Characterisation of Gs activation by dopamine D1 receptors using an antibody capture assay: antagonist properties of clozapine

Eur J Pharmacol. 2004 Feb 6;485(1-3):111-7. doi: 10.1016/j.ejphar.2003.11.077.


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.

MeSH terms

  • Animals
  • Antibodies / metabolism*
  • Antibodies / pharmacology
  • Antibody Specificity
  • Binding Sites, Antibody
  • Binding, Competitive
  • Clozapine / metabolism
  • Clozapine / pharmacology*
  • Dopamine / metabolism
  • Dopamine Antagonists / metabolism
  • Dopamine Antagonists / pharmacology*
  • Dose-Response Relationship, Drug
  • GTP-Binding Protein alpha Subunits, Gs / antagonists & inhibitors*
  • GTP-Binding Protein alpha Subunits, Gs / immunology
  • GTP-Binding Protein alpha Subunits, Gs / metabolism*
  • Humans
  • L Cells
  • Mice
  • Radioligand Assay / methods*
  • Rats
  • Receptors, Dopamine D1 / antagonists & inhibitors*
  • Receptors, Dopamine D1 / physiology*


  • Antibodies
  • Dopamine Antagonists
  • Receptors, Dopamine D1
  • GTP-Binding Protein alpha Subunits, Gs
  • Clozapine
  • Dopamine