Characterization of agonist stimulation of @entity268 -dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the XXXX using @entity8283 -specific antibodies.

@entity268 @entity1 @entity2162 @entity8283 @entity7284 @entity452 @entity6358 @entity1425 @entity42828 @entity437 @entity3045 @entity957 @entity5015 @entity10453 @entity6410 @entity47644 @entity4183 @entity1928
Agonist-stimulated desensitization of the @entity2162 ( @entity2162 ) is caused by both a potent @entity268 -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 @entity1 @entity2162 overexpressed in @entity1 embryonic kidney (HEK) 293 cells were characterized using @entity8283 -specific antibodies. Specificity of the antibodies was shown by their lack of reactivity with mutant beta2ARs lacking the respective sites. In addition, overexpression of @entity437 and @entity47644 increased basal levels of phosphorylation of the GRK sites Ser355, Ser356 in both COS-7 and HEK 293 cells. @entity1425 , @entity5015 , and @entity1928 at maximum concentrations stimulated phosphorylation of the @entity2162 PKA site (Ser262) by 4-fold, whereas PMA stimulated it by 2-fold. @entity1425 stimulated PKA site phosphorylation with an EC50 of 20 to 40 pM. In contrast, @entity1425 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 @entity1425 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 @entity1425 stimulation. The kinetics of @entity1425 -stimulated GRK site phosphorylation were not altered in a mutant of the @entity2162 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 @entity1425 > or = @entity6410 = @entity7284 > @entity4183 = @entity42828 = @entity3045 > @entity10453 > @entity452 > or = @entity6358 . However, after 20 to 30 min of treatment, agonists with intermediate strengths, such as @entity3045 and @entity10453 , stimulate GRK site phosphorylations that are approximately equal to that produced by @entity1425 , and the correlation breaks down. The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous @entity2162 expressed in A431 @entity1 @entity957 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.
