1. 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
- Author
-
Thierry Christophe, Marie Danielle Milcent, François Boulay, Marianne Tardif, and Marie-Josèphe Rabiet
- Subjects
Phosphatase ,Molecular Sequence Data ,Biophysics ,Gene Expression ,Complement C5a ,Biology ,Protein Serine-Threonine Kinases ,Transfection ,Biochemistry ,C5a receptor ,Antigens, CD ,Okadaic Acid ,Animals ,Humans ,Amino Acid Sequence ,Enzyme Inhibitors ,Phosphorylation ,Receptor ,Molecular Biology ,Polyacrylamide gel electrophoresis ,Receptor, Anaphylatoxin C5a ,Kinase ,Autophosphorylation ,Wild type ,Receptor Protein-Tyrosine Kinases ,Cell Biology ,G-Protein-Coupled Receptor Kinases ,Molecular biology ,Cyclic AMP-Dependent Protein Kinases ,Recombinant Proteins ,Receptors, Complement ,Kinetics ,beta-Adrenergic Receptor Kinases ,COS Cells ,Mutation ,Mutagenesis, Site-Directed - 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.
- Published
- 1999