| Abstrakt: |
Phosphorylation by protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs) desensitize β2-adrenergic receptor (β2AR) 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 β2AR 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 β2ARs lacking PKA and/or GRK phosphorylation sites on ASM at ≈4-fold over background. Functional gains in response to β-agonist from the selective loss of these mechanisms were determined in mouse airways. Relaxation kinetics were altered in all mutant airways compared with β2WT. At low receptor occupancy, β2PKA(-) had enhanced agonist-promoted relaxation, while β2GRK(-) airways were unaffected. In contrast, at saturating agonist concentrations, the greatest relaxation enhancement was with β2GRK(-), with no evidence for add itivity when PKA sites were also removed. For the full range of responses, the β2PKA(-)/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 β2AR and GRK-promoted events was identified by β-arrestin-2 recruitment. Thus, these two mechanisms indeed impact a relevant β2AR physiologic function, acting as attenuators of the acute response, and represent specific interfaces where adjunct therapy or biased ligands may improve β-agonist treatment of obstructive lung disease. [ABSTRACT FROM AUTHOR] |
