FIGURE 2.
Effect of overexpressing nonvisual GRKs on basal and agonist-induced D2 DAR phosphorylation. HEK293T cells were transiently transfected with the FLAG-tagged D2L DAR along with the following expression constructs: pcDNA (Mock), GRK2, GRK3, GRK4, GRK5, or GRK6. [32P]H3PO4-labeled cells were stimulated with 1 μm DA for 20 min and processed as described in Fig. 1. A, autoradiogram from a single experiment, representative of four, is shown. B, receptor phosphorylation was quantified as described in Fig. 1. Data are expressed as the mean ± S.E. values from at least three independent experiments. *, p < 0.005, compared with the basal or DA-stimulated values of the mock transfection, respectively, unpaired Student's t test. C, analysis of GRK isozyme expression in transfected HEK293T cells. Cell lysates were prepared from cells transfected with the indicated GRKs and used to determine the expression of each GRK isozyme by immunoblotting with specific anti-GRK antibodies. A representative experiment is shown, which was performed twice with identical results.