GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress Gs-mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways. It is unknown how GPR161, an orphan receptor involved in Hedgehog signaling, is activated. This study identifies a sterol that promotes GPR161-driven cAMP signaling but shows that cAMP is dispensable for Hedgehog pathway repression.