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 G_s. 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 G_s coupling. Mutations that prevent sterol binding to GPR161 suppress G_s-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.

孤儿 G 蛋白偶联受体 (GPCR) GPR161 通过抑制 Hedgehog 信号传导在发育中发挥核心作用。GPR161 如何激活的基本原理仍不清楚。_{在这里，我们确定了与异三聚体G}结合的活性人 GPR161 的低温电子显微镜结构。该结构揭示了占据典型 GPCR 正构配体口袋的细胞外环 2。此外，与跨膜螺旋 6 和 7 相邻结合的甾醇可稳定 G _s偶联所需的 GPR161 构象。阻止甾醇与 GPR161 结合的突变会抑制 G _s介导的信号传导。这些突变体保留了抑制初级纤毛中 GLI2 转录因子积累的能力，这是纤毛 GPR161 的一个关键功能。相比之下，GPR161 C 末端的蛋白激酶 A 结合位点对于抑制 GLI2 纤毛积累至关重要。我们的工作强调了 GPR161 的结构特征如何与 Hedgehog 通路相互作用，并为理解 GPR161 在其他信号通路中的作用奠定了基础。