Ligand-induced phosphorylation of anaphylatoxin receptors C3aR and C5aR ismediated by G protein-coupled receptor kinases

Continuous stimulation of anaphylatoxin receptors C3aR and C5aR with their cognate ligands engenders, within minutes, diminished responsiveness of the se receptors. We tested the hypothesis that agonist-induced desensitization involves C3aR and C5aR phosphorylation by G protein-coupled receptor kinas es (GRK). When expressed in rat basophilic leukemia cells and exposed to C3 a, the C3aR underwent rapid (t(1/2) approximate to 15s), dose-dependent (EC 50 approximate to 10 nM) and reversible phosphorylation by a kinase refract ory to the effects of PKC inhibitors. Phosphoamino acid analysis revealed t hat the C3aR is phosphorylated on serine and threonine, but not on tyrosine residues. Overexpression of GRK2, GRK3, GRK5 or GRK6 together with C3aR in COS-7 cells enhanced the C3a-induced C3aR phosphorylation 1.5-1.9-fold (p < 0.05), but each kinase reduced ligand-stimulated phospholipase C activity differently. Conversely, antibody-mediated inhibition of endogenous GRK2 a nd GRK3 significantly inhibited C3aR phosphorylation in permeabilized cells . GRK overexpression in cells which co-expressed C5aR and were exposed to C 5a resulted in the hyperphosphorylation of the C5aR. These findings are of physiological relevance, since we observed anaphylatoxin-induced phosphoryl ation of C3aR and C5aR endogenously expressed in human mast cells (HMC-1) w hich contain significant intracellular levels of GRK2 and GRK3.