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 guanine 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.
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D000818:Animals; D001552:Benzazepines / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003342:Corpus Striatum / Q000378:metabolism; D004072:Digitonin / Q000378:metabolism; D004285:Dogs; D006150:Guanine Nucleotides / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D009638:Norepinephrine / Q000378:metabolism; D011954:Receptors, Dopamine / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D013237:Stereoisomerism; D013764:Tetrahydronaphthalenes / Q000378:metabolism
D001552:Benzazepines; D006150:Guanine Nucleotides; D011954:Receptors, Dopamine; D013764:Tetrahydronaphthalenes; D012701:Serotonin; D006165:Guanylyl Imidodiphosphate; D012965:Sodium Chloride; C011639:ADTN; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D004072:Digitonin; D009638:Norepinephrine