Targeted transgenesis reveals discrete attenuator functions of GRK and PKA in airway beta2-adrenergic receptor physiologic signaling.
Phosphorylation by protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs) desensitize beta2-adrenergic receptor (beta2AR) 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 beta2AR 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 beta2ARs lacking PKA and/or GRK phosphorylation sites on ASM at approximately 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 beta2WT. At low receptor occupancy, beta2PKA(-) had enhanced agonist-promoted relaxation, while beta2GRK(-) airways were unaffected. In contrast, at saturating agonist concentrations, the greatest relaxation enhancement was with beta2GRK(-), with no evidence for additivity when PKA sites were also removed. For the full range of responses, the beta2PKA(-)/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 beta2AR and GRK-promoted events was identified by beta-arrestin-2 recruitment. Thus, these two mechanisms indeed impact a relevant beta2AR 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.
Wang
Wayne C H
WC
Cardiopulmonary Genomics Program, Department of Medicine, University of Maryland School of Medicine, 20 Penn Street, HSF-II, Baltimore, MD 21201, USA.
Mihlbachler
Kathryn A
KA
Brunnett
Alicyn C
AC
Liggett
Stephen B
SB
D058666:Adrenergic beta-2 Receptor Agonists; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D054768:G-Protein-Coupled Receptor Kinases / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009130:Muscle, Smooth / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D012143:Respiratory Physiological Phenomena / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects
D058666:Adrenergic beta-2 Receptor Agonists; D018343:Receptors, Adrenergic, beta-2; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054768:G-Protein-Coupled Receptor Kinases
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