Characterisation of the RANTES/MIP-1 alpha receptor (CC CKR-1) stably transfected in HEK 293 cells and the recombinant ligands

FEBS Lett. 1995 Nov 27;376(1-2):19-23. doi: 10.1016/0014-5793(95)01235-x.

Abstract

The CC chemokines RANTES and MIP-1 alpha are known to activate certain leucocytes and leucocytic cell lines. We have produced and fully characterised the recombinant proteins expressed in E. coli. They induce chemotaxis of the pro-monocytic cell line, THP-1 and T cells. THP-1 cells express three of the known CC chemokine receptors. In order to study the activation of a single receptor, we have expressed the shared receptor (CC CKR-1) for RANTES and MIP-1 alpha stably in the HEK 293 cell line. We have examined the effects of RANTES and MIP-1 alpha on the CC CKR-1 transfectants by equilibrium binding studies and in a chemotaxis assay. RANTES competes for [125I]RANTES with an IC50 of 0.6 +/- 0.23 nM, whereas MIP-1 alpha competes for its radiolabelled counterpart with an IC50 of 10 +/- 1.6 nM in the transfectants. These affinities are the same as those measured on the THP-1 cell line. The stably transfected HEK 293 cells respond to both these chemokines in the chemotaxis assay with the same EC50 values as those measured for THP-1 cells. This indicates that this cellular response can be mediated through the CC CKR-1 receptor.

MeSH terms

  • Base Sequence
  • Binding Sites
  • Binding, Competitive / genetics
  • Calcium / metabolism
  • Cell Line
  • Chemokine CCL4
  • Chemokine CCL5 / genetics*
  • Chemokine CCL5 / metabolism*
  • Chemokines / genetics*
  • Chemokines / metabolism*
  • Chemotaxis / genetics
  • Cloning, Molecular
  • Cytokines / genetics*
  • Cytokines / metabolism*
  • Electrophoresis, Agar Gel
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial / genetics
  • Humans
  • Leukocytes / metabolism
  • Macrophage Inflammatory Proteins
  • Membrane Proteins / metabolism
  • Molecular Sequence Data
  • Monokines / genetics*
  • Monokines / metabolism*
  • Protein Binding / genetics
  • Receptors, Interleukin / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Transfection / genetics

Substances

  • Chemokine CCL4
  • Chemokine CCL5
  • Chemokines
  • Cytokines
  • Macrophage Inflammatory Proteins
  • Membrane Proteins
  • Monokines
  • Receptors, Interleukin
  • Recombinant Proteins
  • Calcium