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

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Abstract
The CC chemokines RANTES and MIP‐1α 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‐1α stably in the HEK 293 cell line. We have examined the effects of RANTES and MIP‐1α 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‐1α 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.