Title Page: 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 PLC-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 employing subtype selective ligands, revealed that concurrent stimulation is required for coupling. Surprisingly, characterization of a putative D 1-D 2 heteromer-selective ligand, SKF83959, found no stimulation of calcium release, but a broad range of cross-reactivity with other GPCRs. In contrast, SKF83959 appeared to be an antagonist of calcium mobilization. Over-expression of G qα with the D 1 and D 2 DARs enhanced the dopamine-stimulated calcium response. However, this was also observed in cells expressing G qα with only the D 1 R. Inactivation of G i or G s with pertussis or cholera toxin, respectively, was found to largely, but not entirely reduce the calcium response in D 1 R and D 2 R co-transfected cells. Moreover, we found that sequestration of G β γ subunits through over-expression of GRK2 mutants either completely or largely eliminated dopamine-stimulated calcium mobilization. Our data suggests 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.