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

FEBS Letters

Montjovent, Marc-Olivier¹  Hoogewerf, Arlene¹  Power, Christine A.¹  Wells, Timothy N.C.¹  Borlat, Frederic¹  Proudfoot, Amanda E.I.¹ 

[1] Glaxo Institute for Molecular Biology, 14, Ch. des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland

关键词: Recombinant chemokine;    Chemotaxis;    RANTES/MIP-1α receptor;    HEK 293 transfectant;    RANTES;    Regulated on Activation;    Normal T expressed and Secreted;    MIP-1α;    macrophage inflammatory protein-1α;    MCP-1;    monocyte chemotactic protein-1;    Il-8;    interleukin-8;    IPTG;    isopropylthiogalactoside;    EC50;    concentration of agonist giving half the maximal response;    IC50;    concentration of inhibitor giving half the maximal inhibition;   

DOI  :  10.1016/0014-5793(95)01235-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown