TI - Characterization of agonist stimulation of cAMP -dependent protein kinase and G protein -coupled receptor kinase phosphorylation of the beta2-adrenergic receptor using phosphoserine-specific antibodies . AB - Agonist -stimulated desensitization of the beta2-adrenergic receptor ( beta2AR ) is caused by both a potent c-AMP -dependent protein kinase ( PKA ) -mediated phosphorylation and a less potent , occupancy -dependent , G protein-coupled receptor kinase ( GRK ) -mediated phosphorylation that leads to beta-arrestin binding and internalization . In this study the kinetics of phosphorylation of the third intracellular loop PKA site Ser262 and the putative C-tail GRK sites Ser355 , Ser356 of the human beta2AR overexpressed in human embryonic kidney ( HEK ) 293 cells were characterized using phosphoserine-specific antibodies . Specificity of the antibodies was shown by their lack of reactivity with mutant beta2ARs lacking the respective sites . In addition , overexpression of GRK2 and GRK5 increased basal levels of phosphorylation of the GRK sites Ser355 , Ser356 in both COS-7 and HEK 293 cells . Epinephrine , prostaglandin E1 , and forskolin at maximum concentrations stimulated phosphorylation of the beta2AR PKA site (Ser262) by 4-fold , whereas PMA stimulated it by 2-fold . Epinephrine stimulated PKA site phosphorylation with an EC50 of 20 to 40 pM . In contrast , epinephrine stimulated GRK site phosphorylation ( Ser355 , Ser356 ) with an EC50 of 200 nM ( 1-min treatments ) , which is more than 4000-fold higher relative to PKA site phosphorylation , consistent with an occupancy-driven process . After 10 to 30 min , the EC50 for epinephrine stimulation of GRK site phosphorylation was reduced to 10 to 20 nM but was still approximately 200-fold greater than for the PKA site . The EC50 for internalization correlated with GRK site phosphorylation and showed a similar shift with time of epinephrine stimulation . The kinetics of epinephrine-stimulated GRK site phosphorylation were not altered in a mutant of the beta2AR lacking the PKA consensus sites . The initial levels ( 2 min ) of a range of agonist -stimulated GRK site phosphorylations were correlated with their efficacy for activation of adenylyl cyclase , namely epinephrine &gt ; or = formoterol = fenoterol &gt ; terbutaline = zinterol = albuterol &gt ; salmeterol &gt ; dobutamine &gt ; or = ephedrine . However , after 20 to 30 min of treatment , agonists with intermediate strengths , such as albuterol and salmeterol , stimulate GRK site phosphorylations that are approximately equal to that produced by epinephrine , and the correlation breaks down . The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous beta2AR expressed in A431 human epidermoid carcinoma cells . To summarize , our results show a remarkable amplification of PKA site phosphorylation relative to the putative GRK site phosphorylation , heterologous stimulation of the PKA site phosphorylation , no dependence of GRK site phosphorylation on PKA sites , and a reasonable correlation of initial levels of GRK site phosphorylation with the strength of a range of agonists .