CHARACTERIZATION OF THE RANTES MIP-1-ALPHA RECEPTOR (CC-CKR-1) STABLYTRANSFECTED IN HEK-293 CELLS AND THE RECOMBINANT LIGANDS

The CC chemokines RANTES and MIP-1 alpha are known to activate certain leucocytes and leucocytic cell lines. We have produced and fully char acterised the recombinant proteins expressed in E. coli, They induce c hemotaxis of the pro-monocytic cell line, THP-1 and T cells, THP-1 cel ls express three of the known CC chemokine receptors, In order to stud y the activation of a single receptor, we have expressed the shared re ceptor (CC CKR-1) for RANTES and MIP-1 alpha stably in the HEK 293 cel l 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 chemota xis assay, RANTES competes for [I-125]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 tran sfected HEK 293 cells respond to both these chemokines in the chemotax is assay with the same EC(5)0 values as those measured for THP-1 cells , This indicates that this cellular response can be mediated through t he CC CKR-1 receptor.