- Hoppe, Nicholas;
- Harrison, Simone;
- Hwang, Sun-Hee;
- Chen, Ziwei;
- Karelina, Masha;
- Deshpande, Ishan;
- Suomivuori, Carl-Mikael;
- Palicharla, Vivek;
- Berry, Samuel;
- Tschaikner, Philipp;
- Regele, Dominik;
- Covey, Douglas;
- Stefan, Eduard;
- Marks, Debora;
- Reiter, Jeremy;
- Dror, Ron;
- Evers, Alex;
- Mukhopadhyay, Saikat;
- Manglik, Aashish
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.