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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 induce dimerization  of CCR5. (A) CCR5-transfected HEK-293 cells were stimulated  for 60 s with 10 nM RANTES, 10 nM (AOP)-RANTES, or 20 nM  SDF-1α at 37°C, and cross-linked using 1 mM DSS. Cell lysates  were immunoprecipitated with mAb CCR5-03, electrophoresed,  and transferred to nitrocellulose membranes. The Western blot  was analyzed with mAb CCR5-02; as a positive control, CCR5-transfected HEK-293 cell lysates were immunoblotted with mAb  CCR5-02. Arrows indicate the CCR5 monomer (38 kD) and  dimer (76 kD). Upper corner insert shows RANTES-induced  Ca2+ mobilization in CCR5-transfected HEK-293 cells, performed as described in Materials and Methods. Results, expressed as in Fig. 2 A, are a percentage of the maximum chemokine response. (B) CCR5-transfected HEK-293 cells as in A were  immunoprecipitated and analyzed in Western blot with anti-CXCR4 mAb. Arrows indicate the CXCR4 monomer (42 kD)  and dimer (84 kD). Protein loading was controlled using a protein detection kit (Pierce Chemical Co.). Upper corner insert  shows SDF-1α–induced Ca2+ mobilization in CCR5-transfected  HEK-293 cells, performed as described in Materials and Methods. Results are expressed as in A. Note that the response of  CCR5-transfected HEK-293 cells to SDF-1α is weaker than  RANTES, as measured by Ca2+ influx. This lower response correlates with the lower amounts of dimerized CXCR4 receptor,  and explains the longer exposure time required to develop the  Western blot for the dimerized receptor. The 52-kD protein band  corresponds to the heavy chain of the immunoprecipitating antibody.
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Figure 6: RANTES and (AOP)-RANTES induce dimerization of CCR5. (A) CCR5-transfected HEK-293 cells were stimulated for 60 s with 10 nM RANTES, 10 nM (AOP)-RANTES, or 20 nM SDF-1α at 37°C, and cross-linked using 1 mM DSS. Cell lysates were immunoprecipitated with mAb CCR5-03, electrophoresed, and transferred to nitrocellulose membranes. The Western blot was analyzed with mAb CCR5-02; as a positive control, CCR5-transfected HEK-293 cell lysates were immunoblotted with mAb CCR5-02. Arrows indicate the CCR5 monomer (38 kD) and dimer (76 kD). Upper corner insert shows RANTES-induced Ca2+ mobilization in CCR5-transfected HEK-293 cells, performed as described in Materials and Methods. Results, expressed as in Fig. 2 A, are a percentage of the maximum chemokine response. (B) CCR5-transfected HEK-293 cells as in A were immunoprecipitated and analyzed in Western blot with anti-CXCR4 mAb. Arrows indicate the CXCR4 monomer (42 kD) and dimer (84 kD). Protein loading was controlled using a protein detection kit (Pierce Chemical Co.). Upper corner insert shows SDF-1α–induced Ca2+ mobilization in CCR5-transfected HEK-293 cells, performed as described in Materials and Methods. Results are expressed as in A. Note that the response of CCR5-transfected HEK-293 cells to SDF-1α is weaker than RANTES, as measured by Ca2+ influx. This lower response correlates with the lower amounts of dimerized CXCR4 receptor, and explains the longer exposure time required to develop the Western blot for the dimerized receptor. The 52-kD protein band corresponds to the heavy chain of the immunoprecipitating antibody.

Mentions: Similar to growth factor–induced dimerization of tyrosine kinase receptors, some GPCR, including chemokine receptors like CCR2 and CXCR4, undergo ligand-induced dimerization (Hebert et al., 1996; Rodríguez-Frade et al., 1999; Vila-Coro, A.J., J.M. Rodríguez-Frade, A. Martín de Ana, M.C. Moreno-Ortiz, C. Martínez-A., and M. Mellado, manuscript submitted for publication). We have now tested whether RANTES and (AOP)-RANTES trigger CCR5 dimerization. DSS-mediated cross-linking in CCR5-transfected HEK-293 cells was carried out after RANTES or (AOP)-RANTES stimulation. In CCR5-transfected HEK-293 cells, but not in mock-transfected cells, a high molecular mass receptor species (76 kD) was observed following both stimuli. This band corresponds to the expected molecular mass of two CCR5 molecules, as assessed by immunoprecipitation with anti-CCR5 antibodies and Western blot developed with anti-CCR5 antibodies (Fig. 6 A). When CCR5-transfected HEK-293 cells were stimulated with SDF-1α, no CCR5 dimerization was observed under these experimental conditions (Fig. 6 A), despite the fact that HEK-293 cells express a functional CXCR4 receptor as determined by flow cytometry and SDF-1α–induced calcium mobilization assays (Fig. 6 B). When the same experiment was done using anti-CXCR4 mAb for immunoprecipitation and Western blot, a high molecular mass species (84 kD) of the CXCR4 receptor is observed only after SDF-1α, but not after RANTES or (AOP)-RANTES stimulation (Fig. 6 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)

