title: Exploiting functional domains of GRK2/3 to alter the competitive balance of pro- and anticontractile signaling in airway smooth muscle.
abstract: To clarify the potential utility of targeting GRK2/3-mediated desensitization as a means of manipulating airway smooth muscle (ASM) contractile state, we assessed the specificity of GRK2/3 regulation of procontractile and relaxant G-protein-coupled receptors in ASM. Functional domains of GRK2/3 were stably expressed, or siRNA-mediated GRK2/3 knockdown was performed, in human ASM cultures, and agonist-induced signaling was assessed. Regulation of contraction of murine tracheal rings expressing GRK2 C terminus was also assessed. GRK2/3 knockdown or expression of the GRK2 C terminus caused a significant (∼ 30-90%) increase in maximal β-agonist and histamine [phosphoinositide (PI) hydrolysis] signaling, without affecting the calculated EC50. GRK2 C-terminal expression did not affect signaling by methacholine, thrombin, or LTD4. Expression of the GRK2 N terminus or kinase-dead holo-GRK2 diminished (∼ 30-70%) both PI hydrolysis and Ca(2+) mobilization by every Gq-coupled receptor examined. Under conditions of GRK2 C-terminal expression, β-agonist inhibition of methacholine-stimulated PI hydrolysis was greater. Finally, transgenic expression of the GRK2 C terminus in murine ASM enabled ∼ 30-50% greater β-agonist-mediated relaxation of methacholine-induced contraction. Collectively these data demonstrate the relative selectivity of GRKs for the β2AR in ASM and the ability to exploit GRK2/3 functional domains to render ASM hyporesponsive to contractile agents while increasing responsiveness to bronchodilating β-agonist.
authors.affiliation: 2Department of Medicine, Thomas Jefferson University, Room 317, College Bldg., 1025 Walnut St., Philadelphia, PA 19107, USA. raymond.penn@jefferson.edu.
authors.forename: Deepak A
authors.initials: DA
authors.lastname: Deshpande
authors.forename: Huandong
authors.initials: H
authors.lastname: Yan
authors.forename: Kok-Choi
authors.initials: KC
authors.lastname: Kong
authors.forename: Brian C
authors.initials: BC
authors.lastname: Tiegs
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Morgan
authors.forename: Tonio
authors.initials: T
authors.lastname: Pera
authors.forename: Reynold A
authors.initials: RA
authors.lastname: Panettieri
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Eckhart
authors.forename: Raymond B
authors.initials: RB
authors.lastname: Penn
mesh_terms: D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000737:chemistry / Q000378:metabolism*; D054770:G-Protein-Coupled Receptor Kinase 3 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D009130:Muscle, Smooth / Q000378:metabolism*; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D012137:Respiratory System / Q000166:cytology; D015398:Signal Transduction / Q000502:physiology
chemical_list: D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP; D054770:G-Protein-Coupled Receptor Kinase 3; C496567:GRK2 protein, human; C517848:GRK3 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D002118:Calcium
keywords: G-protein-coupled receptor; asthma; bronchodilation; desensitization; β-agonist