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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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Functional responses induced by RANTES and  (AOP)-RANTES. (A) Both RANTES and (AOP)-RANTES– induced Ca2+ mobilization in CCR5-transfected HEK-293 cells  was desensitized by the other ligand. Pretreatment with PTX (0.1  μg/ml, 16 h) completely blocks responses in both cases. One of  three experiments is shown. Results are expressed as a percentage of the maximum chemokine response. (B) RANTES, but not  (AOP)-RANTES (10 nM), induces chemotaxis in CCR5-transfected HEK-293 cells. PTX pretreatment (0.1 μg/ml, 16 h) blocks  the RANTES-induced response. The figure depicts one of five  experiments performed, with the SD indicated.
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Figure 2: Functional responses induced by RANTES and (AOP)-RANTES. (A) Both RANTES and (AOP)-RANTES– induced Ca2+ mobilization in CCR5-transfected HEK-293 cells was desensitized by the other ligand. Pretreatment with PTX (0.1 μg/ml, 16 h) completely blocks responses in both cases. One of three experiments is shown. Results are expressed as a percentage of the maximum chemokine response. (B) RANTES, but not (AOP)-RANTES (10 nM), induces chemotaxis in CCR5-transfected HEK-293 cells. PTX pretreatment (0.1 μg/ml, 16 h) blocks the RANTES-induced response. The figure depicts one of five experiments performed, with the SD indicated.

Mentions: In response to RANTES or (AOP)-RANTES, these CCR5-transfected HEK-293 cells mobilize calcium (Fig. 2 A) and are desensitized to a second stimulation. However, only RANTES triggers migration in these cells (Fig. 2 B). PTX treatment abrogates both calcium release and cell migration in response to RANTES and (AOP)-RANTES (Fig. 2 A), whereas no effect was observed following incubation with cholera toxin (not shown). This is consistent with other studies showing that RANTES downstream signals in other cell lines and T cells are coupled to PTX-sensitive G proteins (Bacon et al., 1995b), and with coupling of other chemokine receptors to Gi in transfected HEK-293 cells (Aragay et al., 1998; Mellado et al., 1998), indicating the utility of this cell line in the study of signaling through GPCR.


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)

Functional responses induced by RANTES and  (AOP)-RANTES. (A) Both RANTES and (AOP)-RANTES– induced Ca2+ mobilization in CCR5-transfected HEK-293 cells  was desensitized by the other ligand. Pretreatment with PTX (0.1  μg/ml, 16 h) completely blocks responses in both cases. One of  three experiments is shown. Results are expressed as a percentage of the maximum chemokine response. (B) RANTES, but not  (AOP)-RANTES (10 nM), induces chemotaxis in CCR5-transfected HEK-293 cells. PTX pretreatment (0.1 μg/ml, 16 h) blocks  the RANTES-induced response. The figure depicts one of five  experiments performed, with the SD indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132943&req=5

Figure 2: Functional responses induced by RANTES and (AOP)-RANTES. (A) Both RANTES and (AOP)-RANTES– induced Ca2+ mobilization in CCR5-transfected HEK-293 cells was desensitized by the other ligand. Pretreatment with PTX (0.1 μg/ml, 16 h) completely blocks responses in both cases. One of three experiments is shown. Results are expressed as a percentage of the maximum chemokine response. (B) RANTES, but not (AOP)-RANTES (10 nM), induces chemotaxis in CCR5-transfected HEK-293 cells. PTX pretreatment (0.1 μg/ml, 16 h) blocks the RANTES-induced response. The figure depicts one of five experiments performed, with the SD indicated.
Mentions: In response to RANTES or (AOP)-RANTES, these CCR5-transfected HEK-293 cells mobilize calcium (Fig. 2 A) and are desensitized to a second stimulation. However, only RANTES triggers migration in these cells (Fig. 2 B). PTX treatment abrogates both calcium release and cell migration in response to RANTES and (AOP)-RANTES (Fig. 2 A), whereas no effect was observed following incubation with cholera toxin (not shown). This is consistent with other studies showing that RANTES downstream signals in other cell lines and T cells are coupled to PTX-sensitive G proteins (Bacon et al., 1995b), and with coupling of other chemokine receptors to Gi in transfected HEK-293 cells (Aragay et al., 1998; Mellado et al., 1998), indicating the utility of this cell line in the study of signaling through GPCR.

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