Characterisation of the RANTES/MIP-1 alpha receptor (CC CKR-1) stably transfected 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 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.
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
A E
AE
Proudfoot
C A
CA
Power
A
A
Hoogewerf
M O
MO
Montjovent
F
F
Borlat
T N
TN
Wells
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D054407:Chemokine CCL4; D018946:Chemokine CCL5 / Q000235:genetics* / Q000378:metabolism*; D018925:Chemokines / Q000235:genetics* / Q000378:metabolism*; D002633:Chemotaxis / Q000235:genetics; D003001:Cloning, Molecular; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D019402:Macrophage Inflammatory Proteins; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D015846:Monokines / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D018123:Receptors, Interleukin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection / Q000235:genetics
D054407:Chemokine CCL4; D018946:Chemokine CCL5; D018925:Chemokines; D016207:Cytokines; D019402:Macrophage Inflammatory Proteins; D008565:Membrane Proteins; D015846:Monokines; D018123:Receptors, Interleukin; D011994:Recombinant Proteins; D002118:Calcium