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

Biochem Biophys Res Commun. 1999 May 27;259(1):224-9. doi: 10.1006/bbrc.1999.0758.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antigens, CD / metabolism*
  • COS Cells
  • Complement C5a / genetics
  • Complement C5a / metabolism
  • Cyclic AMP-Dependent Protein Kinases / genetics*
  • Enzyme Inhibitors / pharmacology
  • G-Protein-Coupled Receptor Kinases
  • Gene Expression / genetics
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation / genetics
  • Okadaic Acid / pharmacology
  • Phosphorylation
  • Protein-Serine-Threonine Kinases*
  • Receptor Protein-Tyrosine Kinases / genetics*
  • Receptor, Anaphylatoxin C5a
  • Receptors, Complement / metabolism*
  • Recombinant Proteins / metabolism
  • Transfection
  • beta-Adrenergic Receptor Kinases

Substances

  • Antigens, CD
  • Enzyme Inhibitors
  • Receptor, Anaphylatoxin C5a
  • Receptors, Complement
  • Recombinant Proteins
  • Okadaic Acid
  • Complement C5a
  • Receptor Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases
  • Cyclic AMP-Dependent Protein Kinases
  • beta-Adrenergic Receptor Kinases
  • G-Protein-Coupled Receptor Kinases
  • G-protein-coupled receptor kinase 6