title: Overexpression of wild-type and catalytically inactive forms of GRK2 and GRK6 fails to alter the agonist-induced phosphorylation of the C5a receptor (CD88): evidence that GRK6 is autophosphorylated in COS-7 cells.
abstract: The G protein-coupled receptor kinase family comprises six members (GRK1 to GRK6) that phosphorylate and desensitize a number of agonist-occupied G protein-coupled receptors. Overexpression of the dominant negative mutant GRK2-K220R is often accompanied by an inhibition of the agonist-mediated phosphorylation of G protein-coupled receptors. In the case of the C5a receptor (C5aR), the overexpression of wild-type GRK2 or GRK6 as well as of catalytically inactive forms of these kinases (GRK2-K220R and GRK6-K215R) failed to increase or to inhibit the agonist-mediated phosphorylation of C5aR, respectively. Replacement of Lys215 by an arginine residue in GRK6 yielded a protein with a relative molecular mass of 63 kDa, whereas wild-type GRK6 had a relative molecular mass of 66 kDa on polyacrylamide gel. The mutations S484D and T485D in the catalytically inactive mutant GRK6-K215R resulted in a protein (GRK6-RDD) with the same electrophoretic mobility as wild-type GRK6. Furthermore, in the absence of phosphatase inhibitors, GRK6 was rapidly converted into the 63 kDa species, whereas GRK6-RDD was not. Overepression of GRK6-RDD failed to alter the agonist-mediated phosphorylation of C5aR. Taken together, the results suggest that C5aR is not a substrate for either GRK2 or GRK6 and that GRK6 is very likely autophosphorylated on Ser484 and Thr485 in vivo.
authors.affiliation: DBMS/Laboratoire de Biochimie et de Biophysique des Systèmes Intégrés (UMR 314 CEA/CNRS), CEA/Grenoble, 17 rue des Martyrs, Grenoble Cedex 9, 38054, France.
authors.forename: M D
authors.initials: MD
authors.lastname: Milcent
authors.forename: T
authors.initials: T
authors.lastname: Christophe
authors.forename: M J
authors.initials: MJ
authors.lastname: Rabiet
authors.forename: M
authors.initials: M
authors.lastname: Tardif
authors.forename: F
authors.initials: F
authors.lastname: Boulay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D019556:COS Cells; D015936:Complement C5a / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054768:G-Protein-Coupled Receptor Kinases; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D015703:Antigens, CD; D004791:Enzyme Inhibitors; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011994:Recombinant Proteins; D019319:Okadaic Acid; D015936:Complement C5a; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6