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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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Both RANTES and (AOP)-RANTES induce early  tyrosine phosphorylation of CCR5. RANTES- or (AOP)- RANTES (10 nM)–induced CCR5-transfected HEK-293 cells  were stimulated with 10 nM RANTES or (AOP)-RANTES,  lysed, immunoprecipitated with anti-CCR5 antibody, and developed in Western blot with the anti-PTyr mAb 4G10. Protein  loading was controlled using a protein detection kit (Pierce  Chemical Co.). To control for equivalent CCR5 loading, the blot  was stripped and reprobed with the anti-CCR5 mAb CCR5-02. A  control of RANTES-stimulated CCR5-transfected HEK-293 cell  lysate, immunoprecipitated with anti–β2-microglobulin antibody,  was analyzed with anti-CCR5 antibody (not shown). One representative experiment is shown of four performed.
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Figure 4: Both RANTES and (AOP)-RANTES induce early tyrosine phosphorylation of CCR5. RANTES- or (AOP)- RANTES (10 nM)–induced CCR5-transfected HEK-293 cells were stimulated with 10 nM RANTES or (AOP)-RANTES, lysed, immunoprecipitated with anti-CCR5 antibody, and developed in Western blot with the anti-PTyr mAb 4G10. Protein loading was controlled using a protein detection kit (Pierce Chemical Co.). To control for equivalent CCR5 loading, the blot was stripped and reprobed with the anti-CCR5 mAb CCR5-02. A control of RANTES-stimulated CCR5-transfected HEK-293 cell lysate, immunoprecipitated with anti–β2-microglobulin antibody, was analyzed with anti-CCR5 antibody (not shown). One representative experiment is shown of four performed.

Mentions: When CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES, a 38-kD protein phosphorylated in tyrosine residues was initially identified as the CCR5 receptor by immunoblot (not shown). Thereafter, lysates from transfected HEK-293 cells were immunoprecipitated with anti-CCR5, and Western blots developed with anti-PTyr antibodies (Fig. 4). An increase in CCR5 receptor phosphorylation in tyrosines is seen as early as 60 s after stimulation. At this time no differences were observed following treatment with either stimulus, indicating that (AOP)-RANTES not only binds to CCR5, but also signals through this receptor. The early, weak tyrosine phosphorylation level may occur because only one tyrosine (Tyr 126 in the DRY motif) was being phosphorylated, analogous to the case of Tyr 139 in the CCR2 receptor (Mellado et al., 1998). Similar results are observed in the inverse experiment, in which anti-PTyr antibody immunoprecipitates are analyzed in Western blot with anti-CCR5 receptor antibody (not shown). Again, tyrosine phosphorylation is detected in CCR5 after stimulation with either RANTES or (AOP)-RANTES. Nevertheless, significant differences are observed in tyrosine phosphorylated CCR5 after longer stimulation periods. Whereas CCR5 phosphorylation persists in RANTES-treated cells after 15 min of treatment, this is not the case after (AOP)- RANTES stimulation, reinforcing the observation that there are differences in the signaling pathways activated by these chemokines.


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 induce early  tyrosine phosphorylation of CCR5. RANTES- or (AOP)- RANTES (10 nM)–induced CCR5-transfected HEK-293 cells  were stimulated with 10 nM RANTES or (AOP)-RANTES,  lysed, immunoprecipitated with anti-CCR5 antibody, and developed in Western blot with the anti-PTyr mAb 4G10. Protein  loading was controlled using a protein detection kit (Pierce  Chemical Co.). To control for equivalent CCR5 loading, the blot  was stripped and reprobed with the anti-CCR5 mAb CCR5-02. A  control of RANTES-stimulated CCR5-transfected HEK-293 cell  lysate, immunoprecipitated with anti–β2-microglobulin antibody,  was analyzed with anti-CCR5 antibody (not shown). One representative experiment is shown of four performed.
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Related In: Results  -  Collection

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Figure 4: Both RANTES and (AOP)-RANTES induce early tyrosine phosphorylation of CCR5. RANTES- or (AOP)- RANTES (10 nM)–induced CCR5-transfected HEK-293 cells were stimulated with 10 nM RANTES or (AOP)-RANTES, lysed, immunoprecipitated with anti-CCR5 antibody, and developed in Western blot with the anti-PTyr mAb 4G10. Protein loading was controlled using a protein detection kit (Pierce Chemical Co.). To control for equivalent CCR5 loading, the blot was stripped and reprobed with the anti-CCR5 mAb CCR5-02. A control of RANTES-stimulated CCR5-transfected HEK-293 cell lysate, immunoprecipitated with anti–β2-microglobulin antibody, was analyzed with anti-CCR5 antibody (not shown). One representative experiment is shown of four performed.
Mentions: When CCR5-transfected HEK-293 cells were stimulated with RANTES or (AOP)-RANTES, a 38-kD protein phosphorylated in tyrosine residues was initially identified as the CCR5 receptor by immunoblot (not shown). Thereafter, lysates from transfected HEK-293 cells were immunoprecipitated with anti-CCR5, and Western blots developed with anti-PTyr antibodies (Fig. 4). An increase in CCR5 receptor phosphorylation in tyrosines is seen as early as 60 s after stimulation. At this time no differences were observed following treatment with either stimulus, indicating that (AOP)-RANTES not only binds to CCR5, but also signals through this receptor. The early, weak tyrosine phosphorylation level may occur because only one tyrosine (Tyr 126 in the DRY motif) was being phosphorylated, analogous to the case of Tyr 139 in the CCR2 receptor (Mellado et al., 1998). Similar results are observed in the inverse experiment, in which anti-PTyr antibody immunoprecipitates are analyzed in Western blot with anti-CCR5 receptor antibody (not shown). Again, tyrosine phosphorylation is detected in CCR5 after stimulation with either RANTES or (AOP)-RANTES. Nevertheless, significant differences are observed in tyrosine phosphorylated CCR5 after longer stimulation periods. Whereas CCR5 phosphorylation persists in RANTES-treated cells after 15 min of treatment, this is not the case after (AOP)- RANTES stimulation, reinforcing the observation that there are differences in the signaling pathways activated by these chemokines.

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