Dopamine D₁ receptors characterized with [³H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D₁ receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D₁ receptors, as assessed by the binding of the selective antagonist [³H]SCH 23390. The binding of [³H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [³H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D₁ receptors. Moreover, agonist high affinity binding to D₁ receptors and its sensitivity to guainine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D₁ receptors labeled with [³H]dopamine (agonist) or [³H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [³H]dopamine but not [³H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D₁ receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D₂ receptor, and the molecular mechanism responsible for this difference remains to be elucidated.