The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605
The Journal of Cell Biology (Impact Factor: 9.83). 01/2010; 188(1):21-8. DOI: 10.1083/jcb.200910096
Source: PubMed


Sensory functions of primary cilia rely on ciliary-localized membrane proteins, but little is known about how these receptors are targeted to the cilium. To further our understanding of this process, we dissected the ciliary targeting sequence (CTS) of fibrocystin, the human autosomal recessive polycystic kidney disease gene product. We show that the fibrocystin CTS is an 18-residue motif localized in the cytoplasmic tail. This motif is sufficient to target green fluorescent protein (GFP) to cilia of ciliated cells and targets GFP to lipid rafts if the cells are not ciliated. Rab8, but not several other Rabs implicated in ciliary assembly, binds to the CTS in a coimmunoprecipitation assay. Dominant-negative Rab8 interacts more strongly than wild-type or constitutively active Rab8, and coexpression of this dominant-negative mutant Rab8 blocks trafficking to the cilium. This suggests that the CTS functions by binding regulatory proteins like Rab8 to control trafficking through the endomembrane system and on to the cilium.

Download full-text


Available from: Gregory J Pazour
    • "Stable control and IFT20KD Jurkat lines were as previously described (Finetti et al., 2009). Human wild-type, T22N (DN) and Q77L (CA) Rab8 in p3XFLAG-myc-CMV-26 (Sigma-Aldrich) (Follit et al., 2010), pCMV-EGFP-C3-Rab11 (kindly provided by M. Zerial, "
    [Show abstract] [Hide abstract]
    ABSTRACT: IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse (IS) assembly in the non-ciliated T cell by promoting TCR recycling. Here we have addressed the role of Rab8, a small GTPase implicated in ciliogenesis, in TCR traffic to the IS. We show that Rab8, which colocalizes with IFT20 in Rab11(+) endosomes, is required for TCR recycling. Interestingly, at variance with IFT20-deficient T cells, TCR(+) endosomes polarized normally beneath the IS membrane in the presence of dominant negative Rab8, but were unable to undergo the final docking/fusion step. This could be accounted for by the inability of the v-SNARE VAMP-3 to cluster at the IS in the absence of functional Rab8, which is responsible for its recruitment. Of note, similar to T cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of Smoothened. The results identify Rab8 as a novel player in vesicular traffic to the IS and provide insight into the pathways co-opted by different cell types for IS assembly and ciliogenesis. © 2015. Published by The Company of Biologists Ltd.
    No preview · Article · Jun 2015 · Journal of Cell Science
  • Source
    • "FPC is detected on the primary cilia with antibodies to both N- and C-termini of FPC [9], [12], we thus believe that FPC undergoes a C-tail cleavage under defined conditions and some of the cleaved fragments translocate into the nucleus [14], [15]. Notably, our human free FPC C-tail was not found on the primary cilia, in contrast to the mouse FPC C-tail constructs used for the study of ciliary targeting sequence which contains additional nine residues (LSCLVCCWF) in the transmembrane segment of FPC [16], although nuclear FPC C-tail signals were also observed in that study [16]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: FPC (fibrocystin or polyductin) is a single transmembrane receptor-like protein, responsible for the human autosomal recessive polycystic kidney disease (ARPKD). It was recently proposed that FPC undergoes a Notch-like cleavage and subsequently the cleaved carboxy(C)-terminal fragment translocates to the nucleus. To study the functions of the isolated C-tail, we expressed the intracellular domain of human FPC (hICD) in renal epithelial cells. By 3-dimensional (3D) tubulogenesis assay, we found that in contrast to tubule-like structures formed from control cells, hICD-expressing cells exclusively formed cyst-like structures. By western blotting, we showed that the Akt/mTOR pathway, indicated by increased phosphorylation of Akt at serine 473 and S6 kinase 1 at threonine 389, was constitutively activated in hICD-expressing cells, similar to that in FPC knockdown cells and ARPKD kidneys. Moreover, application of mTOR inhibitor rapamycin reduced the size of the cyst-like structures formed by hICD-expressing cells. Application of either LY294002 or wortmannin inhibited the activation of both S6K1 and Akt. Expression of full-length FPC inhibited the activation of S6 and S6 kinase whereas co-expression of hICD with full-length FPC antagonized the inhibitory effect of full-length FPC on mTOR. Taken together, we propose that FPC modulates the PI3K/Akt/mTOR pathway and the cleaved C-tail regulates the function of the full-length protein.
    Full-text · Article · May 2014 · PLoS ONE
  • Source
    • "Since most of the differences between VPAC2 and its homologs are concentrated in their C-terminal tails (supplementary material Fig. S1), we tested whether the C-terminus of VPAC2 is sufficient to target the non-ciliary membrane protein CD8α to cilia (Fig. 4B). CD8α is a well-characterized non-ciliary membrane protein that has been used in chimeras to identify ciliary targeting domains (Follit et al., 2010). As shown in Fig. 4D, a chimera protein CD8α-hVPAC2-Cterm-GFP containing the extracellular and transmembrane domains of CD8α and the C-terminus of VPAC2 targeted to primary cilia, while CD8α-GFP, CD8α-hVPAC1-Cterm-GFP, and CD8α-hPAC1-Cterm-GFP failed to do so (Fig. 4C,F,G). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Primary cilia protrude from the cell surface of many cell types in the human body and function as cellular antennae via ciliary membrane localized receptors. Neurons and glial cells in the brain possess primary cilia, and the malfunction of primary cilia may contribute to neurological deficits present in many cilia-associated disorders. Several rhodopsin family G-protein coupled receptors (GPCRs) are specifically localized to a subset of neuronal primary cilia. However, whether other family GPCRs target to neuronal cilia and whether glial primary cilia harbor any GPCRs are not known. We conducted a screening of GPCRs to determine their ability to target to primary cilia, and identified a secretin family member, Vasoactive Intestinal Receptor 2 (VPAC2), as a novel ciliary GPCR. Here, we show that endogenous VPAC2 targets to primary cilia in various brain regions, including the suprachiasmatic nuclei and the thalamus. Surprisingly, VPAC2 not only localizes to neuronal cilia but also to glial cilia. In addition, we show that VPAC2's C-terminus is both necessary and sufficient for its ciliary targeting and we define a novel ciliary targeting signal: the tetrapeptide RDYR motif in the C-terminus of VPAC2. Furthermore, we demonstrate that VPAC2 ciliary targeting is dependent on Tubby, the BBSome (a complex of Bardet-Biedl syndrome proteins) and the BBSome targeting factor, Arl6.
    Full-text · Article · Jul 2013 · Biology Open
Show more