Figure 4.

CCL19-triggered G-protein coupling to CCR7 is barely impaired by Nb1, Nb5, or Nb38. (a) Schematic illustration of the G-protein competition assay based on split-luciferase complementation. (b–e) HEK293 cells transiently expressing β₂AR-SmBiT (b) or CCR7-SmBiT (c–e) together with LgBiT-mGα_i and Nb-YFP1 (Nb-Y1) constructs were incubated with coelenterazine H (5 µM) and subsequently stimulated (indicated by the arrow head) either with 10 µM isoproterenol (b) or 1.5 µg/mL CCL19 (c–e), respectively. As control, HEK293 cells were transiently co-transfected with GPCR-SmBiT, LgBiT-mGα_i and pcDNA3 (indicated in black). In this case, increase in luminescence indicated functional activity of reconstituted split NLuc luciferase upon recruitment and interaction of mGα_i with the GPCR. Replacing empty vector (pcDNA3) with Nb80-YFP1 (b) caused complete blockage in luciferase activity (indicated in green). Expressing Nb1-YFP1 (c), Nb5-YFP1 (d) or Nb38-YFP1 (e) barely interfered with mGα_i-coupling to CCR7 (indicated in red). Results represent the fold increase in luminescence over the baseline, which is set to 1, upon agonist-stimulation. Mean values and SEM of three independent experiments are shown.