RANTES and (AOP)-RANTES induce dimerization  of CCR5. (A) CCR5-transfected HEK-293 cells were stimulated  for 60 s with 10 nM RANTES, 10 nM (AOP)-RANTES, or 20 nM  SDF-1α at 37°C, and cross-linked using 1 mM DSS. Cell lysates  were immunoprecipitated with mAb CCR5-03, electrophoresed,  and transferred to nitrocellulose membranes. The Western blot  was analyzed with mAb CCR5-02; as a positive control, CCR5-transfected HEK-293 cell lysates were immunoblotted with mAb  CCR5-02. Arrows indicate the CCR5 monomer (38 kD) and  dimer (76 kD). Upper corner insert shows RANTES-induced  Ca2+ mobilization in CCR5-transfected HEK-293 cells, performed as described in Materials and Methods. Results, expressed as in Fig. 2 A, are a percentage of the maximum chemokine response. (B) CCR5-transfected HEK-293 cells as in A were  immunoprecipitated and analyzed in Western blot with anti-CXCR4 mAb. Arrows indicate the CXCR4 monomer (42 kD)  and dimer (84 kD). Protein loading was controlled using a protein detection kit (Pierce Chemical Co.). Upper corner insert  shows SDF-1α–induced Ca2+ mobilization in CCR5-transfected  HEK-293 cells, performed as described in Materials and Methods. Results are expressed as in A. Note that the response of  CCR5-transfected HEK-293 cells to SDF-1α is weaker than  RANTES, as measured by Ca2+ influx. This lower response correlates with the lower amounts of dimerized CXCR4 receptor,  and explains the longer exposure time required to develop the  Western blot for the dimerized receptor. The 52-kD protein band  corresponds to the heavy chain of the immunoprecipitating antibody.
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Figure 6: RANTES and (AOP)-RANTES induce dimerization of CCR5. (A) CCR5-transfected HEK-293 cells were stimulated for 60 s with 10 nM RANTES, 10 nM (AOP)-RANTES, or 20 nM SDF-1α at 37°C, and cross-linked using 1 mM DSS. Cell lysates were immunoprecipitated with mAb CCR5-03, electrophoresed, and transferred to nitrocellulose membranes. The Western blot was analyzed with mAb CCR5-02; as a positive control, CCR5-transfected HEK-293 cell lysates were immunoblotted with mAb CCR5-02. Arrows indicate the CCR5 monomer (38 kD) and dimer (76 kD). Upper corner insert shows RANTES-induced Ca2+ mobilization in CCR5-transfected HEK-293 cells, performed as described in Materials and Methods. Results, expressed as in Fig. 2 A, are a percentage of the maximum chemokine response. (B) CCR5-transfected HEK-293 cells as in A were immunoprecipitated and analyzed in Western blot with anti-CXCR4 mAb. Arrows indicate the CXCR4 monomer (42 kD) and dimer (84 kD). Protein loading was controlled using a protein detection kit (Pierce Chemical Co.). Upper corner insert shows SDF-1α–induced Ca2+ mobilization in CCR5-transfected HEK-293 cells, performed as described in Materials and Methods. Results are expressed as in A. Note that the response of CCR5-transfected HEK-293 cells to SDF-1α is weaker than RANTES, as measured by Ca2+ influx. This lower response correlates with the lower amounts of dimerized CXCR4 receptor, and explains the longer exposure time required to develop the Western blot for the dimerized receptor. The 52-kD protein band corresponds to the heavy chain of the immunoprecipitating antibody.
Mentions: Similar to growth factor–induced dimerization of tyrosine kinase receptors, some GPCR, including chemokine receptors like CCR2 and CXCR4, undergo ligand-induced dimerization (Hebert et al., 1996; Rodríguez-Frade et al., 1999; Vila-Coro, A.J., J.M. Rodríguez-Frade, A. Martín de Ana, M.C. Moreno-Ortiz, C. Martínez-A., and M. Mellado, manuscript submitted for publication). We have now tested whether RANTES and (AOP)-RANTES trigger CCR5 dimerization. DSS-mediated cross-linking in CCR5-transfected HEK-293 cells was carried out after RANTES or (AOP)-RANTES stimulation. In CCR5-transfected HEK-293 cells, but not in mock-transfected cells, a high molecular mass receptor species (76 kD) was observed following both stimuli. This band corresponds to the expected molecular mass of two CCR5 molecules, as assessed by immunoprecipitation with anti-CCR5 antibodies and Western blot developed with anti-CCR5 antibodies (Fig. 6 A). When CCR5-transfected HEK-293 cells were stimulated with SDF-1α, no CCR5 dimerization was observed under these experimental conditions (Fig. 6 A), despite the fact that HEK-293 cells express a functional CXCR4 receptor as determined by flow cytometry and SDF-1α–induced calcium mobilization assays (Fig. 6 B). When the same experiment was done using anti-CXCR4 mAb for immunoprecipitation and Western blot, a high molecular mass species (84 kD) of the CXCR4 receptor is observed only after SDF-1α, but not after RANTES or (AOP)-RANTES stimulation (Fig. 6 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