Figure 2.

Chemokine ligands differentially stimulate G protein activation via CCR4, CCR7 and CCR10. (a) The principle of the BRET-based G-protein recruitment assay. RLuc8 is fused at the C-terminus tail of GRK3 (GRKct), which is localized to the plasma membrane, and the YFP variant Venus is tagged at the Gβ₁γ₂ dimer. Upon activation of a chemokine receptor, the activity of the trimeric G protein complex (Gαi2 was overexpressed in this assay) can be detected as the dissociation of Gαi and Gβγ; the latter binds to the GRK3 C-terminal tail, bringing the YFP and RLuc8 into close proximity and enabling resonance energy transfer after addition of coelenterazine h (coel). (b–d) Chemokine-induced G-protein activation at (b) CCR4 (c) CCR7, and (d) CCR10. A dashed line of best fit is shown in the case that the signal does not reach saturation. The presented data are mean ± SEM of at least three independent experiments, each performed in triplicate.