Abstract

The D₁ dopamine receptor (D₁R) has been proposed to form a hetero-oligomer with the D₂ dopamine receptor (D₂R), which in turn results in a complex that couples to phospholipase C–mediated intracellular calcium release. We have sought to elucidate the pharmacology and mechanism of action of this putative signaling pathway. Dopamine dose-response curves assaying intracellular calcium mobilization in cells heterologously expressing the D₁ and D₂ subtypes, either alone or in combination, and using subtype selective ligands revealed that concurrent stimulation is required for coupling. Surprisingly, characterization of a putative D₁-D₂ heteromer-selective ligand, 6-chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3-benzazepine-7,8-diol (SKF83959), found no stimulation of calcium release, but it did find a broad range of cross-reactivity with other G protein–coupled receptors. In contrast, SKF83959 appeared to be an antagonist of calcium mobilization. Overexpression of G_qα with the D₁ and D₂ dopamine receptors enhanced the dopamine-stimulated calcium response. However, this was also observed in cells expressing G_qα with only the D₁R. Inactivation of G_i or G_s with pertussis or cholera toxin, respectively, largely, but not entirely, reduced the calcium response in D₁R and D₂R cotransfected cells. Moreover, sequestration of G_βγ subunits through overexpression of G protein receptor kinase 2 mutants either completely or largely eliminated dopamine-stimulated calcium mobilization. Our data suggest that the mechanism of D₁R/D₂R–mediated calcium signaling involves more than receptor-mediated G_q protein activation, may largely involve downstream signaling pathways, and may not be completely heteromer-specific. In addition, SKF83959 may not exhibit selective activation of D₁-D₂ heteromers, and its significant cross-reactivity to other receptors warrants careful interpretation of its use in vivo.