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Similarities and differences in RANTES- and (AOP)-RANTES-triggered signals: implications for chemotaxis.

Rodríguez-Frade JM, Vila-Coro AJ, Martín A, Nieto M, Sánchez-Madrid F, Proudfoot AE, Wells TN, Martínez-A C, Mellado M - J. Cell Biol. (1999)

Bottom Line: Chemokines mediate their effects via interaction with seven transmembrane G protein-coupled receptors (GPCR).Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Galphai as well as JAK/STAT association to the receptor.The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnolog¿ia, CSIC/UAM, Campus de Cantoblanco, E-28049 Madrid, Spain.

ABSTRACT
Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G protein-coupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Galphai as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.

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Related in: MedlinePlus

CCR5-01, -02, and -03 antibodies are specific for the  CCR5 chemokine receptor. mAb specificity for the CCR5 receptor was assessed by flow cytometry analysis of mock- and CCR5-transfected HEK-293 cells. (A) CCR5-transfected HEK-293 cells  (HEK-293-CCR5) or mock-transfected controls (HEK-293) were  incubated with the biotin-labeled CCR5-01, -02, or -03 antibodies, followed by streptavidin-PE. An isotype-matched mAb was  used as a control. (B) CCR5- or mock-transfected HEK-293 cells  were immunoprecipitated as described in Materials and Methods  using CCR5-03 mAb, then analyzed in Western blot with CCR5-01, -02, -03, or control mAb, as indicated. One representative experiment of five is shown.
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Figure 1: CCR5-01, -02, and -03 antibodies are specific for the CCR5 chemokine receptor. mAb specificity for the CCR5 receptor was assessed by flow cytometry analysis of mock- and CCR5-transfected HEK-293 cells. (A) CCR5-transfected HEK-293 cells (HEK-293-CCR5) or mock-transfected controls (HEK-293) were incubated with the biotin-labeled CCR5-01, -02, or -03 antibodies, followed by streptavidin-PE. An isotype-matched mAb was used as a control. (B) CCR5- or mock-transfected HEK-293 cells were immunoprecipitated as described in Materials and Methods using CCR5-03 mAb, then analyzed in Western blot with CCR5-01, -02, -03, or control mAb, as indicated. One representative experiment of five is shown.

Mentions: We developed anti–human CCR5-specific mAb using synthetic peptides corresponding to the NH2-terminal domain of this receptor (amino acids 13–28), a CCR5-specific sequence not present in other chemokine receptors. These mAbs recognize the CCR5 in human PBMC (not shown) and in CCR5 stably transfected HEK-293 cells, in flow cytometry (Fig. 1 A), as well as in Western blot, and immunoprecipitation analysis (Fig. 1 B). No binding is observed in mock- or CCR2-transfected HEK-293 cells, or in any other cell tested that does not express CCR5.


Similarities and differences in RANTES- and (AOP)-RANTES-triggered signals: implications for chemotaxis.

Rodríguez-Frade JM, Vila-Coro AJ, Martín A, Nieto M, Sánchez-Madrid F, Proudfoot AE, Wells TN, Martínez-A C, Mellado M - J. Cell Biol. (1999)

CCR5-01, -02, and -03 antibodies are specific for the  CCR5 chemokine receptor. mAb specificity for the CCR5 receptor was assessed by flow cytometry analysis of mock- and CCR5-transfected HEK-293 cells. (A) CCR5-transfected HEK-293 cells  (HEK-293-CCR5) or mock-transfected controls (HEK-293) were  incubated with the biotin-labeled CCR5-01, -02, or -03 antibodies, followed by streptavidin-PE. An isotype-matched mAb was  used as a control. (B) CCR5- or mock-transfected HEK-293 cells  were immunoprecipitated as described in Materials and Methods  using CCR5-03 mAb, then analyzed in Western blot with CCR5-01, -02, -03, or control mAb, as indicated. One representative experiment of five is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132943&req=5

Figure 1: CCR5-01, -02, and -03 antibodies are specific for the CCR5 chemokine receptor. mAb specificity for the CCR5 receptor was assessed by flow cytometry analysis of mock- and CCR5-transfected HEK-293 cells. (A) CCR5-transfected HEK-293 cells (HEK-293-CCR5) or mock-transfected controls (HEK-293) were incubated with the biotin-labeled CCR5-01, -02, or -03 antibodies, followed by streptavidin-PE. An isotype-matched mAb was used as a control. (B) CCR5- or mock-transfected HEK-293 cells were immunoprecipitated as described in Materials and Methods using CCR5-03 mAb, then analyzed in Western blot with CCR5-01, -02, -03, or control mAb, as indicated. One representative experiment of five is shown.
Mentions: We developed anti–human CCR5-specific mAb using synthetic peptides corresponding to the NH2-terminal domain of this receptor (amino acids 13–28), a CCR5-specific sequence not present in other chemokine receptors. These mAbs recognize the CCR5 in human PBMC (not shown) and in CCR5 stably transfected HEK-293 cells, in flow cytometry (Fig. 1 A), as well as in Western blot, and immunoprecipitation analysis (Fig. 1 B). No binding is observed in mock- or CCR2-transfected HEK-293 cells, or in any other cell tested that does not express CCR5.

Bottom Line: Chemokines mediate their effects via interaction with seven transmembrane G protein-coupled receptors (GPCR).Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Galphai as well as JAK/STAT association to the receptor.The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnolog¿ia, CSIC/UAM, Campus de Cantoblanco, E-28049 Madrid, Spain.

ABSTRACT
Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G protein-coupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Galphai as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.

Show MeSH
Related in: MedlinePlus