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. Author manuscript; available in PMC: 2018 Jan 12.
Published in final edited form as: Cell. 2016 Dec 22;168(1-2):252–263.e14. doi: 10.1016/j.cell.2016.11.036

Figure 7. Ciliary exit pathways are required for the appropriate regulation of Hedgehog signaling.

Figure 7

(A) Endogenous GPR161 is lost by ectocytosis in retrieval-defective cells. WT, β-arrestin2−/−, or Arrb1/2−/− cells were treated with vehicle or SAG, fixed, immunostained for GPR161 and ciliary fluorescence levels measured. As indicated, cells were treated with blebbistatin (bleb) or cytochalasin D (CytoD). Mean values were normalized to the vehicle condition. Error bars: SEM. (n=61–163 cilia).

(B) Hedgehog-dependent removal of GPR161 is predicted to lower ciliary cAMP levels. In retrieval mutants, GPR161 is removed by ectocytosis still permitting a partial reduction in ciliary cAMP. Simultaneous blockage of retrieval and ectocytosis release is predicted to keep ciliary cAMP levels constant irrespective of Hedgehog pathway activation.

(C–H) Retrieval and ectocytosis ensure appropriate Gli3 processing. Wild-type or Ift27−/− (C–E) IMCD3 or (F–H) MEF cells were starved for 24 h, and then treated with vehicle or SAG and either DMSO or the Drebrin inhibitor BTP2 for 8 h. (C and F) show Western blots for Gli3R (see Fig. S7B–C for full blot). (D and G) plot ratios of Gli3R to Gli3FL from representative experiments. (E and H) report the Gli3 processing index from 3 biological replicates. The Gli3 processing index is the fractional change of the Gli3R:Gli3FL ratio upon SAG treatment (Gli3R:Gli3FLSAG/Gli3R:Gli3FLvehicle). Error bars: SD.