A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. I. Identification by photolabeling or membrane and ternary complex preparation.
The G-protein involved in alpha 1-adrenergic receptor signaling was identified using two different approaches. First, purified rat liver membranes were incubated with [alpha-32P]GTP in the absence or presence of the adrenergic agonist (-)-epinephrine, or in the presence of GTP. After UV irradiation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography, covalent labeling of a number of proteins was apparent and could be blocked by unlabeled GTP. In the preparation treated with (-)-epinephrine alone, labeling of a 74-kDa species was markedly enhanced. Enhanced labeling of 40-50-kDa species was also observed. Labeling of the 74-kDa protein was also evident in similarly treated membranes prepared from FRTL-5 thyroid cells, which contain abundant alpha 1-adrenergic receptors, but not in those prepared from turkey erythrocytes or NIH 3T3 fibroblasts, which are essentially devoid of alpha 1-receptors. Second, alpha 1-agonist-receptor-G-protein ternary complex formation was induced by incubating purified rat liver membranes with (-)-epinephrine. Rauwolscine (10(-7) M) and (+/-)-propranolol (10(-6) M) were included to prevent activation of alpha 2- and beta-adrenergic receptors by (-)-epinephrine. The ternary complex of hormone, receptor, and G-protein was solubilized, partially purified using heparin- and wheat germ agglutinin-agarose, and reconstituted into phospholipid vesicles. The vesicles displayed agonist-stimulated guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding that was blocked by phentolamine (10(-4) M). By contrast, stimulation of GTP gamma S binding was not evident when the vesicles were incubated with the beta-agonist, isoproterenol. Incubation of the vesicles with [alpha-32P]GTP or [alpha-32P]azido-GTP in the presence of (-)-epinephrine, followed by photolysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography, resulted in the covalent labeling of a 74-kDa protein. Labeling of this protein could be blocked by preincubation with phentolamine or unlabeled GTP. These findings provide direct evidence for the coupling of the alpha 1-adrenergic receptor to a previously uncharacterized G-protein (termed Gh), which has an apparent molecular mass of approximately 74 kDa.
Im
M J
MJ
Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Boston.
Graham
R M
RM
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; 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*; D046911:Macromolecular Substances; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011942:Receptors, Adrenergic, alpha / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D011942:Receptors, Adrenergic, alpha; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins