Characterization of β₂-Adrenergic Receptor Dephosphorylation: Comparison with the Rate of Resensitization

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 ␤ 2 -adrenergic receptor (␤ 2 AR) 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 1/2 of 9 min (k ϭ 0.08 Ϯ 0.016/min) in intact cells in the absence of internalization. Dephosphorylation of the GRK sites in intact cells after treatment with 1.0 M isoproterenol for 5 min exhibited a lag phase of Ϸ 5 min, after which dephosphorylation proceeded slowly with a t 1/2 of 18 min (k ϭ 0.039 Ϯ 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 ␤ 2 AR 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 ␤ 2 AR 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 ϭ 0.43 Ϯ 0.009/min; t 1/2 ϭ 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0 -5 min) from dephosphorylation.In any given cell, the concentration of agonist and the rate constants for phosphorylation and dephosphorylation define the level of PKA and GRK site phosphorylation of the ␤ 2 -adrenergic receptor (␤ 2 AR) at various times after agonist stimulation, and these factors in turn affect the level of arrestin binding and receptor internalization. During agonist treatment, the rate constants for internalization and recycling control the level of the ␤ 2 ARs in the plasma membrane. To what extent do internalization and dephosphorylation correlate with functional resensitization? This question has been the subject of intense study. Several earlier studies led to the proposal that internalization was required for dephosphorylation and functional resensitization because it appeared that dephosphorylation occurred in endosomes, but not at the plasma membrane (Sibley et al

Influence of the Accessory Protein SET on M3 Muscarinic Receptor Phosphorylation and G Protein Coupling

“…The different time courses of M3-MR internalization and dephosphorylation in CHO-M3 cells suggest that receptor dephosphorylation events are taking place at the plasma membrane rather than after receptor internalization. Several studies demonstrated that other GPCRs such as the D1 dopamine receptor (Gardner et al, 2001), the thyrotropin-releasing hormone receptor (Jones and Hinkle, 2005), and the ␤ 2 -adrenergic receptor (Tran et al, 2007) are dephosphorylated at the plasma membrane. Dephosphorylation of GPCRs at the plasma membrane rather than in endosomes may provide a more rapid way of regeneration of a functional receptor without the need to enter the internalization and recycling pathways.…”

Desensitization of the Dopamine D1 and D2 Receptor Hetero-Oligomer Mediated Calcium Signal by Agonist Occupancy of Either Receptor

“…We postulate that these kinases may alter the resensitization of the calcium signal. It has been demonstrated that blocking PKA and CAMKII phosphorylation of GPCRs inhibited receptor resensitization and increased desensitization (Hishinuma and Ogura, 2000;Tran et al, 2007). Despite the fact that inhibiting PKA and PKC activities did not affect homologous desensitization, these kinases are involved in heterologous desensitization of this signal.…”

β2-Adrenergic Receptor Signaling and Desensitization Elucidated by Quantitative Modeling of Real Time cAMP Dynamics

“…We assume that GRK only phosphorylates the ligated receptor (21) and that this phosphorylation event is irreversible over the time span of the experiment, since the rate of ␤ 2 AR dephosphorylation after agonist withdrawal has been shown to be very slow (22). We assume that active PKA can phosphorylate both the ligated and ligand-free receptor and that this phosphorylation event is reversible, since dephosphorylation of PKA-phosphorylated ␤ 2 AR occurs much faster than GRKphosphorylated ␤ 2 AR (22).…”