FIGURE 1.
Agonist- and PKC-mediated phosphorylation of the D2 DAR expressed in HEK293T cells. Transfected HEK293T cells were metabolically labeled with [32P]H3PO4 for 45 min prior to stimulation with the indicated drugs for 20 min. The cells were then solubilized, and the samples were subjected to immunoprecipitation as described under “Experimental Procedures.” Receptors were quantified in each transfection, and equal amounts of receptor protein were loaded in each gel lane followed by SDS-PAGE resolution. The extent of receptor phosphorylation was visualized by autoradiography and quantified as described below. A, upper panel shows a representative autoradiogram in which the cells were treated with the indicated drugs: 1st lane, vehicle (Control); 2nd lane, 1 μm PMA; 3rd lane, 10 μm DA. In the lower panel, the receptor phosphorylation was quantified by scanning the autoradiograms followed by analysis with the LabWorks™ software. The data are presented as the percentage of the basal phosphorylation and expressed as the mean ± S.E. values from more than four independent experiments. B, HEK293T cells were transfected with FLAG-tagged D2L DAR only (upper panel) or with a GRK2 expression construct (lower panel). Transfected cells were cultured in the presence or absence of 200 ng/ml of pertussis toxin (PTX) for ∼18 h and then labeled with [32P]H3PO4 before stimulating the cells with or without DA as described in A. This experiment was performed twice with identical results.