Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

G-protein coupled receptors (GPCRs) undergo regulated trafficking into and out of cilia to control cilium-based signaling pathways. β-arrestin 2, a molecular sensor of activated GPCRs, and the BBSome are required for the signal-dependent exit of ciliary GPCRs but it is not known how β-arrestin 2 relays the activation state of GPCRs to the ciliary exit machinery. Here we find that, upon activation, the ciliary GPCRs SSTR3 and GPR161 become tagged with K63-linked ubiquitin (K63Ub) chains in a β-arrestin 2-dependent manner prior to BBSome-mediated exit. Removal of ubiquitin acceptor residues from SSTR3 and GPR161 demonstrates that ubiquitination of ciliary GPCRs is required for their regulated exit from cilia. Furthermore, targeting a K63Ub-specific deubiquitinase to cilia blocks the exit of GPR161, SSTR3 and SMO from cilia. Finally, ubiquitinated proteins accumulate in cilia of mammalian photoreceptors and Chlamydomonas cells. We conclude that K63Ub chains mark GPCRs and other unwanted ciliary proteins for recognition by the ciliary exit machinery.