Potent activation of dopamine D-3/D-2 heterodimers by the antiparkinsonian agents, S32504, pramipexole and ropinirole

Recombinant, human dopamine D-3 and D-2 receptors form functional heterodimers upon co-expression in COS-7 cells. Herein, actions of the antiparkinsonian agents, S32504, ropinirole and pramipexole, at D-3/D-2L heterodimers were compared to their effects at the respective monomers and at split, chimeric D-3trunk/D-2tail and D-2trunk/D-3tail receptors: the trunk incorporated transmembrane domains (TDs) I-V and the tail TDs VI and VII. In binding assays with the antagonist [H-3]nemonapride, all agonists were potent ligands of D-3 receptors showing, respectively, 100-, 18- and 56-fold lower affinity at D-2L receptors, mimicking the selective D-3 receptor antagonist, S33084 (100-fold). At D-3trunk/D-2tail receptors, except for ropinirole, all drugs showed lower affinities than at D-3 sites, whereas for D/(2trunk)/D-3tail receptors, affinities of all drugs were higher than at D-2L sites. The proportion of high affinity binding sites recognized by S32504, pramipexole and ropinirole in membranes derived from cells co-expressing D-3 and D-2L sites was higher than in an equivalent mixture of membranes from cells expressing D-3 or D-2L sites, consistent with the promotion of heterodimer formation. In contrast, the percentage of high and low affinity sites (biphasic isotherms) recognized by S33084 was identical. Functional actions were determined by co-transfection of a chimeric adenylyl cyclase (AC)-V/VI insensitive to D-3 receptors. Accordingly, D-3 receptor-transfected cells were irresponsive whereas, in D-2L receptor-transfected cells, agonists suppressed forskolin-stimulated cAMP production with modest potencies. In cells co-transfected with D-3 and D-2L receptors, S32504, ropinirole and pramipexole potently suppressed AC-V/VI with EC(50)s 33-, 19- and 11-fold lower than at D-2L receptors, respectively. S32504 also suppressed AC-V/VI activity at split D-3trunk/D-2tail and D/(2trunk)/D-3tail chimeras transfected into COS-7 cells. In conclusion, antiparkinson agents behave as potent agonists at D/(3)/D/(2)'heterodimers', though any role in their actions in vivo remains to be demonstrated.