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 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 Alzheimer's 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. 3 (IC3s). This was first found for Sstr3 and Htr6 and was later extended to others like Mchr1, Gpr161, Mc4r or Npy2r 9,[20][21][22][23] .For Sstr3 and Htr6, their CTSs were discovered by generating chimeras between these ciliary GPCRs and their non-ciliary relatives Sstr5 and Htr7. After extensive analyses, replacing the IC3s of Sstr5 or Htr7 with those of Sstr3 or Htr6, respectively, was found to suffice for ciliary targeting of the nonciliary receptors. Furthermore, ciliary targeting of these chimeras was abolished by mutating to phenylalanine the first and last residues of Ax[AS]xQ motifs (hereafter referred to as A-Q motifs) present in the IC3s of both Sstr3 and Htr6 20 . In this study, another interesting observation was noted: the reverse pair of chimeras, in which the IC3s of Sstr3 and Htr6 were replaced by those of Sstr5 and Htr7, still accumulated in cilia, indicating that the newly discovered CTSs, albeit sufficient for ciliary targeting of non-ciliary receptors, were dispensable for ciliary targeting of Sstr3 and Htr6 themselves. This suggested, it was also noted, the presence of additional CTSs in these receptors 20 . A subsequent study confirmed that Htr6 IC3 is dispensable for its ciliary targeting 16 .Herein, we report the identification and characterization of those missing CTSs. We show that, for both Sstr3 and Htr6, ciliary targeting occurs as long as either IC3, CT, or both, are pre...

Comparison of Ciliary Targeting of Two Rhodopsin-Like GPCRs: Role of C-Terminal Localization Sequences in Relation to Cilium Type

“…Our results indicate that the C terminus of SSTR3 is required for TULP3-mediated ciliary localization. Consistent with this observation, Barbeito et al (2021) recently reported that a proximity labeling assay indicated association of TULP3 with the C terminus of SSTR3. The tubby protein family includes TUB and TULP1-4.…”

“…During the course of our study, Barbeito et al (2021) reported the presence of a C-terminal CLS in SSTR3 and HTR6, expressed in IMCD3 cells. This work, which was focused mainly on HTR6, used some different approaches from ours to characterize the CLS, resulting in some different conclusions as well as some in common.…”

“…SSTR3 has been used as a model GPCR to investigate ciliary trafficking mechanisms, such as the role of the BBSome in ciliary retrieval (Ye et al, 2018) and ciliary entry (Berbari et al, 2008b;Jin et al, 2010), as well as the role of b -arrestin (Green et al, 2016) and Rab proteins (Tower-Gilchrist et al, 2011) in ciliary trafficking, and robust ciliary localization has been advantageous in studies of intraciliary protein movement (Ye et al, 2013;Lee et al, 2018). With regard to ciliary localization, studies had focused on the IC3 of SSTR3, which was found to contain an important CLS (Berbari et al, 2008a;Barbeito et al, 2021). The third intracellular loop of SSTR3 was also used to generate a chimeric RHO-i3SR3-EGFP protein to enhance ciliary enrichment of RHO in vitro and thereby study intraciliary trafficking of RHO using single-molecule tracking (Geneva et al, 2017;Lee et al, 2018).…”

Formin-like 1 β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse

“…CD63 is an enriched marker in intraluminal vesicles (ILVs) contained into MVB and exosomes and has been widely used to study MVB secretory traffic in both living and fixed cells(2,17).The FMNL1 interfering vector (shFMNL1-HA-YFP) and FMNL1-interfering, YFP-FMNL1bWT expressing vector (shFMNL1-HA-YFP-FMNL1bWT) were previously described (35) and generously provided by Dr. Billadeau. shFMNL1-HA-YFP-FMNL1βS1086A and shFMNL1-HA-YFP-FMNL1βS1086D mutants were generated by site-directed mutagenesis, as previously reported(74). Briefly, overlap extension PCR was used to introduce the desired mutations into a 900 bp SalI-NotI fragment, which was then used to replace the corresponding wild type sequence in shFMNL1-HA-YFP-FMNL1β-WT.…”

Formin-like 1 β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes

“…CD63 is an enriched marker in intraluminal vesicles (ILVs) contained into MVB and exosomes and has been widely used to study MVB secretory traffic in both living and fixed cells (2, 17).The FMNL1 interfering vector (shFMNL1-HA-YFP) and FMNL1-interfering, YFP-FMNL1bWT expressing vector (shFMNL1-HA-YFP-FMNL1bWT) were previously described (35) and generously provided by Dr. Billadeau. shFMNL1-HA-YFP-FMNL1βS1086A and shFMNL1-HA-YFP-FMNL1βS1086D mutants were generated by site-directed mutagenesis, as previously reported(73). Briefly, overlap extension PCR was used to introduce the desired mutations into a 900 bp SalI-NotI fragment, which was then used to replace the corresponding wild type sequence in shFMNL1-HA-YFP-FMNL1β-WT.…”