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Dopamine receptors reveal an essential role of IFT-B, KIF17, and Rab23 in delivering specific receptors to primary cilia.

Leaf A, Von Zastrow M - Elife (2015)

Bottom Line: This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo.Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane.Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, University of California, San Francisco, San Francisco, United States.

ABSTRACT
Appropriate physiological signaling by primary cilia depends on the specific targeting of particular receptors to the ciliary membrane, but how this occurs remains poorly understood. In this study, we show that D1-type dopaminergic receptors are delivered to cilia from the extra-ciliary plasma membrane by a mechanism requiring the receptor cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non-ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the ciliary membrane. Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process.

No MeSH data available.


Disruption of KIF17 motor activity does not affect Rab23 ciliary localization.Representative live-cell images of cells co-transfected with Flag-Rab23-Q68L, Arl13b-YFP as a cilia marker, and either empty vector (+pcDNA) or motor domain mutant KIF17 (+KIF17-G234A). In merged image, Flag-Rab23-Q68L (Flag) immunoreactivity is shown in red, Arl13b-YFP in green, and HA-KIF17-G234A in blue. Rab23-Q68L was observed in 8/18 cilia in cells expressing pcDNA. Similarly, Rab23-Q68L was clearly visible in 9/15 cilia in cells expressing KIF17-G234A. Scale bar, 5 μm.DOI:http://dx.doi.org/10.7554/eLife.06996.042
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fig8s2: Disruption of KIF17 motor activity does not affect Rab23 ciliary localization.Representative live-cell images of cells co-transfected with Flag-Rab23-Q68L, Arl13b-YFP as a cilia marker, and either empty vector (+pcDNA) or motor domain mutant KIF17 (+KIF17-G234A). In merged image, Flag-Rab23-Q68L (Flag) immunoreactivity is shown in red, Arl13b-YFP in green, and HA-KIF17-G234A in blue. Rab23-Q68L was observed in 8/18 cilia in cells expressing pcDNA. Similarly, Rab23-Q68L was clearly visible in 9/15 cilia in cells expressing KIF17-G234A. Scale bar, 5 μm.DOI:http://dx.doi.org/10.7554/eLife.06996.042

Mentions: We next sought to investigate if the identified protein components required for D1R ciliary targeting function in an integrated pathway. As noted above, disrupting KIF17 motor activity strongly reduced ciliary enrichment of the wild-type D1R. In contrast, ciliary enrichment driven by direct fusion of the activated Rab23 to the D1R (D1Δ381-395-Rab23-Q68L) was unaffected by this manipulation (Figure 8A–C; whole-cell images verifying HA-KIF17-G234A expression are shown in Figure 8—figure supplement 1). Additionally, full ciliary enrichment of D1Δ381-395-Rab23-Q68L remained in the presence of IFT172 knockdown (Figure 8D), suggesting that fusion to activated Rab23 can also override the IFT-B requirement. We also noted that Rab23 knockdown did not prevent or reduce D1R association with IFT-B, as estimated by co-immunoprecipitation of IFT57 (Figure 8E). To the contrary, Rab23 knockdown tended to increase the IFT57-D1R co-IP signal (Figure 8F). In contrast to its clear effect on the ciliary concentration of D1Rs, disrupting KIF17 motor activity did not prevent Rab23 localization to cilia (Figure 8—figure supplement 2). Together, these results suggest that IFT-B/KIF17 and Rab23 are all required for efficient targeting of D1Rs to cilia and may indeed function in an integrated pathway.10.7554/eLife.06996.040Figure 8.Evidence IFT-B, KIF17, and Rab23 function in an integrated ciliary delivery pathway.


Dopamine receptors reveal an essential role of IFT-B, KIF17, and Rab23 in delivering specific receptors to primary cilia.

Leaf A, Von Zastrow M - Elife (2015)

Disruption of KIF17 motor activity does not affect Rab23 ciliary localization.Representative live-cell images of cells co-transfected with Flag-Rab23-Q68L, Arl13b-YFP as a cilia marker, and either empty vector (+pcDNA) or motor domain mutant KIF17 (+KIF17-G234A). In merged image, Flag-Rab23-Q68L (Flag) immunoreactivity is shown in red, Arl13b-YFP in green, and HA-KIF17-G234A in blue. Rab23-Q68L was observed in 8/18 cilia in cells expressing pcDNA. Similarly, Rab23-Q68L was clearly visible in 9/15 cilia in cells expressing KIF17-G234A. Scale bar, 5 μm.DOI:http://dx.doi.org/10.7554/eLife.06996.042
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Related In: Results  -  Collection

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fig8s2: Disruption of KIF17 motor activity does not affect Rab23 ciliary localization.Representative live-cell images of cells co-transfected with Flag-Rab23-Q68L, Arl13b-YFP as a cilia marker, and either empty vector (+pcDNA) or motor domain mutant KIF17 (+KIF17-G234A). In merged image, Flag-Rab23-Q68L (Flag) immunoreactivity is shown in red, Arl13b-YFP in green, and HA-KIF17-G234A in blue. Rab23-Q68L was observed in 8/18 cilia in cells expressing pcDNA. Similarly, Rab23-Q68L was clearly visible in 9/15 cilia in cells expressing KIF17-G234A. Scale bar, 5 μm.DOI:http://dx.doi.org/10.7554/eLife.06996.042
Mentions: We next sought to investigate if the identified protein components required for D1R ciliary targeting function in an integrated pathway. As noted above, disrupting KIF17 motor activity strongly reduced ciliary enrichment of the wild-type D1R. In contrast, ciliary enrichment driven by direct fusion of the activated Rab23 to the D1R (D1Δ381-395-Rab23-Q68L) was unaffected by this manipulation (Figure 8A–C; whole-cell images verifying HA-KIF17-G234A expression are shown in Figure 8—figure supplement 1). Additionally, full ciliary enrichment of D1Δ381-395-Rab23-Q68L remained in the presence of IFT172 knockdown (Figure 8D), suggesting that fusion to activated Rab23 can also override the IFT-B requirement. We also noted that Rab23 knockdown did not prevent or reduce D1R association with IFT-B, as estimated by co-immunoprecipitation of IFT57 (Figure 8E). To the contrary, Rab23 knockdown tended to increase the IFT57-D1R co-IP signal (Figure 8F). In contrast to its clear effect on the ciliary concentration of D1Rs, disrupting KIF17 motor activity did not prevent Rab23 localization to cilia (Figure 8—figure supplement 2). Together, these results suggest that IFT-B/KIF17 and Rab23 are all required for efficient targeting of D1Rs to cilia and may indeed function in an integrated pathway.10.7554/eLife.06996.040Figure 8.Evidence IFT-B, KIF17, and Rab23 function in an integrated ciliary delivery pathway.

Bottom Line: This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo.Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane.Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, University of California, San Francisco, San Francisco, United States.

ABSTRACT
Appropriate physiological signaling by primary cilia depends on the specific targeting of particular receptors to the ciliary membrane, but how this occurs remains poorly understood. In this study, we show that D1-type dopaminergic receptors are delivered to cilia from the extra-ciliary plasma membrane by a mechanism requiring the receptor cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non-ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the ciliary membrane. Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process.

No MeSH data available.