Htr6 and Sstr3 ciliary targeting relies on both IC3 loops and C-terminal tails

G protein-coupled receptors (GPCRs) are the most common pharmacological target in clinical practice. To perform their signaling functions, many GPCRs must accumulate at primary cilia, microtubule-based plasma membrane protrusions that work as cellular antennae. Despite their great importance, the molecular mechanisms underlying GPCR ciliary targeting remain poorly understood. Serotonin receptor 6 (Htr6) and somatostatin receptor 3 (Sstr3) are two brain-enriched ciliary GPCRs controlling cognition and involved in multiple pathologies such as Alzheimers disease and cancer. We previously showed that the third intracellular loops (IC3s) of Htr6 and Sstr3 contain ciliary targeting sequences (CTSs) that are sufficient to confer ciliary localization to non-ciliary GPCRs. However, these CTSs are dispensable for the ciliary targeting of Htr6 and Sstr3 themselves, suggesting these GPCRs have additional CTSs. Herein, we show that the C-terminal tails of Htr6 and Sstr3 also contain CTSs, which act redundantly with those in the IC3s. Accordingly, simultaneous disruption of CTS1 (IC3) and CTS2 (C-terminal tail) abolishes ciliary targeting of both receptors. Mapping the individual residues required for Htr6 ciliary targeting reveals RKQ and LPG motifs critical for CTS1 and CTS2 function, respectively. In Sstr3, CTS1 function relies on the tandem AP[AS]CQ motifs and a subsequent arginine-rich stretch, whereas CTS2 operation requires the juxtamembrane residues. Furthermore, we shed light on the mechanisms of action of Htr6 CTSs by showing how they regulate binding to Tulp3 and Rabl2, two adapters needed for ciliary GPCR targeting.