Characterization of beta2-adrenergic receptor dephosphorylation: Comparison with the rate of resensitization.
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 beta2-adrenergic receptor (beta2AR) 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 = 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 microM isoproterenol for 5 min exhibited a lag phase of approximately 5 min, after which dephosphorylation proceeded slowly with a t(1/2) 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 beta2AR 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 beta2AR 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(1/2) = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.
Tran
Tuan M
TM
Department of Integrative Biology and Pharmacology, University of Texas Health Science Center Houston, Medical School, 6431 Fannin, Houston, TX, USA.
Friedman
Jacqueline
J
Baameur
Faiza
F
Knoll
Brian J
BJ
Moore
Robert H
RH
Clark
Richard B
RB
D000262:Adenylyl Cyclases / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000378:metabolism; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D010768:Phosphoserine / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects / Q000378:metabolism*
D018343:Receptors, Adrenergic, beta-2; D010768:Phosphoserine; D017868:Cyclic AMP-Dependent Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D000262:Adenylyl Cyclases; D007545:Isoproterenol