title: Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1.
abstract: The beta 2-adrenergic receptor (beta 2AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a beta 2AR mutant (beta 2AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type beta 2AR. However, this mutant receptor not only desensitized in response to agonist stimulation but down-regulated normally. In an attempt to understand the basis for the properties of this mutant, we have examined the ability of this regulation-defective mutant to undergo agonist-mediated phosphorylation. When expressed in 293 cells, the maximal response for phosphorylation of the beta 2AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient beta 2AR-Y326A mutants, demonstrated that the beta 2AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the beta-adrenergic receptor kinase 1 (beta ARK1). However, overexpression of beta ARK1 led to the agonist-dependent phosphorylation of the beta 2AR-Y326A mutant and rescue of its sequestration. beta ARK1-mediated rescue of beta 2AR-Y326A sequestration could be prevented by mutating putative beta ARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type beta 2AR could be attenuated by overexpressing a dominant-negative mutant of beta ARK1 (C20 beta ARK1-K220M). These findings implicate a role for beta ARK1-mediated phosphorylation in facilitating wild-type beta 2AR sequestration.
authors.affiliation: Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Ferguson
authors.forename: L
authors.initials: L
authors.lastname: Ménard
authors.forename: L S
authors.initials: LS
authors.lastname: Barak
authors.forename: W J
authors.initials: WJ
authors.lastname: Koch
authors.forename: A M
authors.initials: AM
authors.lastname: Colapietro
authors.forename: M G
authors.initials: MG
authors.lastname: Caron
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D009154:Mutation; D010766:Phosphorylation; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases