FIGURE 6.
Effect of mutating the GRK phosphorylation sites on D2 DAR expression and function. HEK293T cells were transiently transfected with either WT or GRK(-) mutant D2L DAR. A, total cellular D2L DAR expression was measured by [3H]methylspiperone (2 nm) binding to HEK293T cell membranes. The data represent the mean ± S.E. values from 12 experiments. B, dopamine inhibition of cAMP accumulation was measured in intact HEK293T cells. Cells were incubated with various concentrations of DA for 10 min in the presence of 3 μm forskolin. cAMP accumulation was then assessed as described under “Experimental Procedures.” The data shown represent the mean ± S.E. values from six experiments and are expressed as a percentage of forskolin-stimulated cAMP accumulation in the absence of DA. C, DA-stimulated [35S]GTPγS binding was determined in membranes prepared from HEK293T cells transiently expressing WT or GRK(-) mutant D2L DARs. Dose-response data are expressed as agonist-stimulated binding over basal. D, average values for 100 μm DA-stimulated [35S]GTPγS binding are shown. Data represent the means ± S.E. from 12 experiments.