Abstract

Structural studies show that the activity of the G-protein-coupled receptor Smoothened is modulated by ligand-regulated interactions between its extracellular and transmembrane domains. Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD–linker domain–TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains. Smoothened (SMO) is a G-protein-coupled receptor that transduces Hedgehog (Hh) signals across the membrane in all animals. Despite being a key developmental regulator, oncoprotein and drug target in oncology, the mechanism by which SMO is activated has remained unknown. These authors solve the 3.2 Å crystal structure of SMO containing its extracellular cysteine-rich (CRD), linker and heptahelical G-protein-coupled receptor (TMD) domains. Surprisingly, a cholesterol molecule was bound to SMO in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of SMO to transmit native Hh signals. Binding of the potent antagonist and anti-cancer drug vismodegib leads to a number of conformational changes and the loss of cholesterol from the CRD–linker domain–TMD interface.