Proudfoot A E, Power C A, Hoogewerf A, Montjovent M O, Borlat F, Wells T N
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
FEBS Lett. 1995 Nov 27;376(1-2):19-23. doi: 10.1016/0014-5793(95)01235-x.
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.
已知CC趋化因子RANTES和MIP-1α可激活某些白细胞和白细胞系。我们已经制备并全面表征了在大肠杆菌中表达的重组蛋白。它们可诱导前单核细胞系THP-1和T细胞的趋化性。THP-1细胞表达三种已知的CC趋化因子受体。为了研究单一受体的激活情况，我们已在HEK 293细胞系中稳定表达了RANTES和MIP-1α的共享受体（CC CKR-1）。我们通过平衡结合研究和趋化性测定，研究了RANTES和MIP-1α对CC CKR-1转染子的影响。在转染子中，RANTES与[125I]RANTES竞争，IC50为0.6±0.23 nM，而MIP-1α与其放射性标记物竞争，IC50为10±1.6 nM。这些亲和力与在THP-1细胞系上测得的相同。在趋化性测定中，稳定转染的HEK 293细胞对这两种趋化因子的反应与THP-1细胞测得的EC50值相同。这表明这种细胞反应可通过CC CKR-1受体介导。
