TY - JOUR
T1 - Functional activation of β-adrenergic receptors by thiols in the presence or absence of agonists
AU - Pedersen, S. E.
AU - Ross, E. M.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1985
Y1 - 1985
N2 - Treatment of β-adrenergic receptor with dithiothreitol (DTT) or other thiol compounds caused its functional activation in the presence or absence of agonist ligands. Such activation was observed in reconstituted unilamellar phospholipid vesicles that contained β-adrenergic receptors, purified to ≥ 95% homogeneity from turkey erythrocyte plasma membranes, and the stimulatory GTP-binding protein of the adenylate cyclase system (G(s)) purified from rabbit liver. Incubation of the vesicles with 2-10 mM DTT at 0° C for 1 h increased the rate (4-5-fold) and the extent (3-4-fold) of activation of G(s) by guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding, an effect about equivalent to the addition of β-adrenergic agonists. Treatment with DTT also markedly potentiated the ability of agonists to stimulate GTPγS binding, increasing the initial rate about 10-fold. DTT treatment was as effective as agonist in stimulating GTPase activity and maximal stimulation was obtained when DTT-treated vesicles were assayed in the presence of agonist. Other thiol compounds produced effects similar to those of DTT but were at least 10-fold less potent. Stimulation of GTPγS binding or GTPase activity required active receptor, and treatment of the receptor with DTT prior to reconstitution also increased its efficacy. There was no effect of DTT on G(s) alone. Thus, the site of action of DTT appears to be on the β-adrenergic receptor itself, and the reduction of disulfides and the binding of agonist act synergistically to activate the receptor. DTT treatment made the receptor more labile to thermal denaturation. Inclusion of cholesterol or cholesteryl-hemisuccinate (5-25%) in the vesicles protected the reduced receptor against such denaturation and enhanced its recovery during reconstitution. No effect of cholesterol or cholesteryl-hemisuccinate was observed on the stability of the nonreduced receptor, which was comparable to that observed in native membranes.
AB - Treatment of β-adrenergic receptor with dithiothreitol (DTT) or other thiol compounds caused its functional activation in the presence or absence of agonist ligands. Such activation was observed in reconstituted unilamellar phospholipid vesicles that contained β-adrenergic receptors, purified to ≥ 95% homogeneity from turkey erythrocyte plasma membranes, and the stimulatory GTP-binding protein of the adenylate cyclase system (G(s)) purified from rabbit liver. Incubation of the vesicles with 2-10 mM DTT at 0° C for 1 h increased the rate (4-5-fold) and the extent (3-4-fold) of activation of G(s) by guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding, an effect about equivalent to the addition of β-adrenergic agonists. Treatment with DTT also markedly potentiated the ability of agonists to stimulate GTPγS binding, increasing the initial rate about 10-fold. DTT treatment was as effective as agonist in stimulating GTPase activity and maximal stimulation was obtained when DTT-treated vesicles were assayed in the presence of agonist. Other thiol compounds produced effects similar to those of DTT but were at least 10-fold less potent. Stimulation of GTPγS binding or GTPase activity required active receptor, and treatment of the receptor with DTT prior to reconstitution also increased its efficacy. There was no effect of DTT on G(s) alone. Thus, the site of action of DTT appears to be on the β-adrenergic receptor itself, and the reduction of disulfides and the binding of agonist act synergistically to activate the receptor. DTT treatment made the receptor more labile to thermal denaturation. Inclusion of cholesterol or cholesteryl-hemisuccinate (5-25%) in the vesicles protected the reduced receptor against such denaturation and enhanced its recovery during reconstitution. No effect of cholesterol or cholesteryl-hemisuccinate was observed on the stability of the nonreduced receptor, which was comparable to that observed in native membranes.
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M3 - Article
C2 - 2997196
AN - SCOPUS:0022356539
SN - 0021-9258
VL - 260
SP - 14150
EP - 14157
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -