G protein-coupled receptor kinases promote phosphorylation and beta-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-DeltaNT 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-DeltaNT 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 (125)I-CCL5. Co-expression of C5aR, but not of AT(1a)R, 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.
Hüttenrauch
Friederike
F
Department of Cellular and Molecular Immunology, Georg-August-University, Göttingen, Germany.
Pollok-Kopp
Beatrix
B
Oppermann
Martin
M
D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D002478:Cells, Cultured; D019281:Dimerization; D004705:Endocytosis; D005786:Gene Expression Regulation; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D044087:Receptor, Anaphylatoxin C5a; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011951:Receptors, Complement / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D000071557:beta-Arrestins
D019390:Arrestins; C488033:C5AR1 protein, human; D008565:Membrane Proteins; D044087:Receptor, Anaphylatoxin C5a; D019713:Receptors, CCR5; D011951:Receptors, Complement; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins; D011493:Protein Kinase C