Fig 7

Fig 7

(A) GLI transcriptional reporter assay in Smo^−/− MEFs expressing wild-type SMO or SMOΔ570–581, treated with conditioned medium containing the N-terminal signaling domain of Sonic hedgehog (ShhN, green) or control, non-ShhN-containing conditioned medium (Vehicle, black). GFP serves as a negative control (“Neg.”). (B) BRET in HEK293 cells between nanoluc-tagged wild-type or Δ570–581 forms of SMO657 as donor, with YFP-tagged NbSmo2 (gray) or PKA-C (blue) as acceptor. (C) Left: Schematic of SMO-NbSmo2 fusion, predicted to block interactions with SMO that require the intracellular face of the 7TM domain. Right: YFP-tagged PKA-C was coexpressed in HEK293 cells with FLAG-tagged SMO674 (lane 1), SMO566 (lane 2), SMO657-NbSmo2 (lane 3), or SMO657-Nbβ2AR80 (lane 4). Following cell lysis and FLAG purification of SMO complexes, samples were separated on SDS-PAGE. Fluorescence scans of total protein (top) and YFP (bottom) in FLAG eluates are shown. Note that DSP crosslinker was not used in this experiment; thus, copurification of PKA-C was less efficient than in Fig 3E. Molecular masses are in kDa. (D) GLI transcriptional reporter assay in Smo^−/− MEFs expressing fusions to NbSmo2 or Nbβ2AR80. Non-Nb-fused SMO (“None”) serves as a positive control. (E) Confocal images of whole-mount wild-type zebrafish embryos or smo^−/− mutants injected with mRNAs encoding either wild-type SMO, SMO-Nbβ2AR80, or SMO-NbSmo2. Embryos 26 hpf were fixed and stained with antibodies against Prox1 (magenta) or En (green) to mark populations of muscle fiber nuclei. (F) GLI transcriptional reporter assay in Smo^−/− MEFs expressing wild-type SMO657 (“WT”) or SMO657Ala. (G) Zebrafish were injected with mRNAs encoding wild-type SMO657 or SMO657Ala, then stained for muscle fiber nuclei as described in (E). (H) Proposed model for SMO activation of GLI via PKA-C membrane sequestration: (1) Hh proteins bind to and inhibit PTCH1, inducing an activating conformational change in SMO; (2) Active SMO is recognized and phosphorylated by GRKs; (3) Phosphorylated SMO recruits PKA-C to the membrane, preventing PKA-C from phosphorylating and inhibiting GLI; (4) GPCRs that couple to Gα_s (such as GPR161) [92] or Gα_i/o/z (perhaps including SMO itself, which can couple to Gα_i/o/z [32,33,40–42]) can raise or lower cAMP levels, respectively, thereby affecting SMO/PKA-C interactions by regulating the size of the free PKA-C pool. (5) GLI is converted from a repressed (GLI_R) to an active (GLI_A) form and regulates transcription of Hh target genes. Data in (A), (B), (D), and (F): n = 3 biological replicates per condition. Error bars = SEM. Data in (E) and (G): n = 78 (SMO), 61 (SMO-NbSmo2), 63 (SMO-Nbβ2AR80), 62 (SMO657), 70 (SMO657Ala), and 100 (uninjected). The underlying data for this figure can be found under S7 Data. The uncropped protein gels are included in S8 Data. See S1 Table for statistical analysis. cAMP, cyclic AMP; DSP, dithiobis(succinimidyl propionate); En, Engrailed; GLI, glioma-associated; GPCR, G protein–coupled receptor; Hh, Hedgehog; hpf, hours postfertilization; MEF, mouse embryonic fibroblast; PKA-C, PKA catalytic subunits; RLU, relative luciferase unit; ShhN, N-terminal signaling domain of Sonic hedgehog; SMO, Smoothened.