Targeted transgenesis reveals discrete attenuator functions of GRK and PKA in airway beta(2)-adrenergic receptor physiologic signaling

Phosphorylation by protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs) desensitize beta(2)-adrenergic receptor (beta(2)AR) signaling, and these are thought to be mechanisms involved with cell and organ homeostasis and tolerance to agonists. However, there is little direct evidence that these events are relevant to beta(2)AR physiological function, such as airway smooth muscle (ASM) relaxation leading to bronchodilation. To maintain cell- and receptor-specificity without altering the natural complement of kinases/arrestins, transgenic mice were generated expressing the human WT and mutated beta(2)ARs lacking PKA and/or GRK phosphorylation sites on ASM at approximate to 4-fold over background. Functional gains in response to beta-agonist from the selective loss of these mechanisms were determined in mouse airways. Relaxation kinetics were altered in all mutant airways compared with beta 2WT. At low receptor occupancy, beta(2)PKA(-) had enhanced agonist-promoted relaxation, while beta(2)GRK(-) airways were unaffected. In contrast, at saturating agonist concentrations, the greatest relaxation enhancement was with beta(2)GRK(-), with no evidence for additivity when PKA sites were also removed. For the full range of responses, the beta(2)PKA(-)/GRK(-) airways had the greatest relaxation efficiency, indicating a graded effect of GRKs as agonist concentration increased. ASM cAMP levels paralleled relaxation phenotypes. No interaction between PKA phosphorylation of beta(2)AR and GRK-promoted events was identified by beta-arrestin-2 recruitment. Thus, these two mechanisms indeed impact a relevant beta(2)AR physiologic function, acting as attenuators of the acute response, and represent specific interfaces where adjunct therapy or biased ligands may improve beta-agonist treatment of obstructive lung disease.