title: A beta-adrenergic receptor kinase dominant negative mutant attenuates desensitization of the beta 2-adrenergic receptor.
abstract: The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the activated form of the beta 2-adrenergic receptor (beta 2AR) and related G protein-coupled receptors. To further elucidate the role of beta ARK in receptor desensitization, we generated a beta ARK dominant negative mutant by converting an invariant lysine residue in the protein kinase catalytic domain to an arginine. Expressed and purified beta ARK-K220R was able to inhibit wild type beta ARK phosphorylation of the beta 2AR in vitro. When stably transfected into human bronchial epithelial BEAS-2B cells, beta ARK-K220R promoted a > 2-fold increase in beta-agonist-stimulated cAMP production without affecting beta 2AR sequestration. In contrast, beta ARK-K220R had no effect on the desensitization of the prostaglandin E2 receptor response in BEAS-2B cells. These findings directly demonstrate a role for beta ARK in desensitization of the beta 2AR in intact cells and establish the potential utility of using dominant negative mutants to elucidate the substrate specificity of G protein-coupled receptor kinases.
authors.affiliation: Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: G
authors.initials: G
authors.lastname: Kong
authors.forename: R
authors.initials: R
authors.lastname: Penn
authors.forename: J L
authors.initials: JL
authors.lastname: Benovic
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D018078:Receptors, Prostaglandin E / Q000378:metabolism; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D009838:Oligodeoxyribonucleotides; D018343:Receptors, Adrenergic, beta-2; D018078:Receptors, Prostaglandin E; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases