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

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

doi:10.1101/2023.05.23.540554

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

bioRxiv [Preprint]. 2023 May 24:2023.05.23.540554. doi: 10.1101/2023.05.23.540554.

Authors

Nicholas Hoppe^{1

2}, Simone Harrison^{1

2}, Sun-Hee Hwang³, Ziwei Chen^{4

5}, Masha Karelina^{6

7

8

9

10}, Ishan Deshpande¹, Carl-Mikael Suomivuori^{7

8

9

10}, Vivek R Palicharla³, Samuel P Berry¹¹, Philipp Tschaikner^{12

13}, Dominik Regele¹², Douglas F Covey^{4

5

14

15}, Eduard Stefan^{12

13}, Debora S Marks¹¹, Jeremy Reiter¹⁶, Ron O Dror^{6

7

8

9

10}, Alex S Evers^{4

5

15}, Saikat Mukhopadhyay³, Aashish Manglik^{1

17

18}

Affiliations

¹ Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA.
² Biophysics Graduate Program, University of California, San Francisco, CA, USA.
³ Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁴ Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
⁵ Taylor Institute for Innovative Psychiatric Research, St. Louis, MO 63110, USA.
⁶ Biophysics Program, Stanford University, Stanford, CA 94305, USA.
⁷ Department of Computer Science, Stanford University, Stanford, CA, USA.
⁸ Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA.
⁹ Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA.
¹⁰ Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA, USA.
¹¹ Department of Systems Biology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA.
¹² Institute of Molecular Biology and Center for Molecular Biosciences, University of Innsbruck, Innsbruck 6020, Austria.
¹³ Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck; Tyrolean Cancer Research Institute (TKFI), Innsbruck 6020, Austria.
¹⁴ Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110, USA.
¹⁵ Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USA.
¹⁶ Department of Biochemistry and Biophysics, Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 94158.
¹⁷ Department of Anesthesia and Perioperative Care, University of California, San Francisco, CA, USA.
¹⁸ Chan Zuckerberg Biohub, San Francisco, CA, USA.

Abstract

The orphan G protein-coupled receptor (GPCR) GPR161 is enriched in primary cilia, where it plays a central role in suppressing Hedgehog signaling¹. GPR161 mutations lead to developmental defects and cancers^2,3,4. The fundamental basis of how GPR161 is activated, including potential endogenous activators and pathway-relevant signal transducers, remains unclear. To elucidate GPR161 function, we determined a cryogenic-electron microscopy structure of active GPR161 bound to the heterotrimeric G protein complex G_s. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, we identify a sterol that binds to a conserved extrahelical site adjacent to transmembrane helices 6 and 7 and stabilizes a GPR161 conformation required for G_s coupling. Mutations that prevent sterol binding to GPR161 suppress cAMP pathway activation. Surprisingly, these mutants retain the ability to suppress GLI2 transcription factor accumulation in cilia, a key function of ciliary GPR161 in Hedgehog pathway suppression. By contrast, a protein kinase A-binding site in the GPR161 C-terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how unique structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the broader role of GPR161 function in other signaling pathways.

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Abstract

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