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Cell lines expressing recombinant aequorin and a G-protein coupled receptor for functional screening

using the MicroLumat single-detector, singleinjector luminometer.

CHO-CCR5-Gα16-apoaequorin cells were injected onto increasing (from column 1 to 12) concentrations of RANTES (rows A and B, same dilutions in duplicate), MIP1α (rows C and D, duplicates), MIP1β (rows E and F, duplicates), and another analog of RANTES (rows G and H, duplicates). The emitted light was recorded for 30 seconds and the intensity of the emitted light was plotted as a function of time. The scaling is the same for all the graphs presented here.

Agonist-induced light emission in CHO cells expressing the CCR5 receptor

Human chemokine CCR3 receptor:

A K562 cell line expressing the chemokine CCR3 receptor, the Gα16 coupling protein and apoaequorin was established. Dose-response curves were obtained as described above and results are shown in figure 4.

Human chemokine CCR8 receptor:

CHO cells expressing the chemokine CCR8 receptor were transfected with a plasmid encoding the Gα16 coupling protein and apoaequorin and stable transfectants were selected with antibiotic. The mix of clones obtained from this transfection was used to generate a dose-response curve with I-309 as described above. Results are shown in figure 5.

Human serotonin 5HT2B receptor:

A CHO cell line expressing the serotonin 5HT2B receptor, the Gα16 coupling protein and apoaequorin was established.

A. Detection of agonists

Dose-response curves for agonists of the 5HT2B receptor were obtained as described above and results are shown in figure 6A.

B. Dectection of antagonists

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