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
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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.

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    • "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, "
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    Journal of Cell Science 06/2015; 128(14). DOI:10.1242/jcs.171652 · 5.43 Impact Factor
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    • "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]. "
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    • "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). "
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