Fig. 5.

Effector-stimulated activity of SYNAC in live cells. (A) cAMP generation in untransfected (Sf9) or SYNAC-expressing Sf9 cells (SYNAC) after treatment with indicated concentrations of FSK. cAMP levels are reported relative to untransfected, untreated cells. (B) Basal cAMP levels are elevated in Sf9 cells coexpressing SYNAC and Gαs, but not SYNAC or Gαs independently. (C) cAMP levels are basally elevated in cells coexpressing SYNAC and a β2-AR-Gαs fusion protein, and are further increased by addition of isoproterenol (ISO). cAMP levels in Sf9 cells expressing SYNAC, β2-AR-Gαs fusion, or coexpressing SYNAC and β2-AR-Gαs fusion with or without 100 μM isoproterenol treatment (10 minutes). (D) SYNAC FRET response in Sf9 cells after FSK (100 μM) and isoproterenol (100 μM) treatment with and without coexpression of Gαs and β2-AR-Gαs. Both conditions were incubated for 5 minutes with their respective conditions. Cells were held at equivalent optical density and expression as judged by mCerulean fluorescence. Activity data are mean ± S.E.M. of three independent repeats using three different preparations of protein. FRET data are mean ± S.D. of four spectra. Significance assessed by Student’s t test (unpaired). n.s. = not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001.