The binding of
CC chemokines to
CC chemokine receptor 5 (CCR5) triggers cellular responses that, generally, are only transient in nature. To explore the potential role of
G protein-coupled receptor kinases (GRKs) in the regulation of CCR5, we performed phosphorylation experiments in a rat basophilic
leukemia cell line stably expressing CCR5. The ability of various CCR5
ligands to stimulate
calcium mobilization in these cells correlated with their ability to induce receptor phosphorylation, desensitization, internalization, and GRK association with the receptor.
Aminooxypentane-RANTES, a potent inhibitor of human immunodeficiency virus
infection, has been proposed to act through enhanced CCR5 internalization and inhibition of receptor recycling.
Aminooxypentane-RANTES profoundly induced CCR5 phosphorylation, but had no effect on CCR1. In permeabilized rat basophilic
leukemia CCR5 cells,
monoclonal antibodies with specificity for GRK2/3 inhibited
RANTES-induced receptor phosphorylation. Consistent with a role for these
kinases in CCR5 regulation, 1-2 x 10(5) copies of GRK2 or GRK3 were found to be expressed in peripheral blood leukocytes. Phosphoamino
acid analysis revealed that
RANTES-induced CCR5 phosphorylation selectively occurs on
serine residues. Our findings with receptor mutants indicate that
serine residues at positions 336, 337, 342, and 349 represent GRK phosphorylation sites on CCR5. This study demonstrates that
chemokines differ in their ability to induce CCR5 phosphorylation and desensitization and provides a molecular mechanism for the agonist-induced attenuation of CCR5 signaling.