HTR6 and SSTR3 ciliary targeting relies on both IC3 loops and C-terminal tails

Abstract

G protein-coupled receptors (GPCRs) are the most common pharmacological target in human clinical practice. To perform their functions, many GPCRs must accumulate inside primary cilia, microtubule-based plasma membrane protrusions working as cellular antennae. Nevertheless, 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 involved in cognition and pathologies such as Alzheimer’s disease and cancer. Although the third intracellular loops (IC3) of HTR6 and SSTR3 suffice to target non-ciliary GPCRs to cilia, these IC3s are dispensable for ciliary targeting of HTR6 and SSTR3 themselves, suggesting these GPCRs contain additional ciliary targeting sequences (CTSs). Herein, we discover and characterize novel CTSs in HTR6 and SSTR3 C-terminal tails (CT). These CT-CTSs (CTS2) act redundantly with IC3-CTSs (CTS1), each being sufficient for ciliary targeting. In HTR6, RKQ and LPG motifs are critical for CTS1 and CTS2 function, respectively, whereas in SSTR3 these roles are mostly fulfilled by AP[AS]CQ motifs in IC3 and juxtamembrane residues in CT. Furthermore, we shed light on how these CTSs promote ciliary targeting by modulating binding to ciliary trafficking adapters TULP3 and RABL2.

HTR6 and SSTR3 targeting to primary cilia

“…More specifically, using the same chimera with which they proved that HTR6-IC3 targets HTR7 to cilia (Figure 2a, Chimera N), they showed that introducing the A242F + Q246F mutation into HTR6-IC3 prevents ciliary localization [51]. Although we fully confirmed this result, we also found that this mutation strongly reduced the ability of this chimera to traffic to the cell surface, instead accumulating intracellularly, most likely in the endoplasmic reticulum [59]. Thus, this mutant chimera failed to fold properly, explaining its inability to accumulate in cilia [59].…”

“…Furthermore, both CLS1 and CLS2 can target other transmembrane proteins to cilia, including not only HTR7 (Figure 2a,c), but also CD8α, a non-ciliary single-pass transmembrane protein (Figure 2d). In contrast, CLS2 fusion to soluble EGFP did not suffice for ciliary localization, indicating a requirement for membrane association [59].…”

“…More recently, similar chimera studies indicated that two other ciliary GPCRs, NPY2R, and GPR83, contain CLSs not only in IC3 but also in CT [53]. To identify the missing CLSs in HTR6, we performed additional chimera studies with the HTR6/HTR7 pair [59]. This made it clear that HTR6 also contains CLSs in both IC3 and CT.…”

“…Indeed, HTR6 ciliary localization is completely lost if both its IC3 and CT are replaced by those of HTR7 (Figure 2b) [59]. This chimera, however, readily reaches the plasma membrane (PM), showing that its folding and intracellular trafficking are not perturbed [59]. Thus, HTR6 ciliary targeting relies on two CLSs, one in IC3 (CLS1) and one in CT (CLS2) (Figure 2b).…”

“…In contrast, deleting HTR6-CT's second half abolished ciliary localization. Finer deletion mapping, followed by alanine-scanning mutagenesis of individual residues, led to the identification of three critical residues for CLS2 function: the LPG motif (aa 400-402 of mouse HTR6, fully conserved in humans) (Figure 3a) [59].…”