β-Arrestin mediates the export of ciliary GPR161 but not Smoothened together with the BBSome and intraflagellar transport machinery.

Specific G-protein-coupled receptors (GPCRs) exist on the ciliary membrane. Hedgehog signaling activation triggers the import of Smoothened into and export of GPR161 from cilia. The BBSome, which comprises eight Bardet-Biedl syndrome (BBS) proteins, mediates GPCR export, together with the intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes. The absence of any BBSome subunit or IFT27 (also known as BBS19) (an IFT-B subunit) impairs ciliary GPCR export, including that of GPR161. Plasma membrane GPCRs undergo phosphorylation by GPCR kinases (GRKs) and subsequent binding of β-arrestins [β-arrestin1 (ARRB1) and β-arrestin2 (ARRB2)], which is crucial for clathrin-mediated endocytosis. We here confirmed that GPR161 and β-arrestin are accumulated within cilia in the absence of IFT27 or the BBSome, and that ARRB1 and ARRB2 double-knockout impairs GPR161 export. Notably, we found that activation-mimetic β-arrestin mutants can interact with both the BBSome and ciliary GPCRs, and cause constitutive export of GPR161. Moreover, we demonstrated that GRK2 plays a crucial role in GPR161 export. We here propose that phosphorylated GPR161 recruits β-arrestins, converting them into their activated conformation. Activated β-arrestins then interact with the BBSome, which connects them to the IFT machinery to facilitate GPR161 export.