Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization. Export

@article{citeulike:353532, abstract = {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.}, address = {Department of Immunology, University of G\"{o}ttingen, 37075 G\"{o}ttingen, Germany.}, author = {Kraft, K. and Olbrich, H. and Majoul, I. and Mack, M. and Proudfoot, A. and Oppermann, M.}, citeulike-article-id = {353532}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M102782200}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/11448957}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=11448957}, day = {14}, doi = {10.1074/jbc.M102782200}, issn = {0021-9258}, journal = {J Biol Chem}, keywords = {algorithm, fret}, month = {September}, number = {37}, pages = {34408--34418}, posted-at = {2006-06-23 12:37:01}, priority = {2}, title = {Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization.}, url = {http://dx.doi.org/10.1074/jbc.M102782200}, volume = {276}, year = {2001} }

TY - JOUR ID - citeulike:353532 L3 - citeulike-article-id:353532 N2 - 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. IS - 37 JF - J Biol Chem SN - 0021-9258 AD - Department of Immunology, University of Göttingen, 37075 Göttingen, Germany. EP - 34418 TI - Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization. VL - 276 SP - 34408 KW - algorithm KW - fret AU - Kraft, K AU - Olbrich, H AU - Majoul, I AU - Mack, M AU - Proudfoot, A AU - Oppermann, M PY - 2001/09/14/ UR - http://dx.doi.org/10.1074/jbc.M102782200 ER -