Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization.
The CC chemokine receptor CCR5 mediates chemotaxis of leukocytes and serves as a principal co-receptor for macrophage-tropic human immunodeficiency virus type 1. To identify determinants on the CCR5 carboxyl-terminal domain that regulate receptor signaling and internalization, we generated several CCR5 mutants, which were progressively shortened from the COOH terminus or had carboxyl-terminal serine, cysteine, or leucine residues substituted by alanine and expressed them in RBL-2H3 cells. Using fluorescence resonance energy transfer between beta-arrestin and CCR5 tagged with cyan and yellow variants of green fluorescent protein, we show that high affinity association of the two molecules in living cells requires intact carboxyl-terminal serine phosphorylation sites. Phosphorylation-deficient truncation or Ser/Ala replacement mutants of CCR5 mediated a sustained calcium response and enhanced granular enzyme release in RANTES-stimulated cells. Carboxyl-terminal serine residues are critically involved in CCR5 endocytosis and a dileucine motif, similar to that implicated in the regulation of CXCR2 and CXCR4, contributes to the internalization of CCR5 in a phosphorylation-independent manner. Despite their prominent role in receptor desensitization and internalization, beta-arrestins are dispensable for the CCR5-mediated stimulation of mitogen-activated protein kinase pathways in RBL-2H3 cells. We also show that CCR5 is palmitoylated on carboxyl-terminal cysteine residues. Inhibition of CCR5 palmitoylation by alanine mutagenesis of cysteines or treatment with a palmitate analogue inhibitor profoundly reduces phorbol 12-myristate 13-acetate- and RANTES-induced receptor phosphorylation, homologous desensitization, and internalization. Alanine mutagenesis of serine, cysteine, or leucine residues or the limited carboxyl-terminal truncation of CCR5 did not impair chemokine-stimulated migration of RBL-2H3 cells. Together these results indicate that post-translational modifications of carboxyl-terminal serine and cysteine residues have a significant impact on receptor deactivation and internalization.
Kraft
K
K
Department of Immunology, University of Göttingen, 37075 Göttingen, Germany.
Olbrich
H
H
Majoul
I
I
Mack
M
M
Proudfoot
A
A
Oppermann
M
M
D000595:Amino Acid Sequence; D019393:Arrestin / Q000378:metabolism; D002118:Calcium; D002460:Cell Line; D018946:Chemokine CCL5 / Q000494:pharmacology; D004789:Enzyme Activation; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000378:metabolism; D010766:Phosphorylation; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D019393:Arrestin; D018946:Chemokine CCL5; D019713:Receptors, CCR5; D019308:Palmitic Acid; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium