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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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RANTES and (AOP)-RANTES promote Gαi and  p125FAK association to the CCR5 receptor. (A) 10 nM RANTES  or (AOP)-RANTES–induced CCR5-transfected HEK-293 cell  lysates were immunoprecipitated with anti-CCR5 (CCR5-03) or  with an anti–β2-microglobulin control mAb, and the Western  blot developed with anti-Gαi antibody. As a control for CCR5  loading equivalence, the blot was stripped and reprobed with the  anti-CCR5 mAb CCR5-02. As a positive control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the  same anti-Gαi or anti-CCR5 antibodies. (B) CCR5-transfected  HEK-293 cells were stimulated with RANTES or (AOP)- RANTES as in A. Cell lysates were immunoprecipitated with  anti-p125FAK, or anti-JAK2 antibodies as control, the Western  blot developed with anti-CCR5 mAb CCR5-02. Protein loading  was controlled by stripping the membrane and reprobing with  anti-p125FAK antibody.
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Figure 7: RANTES and (AOP)-RANTES promote Gαi and p125FAK association to the CCR5 receptor. (A) 10 nM RANTES or (AOP)-RANTES–induced CCR5-transfected HEK-293 cell lysates were immunoprecipitated with anti-CCR5 (CCR5-03) or with an anti–β2-microglobulin control mAb, and the Western blot developed with anti-Gαi antibody. As a control for CCR5 loading equivalence, the blot was stripped and reprobed with the anti-CCR5 mAb CCR5-02. As a positive control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the same anti-Gαi or anti-CCR5 antibodies. (B) CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)- RANTES as in A. Cell lysates were immunoprecipitated with anti-p125FAK, or anti-JAK2 antibodies as control, the Western blot developed with anti-CCR5 mAb CCR5-02. Protein loading was controlled by stripping the membrane and reprobing with anti-p125FAK antibody.

Mentions: CCR5- or mock-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES, immunoprecipitated with anti-CCR5 or isotype-matched control antibodies, followed by a Western blot with anti-Gαi antibodies. After both RANTES and (AOP)-RANTES stimulation, Gαi associated to CCR5 (Fig. 7 A). Whereas association persisted for longer than 15 min when cells were RANTES-stimulated, Gαi is dissociated from the receptor after 5 min of (AOP)-RANTES treatment (Fig. 7 A). The CCR5 expression level was controlled in both immunoprecipitates by stripping and reblotting membranes with anti-CCR5 antibody (Fig. 7 A). These data concur with the similar Ca2+ mobilization promoted by both of these ligands (Fig. 2 A), as Gαi association to CCR5 is unaltered at the times employed for these experiments (1–3 min). Rapid Gαi dissociation from the CCR5, induced only by (AOP)-RANTES, implies a role for Gi in later chemokine-triggered events such as chemotaxis. This result concurs with data showing the importance of βγ release from Gi in chemotaxis (Neptune and Bourne, 1997).


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)

RANTES and (AOP)-RANTES promote Gαi and  p125FAK association to the CCR5 receptor. (A) 10 nM RANTES  or (AOP)-RANTES–induced CCR5-transfected HEK-293 cell  lysates were immunoprecipitated with anti-CCR5 (CCR5-03) or  with an anti–β2-microglobulin control mAb, and the Western  blot developed with anti-Gαi antibody. As a control for CCR5  loading equivalence, the blot was stripped and reprobed with the  anti-CCR5 mAb CCR5-02. As a positive control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the  same anti-Gαi or anti-CCR5 antibodies. (B) CCR5-transfected  HEK-293 cells were stimulated with RANTES or (AOP)- RANTES as in A. Cell lysates were immunoprecipitated with  anti-p125FAK, or anti-JAK2 antibodies as control, the Western  blot developed with anti-CCR5 mAb CCR5-02. Protein loading  was controlled by stripping the membrane and reprobing with  anti-p125FAK antibody.
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Figure 7: RANTES and (AOP)-RANTES promote Gαi and p125FAK association to the CCR5 receptor. (A) 10 nM RANTES or (AOP)-RANTES–induced CCR5-transfected HEK-293 cell lysates were immunoprecipitated with anti-CCR5 (CCR5-03) or with an anti–β2-microglobulin control mAb, and the Western blot developed with anti-Gαi antibody. As a control for CCR5 loading equivalence, the blot was stripped and reprobed with the anti-CCR5 mAb CCR5-02. As a positive control, CCR5-transfected HEK-293 cell lysates were tested in Western blot with the same anti-Gαi or anti-CCR5 antibodies. (B) CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)- RANTES as in A. Cell lysates were immunoprecipitated with anti-p125FAK, or anti-JAK2 antibodies as control, the Western blot developed with anti-CCR5 mAb CCR5-02. Protein loading was controlled by stripping the membrane and reprobing with anti-p125FAK antibody.
Mentions: CCR5- or mock-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES, immunoprecipitated with anti-CCR5 or isotype-matched control antibodies, followed by a Western blot with anti-Gαi antibodies. After both RANTES and (AOP)-RANTES stimulation, Gαi associated to CCR5 (Fig. 7 A). Whereas association persisted for longer than 15 min when cells were RANTES-stimulated, Gαi is dissociated from the receptor after 5 min of (AOP)-RANTES treatment (Fig. 7 A). The CCR5 expression level was controlled in both immunoprecipitates by stripping and reblotting membranes with anti-CCR5 antibody (Fig. 7 A). These data concur with the similar Ca2+ mobilization promoted by both of these ligands (Fig. 2 A), as Gαi association to CCR5 is unaltered at the times employed for these experiments (1–3 min). Rapid Gαi dissociation from the CCR5, induced only by (AOP)-RANTES, implies a role for Gi in later chemokine-triggered events such as chemotaxis. This result concurs with data showing the importance of βγ release from Gi in chemotaxis (Neptune and Bourne, 1997).

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