Multiple ligand binding sites regulate the Hedgehog signal transducer Smoothened in vertebrates.

The Hedgehog (Hh) pathway plays a central role in the development of multicellular organisms, guiding cell differentiation, proliferation and survival. While many components of the vertebrate pathway were discovered two decades ago, the mechanism by which the Hh signal is transmitted across the plasma membrane remains mysterious. This fundamental task in signalling is carried out by Smoothened (SMO), a human oncoprotein and validated cancer drug target that is a member of the G-protein coupled receptor protein family. Recent structural and functional studies have advanced our mechanistic understanding of SMO activation, revealing its unique regulation by two separable but allosterically-linked ligand-binding sites. Unexpectedly, these studies have nominated cellular cholesterol as having an instructive role in SMO signalling.

Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling.

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.