RABL2 positively controls localization of GPCRs in mammalian primary cilia

Dateyama, Izumi; Sugihara, Yoshihiro; Chiba, Shuhei; Ota, Reo; Nakagawa, Risa; Kobayashi, Tetsuo; Itoh, Hiroshi

doi:10.1242/jcs.224428

Cited by 27 publications

(51 citation statements)

References 48 publications

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“…Rabl2 is an atypical Rab small GTPase that promotes anterograde IFT from the ciliary base 26,[32][33][34] . Recently, we have shown that Rabl2 is required for ciliary targeting of Htr6 and Gpr161, with which it interacts 26 . Since Htr6 and Rabl2 are readily seen to interact by coimmunoprecipitation, we performed these assays to test whether Htr6 binding to Rabl2 depends on our newly identified CTSs.…”

Section: Htr6 Cts1 and Cts2 Contribute To Rabl2 Bindingmentioning

confidence: 99%

“…Rat monoclonal antibody: HA (Chromotek, 7c9, WB: 1:1000). Rabbit polyclonal antibodies: EGFP (Proteintech, 50403-2-AP, IF: 1:200, WB: 1:1000), Flag (Sigma, F7425, WB: 1:1000), adenylyl cyclase III (Santa Cruz, C-20, IF: 1:100), B9D1 (Novus Biologicals, NBP2-13 84489, WB: 1:1000) and Htr6 26 . Goat polyclonal antibodies: γ-Tubulin (Santa Cruz, sc-7396, IF: 1:200).…”

Section: Reagents and Antibodiesmentioning

confidence: 99%

“…However, whether this is also true for Htr6 has not yet been determined. To clarify this issue, we used HTR6-IMCD3 cells, which we previously generated to stably express Htr6 26 . Expression of two independent Tulp3 siRNAs (siTulp3 #1 and #2) in these cells caused a strong reduction in ciliary Htr6 intensity, which was not observed when a negative control luciferase siRNA (siLuc) was expressed instead ( Fig.1a-b).…”

Section: Introductionmentioning

confidence: 99%

“…In contrast, the tandem AP[AS]CQ motifs in Sstr3-IC3 do affect CTS1 function, in conjunction with a neighboring argininerich tract. On the other hand, Sstr3 CTS2 function mostly depends on the residues immediately following its seventh transmembrane helix, including LLxP and FK motifs, the latter homologous to the WR motif driving Smo ciliary targeting 24 .Finally, we studied how these newly identified CTSs control binding to well-established ciliary trafficking adapters like Tulp3 and Rabl2, both of which interact with the intraflagellar transport (IFT) machinery 25,26 . We show that Htr6 ciliary targeting is Tulp3-dependent, as previously shown for Sstr3 and other ciliary GPCRs 22,25,27 .…”

mentioning

confidence: 99%

“…Finally, we studied how these newly identified CTSs control binding to well-established ciliary trafficking adapters like Tulp3 and Rabl2, both of which interact with the intraflagellar transport (IFT) machinery 25,26 . We show that Htr6 ciliary targeting is Tulp3-dependent, as previously shown for Sstr3 and other ciliary GPCRs 22,25,27 .…”

mentioning

confidence: 99%

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

Barbeito

Tachibana

Martin-Morales

et al. 2020

Preprint

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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...

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