Figure 1.

Alanine mutations at a βγ-binding interface of α_z cause the mutant protein, α_zMUT, to mislocalize to intracellular membranes. (A) Immunofluorescence microscopy of CHO cells transfected with wild-type α_z (a) or α_zMUT (b). Although the fluorescent signal for wild-type α_z (a) is not confined to the outer perimeter of the cell, the staining pattern indicates localization at the PM. This pattern is due to the extremely flat and thin shape of CHO cells under our culture conditions; using confocal microscopy, we previously observed the same pattern of fluorescence for PM-localized proteins in CHO cells (1). (Bar = 20 μm.) (B) MAPK assays were performed on CHO cells transfected 48 h earlier with the D₂ dopamine receptor and HA-MAPK, together with wild-type α_z, α_zMUT, or α_zMUT plus β₁ and wild-type γ₂. MAPK activity was determined after a 4-h treatment with pertussis toxin (100 ng/ml) and a 7-min exposure to the D₂ agonist (10 μM quinpirole, black bars) or no agonist (white bars). Data shown are the means ±2 SEM of four experiments.