FIGURE 3.

G_s-biased pepducins do not promote GRK-mediated β₂AR phosphorylation. A, schematic diagram of kinase-specific phosphorylation sites on the β₂AR upon β-agonist stimulation. GRK5/6 phosphorylates Ser^355/6 on the β₂AR C-terminal tail, whereas PKA can phosphorylate Ser^261/2 in ICL3. B and C, HEK 293 cells stably overexpressing FLAG-β₂AR were stimulated with 1 μm isoproterenol, 5 μm salbutamol, or 10 μm pepducin in DMEM with 10% FBS. 0.05% DMSO was included in non-pepducin-stimulated cells. B, GRK5/6 phosphorylation at Ser^355/6 was monitored on immunoprecipitated FLAG-β₂AR at the indicated time points by Western blotting using α-Ser(P)-³⁵⁵/Ser³⁵⁶. C, PKA phosphorylation at Ser^261/2 was monitored on immunoprecipitated FLAG-β₂AR at the indicated time points by Western blotting using α-Ser(P)²⁶².