Figure 2

Functional assay to identify chemokine neutraligands.

(a) Functional expression of Gqi5 in EGFP-CCR4-expressing HEK cells. Intracellular calcium responses were recorded after stimulation with 5 nM CCL17 (at t = 50 s) in EGFP-CCR4⁺HEK cells transfected with Gqi5 (⋄) at 21 °C. Gqi5-non transfected EGFP-CCR4⁺HEK cells were used as controls at 37 °C (◆) and 21 °C (•). Dose-response curves of CCL17 (b) and CCL22 (c)-induced calcium responses were performed in EGFP-CCR4⁺Gqi5⁺ HEK cells at 21 °C. Data represent the means ± SD of three independent experiments. (d) “Neutraligand” TRIC-r protocol. The identification of neutraligands relies on the measurement of kinetic-based calcium responses. To prove this concept, we used chalcone 4, a fully validated CXCL12 neutraligand. After stimulation with CXCL12 (at t = 60 s), EGFP-CXCR4⁺ HEK cells exhibited calcium responses (solid black line). When chalcone 4 was preincubated with CXCL12 for 1 h before addition to the cells (dotted black line), the amplitude of calcium responses was smaller than when the neutraligand was preincubated with the receptor, prior to chemokine addition (solid grey line). (e) “Antagonist” TRIC-r protocol. After stimulation with CXCL12 (at t = 90 s), EGFP-CXCR4⁺ HEK cells exhibited calcium responses (solid black line). When the CXCR4 antagonist (AMD3100) was preincubated with CXCL12 (solid grey line) for 1 h before addition to the cells, the amplitude of calcium responses was much greater than when the antagonist was preincubated with the receptor prior to chemokine addition (dotted black line). A: antagonist; CK: chemokine; N: neutraligand; R: receptor; the preincubation mixture is given in brackets. a.u.; arbitrary unit.