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. 2008 Jun 27;320(5884):1777-81.
doi: 10.1126/science.1157983. Epub 2008 May 22.

Beta-arrestin-mediated localization of smoothened to the primary cilium

Jeffrey J Kovacs  1 , Erin J Whalen, Renshui Liu, Kunhong Xiao, Jihee Kim, Minyong Chen, Jiangbo Wang, Wei Chen, Robert J Lefkowitz
Affiliations

Beta-arrestin-mediated localization of smoothened to the primary cilium

Jeffrey J Kovacs et al. Science. .

Abstract

beta-Arrestins have important roles in the regulation of seven-transmembrane receptors (7TMRs). Smoothened (Smo) is a 7TMR that mediates effects of Hedgehog on developmental processes and whose dysregulation may cause tumorigenesis. beta-Arrestins are required for endocytosis of Smo and signaling to Gli transcription factors. In mammalian cells, Smo-dependent signaling requires translocation to primary cilia. We demonstrated that beta-arrestins mediate the activity-dependent interaction of Smo and the kinesin motor protein Kif3A. This multimeric complex localized to primary cilia and was disrupted in cells transfected with beta-arrestin small interfering RNA. beta-Arrestin 1 or beta-arrestin 2 depletion prevented the localization of Smo to primary cilia and the Smo-dependent activation of Gli. These results suggest roles for beta-arrestins in mediating the intracellular transport of a 7TMR to its obligate subcellular location for signaling.

