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

Abstract

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.

Fig. 1.

Agonist-induced calcium mobilization in DA receptor–transfected cells. HEK293T cells were transiently transfected with D₁R, D_2LR, D_2SR, D₁R + D_2LR, or D₁R + D_2SR, as indicated and described in Materials and Methods. Twenty-four hours later, cells were plated in 384-well plates and assayed the following day for calcium mobilization after stimulation by DA (D₁R + D_2LR EC₅₀ = 73.8 nM, D₁R + D_2SR, EC₅₀ = 58.2 nM). Data are representative of three independent experiments done with the same assay conditions on different days. Data are expressed as percentage of control, normalized to the maximum signal seen via DA stimulation of D₁R + D_2LR transfected cells. Error bars indicate S.E.M. from multiple wells within the representative experiment.

Fig. 2.

Inhibition of D₁R + D_2LR–mediated calcium mobilization by either D₁R- or D₂R-selective antagonists. HEK293T cells were transfected with D₁R + D_2LR as described and 24 hours later were plated in 384-well plates. Cells were incubated with the indicated concentrations of the D₁R-selective antagonist SCH23390 (A) or the D₂R-selective antagonist sulpiride (B) and then stimulated with an ∼EC₈₀ of DA (1 μM; SCH23390 IC₅₀ = 8.0 nM, sulpiride, IC₅₀ = 0.7 nM). Data are expressed as a percentage of the control (10 μM) DA response and are representative of two independent experiments performed with the same assay conditions on different days. Error bars indicate S.E.M. from multiple wells within the representative experiment.

Fig. 3.

Stimulation of D₁R + D_2LR–mediated calcium mobilization by either D₁R- or D₂R-selective agonists. HEK293T cells were transfected with D₁ R+ D_2LR as described, plated 24 hours later in 384-well plates, and assayed for calcium accumulation the following day. Cells were stimulated with one of the following agonists as indicated: DA, the D₁R-selective agonist SKF83822, the D₂R-selective agonist quinpirole, or both SKF83822 and quinpirole (D₁R + D_2LR EC₅₀ = 610.8 nM) combined. Control cells expressing the D₁R, D_2SR, or D_2LR individually did not show a significant calcium response to concurrent agonist administration. Data are expressed as a percentage of control maximum DA-stimulated response and are representative of two independent experiments performed with the same assay conditions on different days. Error bars indicate S.E.M. from multiple wells within the representative experiment.

Fig. 4.

Pharmacological characterization of SKF83959 on D₁R + D_2LR–mediated calcium mobilization. HEK293T cells were transfected with D₁R + D_2LR as described, plated 24 hours later in 384-well plates, and assayed for calcium accumulation the following day. (A) Cells were stimulated with one of the following conditions as indicated: DA, SKF83959, the D₂R-selective agonist quinpirole, or both SKF83959 and quinpirole combined. (B) Cells were incubated with SKF83959 or the D₁R-selective antagonist SCH23390, then stimulated with an ∼EC₈₀ of DA (1 μM). Data are expressed as a percentage of control maximum DA-stimulated response and are representative of two or three independent experiments performed with the same assay conditions on different days. Error bars indicate S.E.M. from multiple wells within the representative experiment. (C) HEK293 cells stably transfected with D₁R (Codex Biosolutions, Inc., Gaithersburg, MD) were grown and membranes harvested as described in Materials and Methods. Membranes were incubated with various concentrations of SKF83959 and 0.5 nM [³H]SCH23390 as indicated. Graph is representative of two independent experiments done on different days. Data are expressed as specific binding in units of fmol/mg. K_i value was calculated using the Cheng-Prushoff equation and a radioligand K_d value of 0.5 nM as determined via saturation binding isotherms (unpublished data). Average K_i for SKF83959 on D₁R was 2.6 nM ± 0.7.

Fig. 5.

