Figure 1.

Strong Smo activation stimulates cAMP production in S2 cells. Graphed data represent mean ± S.D. A, EPAC-BRET assay to measure changes in cAMP levels in S2-R+ cells in response to expression of constitutively active forms of Gα_s (Gαs^Q215L) or Gα_i (Gαi^Q205L). t test versus cells transfected with empty vector (−): **, p < 0.01; ***, p < 0.001. B, ptc-luc reporter assay monitoring target gene activation in mock- and Hh^N-transfected S2-R+ cells. t test: ***, p < 0.001. C, measurement of cAMP levels by EPAC-BRET assay in mock- and Hh^N-transfected S2-R+ cells. t test: n.s., not significant. D, schematic diagram of truncated/mutated Smo variants used in this study (not to scale). The relative locations of the 7-transmembrane domains (black boxes), the fours clusters of Gprk2 phosphorylation sites (green boxes), and the SAID containing three clusters of PKA/CkI phosphorylation sites (blue box) are indicated. Numbers refer to amino acid positions in Smo. E, ptc-luc reporter assay monitoring target gene activation in control and Smo^WT-GFP transfected cells, with and without Hh^N co-transfection. t test: ***, p < 0.001. F, measurement of cAMP levels by EPAC-BRET assay in cells transfected with empty vector (−), Smo^WT-GFP, or Smo^WT-GFP plus Hh^N. t test: ***, p < 0.001; n.s., not significant. G, ptc-luc reporter activity in cells transfected with empty vector (−) or Smo^SD-GFP. H, EPAC-BRET assay of cAMP levels in cells transfected with empty vector (−) or Smo^SD-GFP. t test: ***, p < 0.001. I, EPAC-BRET assay of cAMP levels in Smo^WT-GFP-expressing cells treated for 0.5, 1, 2, or 4 h with medium conditioned by control cells or cells expressing Hh^N, or co-transfected with Hh^N expression plasmid (Smo + Hh). t test: **, p < 0.01; ***, p < 0.001; n.s., not significant. J, immunoblot (IB) analysis of Smo^WT-GFP in cells treated as in I.