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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

ICAM-3 and CCR5 polarization induced by RANTES and (AOP)- RANTES in T lymphoblasts. (A) Time course of RANTES- and (AOP)- RANTES–induced ICAM-3 redistribution to the uropod of T lymphoblasts. FN-adhered (30 μg/ml) T lymphoblasts were stimulated with 10 ng/ml of RANTES or  (AOP)-RANTES for different time periods. The percentage of cells in which  ICAM-3 was redistributed was calculated as described in Materials and Methods.  One representative experiment of three is shown. (B) Membrane CCR5 expression is not detectable in (AOP)-RANTES–stimulated polarized T lymphoblasts.  Cells adhered to FN (30 μg/ml) were stimulated with RANTES (10 ng/ml; a and  b) or (AOP)-RANTES (10 ng/ml; c and d), fixed, and stained for CCR5 (red fluorescence) and ICAM-3 (green fluorescence). Cells were photographed under epifluorescence (a and c) and bright-field conditions (b and d). (C) Inhibition of  CCR5 redistribution to the leading edge of (AOP)-RANTES–polarized T lymphoblasts. Cells adhered to FN (30 μg/ml) were stimulated with 10 ng/ml of  RANTES, (AOP)-RANTES, or MCP-1 for 30 min. The percentage of cells in  which CCR5 was distributed was then calculated as described in Materials and  Methods. The arithmetic mean ± SD of three independent experiments is shown.
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Figure 3: ICAM-3 and CCR5 polarization induced by RANTES and (AOP)- RANTES in T lymphoblasts. (A) Time course of RANTES- and (AOP)- RANTES–induced ICAM-3 redistribution to the uropod of T lymphoblasts. FN-adhered (30 μg/ml) T lymphoblasts were stimulated with 10 ng/ml of RANTES or (AOP)-RANTES for different time periods. The percentage of cells in which ICAM-3 was redistributed was calculated as described in Materials and Methods. One representative experiment of three is shown. (B) Membrane CCR5 expression is not detectable in (AOP)-RANTES–stimulated polarized T lymphoblasts. Cells adhered to FN (30 μg/ml) were stimulated with RANTES (10 ng/ml; a and b) or (AOP)-RANTES (10 ng/ml; c and d), fixed, and stained for CCR5 (red fluorescence) and ICAM-3 (green fluorescence). Cells were photographed under epifluorescence (a and c) and bright-field conditions (b and d). (C) Inhibition of CCR5 redistribution to the leading edge of (AOP)-RANTES–polarized T lymphoblasts. Cells adhered to FN (30 μg/ml) were stimulated with 10 ng/ml of RANTES, (AOP)-RANTES, or MCP-1 for 30 min. The percentage of cells in which CCR5 was distributed was then calculated as described in Materials and Methods. The arithmetic mean ± SD of three independent experiments is shown.

Mentions: We have described the ability of RANTES to polarize peripheral blood T cells, such that the CCR5 receptor migrates to the cell's leading edge while the ICAM-1, ICAM-3/ CD43, CD44 molecules localize at the uropod (Nieto et al., 1997). Therefore, we tested the ability of both of these ligands to induce polarization in peripheral T cell blasts. Time-course studies of ICAM-3 redistribution to the cell uropod of FN-adhered T lymphoblasts showed that RANTES induced ICAM-3 redistribution in a significant proportion of the cells as early as 15 min after stimulation. Maximum redistribution, seen at 30 min, persisted until at least 90 min of incubation with the chemokine. In contrast, (AOP)-RANTES–induced ICAM-3 polarization showed a much slower, weaker response, with a peak at 30 min that vanished thereafter (Fig. 3 A).


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)

ICAM-3 and CCR5 polarization induced by RANTES and (AOP)- RANTES in T lymphoblasts. (A) Time course of RANTES- and (AOP)- RANTES–induced ICAM-3 redistribution to the uropod of T lymphoblasts. FN-adhered (30 μg/ml) T lymphoblasts were stimulated with 10 ng/ml of RANTES or  (AOP)-RANTES for different time periods. The percentage of cells in which  ICAM-3 was redistributed was calculated as described in Materials and Methods.  One representative experiment of three is shown. (B) Membrane CCR5 expression is not detectable in (AOP)-RANTES–stimulated polarized T lymphoblasts.  Cells adhered to FN (30 μg/ml) were stimulated with RANTES (10 ng/ml; a and  b) or (AOP)-RANTES (10 ng/ml; c and d), fixed, and stained for CCR5 (red fluorescence) and ICAM-3 (green fluorescence). Cells were photographed under epifluorescence (a and c) and bright-field conditions (b and d). (C) Inhibition of  CCR5 redistribution to the leading edge of (AOP)-RANTES–polarized T lymphoblasts. Cells adhered to FN (30 μg/ml) were stimulated with 10 ng/ml of  RANTES, (AOP)-RANTES, or MCP-1 for 30 min. The percentage of cells in  which CCR5 was distributed was then calculated as described in Materials and  Methods. The arithmetic mean ± SD of three independent experiments is shown.
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Figure 3: ICAM-3 and CCR5 polarization induced by RANTES and (AOP)- RANTES in T lymphoblasts. (A) Time course of RANTES- and (AOP)- RANTES–induced ICAM-3 redistribution to the uropod of T lymphoblasts. FN-adhered (30 μg/ml) T lymphoblasts were stimulated with 10 ng/ml of RANTES or (AOP)-RANTES for different time periods. The percentage of cells in which ICAM-3 was redistributed was calculated as described in Materials and Methods. One representative experiment of three is shown. (B) Membrane CCR5 expression is not detectable in (AOP)-RANTES–stimulated polarized T lymphoblasts. Cells adhered to FN (30 μg/ml) were stimulated with RANTES (10 ng/ml; a and b) or (AOP)-RANTES (10 ng/ml; c and d), fixed, and stained for CCR5 (red fluorescence) and ICAM-3 (green fluorescence). Cells were photographed under epifluorescence (a and c) and bright-field conditions (b and d). (C) Inhibition of CCR5 redistribution to the leading edge of (AOP)-RANTES–polarized T lymphoblasts. Cells adhered to FN (30 μg/ml) were stimulated with 10 ng/ml of RANTES, (AOP)-RANTES, or MCP-1 for 30 min. The percentage of cells in which CCR5 was distributed was then calculated as described in Materials and Methods. The arithmetic mean ± SD of three independent experiments is shown.
Mentions: We have described the ability of RANTES to polarize peripheral blood T cells, such that the CCR5 receptor migrates to the cell's leading edge while the ICAM-1, ICAM-3/ CD43, CD44 molecules localize at the uropod (Nieto et al., 1997). Therefore, we tested the ability of both of these ligands to induce polarization in peripheral T cell blasts. Time-course studies of ICAM-3 redistribution to the cell uropod of FN-adhered T lymphoblasts showed that RANTES induced ICAM-3 redistribution in a significant proportion of the cells as early as 15 min after stimulation. Maximum redistribution, seen at 30 min, persisted until at least 90 min of incubation with the chemokine. In contrast, (AOP)-RANTES–induced ICAM-3 polarization showed a much slower, weaker response, with a peak at 30 min that vanished thereafter (Fig. 3 A).

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