D1-D2 dopamine receptor synergy promotes calcium signaling via multiple mechanisms

The D(1) dopamine receptor (D(1)R) has been proposed to form a hetero-oligomer with the D(2) dopamine receptor (D(2)R), 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 D(1) and D(2) subtypes, either alone or in combination, and using subtype selective ligands revealed that concurrent stimulation is required for coupling. Surprisingly, characterization of a putative D(1)-D(2) 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 D(1) and D(2) dopamine receptors enhanced the dopamine-stimulated calcium response. However, this was also observed in cells expressing G(qα) with only the D1R. Inactivation of Gi or Gs with pertussis or cholera toxin, respectively, largely, but not entirely, reduced the calcium response in D(1)R and D(2)R 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 D(1)R/D(2)R-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 D(1)-D(2) heteromers, and its significant cross-reactivity to other receptors warrants careful interpretation of its use in vivo.