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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

Both RANTES and (AOP)-RANTES activate the JAK/STAT pathway in CCR5-transfected HEK-293 cells. (A) CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES (10 nM). Lysates were immunoprecipitated with mAb  CCR5-03 and analyzed in Western blot with anti-JAK1 antibodies. The figure shows control RANTES-stimulated (10 nM) CCR5-transfected HEK-293 cell lysates immunoprecipitated with anti–β2-microglobulin mAb and analyzed with anti-JAK1 antibodies. As a  control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the same anti-JAK1 antibodies. CCR5 protein loading was controlled by stripping and reprobing membranes with mAb CCR5-02 (bottom). (B) Cells as in A were immunoprecipitated  with anti-PTyr antibody and tested in Western blot with anti-JAK1 antibodies. As a positive control, EGF-stimulated A431 cell lysates  were tested in Western blot with anti-JAK1 antibodies as above. (C) Cells as in A were immunoprecipitated with CCR5-03 mAb and  analyzed in Western blot with anti-STAT5 antibody. CCR5 protein loading was controlled as before. As a positive control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the same anti-STAT5 antibody. (D) Cells as in C were immunoprecipitated with anti-PTyr antibody and analyzed in Western blot with anti-STAT5 antibody. Protein loading was carefully controlled using a  protein detection kit as above. The figure shows control RANTES-stimulated (10 nM) CCR5-transfected HEK-293 cell lysates immunoprecipitated with anti–MHC class I mAb and analyzed with anti-STAT5 antibodies. Arrow indicates the position of STAT5. In all  cases, one experiment of five is shown.
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Figure 5: Both RANTES and (AOP)-RANTES activate the JAK/STAT pathway in CCR5-transfected HEK-293 cells. (A) CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES (10 nM). Lysates were immunoprecipitated with mAb CCR5-03 and analyzed in Western blot with anti-JAK1 antibodies. The figure shows control RANTES-stimulated (10 nM) CCR5-transfected HEK-293 cell lysates immunoprecipitated with anti–β2-microglobulin mAb and analyzed with anti-JAK1 antibodies. As a control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the same anti-JAK1 antibodies. CCR5 protein loading was controlled by stripping and reprobing membranes with mAb CCR5-02 (bottom). (B) Cells as in A were immunoprecipitated with anti-PTyr antibody and tested in Western blot with anti-JAK1 antibodies. As a positive control, EGF-stimulated A431 cell lysates were tested in Western blot with anti-JAK1 antibodies as above. (C) Cells as in A were immunoprecipitated with CCR5-03 mAb and analyzed in Western blot with anti-STAT5 antibody. CCR5 protein loading was controlled as before. As a positive control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the same anti-STAT5 antibody. (D) Cells as in C were immunoprecipitated with anti-PTyr antibody and analyzed in Western blot with anti-STAT5 antibody. Protein loading was carefully controlled using a protein detection kit as above. The figure shows control RANTES-stimulated (10 nM) CCR5-transfected HEK-293 cell lysates immunoprecipitated with anti–MHC class I mAb and analyzed with anti-STAT5 antibodies. Arrow indicates the position of STAT5. In all cases, one experiment of five is shown.

Mentions: To ascertain which kinase is responsible for the rapid CCR5 chemokine receptor phosphorylation, CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES, and cell lysates immunoprecipitated with anti-CCR5, or with anti–β2-microglobulin as an isotype-matched antibody control. The use of a specific anti-JAK1 antibody identified a 130-kD protein in the anti-CCR5 immunoprecipitates (Fig. 5 A). Furthermore, the JAK1 tyrosine kinase is phosphorylated on tyrosine residues after stimulation with either ligand, since anti-PTyr immunoprecipitates can be developed with anti-JAK1 antibodies in Western blot (Fig. 5 B). JAK1 association to the CCR5 receptor takes place as early as 30 s after RANTES or (AOP)-RANTES stimulation (Fig. 5 A). Small amounts of JAK1 were also found associated to the CCR5 receptor in the absence of added ligand, consistent with receptor phosphorylation in the absence of exogenous ligand in CCR5-transfected HEK-293 cells (Fig. 4, A and B).


