Abstract

Dephosphorylation of the cyclic AMP-dependent protein kinase (PKA) site phosphoserine 262 and the G protein-coupled receptor kinase (GRK) site phosphoserines 355 and 356 of the β₂-adrenergic receptor (β₂AR) were characterized in both intact human embryonic kidney 293 cells and subcellular fractions and were correlated with the rate of resensitization of isoproterenol stimulation of adenylyl cyclase after treatment with isoproterenol and blockade by antagonist. Dephosphorylation of the PKA site after stimulation with 300 pM isoproterenol occurred with a t_½ of 9 min (k = 0.08 ± 0.016/min) in intact cells in the absence of internalization. Dephosphorylation of the GRK sites in intact cells after treatment with 1.0 μM isoproterenol for 5 min exhibited a lag phase of ≈ 5 min, after which dephosphorylation proceeded slowly with a t_½ of 18 min (k = 0.039 ± 0.006/min). Consistent with the slow rate of GRK site dephosphorylation, the phosphatase inhibitors calyculin A and okadaic acid failed to augment phosphorylation in intact cells during continuous agonist stimulation indicating that GRK site dephosphorylation was minimal. However, both inhibited dephosphorylation of the GRK sites after the addition of antagonist. Slow GRK site dephosphorylation after antagonist treatment was also demonstrated by the relative stability of internalized phosphorylated β₂AR in cells as observed both by immunofluorescence microscopy using a phospho-site-specific antibody and by studies of the subcellular localization of the GRK-phosphorylated β₂AR on sucrose gradients that revealed nearly equivalent levels of GRK site phosphorylation in the plasma membrane and vesicular fractions. In addition, dephosphorylation of the GRK sites by intrinsic phosphatase activity occurred only in the heavy vesicle fractions. In contrast to the slow rates of dephosphorylation, the rate of resensitization of isoproterenol stimulation of adenylyl cyclase was 5- and 10-fold faster (k = 0.43 ± 0.009/min; t_½ = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.