C5a-stimulated recruitment of beta-arrestin2 to the nonsignaling 7-transmembrane decoy receptor C5L2

J Biomol Screen. 2009 Oct;14(9):1067-75. doi: 10.1177/1087057109341407. Epub 2009 Jul 29.

Abstract

C5L2 (or GPR77) is a high-affinity receptor for the complement fragment C5a and its desarginated product, C5a-desArg. Unlike the classical C5a receptor CD88, C5L2 does not couple to intracellular G-protein-signaling pathways but is thought to function as a decoy receptor. The authors show that stimulation of C5L2 with C5a and C5a-desArg induces redistribution of green fluorescent protein-labeled beta-arrestin2 to cytoplasmic vesicles. C3a and C3a-desArg were inactive in the beta-arrestin translocation assay. Direct interaction of ligand-stimulated C5L2 with beta-arrestin was confirmed using a novel beta-galactosidase fragment complementation assay. In this assay, C5L2 was labeled with a mutationally altered peptide fragment of beta-galactosidase, whereas beta-arrestin2 was labeled with a corresponding deletion mutant of the enzyme. Stable transfection of the modified C5L2 and subsequent stimulation with C5a or C5a-desArg restored beta-galactosidase activity in a dose-dependent manner. The subnanomolar potency of beta-arrestin coupling in the beta-galactosidase fragment complementation assay is in agreement with the affinity of the receptor-ligand interaction. C5L2 is the first example of a 7-transmembrane decoy receptor that couples to beta-arrestin in a ligand-dependent manner. This observation supports the notion that G-protein-signaling and beta-arrestin coupling can be 2 separate activities of 7-transmembrane receptors. Furthermore, the beta-arrestin assays described in this article provide methods of screening for selective C5L2 modulators.

MeSH terms

  • Animals
  • Arrestins / genetics
  • Arrestins / metabolism*
  • CHO Cells
  • Complement C5a / genetics
  • Complement C5a / metabolism*
  • Cricetinae
  • Cricetulus
  • Dose-Response Relationship, Drug
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Humans
  • Receptor, Anaphylatoxin C5a
  • Receptors, Chemokine / genetics
  • Receptors, Chemokine / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction / physiology*
  • beta-Arrestins

Substances

  • Arrestins
  • C5aR2 protein, human
  • Receptor, Anaphylatoxin C5a
  • Receptors, Chemokine
  • Recombinant Fusion Proteins
  • beta-Arrestins
  • Complement C5a
  • Extracellular Signal-Regulated MAP Kinases