Potent activation of dopamine D3/D2 heterodimers by the antiparkinsonian agents, S32504, pramipexole and ropinirole.
Recombinant, human dopamine D3 and D2 receptors form functional heterodimers upon co-expression in COS-7 cells. Herein, actions of the antiparkinsonian agents, S32504, ropinirole and pramipexole, at D3/D2L heterodimers were compared to their effects at the respective monomers and at split, chimeric D3trunk/D2tail and D2trunk/D3tail receptors: the trunk incorporated transmembrane domains (TDs) I-V and the tail TDs VI and VII. In binding assays with the antagonist [3H]nemonapride, all agonists were potent ligands of D3 receptors showing, respectively, 100-, 18- and 56-fold lower affinity at D2L receptors, mimicking the selective D3 receptor antagonist, S33084 (100-fold). At D3trunk/D2tail receptors, except for ropinirole, all drugs showed lower affinities than at D3 sites, whereas for D2trunk/D3tail receptors, affinities of all drugs were higher than at D2L sites. The proportion of high affinity binding sites recognized by S32504, pramipexole and ropinirole in membranes derived from cells co-expressing D3 and D2L sites was higher than in an equivalent mixture of membranes from cells expressing D3 or D2L 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 D3 receptors. Accordingly, D3 receptor-transfected cells were irresponsive whereas, in D2L receptor-transfected cells, agonists suppressed forskolin-stimulated cAMP production with modest potencies. In cells co-transfected with D3 and D2L receptors, S32504, ropinirole and pramipexole potently suppressed AC-V/VI with EC50s 33-, 19- and 11-fold lower than at D2L receptors, respectively. S32504 also suppressed AC-V/VI activity at split D3trunk/D2tail and D2trunk/D3tail chimeras transfected into COS-7 cells. In conclusion, antiparkinson agents behave as potent agonists at D3/D2'heterodimers', though any role in their actions in vivo remains to be demonstrated.
Maggio
Roberto
R
Department of Neuroscience, University of Pisa, Italy. r.maggio@drugs.med.unipi.it
Scarselli
Marco
M
Novi
Francesca
F
Millan
Mark J
MJ
Corsini
Giovanni U
GU
D000262:Adenylyl Cyclases / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D000978:Antiparkinson Agents / Q000494:pharmacology*; D052160:Benzothiazoles; D001667:Binding, Competitive / Q000187:drug effects; D019556:COS Cells; D005576:Colforsin / Q000494:pharmacology; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D008024:Ligands; D009714:Nucleus Accumbens / Q000378:metabolism; D010078:Oxazines / Q000494:pharmacology*; D037342:Pertussis Toxin / Q000494:pharmacology; D000077487:Pramipexole; D011485:Protein Binding / Q000187:drug effects; D017448:Receptors, Dopamine D2 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D050637:Receptors, Dopamine D3; D013844:Thiazoles / Q000494:pharmacology*; D014162:Transfection
D000978:Antiparkinson Agents; D052160:Benzothiazoles; C493593:DRD3 protein, human; D007211:Indoles; D008024:Ligands; D010078:Oxazines; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; C478818:S 32504; D013844:Thiazoles; C113776:dopamine D2L receptor; C046649:ropinirole; D005576:Colforsin; D000077487:Pramipexole; D037342:Pertussis Toxin; D000262:Adenylyl Cyclases