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 t½ 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 t½ 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; t½ = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.
Footnotes
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This work was supported by National Institutes of Health grants GM031208 (to R.B.C.), HL64934 (to R.H.M.), and 0455072Y (to B.J.K.).
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This work has been presented previously in abstract form at the Keystone Meeting on G Protein-Coupled Receptors: Evolving Concepts and New Techniques: Feb 12-17, 2006; Keystone, CO.
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ABBREVIATIONS: PKA, cyclic AMP-dependent protein kinase; GRK, G protein-coupled receptor kinase; β2AR, β2-adrenergic receptor; PP2A, protein phosphatase 2A; PP1, protein phosphatase 1; HEK, human embryonic kidney; PNGase F, peptide N-glycosidase F; WT, wild type; DMEM, Dulbecco's modified Eagle's medium; AT, ascorbate/thiourea; HE, HEPES/EDTA; ICI-118,551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol; PBS, phosphate-buffered saline; CGP-12177, 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one; PAGE, polyacrylamide gel electrophoresis; HV, heavy vesicle; LV, light vesicle.
- Received June 29, 2006.
- Accepted September 28, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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