Functional activation of beta-adrenergic receptors by thiols in the presence or absence of agonists.

S E Pedersen,E M Ross

doi:10.1016/s0021-9258(17)38696-9

Abstract

Treatment of beta-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 beta-adrenergic receptors, purified to greater than or equal to 95% homogeneity from turkey erythrocyte plasma membranes, and the stimulatory GTP-binding protein of the adenylate cyclase system (Gs) purified from rabbit liver. Incubation of the vesicles with 2-10 mM DTT at 0 degrees C for 1 h increased the rate (4-5-fold) and the extent (3-4-fold) of activation of Gs by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding, an effect about equivalent to the addition of beta-adrenergic agonists. Treatment with DTT also markedly potentiated the ability of agonists to stimulate GTP gamma S 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 GTP gamma S 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 Gs alone. Thus, the site of action of DTT appears to be on the beta-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.

Highlights

Treatment of @-adrenergicreceptor with dithio- the activity and regulation of hormone-sensitive adenylate threitol (DTT) or other thiol compoundscaused its cyclase have been recognized for some time
The catalytic unit is inactivated by prolonged incubation with relatively high concentrations of dithiothreitol; indicating the presence of required disulfide bonds
The isolated receptor is insensitive was as effective as agonist in stimulating GTPase ac- to sulfhydryl-alkylating agents [5], and N-ethylmaleimide by tivity, and maximal stimulation was obtained when itself does not inactivate the receptor in plasma membranes

Summary

RESULTS

Brandt et al [5]. Briefly, the affinity column was washed with In the course of developing a generally useful protocol for buffer containing 20 mM NaHepes (pH 8.0), 5 mM EDTA, 0.1 mM the reconstitution of @-adrenergicreceptors and G, into phos-. Rate or extent of activation of purified G. alone, either in detergent solution [5] or after its reconstitution into phospholipid vesicles (datanot shown).whenincreasing amounts of receptor were included in the reconstitution, the ability of DTT to stimulate G. activation was enhanced essentially proportionately (Fig. 6). This requirement for active receptorswas observed when vesicles wperrepared using several of our most highly purified preparations of receptors, which weregreater than 95% pure accordingto silver-stained.

GTPrS binding

DTT Basal

DISCUSSION