title: Endogenous Gs-coupled receptors in smooth muscle exhibit differential susceptibility to GRK2/3-mediated desensitization.
abstract: Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed G s-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gbetagamma sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via beta-agonist stimulation of the beta-2-adrenergic receptor (beta 2AR). Conversely, neither 5'-( N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE 2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE 2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE 2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the beta-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to G s-coupled receptors in ASM, GRK2/3 selectively attenuates beta 2AR signaling, yet relief of GRK2/3-dependent beta 2AR desensitization does not influence at least one important physiological function of the receptor.
authors.affiliation: Department of Internal Medicine and Center for Human Genomics, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, USA.
authors.forename: Kok Choi
authors.initials: KC
authors.lastname: Kong
authors.forename: Uma
authors.initials: U
authors.lastname: Gandhi
authors.forename: T J
authors.initials: TJ
authors.lastname: Martin
authors.forename: Candace B
authors.initials: CB
authors.lastname: Anz
authors.forename: Huandong
authors.initials: H
authors.lastname: Yan
authors.forename: Anna M
authors.initials: AM
authors.lastname: Misior
authors.forename: Rodolfo M
authors.initials: RM
authors.lastname: Pascual
authors.forename: Deepak A
authors.initials: DA
authors.lastname: Deshpande
authors.forename: Raymond B
authors.initials: RB
authors.lastname: Penn
mesh_terms: D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism*; D054770:G-Protein-Coupled Receptor Kinase 3 / Q000235:genetics / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006801:Humans; D009130:Muscle, Smooth / Q000201:enzymology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; 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; D019205:GTP-Binding Protein alpha Subunits, Gs