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)

Both RANTES and (AOP)-RANTES activate the JAK/STAT pathway in CCR5-transfected HEK-293 cells. (A) CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES (10 nM). Lysates were immunoprecipitated with mAb  CCR5-03 and analyzed in Western blot with anti-JAK1 antibodies. The figure shows control RANTES-stimulated (10 nM) CCR5-transfected HEK-293 cell lysates immunoprecipitated with anti–β2-microglobulin mAb and analyzed with anti-JAK1 antibodies. As a  control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the same anti-JAK1 antibodies. CCR5 protein loading was controlled by stripping and reprobing membranes with mAb CCR5-02 (bottom). (B) Cells as in A were immunoprecipitated  with anti-PTyr antibody and tested in Western blot with anti-JAK1 antibodies. As a positive control, EGF-stimulated A431 cell lysates  were tested in Western blot with anti-JAK1 antibodies as above. (C) Cells as in A were immunoprecipitated with CCR5-03 mAb and  analyzed in Western blot with anti-STAT5 antibody. CCR5 protein loading was controlled as before. As a positive control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the same anti-STAT5 antibody. (D) Cells as in C were immunoprecipitated with anti-PTyr antibody and analyzed in Western blot with anti-STAT5 antibody. Protein loading was carefully controlled using a  protein detection kit as above. The figure shows control RANTES-stimulated (10 nM) CCR5-transfected HEK-293 cell lysates immunoprecipitated with anti–MHC class I mAb and analyzed with anti-STAT5 antibodies. Arrow indicates the position of STAT5. In all  cases, one experiment of five is shown.
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Figure 5: Both RANTES and (AOP)-RANTES activate the JAK/STAT pathway in CCR5-transfected HEK-293 cells. (A) CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES (10 nM). Lysates were immunoprecipitated with mAb CCR5-03 and analyzed in Western blot with anti-JAK1 antibodies. The figure shows control RANTES-stimulated (10 nM) CCR5-transfected HEK-293 cell lysates immunoprecipitated with anti–β2-microglobulin mAb and analyzed with anti-JAK1 antibodies. As a control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the same anti-JAK1 antibodies. CCR5 protein loading was controlled by stripping and reprobing membranes with mAb CCR5-02 (bottom). (B) Cells as in A were immunoprecipitated with anti-PTyr antibody and tested in Western blot with anti-JAK1 antibodies. As a positive control, EGF-stimulated A431 cell lysates were tested in Western blot with anti-JAK1 antibodies as above. (C) Cells as in A were immunoprecipitated with CCR5-03 mAb and analyzed in Western blot with anti-STAT5 antibody. CCR5 protein loading was controlled as before. As a positive control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the same anti-STAT5 antibody. (D) Cells as in C were immunoprecipitated with anti-PTyr antibody and analyzed in Western blot with anti-STAT5 antibody. Protein loading was carefully controlled using a protein detection kit as above. The figure shows control RANTES-stimulated (10 nM) CCR5-transfected HEK-293 cell lysates immunoprecipitated with anti–MHC class I mAb and analyzed with anti-STAT5 antibodies. Arrow indicates the position of STAT5. In all cases, one experiment of five is shown.
Mentions: To ascertain which kinase is responsible for the rapid CCR5 chemokine receptor phosphorylation, CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES, and cell lysates immunoprecipitated with anti-CCR5, or with anti–β2-microglobulin as an isotype-matched antibody control. The use of a specific anti-JAK1 antibody identified a 130-kD protein in the anti-CCR5 immunoprecipitates (Fig. 5 A). Furthermore, the JAK1 tyrosine kinase is phosphorylated on tyrosine residues after stimulation with either ligand, since anti-PTyr immunoprecipitates can be developed with anti-JAK1 antibodies in Western blot (Fig. 5 B). JAK1 association to the CCR5 receptor takes place as early as 30 s after RANTES or (AOP)-RANTES stimulation (Fig. 5 A). Small amounts of JAK1 were also found associated to the CCR5 receptor in the absence of added ligand, consistent with receptor phosphorylation in the absence of exogenous ligand in CCR5-transfected HEK-293 cells (Fig. 4, A and B).

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