Regulation of ligand binding to pituitary D-2 dopaminergic receptors. Effects of divalent cations and functional...

The effects of divalent cations and protein-modifying reagents on the binding of agonist and antagonist ligands to D-2 dopaminergic receptors were studied. The divalent cations Mg2+, Ca2+, and Mn2+ markedly increase the binding of the agonist [3H]N-n-propylnorapomorphine ([3H]NPA) to the high affinity state of the pituitary D-2 receptor. The increase in [3H]NPA binding is due to both an increase in binding capacity (Bmax) and a decrease in the dissociation constant (KD). Monovalent cations have no effect on agonist binding either in the presence or absence of divalent cations. Neither divalent nor monovalent cations exert any effects on the binding of the antagonist, [3H]spiroperidol to pituitary D-2 receptors. Modification of sulfhydryl groups by N-ethylmaleimide and p-hydroxymercuribenzoate inactivates both [3H]NPA and [3H]spiroperidol binding; however, [3H]NPA binding exhibits a 50-fold greater sensitivity to N-ethylmaleimide. Inactivation of both [3H]NPA and [3]spiroperidol binding by N-ethylmaleimide and p-hydroxymercuribenzoate results in a diminished binding capacity for both radioligands. The inactivation of [3H] spiroperidol-binding by N-ethylmaleimide and p-hydroxymercuribenzoate can be protected against by occupying the receptor site with either antagonists or agonists, however, such treatments do not protect against inactivation of [3H]NPA binding. The effects of selectively decreasing the receptor subunit concentration on the proportions of high and low affinity agonist-binding states was additionally examined. A phenoxybenzamine-induced decrease of up to 63% of the receptor-binding sites has no effect on the proportions of agonist-binding states as detected with agonist/[3H]spiroperidol-competition curves. These results can all be incorporated into a two-step, three-component ternary complex model in which the second binding reaction appears to be under equilibrium restraint.