of this 74-kDa G-protein (termed Gh) isolated de novo from rat liver membranes. After solubilization of rat liver membranes with the detergent sucrose monolaurate, Gh was isolated by sequential chromatography using heparin-agarose, Ultrogel AcA 34, hydroxylapatite, and heptylamine-Sepharose columns. The protein, thus isolated, is not a substrate for cholera or pertussis toxin but displays GTPase activity (turnover number, 3-5 min-1) and high-affinity guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding (half-maximal binding = 0.25-0.3 microM), which is Mg2(+)-dependent and saturable. The relative order of nucleotide binding by Gh is GTP gamma S greater than GTP greater than GDP greater than ITP much much greater than ATP greater than or equal to adenyl-5'-yl imidodiphosphate, which is similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, specific alpha 1-agonist-stimulated GTPase (turnover number, 10-15 min-1) and GTP gamma S binding activity could be demonstrated after reconstitution of purified Gh with partially purified alpha 1-adrenergic receptor into phospholipid vesicles. The alpha 1-agonist stimulation of GTP gamma S binding and GTPase activity was inhibited by the alpha-antagonist phentolamine. A 50-kDa protein co-purifies with the 74-kDa G-protein. This protein does not bind guanine nucleotides and may be a subunit (beta-subunit) of Gh. These findings indicate that Gh is a G-protein that functionally couples to the alpha 1-adrenergic receptor.
Im
M J
MJ
Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Boston.
Riek
R P
RP
Graham
R M
RM
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D005260:Female; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D011942:Receptors, Adrenergic, alpha / Q000302:isolation & purification* / Q000378:metabolism
D011942:Receptors, Adrenergic, alpha; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D008274:Magnesium