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J. Biol. Chem., Vol. 280, Issue 45, 37503-37515, November 11, 2005

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G Protein-coupled Receptor Kinases Promote Phosphorylation and -Arrestin-mediated Internalization of CCR5 Homo- and Hetero-oligomers^*

From the Department of Cellular and Molecular Immunology, Georg-August-University, 37073 Göttingen, Germany

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_1aR in living cells. Stimulation with CCL5 of RBL cells, which co-expressed CCR5 together with an N-terminally truncated CCR5-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_1aR stimulation resulted only in classical PKC-mediated CCR5 phosphorylation. Co-expression of CCR5-NT together with a phosphorylation-deficient CCR5 mutant that neither binds -arrestin nor undergoes internalization partially restored the CCL5-induced association of -arrestin with the homo-oligomeric receptor complex and augmented cellular uptake of ¹²⁵I-CCL5. Co-expression of C5aR, but not of AT_1aR, 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 -arrestin are involved in heterologous receptor regulation by cross-phosphorylating and co-internalizing unligated receptors within homo- or hetero-oligomeric protein complexes.

Received for publication, January 18, 2005 , and in revised form, August 12, 2005.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 523 TPA10. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Cellular and Molecular Immunology, University of Göttingen, Humboldtallee 34 37073 Göttingen, Germany. Tel.: 49-551-395822; Fax: 49-551-395843; E-mail: mopperm{at}gwdg.de.

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