Characterisation of Gs activation by dopamine D1 receptors using an antibody capture assay: antagonist properties of clozapine.
Herein, we examined the direct coupling of human dopamine D1 receptors to G(s) proteins using an antibody capture assay together with a detection technique employing scintillation proximity assay beads. Using a specific antibody, dopamine (DA) and the selective dopamine D1 receptor agonists, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF81297) and 3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82958), behaved as high-efficacy agonists ( approximately 100%) in stimulating guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding to G(s) in L-cells, whereas 2,3,4,5,-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF38393) displayed partial agonist properties (70%). The action of dopamine was specifically mediated by human dopamine D1 receptors inasmuch as the selective human dopamine D1 receptor antagonist, (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol (SCH23390), blocked dopamine-induced [35S]GTP gamma S binding to G(s) with a pK(B) (9.29) close to its pK(i) (9.33). The antipsychotic agents, clozapine and haloperidol, displayed no intrinsic activity when tested alone and inhibited dopamine-stimulated G(s) activation with pK(B)'s of 6.7 and 7.3, respectively, values close to their pK(i) values at these sites. In conclusion, the use of an anti-G(s) protein immunoprecipitation assay coupled to scintillation proximity assays allows direct evaluation of the functional activity of dopamine D1 receptors ligands at the G protein level. Employing this novel technique, the typical and atypical antipsychotics, clozapine and haloperidol, respectively, both exhibited antagonist properties at dopamine D1 receptors.
Cussac
Didier
D
Psychopharmacology Department, Institut de Recherches Servier, Centre de Recherches de Croissy, 125, Chemin de Ronde, 78290 Croissy-sur-Seine, France.
Pasteau
Valérie
V
Millan
Mark J
MJ
D000818:Animals; D000906:Antibodies / Q000378:metabolism* / Q000494:pharmacology; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D003024:Clozapine / Q000378:metabolism / Q000494:pharmacology*; D004298:Dopamine / Q000378:metabolism; D018492:Dopamine Antagonists / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007739:L Cells; D051379:Mice; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D017447:Receptors, Dopamine D1 / Q000037:antagonists & inhibitors* / Q000502:physiology*
D000906:Antibodies; D018492:Dopamine Antagonists; D017447:Receptors, Dopamine D1; D019205:GTP-Binding Protein alpha Subunits, Gs; D003024:Clozapine; D004298:Dopamine