title: Altered activity of palmitoylation-deficient and isoprenylated forms of the G protein-coupled receptor kinase GRK6.
abstract: G protein-coupled receptor kinases (GRKs) utilize diverse mechanisms to associate with the plasma membrane and mediate phosphorylation of agonist-occupied receptors. For example, two members of this family, GRK4 and GRK6, contain C-terminal cysteine residues that are palmitoylated. To address whether the activity and membrane association of GRK6 is regulated by palmitoylation, we overexpressed and characterized wild-type GRK6 and two GRK6 mutants, one with the palmitoylation sites mutated to serines (GRK6-pal-) and one containing a C-terminal CAAX motif to promote geranylgeranylation (GRK6-GG). Compared with wild-type GRK6, GRK6-pal- had a approximately 5-fold higher Km and approximately 2-fold lower Vmax for phosphorylating rhodopsin, whereas GRK6-GG exhibited a approximately 2-fold lower Km and approximately 14-fold higher Vmax for rhodopsin. In contrast, wild-type GRK6 and GRK6-pal- displayed similar activity toward the nonreceptor substrate phosvitin, indicating that nonpalmitoylated GRK6 is catalytically active. Wild-type GRK6 and GRK6-GG, but not GRK6-pal-, also bound significantly to phosphatidylcholine vesicles (36 +/- 3, 79 +/- 4, and 4 +/- 2%, respectively) suggesting that GRK6 activity is dependent upon its ability to interact with the plasma membrane. When assayed in COS-1 cells GRK6-pal- promoted minimal agonist-dependent sequestration of the beta2-adrenergic receptor, while sequestration was significantly increased in cells expressing either wild-type GRK6 or GRK6-GG. These data demonstrate an important functional link between the ability of GRK6 to bind to the plasma membrane, a process that appears to be regulated by palmitoylation, and its activity toward receptor substrates.
authors.affiliation: Department of Biochemistry, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
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; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002417:Cattle; D003545:Cysteine; D017931:DNA Primers; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D054768:G-Protein-Coupled Receptor Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism*; D010713:Phosphatidylcholines; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000378:metabolism; D017368:Protein Prenylation; D017346:Protein Serine-Threonine Kinases; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D017384:Sequence Deletion; D012694:Serine; D014162:Transfection
chemical_list: D017931:DNA Primers; D005136:Eye Proteins; D008081:Liposomes; D010713:Phosphatidylcholines; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D019308:Palmitic Acid; D012694:Serine; D012243:Rhodopsin; D011494:Protein Kinases; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; D019204:GTP-Binding Proteins; D003545:Cysteine; D007545:Isoproterenol