GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.
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Human GPR161 adopts the canonical active, G protein-coupled class A conformation when bound to heterotrimeric Gs, resolved at 2.74 A by cryo-EM.
"We conclude that our structure of GPR161-miniGs captures the Gs coupled, active conformation of the receptor."
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GPR161 has no orthosteric agonist: its own extracellular loop 2 folds into the ligand pocket, and the receptor is constitutively active.
"the ECL2 of GPR161 forms a beta hairpin that folds over the extracellular face of the receptor to completely occlude the canonical orthosteric ligand binding pocket ... The results from these two orthogonal assays demonstrate that GPR161 is constitutively active"
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Cholesterol binds an extrahelical TM6/TM7 site and promotes Gs coupling and cAMP production.
"Our combined biochemical, simulation, and signaling studies show that cholesterol, and potentially other sterols, can bind GPR161 to support interactions with Gs, thereby promoting cAMP production."
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GPR161 is the only GPCR with an AKAP domain, an amphipathic C-terminal helix that binds PKA type I regulatory subunits directly.
"GPR161 is unique among many GPCRs in that it contains a PKA-binding AKAP domain. ... Previous studies have identified an amphipathic helix in the C-terminus of GPR161 that directly binds PKA regulatory subunits type I (RI)"
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PKA anchoring rather than cAMP production is what GPR161 needs in order to keep GLI2 out of the ciliary tip; the two activities separate cleanly in mutant rescue experiments.
"We conclude that GPR161 binding to PKA-RI is essential for Hedgehog repression in the primary cilia, while GPR161-induced Gs signaling is dispensable."
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Extraciliary GPR161, in the plasma membrane and the periciliary recycling endosome, also contributes to GLI repressor formation.
"Both ciliary and extraciliary pools of GPR161 contribute to GLI-R formation and regulate tissue-specific repression of Hedgehog pathway in mice ... Therefore, both the plasma membrane and periciliary endosomal pools could support cAMP production by GPR161."
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Gpr161 knockout is embryonically lethal in mouse with limb, facial and nervous system defects consistent with hyperactive Hedgehog signalling.
"Knockout of Gpr161 in mice is embryonically lethal, and the embryos display severe limb, facial, and early nervous system defects indicative of hyperactive Hedgehog signaling"