title: Targeted transgenesis reveals discrete attenuator functions of GRK and PKA in airway beta2-adrenergic receptor physiologic signaling.
abstract: 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.
authors.affiliation: Cardiopulmonary Genomics Program, Department of Medicine, University of Maryland School of Medicine, 20 Penn Street, HSF-II, Baltimore, MD 21201, USA.
authors.forename: Wayne C H
authors.initials: WC
authors.lastname: Wang
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Mihlbachler
authors.forename: Alicyn C
authors.initials: AC
authors.lastname: Brunnett
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Liggett
mesh_terms: 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
chemical_list: 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
references.citation: J Clin Invest. 2003 Aug;112(4):619-26
references.pmid: 12925702
references.citation: Am J Physiol Lung Cell Mol Physiol. 2004 Feb;286(2):L312-9
references.pmid: 14565944
references.citation: J Clin Invest. 2003 Aug;112(4):566-74
references.pmid: 12925697
references.citation: Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6201-4
references.pmid: 1648731
references.citation: Science. 1995 Jun 2;268(5215):1350-3
references.pmid: 7761854
references.citation: J Clin Invest. 2006 May;116(5):1400-9
references.pmid: 16670773
references.citation: J Biol Chem. 1999 Nov 5;274(45):32241-7
references.pmid: 10542262
references.citation: Proc Natl Acad Sci U S A. 1987 Dec;84(24):8879-82
references.pmid: 2827157
references.citation: Proc Natl Acad Sci U S A. 1986 May;83(9):2797-801
references.pmid: 2871555
references.citation: FASEB J. 2008 Jul;22(7):2134-41
references.pmid: 18337459
references.citation: J Biol Chem. 1989 Oct 5;264(28):16786-92
references.pmid: 2476447
references.citation: Nat Rev Mol Cell Biol. 2002 Sep;3(9):639-50
references.pmid: 12209124
references.citation: J Biol Chem. 2000 Sep 15;275(37):28989-93
references.pmid: 10893245
references.citation: Biochemistry. 1992 Mar 31;31(12):3193-7
references.pmid: 1348186
references.citation: Science. 1999 Dec 24;286(5449):2495-8
references.pmid: 10617462
references.citation: J Biol Chem. 2008 Feb 1;283(5):2949-61
references.pmid: 18045878
references.citation: J Biol Chem. 1989 Jul 25;264(21):12657-65
references.pmid: 2545714
references.citation: Pharmacogenet Genomics. 2008 Aug;18(8):729-32
references.pmid: 18622265
references.citation: J Biol Chem. 2008 Feb 29;283(9):5669-76
references.pmid: 18086673
references.citation: J Biol Chem. 1990 Feb 25;265(6):3202-11
references.pmid: 2154473
references.citation: Eur J Pharmacol. 2001 Jun 15;421(3):141-7
references.pmid: 11516429
references.citation: Am J Physiol. 1999 Apr;276(4):R1214-21
references.pmid: 10198406