Influence of G_qα protein overexpression on D₁R + D_2LR–mediated calcium mobilization. (A) HEK293T cells were transfected with D₁R + D_2LR with and without G_qα or with G_qα alone (D₁R + D₂R EC₅₀ = 168.3 nM, EC_max = 100%; D₁R + D₂R + G_q EC₅₀ = 16.8 nM, EC_max = 300.1%). (B) HEK293T cells were transfected with D₁R + D_2LR, D₁R, or D₂R with and without G_qα (D₁R + G_q EC₅₀ = 10.3 nM, EC_max = 152.2%). Twenty-four hours later, cells were plated in 384-well plates and assayed the following day for calcium mobilization after stimulation by the indicated concentrations of DA. Data are expressed as a percentage of control maximum DA stimulation for D₁R + D_2LR alone and are representative of two or three independent experiments performed with the same assay conditions on different days. Error bars indicate S.E.M. from multiple wells within the representative experiment.

Fig. 6.

SKF83959 stimulates D₁R-dependent calcium mobilization in the presence of G_qα. HEK293T cells were transfected with D₁R + D_2LR, G_qα or with G_qα alone as described, plated 24 hours later in 384-well plates, and assayed for calcium accumulation the following day. (A) Cells were stimulated with SKF83959. The line at 100% denotes the maximal DA response of D₁R + D_2LR cells. (B) Cells were incubated with the D₁R-selective antagonist SCH23390 (1 μM) or the D₂R-selective antagonist sulpiride (1 μM) and then stimulated with an ∼EC₈₀ of SKF83959 (100 nM). Error bars indicate S.E.M. from multiple wells within the representative experiment, which was replicated twice with similar results. DMSO, dimethylsulfoxide.

Fig. 7.

G protein dependency of D₁R + D_2LR–mediated calcium mobilization. HEK293T cells were transfected with D₁R + D_2LR. Cells were incubated overnight in 1 μg/ml PTX or 1 μg/ml CTX; 48 hours post transfection, cells were assayed for calcium mobilization by stimulation with the indicated concentrations of DA (CTX EC_max = 14%, inhibition = 86% control, PTX EC_max = 24%, inhibition = 76% control). Data are expressed as a percentage of control maximum DA stimulation seen in untreated D₁R + D_2LR cells and are representative of two or three independent experiments performed with the same assay conditions on different days. Error bars indicate S.E.M. from multiple wells within the representative experiment.

Fig. 8.

Dopamine does not elicit a calcium response in cells co-expressing the D₁R and D₄R. HEK293T cells were transiently transfected with D₁R + D_2LR or D₁R + D₄R, as indicated and described in Materials and Methods. Twenty-four hours later, cells were plated in 384-well plates and assayed for calcium mobilization through stimulation by the indicated concentrations of DA. Data are expressed as a percentage of control maximum DA stimulation seen in cells transfected with D₁R + D_2LR only (EC₅₀ = 162.0 nM) and are representative of two or three independent experiments done with the same assay conditions on different days. Expression of the D₄R was confirmed using radioligand binding assays as described in Materials and Methods and was similar to that of the D₂R. Error bars indicate S.E.M. from multiple wells within the representative experiment.

Fig. 9.

GRK2 influence on DA-mediated D₁R + D_2LR calcium mobilization. HEK293T cells were transiently transfected with D₁R + D_2LR and either empty pcDNA vector the GRK2 catalytically inactive mutant GRK2 K220R (A) (D₁R + D₂R EC₅₀ = 269.1 nM) or the GRK2 C-terminal 495–689 fragment (B; GRK2 c-term; D₁R + D₂R EC₅₀ = 90.4 nM, EC_max = 100% control; D₁R+D₂R + pcDNA EC₅₀ = 188.5 nM, EC_max = 106%; D₁R + D₂R +GRK2 c-term EC₅₀ = 288.1 nM, EC_max = 30% control, 70% inhibition), as indicated and described in Materials and Methods. Twenty-four hours later, cells were plated in 384-well plates and assayed the following day for calcium mobilization after stimulation by the indicated concentrations of DA. Data are expressed as a percentage of control maximum DA stimulation seen in cells transfected with D₁R + D_2LR only and are representative of two or three independent experiments done with the same assay conditions on different days. Error bars indicate S.E.M. from multiple wells within the representative experiment.

Fig. 10.

Various mechanisms of PLCβ activation that may occur when the D₁R and D₂R are coexpressed and coactivated.