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Figures

Fig. 1
Fig. 1. Localization of β-arrestins, Smoothened and Kif3A to the primary cilia
(A) NIH-3T3 cells were immunostained using antibodies to acetylated tubulin or βarr1 and βarr2 (top right panel – mouse monoclonal, bottom middle – rabbit polyclonal), to visualize primary cilia (arrowheads). Colocalization is shown in yellow (overlay). (B) NIH-3T3 cells were stained with βarr antibody (red) and pericentrin antibody (green) which marks the basal bodies of primary cilia. (C) (Top panels) NIH-3T3 cells overexpressing GFP-Smo (green) were immunostained with βarr antibody (red). Colocalization of GFP-Smo and endogenous βarr1 and βarr2 in the primary cilia (arrowhead) can be visualized (yellow). (Bottom panels) NIH-3T3 cells were immunostained antibodies to βarr (red) and Kif3A (green). Colocalization in the primary cilia (arrowhead) can be observed (yellow).
Fig. 2
Fig. 2. Multimeric complex formation between β-arrestins, Kif3A, and Smoothened
(A) Effects of Shh on association of Smo and Kif3A with βarrs. NIH 3T3 cells treated with Shh-conditioned media +/- cyclopamine were lysed and immunoprecipitated with an antibody that recognizes both βarr1 and βarr2. Samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) analysis and immunoblotted for the presence of endogenous Smo or Kif3A. (WCL = whole cell lysate) *p< 0.05, **p<0.005, and ***p<0.001 compared to Shh treated maximum, n=4. (B) Effect of overexpressed Smo on association of Kif3A and βarrs. NIH-3T3 cells were transiently transfected with pCDNA control vector or various amounts of GFP-Smo. Proteins from lysates were immunoprecipitated with Kif3A antibody and immunoblotted for the presence of endogenous βarr or Kif3A. In the βarr blot, the top band is endogenous βarr1, and the lower band is endogenous βarr2. *p< 0.05 compared to pCDNA, n=3. (C) Effect of depletion of βarrs on association of Kif3A with GFP-Smo. NIH-3T3 cells stably overexpressing GFP-Smo were transfected with control (CTL), βarr1 or βarr2 siRNA oligonucleotides. Samples were left untreated (DMSO) or treated with 6μM Cyclopamine for 1 hour. Proteins from lysates were immunoprecipitated with GFP antibody followed by immunoblotting for endogenous Kif3A and GFP-Smo. *p< 0.001 compared to control siRNA untreated sample, n=4. (D) Effect of depletion of βarrs on association of FLAG-Smo with Kif3A. NIH-3T3 cells stably overexpressing FLAG-Smo were transfected with control (CTL), βarr1 or βarr2 siRNA oligonucleotides. Samples were left untreated (DMSO) or treated with 6μM cyclopamine for 1 hour. Cells were lysed and proteins were immunoprecipitated with antibody to Kif3A and immunoblotted for FLAG-Smo and endogenous Kif3A. *p< 0.05, **p< 0.001 when compared to control siRNA, n=3. All p values analyzed by one-way ANOVA with Bonferoni correction. Data are shown as mean +/- SEM.
Fig. 3
Fig. 3. Disrupted localization of Smo after siRNA mediated depletion of β – arrestin 1, 2, or Kif3A
(A) Smo was visualized with FLAG antibody in NIH-3T3 cells overexpressing FLAG-Smo and transfected with control (CTL), βarr1 (βarr1-A) or βarr2 (βarr2-A) siRNA oligonucleotides. Representative fields were examined for each and the total number of cilia (arrowheads) was counted along with total number of nuclei to delineate total cell number. (B) The percentage of cells that had FLAG-Smo positive cilia was calculated and normalized to control samples. * p < .001, **p < 0.01 vs. control, n=4. Inset: Western blot of representative samples for βarr1 (top band) and βarr2 (bottom band). (C) Samples were prepared as in (A) with multiple siRNA oligos for each protein (βarr1-A - D, βarr2-A - C, and Kif3A-A - C) as indicated, and immunostained with both antibody to FLAG (green) and acetylated tubulin (red). (D) Percentage of total cilia in which Smo was detected. FLAG-Smo containing cilia and acetylated tubulin-stained cilia were counted. *p < .001 vs. control, n=3. (E and F) Effects of rescue constructs containing silent mutations for the region targeted by the indicated siRNA. Cells were stained for GFP-Smo (green) or FLAG (red) to indicate expression of the rescue construct (E) with βarr1 or βarr2 siRNA as indicated. After SDS-PAGE analysis, membranes were blotted for the presence of βarr1 and βarr2 (F). (G) Percentage of cells with Smo–containing cilia. For rescued samples, one hundred cells in which FLAG expression was detected were counted and examined for the presence of Smo in cilia. *p < .001 vs. control, n=3. All p values analyzed by one-way ANOVA with Bonferoni correction. Data are shown as mean +/- SEM.
Fig. 3
Fig. 3. Disrupted localization of Smo after siRNA mediated depletion of β – arrestin 1, 2, or Kif3A
(A) Smo was visualized with FLAG antibody in NIH-3T3 cells overexpressing FLAG-Smo and transfected with control (CTL), βarr1 (βarr1-A) or βarr2 (βarr2-A) siRNA oligonucleotides. Representative fields were examined for each and the total number of cilia (arrowheads) was counted along with total number of nuclei to delineate total cell number. (B) The percentage of cells that had FLAG-Smo positive cilia was calculated and normalized to control samples. * p < .001, **p < 0.01 vs. control, n=4. Inset: Western blot of representative samples for βarr1 (top band) and βarr2 (bottom band). (C) Samples were prepared as in (A) with multiple siRNA oligos for each protein (βarr1-A - D, βarr2-A - C, and Kif3A-A - C) as indicated, and immunostained with both antibody to FLAG (green) and acetylated tubulin (red). (D) Percentage of total cilia in which Smo was detected. FLAG-Smo containing cilia and acetylated tubulin-stained cilia were counted. *p < .001 vs. control, n=3. (E and F) Effects of rescue constructs containing silent mutations for the region targeted by the indicated siRNA. Cells were stained for GFP-Smo (green) or FLAG (red) to indicate expression of the rescue construct (E) with βarr1 or βarr2 siRNA as indicated. After SDS-PAGE analysis, membranes were blotted for the presence of βarr1 and βarr2 (F). (G) Percentage of cells with Smo–containing cilia. For rescued samples, one hundred cells in which FLAG expression was detected were counted and examined for the presence of Smo in cilia. *p < .001 vs. control, n=3. All p values analyzed by one-way ANOVA with Bonferoni correction. Data are shown as mean +/- SEM.
Figure 4
Figure 4. Inhibition of Shh signaling after siRNA-mediated depletion of β-arrestin 1, β-arrestin 2 or Kif3A
(A) Gli-luciferase reporter activity was assayed in NIH-3T3 cells transfected with either control (CTL), βarr1 (βarr1-A - D) or βarr2 (βarr2-A - C) siRNA oligonucleotides, a Gli-luciferase reporter and a firefly renilla plasmid. Samples were plated, grown and then treated with mock- or Shh-containing medium. Dual luciferase assay data were pooled from 4 separate experiments with each sample repeated in triplicate. *p < 0.001 vs. control / Shh treated, n=5. Inset: western blot for βarr knockdown. (B) Effect of depletion of Kif3A in NIH-3T3 cells transfected with either control (CTL), or Kif3A (Kif3A-A - C) siRNA oligonucleotides, a Gli-luciferase reporter and a firefly renilla plasmid. Lysates were examined as above. *p < 0.001 vs. control / Shh treated, n=3. Inset: Western blot for Kif3A knockdown. (C) Rescue of siRNA treated NIH-3T3 cells transfected with either control (CTL), βarr1 (βarr1-A) or βarr2 (βarr2-A) siRNA oligonucleotides alone or with the corresponding rescue construct, a Gli-luciferase reporter and a firefly renilla plasmid as an internal control. Samples were plated, grown and then treated with mock- or Shh-containing media. For the left panel data were plotted as in (A) and (B). *p < 0.05 vs. control / untreated as analyzed by one-way ANOVA with Bonferoni correction, n=4. For the right panel, values in each individual experiment were analyzed for the change in RLUs after Shh treatment as a relative measure of induction over basal for the indicated condition. **p < 0.001 vs. control / Shh treated, n=4. All p analyzed by one-way ANOVA with Bonferoni correction. Data are shown as mean +/- SEM.
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References

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