We next tested for dopamine receptor agonist specificity. The DRD2 selective agonist quinpirole dose-dependently increases [Ca2+]i in cells coexpressing GHSR1a and DRD2, but not in cells expressing either GHSR1a or DRD2 alone (Figure 3C). To determine DRD2 agonist specificity, cells coexpressing GHSR1a and DRD2 were treated with DRD2 selective antagonist raclopride or the DRD1 antagonist SCH23390. The former inhibited dopamine-induced Ca2+ signaling,
but the latter was ineffective (Figure 3D), illustrating DRD2 selectivity. To probe the mechanism of dopamine-induced Ca2+ signal generation we tested specific inhibitors of GPCR signal transduction. The dopamine-induced Ca2+ signal is blocked by: pertussis toxin (PTX), an inhibitor of Gαi signaling (Figure 3E); the PLC inhibitor (U73122) (Figure 3F); 2-aminoethoxydiphenyl borate (2-APB) click here an antagonist of intracellular IP3 receptors (Figure 3G); thapsigargin
(TG), a depletor of intracellular calcium stores (Figure 3H). Hence, GHSR1a and DRD2 coexpression results in dopamine-induced coupling to Gαi, activation of PLC, and production of IP3 that acts on IP3 receptors to release Ca2+ from the endoplasmic reticulum. Since dopamine-induced Ca2+ release is blocked by PTX (Figure 3E), we reasoned the mechanism might involve direct stimulation of PLC by Gβγ subunits derived from Gαi/o and direct association of Gβγ with GRK2 (Inglese et al., 1994 and Koch et al., 1994). BIBW2992 cost To test for a role of GRK2, we expressed wild-type GRK2 or a mutant GRK2 (GRK2-K220R) lacking kinase activity in cells coexpressing GHSR1a and DRD2 (Namkung and Sibley, 2004). Both WT and mutant GRK2 inhibit dopamine-induced increases in Ca2+ (Figure 3I, p < 0.001). Expression of the Gβγ scavenger, βARK1ct (Koch et al., 1994) dose-dependently inhibits dopamine-induced Ca2+ mobilization (Figure 3J). Pretreating Unoprostone cells expressing GHSR1a and DRD2 with small molecule inhibitors (M119 and M158C) of Gβγ subunit signaling (Bonacci et al., 2006) attenuates dopamine-induced modification of Ca2+ release, whereas the inactive control compound (M119B) is ineffective (Figure 3K). Hence, GHSR1a modification of dopamine-induced signal transduction
is mediated by Gβγ subunits. When GHSR1a is expressed at high concentrations under heterologous nonnative conditions, it exhibits basal activity. Therefore, in all experiments we expressed GHSR1a at concentrations commensurate with the low levels exhibited in vivo and under these conditions GHSR1a does not exhibit detectable basal activity; nevertheless, it was important to rigorously test the possibility that basal activity of GHSR1a might be responsible for altering canonical DRD2 signaling by receptor crosstalk. We coexpressed DRD2 with WT-GHSR1a and with three point mutants that exhibit equivalent basal activity to WT-GHSR1a (S123A-GHSR1a and M213K-GHSR1a), or lack basal activity (F279L-GHSR1a) altogether (Feighner et al.
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