Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus

The binding of CC chemokines to CC chemokine receptor 5 (CCR5) triggers cel lular responses that, generally, are only transient in nature. To explore t he potential role of G protein coupled receptor kinases (GRKs) in the regul ation of CCR5, we performed phosphorylation experiments in a rat basophilic leukemia cell line stably expressing CCR5, The ability of various CCR5 lig ands to stimulate calcium mobilization in these cells correlated with their ability to induce receptor phosphorylation, desensitization, internalizati on, 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 recycl ing. Aminooxypentane-RANTES profoundly induced CCR5 phosphorylation, but ha d no effect on CCR1, In permeabilized rat basophilic leukemia CCR5 cells, m onoclonal antibodies with specificity for GRK2/3 inhibited RANTES-induced r eceptor phosphorylation, Consistent with a role for these kinases in CCR5 r egulation, 1-2 x 10(5) copies of GRK2 or GRK3 were found to be expressed in peripheral blood leukocytes. Phosphoamino acid analysis revealed that RANT ES-induced CCR5 phosphorylation selectively occurs on serine residues. Our findings with receptor mutants indicate that serine residues at positions 3 36, 337, 342, and 349 represent GRK phosphorylation sites on CCR5, This stu dy demonstrates that chemokines differ in their ability to induce CCR5 phos phorylation and desensitization and provides a molecular mechanism for the agonist-induced attenuation of CCR5 signaling.