G protein-coupled receptor kinases promote phosphorylation and β-arrestin-mediated internalization of CCR5 homo- and hetero-oligomers

Expression levels of the chemokine receptor, CC chemokine receptor 5 (CCR5), at the cell surface determine cell susceptibility to HIV entry and infection. Cellular activation by CCR5 itself, but also by unrelated receptors leads to cross-phosphorylation and cross-internalization of CCR5. This study addresses the underlying molecular mechanisms of homologous and heterologous CCR5 regulation. As shown by bioluminescence resonance energy transfer experiments, CCR5 formed constitutive homo- as well as heterooligomeric complexes together with C5aR but not with the unrelated AT(1a)R in living cells. Stimulation with CCL5 of RBL cells, which co-expressed CCR5 together with an N-terminally truncated CCR5-Delta NT mutant, resulted in both protein kinase C(PKC)- and G protein-coupled receptor (GPCR) kinase (GRK)-mediated cross-phosphorylation of the mutant unligated receptor, as determined by phosphosite-specific monoclonal antibody. Similarly, both PKC and GRK cross-phosphorylated CCR5 in a heterologous manner after C5a stimulation of RBL-CCR5/ C5aR cells, whereas AT(1a)R stimulation resulted only in classical PKC-mediated CCR5 phosphorylation. Co-expression of CCR5-Delta NT together with a phosphorylation-deficient CCR5 mutant that neither binds beta-arrestin nor undergoes internalization partially restored the CCL5-induced association of beta-arrestin with the homo- oligomeric receptor complex and augmented cellular uptake of I-125-CCL5. Co-expression of C5aR, but not of AT1aR, promoted CCR5 co-internalization upon agonist stimulation by a mechanism independent of CCR5 phosphorylation. Co-internalization of phosphorylated CCR5 was also observed in C5a-stimulated macrophages. Finally, co-expression of a constitutively internalized C5aR-US28(CT) mutant led to intracellular accumulation of CCR5 in the absence of ligand stimulation. These results show that GRKs and beta-arrestin are involved in heterologous receptor regulation by cross-phosphorylating and co-internalizing unligated receptors within homo- or hetero-oligomeric protein complexes.