QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.106.028456v1 71/1/47    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tran, T. M. Articles by Clark, R. B. Search for Related Content PubMed PubMed Citation Articles by Tran, T. M. Articles by Clark, R. B.

Characterization of ₂-Adrenergic Receptor Dephosphorylation: Comparison with the Rate of Resensitization

Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Medical School, Houston, Texas (T.M.T., J.F., F.B., R.B.C.); Department of Pediatrics, Baylor College of Medicine, Houston, Texas (R.H.M.); and Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, Texas (B.J.K.)

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 (₂AR) 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 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 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 ₂AR 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 ₂AR 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 = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.

Received for publication June 29, 2006.

Accepted for publication September 28, 2006.

Address correspondence to: Richard B. Clark, Dept. of Integrative Biology and Pharmacology, University of Texas Health Science Center Houston, Medical School, 6431 Fannin, Houston, TX. E-mail: richard.b.clark{at}uth.tmc.edu

This article has been cited by other articles:

				X. He, L. Fang, J. Wang, Y. Yi, S. Zhang, and X. Xie Bryostatin-5 Blocks Stromal Cell-Derived Factor-1 Induced Chemotaxis via Desensitization and Down-regulation of Cell Surface CXCR4 Receptors Cancer Res., November 1, 2008; 68(21): 8678 - 8686. [Abstract] [Full Text] [PDF]

				M. D. Bruss, W. Richter, K. Horner, S.-L. C. Jin, and M. Conti Critical Role of PDE4D in {beta}2-Adrenoceptor-dependent cAMP Signaling in Mouse Embryonic Fibroblasts J. Biol. Chem., August 15, 2008; 283(33): 22430 - 22442. [Abstract] [Full Text] [PDF]

				W. Xin, T. M. Tran, W. Richter, R. B. Clark, and T. C. Rich Roles of GRK and PDE4 Activities in the Regulation of {beta}2 Adrenergic Signaling J. Gen. Physiol., March 31, 2008; 131(4): 349 - 364. [Abstract] [Full Text] [PDF]

				J. D. Violin, L. M. DiPilato, N. Yildirim, T. C. Elston, J. Zhang, and R. J. Lefkowitz {beta}2-Adrenergic Receptor Signaling and Desensitization Elucidated by Quantitative Modeling of Real Time cAMP Dynamics J. Biol. Chem., February 1, 2008; 283(5): 2949 - 2961. [Abstract] [Full Text] [PDF]

				C. H. So, V. Verma, B. F. O'Dowd, and S. R. George Desensitization of the Dopamine D1 and D2 Receptor Hetero-Oligomer Mediated Calcium Signal by Agonist Occupancy of Either Receptor Mol. Pharmacol., August 1, 2007; 72(2): 450 - 462. [Abstract] [Full Text] [PDF]