Smoothened (SMO), a class-Frizzled G protein-coupled receptor (class-F GPCR), transduces the Hedgehog signal across cell membrane. Sterols can bind to its extracellular cysteine rich domain (CRD) and to several sites in the 7 transmembrane helices (7-TMs) of SMO. However, the mechanism how sterols regulate SMO via multiple sites is unknown. Here we determined 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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.