Abstract
Muscarinic receptors purified from porcine atria and devoid of G protein underwent a 9-27-fold decrease in their apparent affinity for the antagonists quinuclidinyl benzilate, N-methylscopolamine, and scopolamine when treated with the thiol-selective reagent N-ethylmaleimide. Their apparent affinity for the agonists carbachol and oxotremorine-M was unchanged. Conversely, the rate of alkylation by N-ethylmaleimide, as monitored by the binding of [(3)H]quinuclidinyl benzilate, was decreased by antagonists while agonists were without effect. The receptor also underwent a time-dependent inactivation that was hastened by N-ethylmaleimide but slowed by quinuclidinyl benzilate and N-methylscopolamine. The destabilizing effect of N-ethylmaleimide was counteracted fully or nearly so at saturating concentrations of each antagonist and the agonist carbachol. Similar effects occurred with human M(2) receptors differentially tagged with the c-Myc and FLAG epitopes, coexpressed in Sf9 cells, and extracted in digitonin/cholate. The degree of coimmunoprecipitation was unchanged by N-ethylmaleimide, which therefore was without discernible effect on oligomeric size. The data are quantitatively consistent with a model in which the purified receptor from porcine atria interconverts spontaneously between two states (i.e. R R*). Antagonists favor the R state; agonists and N-ethylmaleimide favor the comparatively unstable R* state, which predominates after purification. Occupancy by a ligand stabilizes both states, and antagonists impede alkylation by favoring R over R*. Similarities with constitutively active receptors suggest that R and R* are akin to the inactive and active states, respectively. Purified M(2) receptors therefore appear to exist predominantly in their active state.
Highlights
Sulfhydryl-specific reagents have been useful probes of the relationship between structure and function in muscarinic and other G protein-coupled receptors (e.g. Refs. 1–5)
Detergents, Antisera, and Other Materials—N-[3H]Methylscopolamine was obtained as the chloride salt from PerkinElmer Life Sciences and as the bromide salt from Amersham Biosciences. (Ϫ)-[3H]Quinuclidinyl benzilate was obtained from PerkinElmer Life Sciences
Binding of Radiolabeled Antagonists and the Stability of Native and Alkylated Receptors—Upon treatment with N-ethylmaleimide for 24 h at 0 °C, M2 receptors purified from porcine atria underwent a 20-fold reduction in their affinity for [3H]quinuclidinyl benzilate and a decrease of 10 –30% in the corresponding capacity
Summary
Sulfhydryl-specific reagents have been useful probes of the relationship between structure and function in muscarinic and other G protein-coupled receptors (e.g. Refs. 1–5). Further support for the existence of multiple interconverting states, only one or comparatively few of which are functionally active, derives from the occurrence of constitutive activity as seen, for example, in mutants of the 2 adrenergic receptor [15] or the M5 muscarinic receptor [16]. In the latter case, it was demonstrated that the ligand-regulated activity of 13 constitutively active mutants, differing only at position 465 in helix number 6, can be accounted for by shifts in a single equilibrium between an inactive and an active state [16]. N-Ethylmaleimide appears to favor the state that is of weaker affinity for antagonists and undergoes a comparatively rapid inactivation, a pattern that is shared by constitutively active receptors (e.g. Refs. 4, 19, and 20)
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