Differential phosphorylation and dephosphorylation of beta2-adrenoceptor sites Ser262 and Ser355,356.
Activated beta2-adrenoceptors are rapidly desensitized by phosphorylation of Ser262 by protein kinase A (PKA) and of Ser355,356 by G-protein-coupled receptor kinase (GRK). We sought to determine whether the phosphorylation and subsequent dephosphorylation of these sites had similar kinetics and requirements for receptor endocytosis. The phosphorylation of the PKA and GRK sites were measured using antibodies that recognize phosphoserine 262 and phosphoserine 355,356. Endocytosis in stably transfected HEK293 cells was blocked by inducible expression of dominant-negative dynamin-1 K44A or by treatment with hypertonic sucrose. The phosphorylation of the GRK site Ser355,356 during a 10 microM isoprenaline treatment rapidly reached a steady state, and the extent of kinetics of phosphorylation were unaffected by dynamin-1 K44A expression, and minimally by hypertonic sucrose. In contrast, phosphorylation of the PKA site Ser262 during a 10 microM isoprenaline treatment peaked after 2 min and then rapidly declined, while inhibition of endocytosis enhanced and prolonged phosphorylation. Treatment with 300 pM isoprenaline, a concentration too low to provoke endocytosis, also resulted in prolonged PKA site phosphorylation. The dephosphorylation of these sites was measured after removal of agonist. Significant dephosphorylation of phosphoserines 262 and 355,356 was observed under conditions of very low endocytosis, however dephosphorylation of the GRK site was greater if antagonist was present after removal of agonist. The results indicate that the kinetics of beta2-adrenoceptor GRK and PKA site phosphorylation are distinct and differently affected by endocytosis, and that receptor dephosphorylation can occur either at the plasma membrane or in internal compartments.
Iyer
Varsha
V
Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Science and Research Bldg 2, Houston, TX 77204, USA.
Tran
Tuan M
TM
Foster
Estrella
E
Dai
Wenping
W
Clark
Richard B
RB
Knoll
Brian J
BJ
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D034284:Dynamin I / Q000032:analysis; D004705:Endocytosis / Q000187:drug effects; D054769:G-Protein-Coupled Receptor Kinase 2; D054770:G-Protein-Coupled Receptor Kinase 3; D006801:Humans; D010766:Phosphorylation; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases / Q000502:physiology
D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D018343:Receptors, Adrenergic, beta-2; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C496567:GRK2 protein, human; C517848:GRK3 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D034284:Dynamin I