CHARACTERIZATION OF THE FUNCTIONAL-ACTIVITY OF DOPAMINE LIGANDS AT HUMAN RECOMBINANT DOPAMINE D-4 RECEPTORS

The human D-4 dopamine receptor has been expressed in Sf9 insect cells where it appears to couple to endogenous G proteins. Increased guanin e nucleotide exchange to G proteins is a reflection of receptor activa tion and can be followed using a [S-35]GTP gamma S binding assay. By m easuring D-4 receptor stimulation of [S-35]GTP gamma S binding we have been able to characterize several dopaminergic compounds for their fu nctional activity at this receptor. In Sf9 cells expressing the D-4 re ceptor, dopamine, quinpirole, and dp-2-aminodihydroxy-1,2,3,4-tetrahyd ronaphthalene were all full agonists, whereas (-)apomorphine appeared to be a partial agonist. No increase in [S-35]GTP gamma S binding was observed for noninfected cells or cells infected with an unrelated seq uence. The quinpirole-stimulated [S-35]GTP gamma S binding could be in hibited by the antagonists clozapine, eticlopride, and haloperidol, an d a Schild analysis of these data showed that all three compounds were acting as competitive antagonists of D-4 receptors. The rank order of affinities derived from the Schild analysis correlated with that obta ined from [H-3]spiperone competition binding assays. In conclusion, we have shown that, using this assay system, it is possible to investiga te functionally the pharmacology of a recombinant G protein-coupled re ceptor in the absence of any information regarding the eventual second messenger pathways involved.