Fig. 4.

G-protein coupled receptor kinase-independent arrestin-dependent uncoupling of D2 like dopamine receptor (D2R) is blocked by staurosporine treatment and by alanine substitution of putative protein kinase C phosphorylation sites. (a) Average desensitization rates (± SEM) of the K_ir3 responses elicited by dopamine in oocytes injected with cRNA for D2R and K_ir3 channel subunits (control, white bars) and in oocytes that were also injected with cRNA for β-arrestin2 (ARRB2, black bars). Staurosporine (10 μM, 10 min) treatment was performed immediately prior to measuring the dopamine response. ARRB2 cRNA injection significantly increased desensitization rate of D2R compared with control (*p < 0.005, t-test, n = 4–6). After staurosporine treatment, however, desensitization rates were not significantly different between control and ARRB2 injected groups. (b) Average desensitization rates (± SEM) of the K_ir3 responses elicited by dopamine in oocytes injected with cRNA for the indicated receptor constructs (D2R or D2R_{T225,S228,S229→A}) and K_ir3 channels (control, white bars) and in oocytes that were also injected with cRNA for ARRB2 (black bars). Data represent two independent experiments from two separate oocyte donors, n = 8–10. ARRB2 cRNA injection significantly increased desensitization rate compared with control for the D2R group (*p < 0.005, t-test). However, for the oocyte groups that were injected with cRNA for the D2R_{T225,S228,S229→A} construct, the desensitization rates were not significantly different between the ARRB2 cRNA injected group and the corresponding control. C. Dopamine dose-response curves for D2R or D2R_{T225,S228,S229→A}-mediated K_ir3 activation. The response at each dopamine dose is expressed as a percent of the average maximal response that was obtained (n = 4). The EC₅₀ (nM) for dopamine activation of D2R and D2R_{T225,S228,S229→A} were significantly different (p < 0.05, t-test) and were 61.5 ± 15.5, and 36.4 ± 10.4 nM, respectively.