Smoothened (SMO), a class Frizzled G protein-coupled receptor (class F GPCR), transduces the Hedgehog signal across the cell membrane. Sterols can bind to its extracellular cysteine-rich domain (CRD) and to several sites in the seven transmembrane helices (7-TMs) of SMO. However, the mechanism by which sterols regulate SMO via multiple sites is unknown. Here we determined the structures of SMO–G_i complexes bound to the synthetic SMO agonist (SAG) and to 24(S),25-epoxycholesterol (24(S),25-EC). A novel sterol-binding site in the extracellular extension of TM6 was revealed to connect other sites in 7-TMs and CRD, forming an intramolecular sterol channel from the middle side of 7-TMs to CRD. Additional structures of two gain-of-function variants, SMO^D384R and SMO^G111C/I496C, showed that blocking the channel at its midpoints allows sterols to occupy the binding sites in 7-TMs, thereby activating SMO. These data indicate that sterol transport through the core of SMO is a major regulator of SMO-mediated signaling.

Smoothened (SMO) 是一种卷曲 G 蛋白偶联受体（F 类 GPCR），可跨细胞膜转导 Hedgehog 信号。甾醇可以与其胞外富含半胱氨酸的结构域 (CRD) 以及 SMO 的七个跨膜螺旋 (7-TM) 中的多个位点结合。然而，甾醇通过多个位点调节 SMO 的机制尚不清楚。在这里，我们确定了与合成 SMO 激动剂 (SAG) 和 24( S ),25-环氧胆固醇 (24( S ),25-EC)结合的 SMO-G i 复合_{物的结构。}TM6细胞外延伸中的一个新的甾醇结合位点被发现连接7-TMs和CRD中的其他位点，形成从7-TMs的中间侧到CRD的分子内甾醇通道。两种功能获得性变体 SMO ^D384R和 SMO ^G111C/I496C的附加结构表明，在中点阻断通道可使甾醇占据 7-TM 中的结合位点，从而激活 SMO。这些数据表明，通过 SMO 核心的甾醇转运是 SMO 介导的信号传导的主要调节剂。