Fig. 3

Characterisation of signalling properties of Gαs-coupled JellyOp and its structural arrestin-biased mutant JellyOp F112A. a HEK293 cells expressing JellyOp F112A mutant (red squares) show a much reduced increase in luminescent cAMP reporter activity following exposure to a flash of light at 2 min (yellow arrow) compared to cells expressing JellyOp (black circles, n = 6). The response of cells expressing an inactivation mutant (K296A bovine numbering; blue triangles) are shown for comparison (n = 3; values in Additional file 1). b A significant increase in aequorin calcium reporter luminescence is seen in response to carbachol (100 μM) treatment of HEK293 cells (blue triangles, n = 5). No calcium reporter increase above background is seen in cells expressing JellyOp F112A (red squares) and exposed to light (P > 0.05, t test, n = 4; values in Additional file 1). c Fluorescence immunohistochemical labelling of the 1D4 epitope in HEK293 cells transiently expressing JellyOp (panel on the left) and F112A mutant (panel on the right). Scale bar: 20 μm. d A schematic presentation of an arrestin-based luminescent probe. Two fragments of a luciferase were fused to an opsin (Luc-C) and β-arrestin (Luc-N), respectively. When opsins are activated, β-arrestin is recruited to the receptors and the fragments recover their luminescence property. e β-arrestin luminescent assay reveals that light stimulation significantly enhances luminescence for β-arrestin 1 and 2 in HEK293 cells expressing positive control opsins known to have light-induced arrestin interaction (melanopsin and rhodopsin; n = 4 biological replicates; values are reported in Additional file 1). f β-arrestin luminescent assay reveals light triggered luminescence induction in JellyOp and F112A mutant only in the presence of 9-cis retinal. (*P < 0.5, **P < 0.1, ***P < 0.01, Student’s t test, n = 4; values in Additional file 1). g–h Kinetics of JellyOp (black circles and line) and F112A mutant (red square and line) interaction with β-arrestin 1 (g) and β-arrestin 2 (h) after a light flash (3 mW cm^–2) indicated by a yellow arrow (n = 3; values in Additional file 1)