BBS1 is involved in retrograde trafficking of ciliary GPCRs in the context of the BBSome complex

Nozaki, Shohei; Katoh, Yohei; Kobayashi, Takuya; Nakayama, Kazuhisa

doi:10.1371/journal.pone.0195005

Cited by 57 publications

(69 citation statements)

References 38 publications

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“…The strategy of CRISPR/Cas9-mediated gene disruption of hTERT-RPE1 cells using homology-independent DNA repair was previously reported in detail ( Katoh et al, 2017 ); also see ( Funabashi et al, 2017 ; Hirano et al, 2017 ; Nishijima et al, 2017 ; Nozaki et al, 2018 , 2017 ; Takahara et al, 2018 ). Single guide RNA (sgRNA) sequences targeting the IFT22 gene or those targeting both the IFT70A and IFT70B genes (see ) were designed using CRISPR design ( Hsu et al, 2013 ).…”

Section: Methodsmentioning

confidence: 99%

Robust interaction of IFT70 with IFT52–IFT88 in the IFT-B complex is required for ciliogenesis

2018

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In the intraflagellar transport (IFT) machinery, the IFT-B and IFT-A complexes mediate anterograde and retrograde ciliary protein trafficking, respectively. Among the 16 subunits of the IFT-B complex, several subunits are essential for ciliogenesis, whereas others, which are associated peripherally with the complex, are dispensable for ciliogenesis but play a role in protein trafficking. IFT22-knockout (KO) cells established in this study demonstrated no defects in ciliogenesis or ciliary protein trafficking. In stark contrast, IFT70A and IFT70B double-knockout cells did not form cilia, even though IFT70 is associated peripherally with the IFT-B complex via the IFT52–IFT88 dimer, and other IFT-B subunits assembled at the ciliary base in the absence of IFT70. Exogenous expression of either IFT70A or IFT70B restored the ciliogenesis defect of IFT70-KO cells, indicating their redundant roles. IFT70 has 15 consecutive tetratricopeptide repeats (TPRs) followed by a short helix (α36). Deletion of the first TPR or α36 of IFT70A greatly reduced its ability to interact with the IFT52–IFT88 dimer. Exogenous expression of any of the IFT70A deletion mutants in IFT70-KO cells could not restore ciliogenesis. These results show that IFT70 plays an essential role in ciliogenesis, although it is dispensable for assembly of the residual IFT-B subunits.

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Section: Methodsmentioning

confidence: 99%

Robust interaction of IFT70 with IFT52–IFT88 in the IFT-B complex is required for ciliogenesis

2018

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“…Cells with no signs of cell surface expression were not counted (such cells were very rare, less than 1%, unless otherwise noted in the text). Quantification of Htr6 intensity in RNAi experiments was performed with Image J as described 43 . To quantify ciliary signal intensity of Sstr3 mutants, at least 25 cilia per condition were imaged from a representative experiment.…”

Section: Immunofluorescence and Quantitationsmentioning

confidence: 99%

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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“…Cilia are necessary for normal activation as well as repression of the Hh signaling pathway (Goetz & Anderson, ; Huang & Schier, ; Wolff et al, ). It was shown that in the absence of the ciliary protein BBS1 and under stimulation of Hh, the ciliary localization of smoothened (Smo) was further enhanced compared with wild‐type cells (Nozaki, Katoh, Kobayashi, & Nakayama, ). We, thus, examined the expression of the Hh receptor Patch1 ( Ptc1 ) and the downstream gene eng in the developing somites in iqce morphants.…”

Section: Resultsmentioning

confidence: 99%

“…It is well known that the primary cilium acts as a Hh signal transduction machine (Goetz, Ocbina, & Anderson, ). Recently, it was shown that in a bbs1 KO cell line, the entry of Smo into cilia upon Hh stimulation was increased compared with the wild‐type cell line (Nozaki et al, ). This, in turn, activated ectopic eng expression as observed in iqce zebrafish morphants.…”

Section: Discussionmentioning

confidence: 99%

NovelIQCEvariations confirm its role in postaxial polydactyly and cause ciliary defect phenotype in zebrafish

et al. 2019

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Polydactyly is one of the most frequent inherited defects of the limbs characterized by supernumerary digits and high‐genetic heterogeneity. Among the many genes involved, either in isolated or syndromic forms, eight have been implicated in postaxial polydactyly (PAP). Among those, IQCE has been recently identified in a single consanguineous family. Using whole‐exome sequencing in patients with uncharacterized ciliopathies, including PAP, we identified three families with biallelic pathogenic variations in IQCE. Interestingly, the c.895_904del (p.Val301Serfs*8) was found in all families without sharing a common haplotype, suggesting a recurrent mechanism. Moreover, in two families, the systemic phenotype could be explained by additional pathogenic variants in known genes (TULP1, ATP6V1B1). RNA expression analysis on patients’ fibroblasts confirms that the dysfunction of IQCE leads to the dysregulation of genes associated with the hedgehog‐signaling pathway, and zebrafish experiments demonstrate a full spectrum of phenotypes linked to defective cilia: Body curvature, kidney cysts, left–right asymmetry, misdirected cilia in the pronephric duct, and retinal defects. In conclusion, we identified three additional families confirming IQCE as a nonsyndromic PAP gene. Our data emphasize the importance of taking into account the complete set of variations of each individual, as each clinical presentation could finally be explained by multiple genes.

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BBS1 is involved in retrograde trafficking of ciliary GPCRs in the context of the BBSome complex

Cited by 57 publications

References 38 publications

Robust interaction of IFT70 with IFT52–IFT88 in the IFT-B complex is required for ciliogenesis

Robust interaction of IFT70 with IFT52–IFT88 in the IFT-B complex is required for ciliogenesis

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

NovelIQCEvariations confirm its role in postaxial polydactyly and cause ciliary defect phenotype in zebrafish

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