title: Expression, purification, and characterization of the G protein-coupled receptor kinase GRK6.
abstract: G protein-coupled receptor kinases (GRKs), such as rhodopsin kinase and beta-adrenergic receptor kinase (beta ARK), are involved in mediating agonist-specific phosphorylation and desensitization of G protein-coupled receptors. GRK6 is the most recently identified member of the GRK family and displays higher homology with GRK5 (70.1% amino acid identity) and IT11 (68.5%) compared to beta ARK (37.4%) and rhodopsin kinase (47.1%). To further characterize GRK6, it has been overexpressed in Sf9 cells and purified to homogeneity by sequential chromatography on SP-Sepharose and heparin-Sepharose columns. GRK6 shares a number of in vitro characteristics with GRK5, including potent inhibition by heparin and dextran sulfate (IC50 values of approximately 15 and approximately 7 nM, respectively), hyperstimulation by polycations, and preference for phosphorylation of non-acidic peptides. Rhodopsin and the beta 2-adrenergic and m2 muscarinic cholinergic receptors serve as stimulus-dependent substrates for GRK6, but with stoichiometries significantly lower than achieved by GRK5 and beta ARK. Additionally, GRK6 does not undergo significant autophosphorylation even though it contains residues identical to those that are autophosphorylated in GRK5 and rhodopsin kinase. These data extend our knowledge of a growing family of receptor-specific kinases and suggest that GRK6 has a substrate specificity distinct from beta ARK, rhodopsin kinase, and GRK5.
authors.affiliation: Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: R P
authors.initials: RP
authors.lastname: Loudon
authors.forename: J L
authors.initials: JL
authors.lastname: Benovic
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D054768:G-Protein-Coupled Receptor Kinases; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D002413:Cations, Divalent; D009842:Oligopeptides; D008345:Manganese; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; D019204:GTP-Binding Proteins; D008274:Magnesium