Figure 4. The Smoothened cysteine-rich domain is required for cholesterol-mediated activation of Hh signaling.
(A) Structure of human SMO (PDB 5L7D), with the CRD in orange, the 7TMD in blue, the linker domain in pink, and the cholesterol ligand bound to the CRD in green. The Cα positions of the gatekeeper residues in the two ligand binding sites are highlighted as yellow spheres and numbered, with the mouse numbering shown in parenthesis. The inset shows a close-up of the cholesterol-binding site. D95 and Y130 form part of a hydrogen-bonding network (dotted lines) with the 3-hydroxyl of cholesterol, G111 abuts the iso-octyl chain of cholesterol, and D473 is a critical residue in the 7TMD binding-site. (B, C and D) Dose-response curves for the indicated agonists in Smo-/- cells stably expressing WT SMO (always solid black circles) or the indicated SMO variants (open circles) carrying mutations in the 7TMD ligand-binding site (B) or at two opposite ends of the CRD binding groove (C and D). All agonists were applied to cells for 12 hr and mean (±SD) values for Gli1 mRNA are plotted based on 3 replicates. In C and D, values on the abscissa represent Log ([Agonist] in M) and the ordinate for all four graphs is only shown once at the left.
Figure 4—figure supplement 1. Role of the cysteine-rich domain of Smoothened in responses to cholesterol and side-chain oxysterols.
