Recruitment of β-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization

Abstract

Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates β-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking β-arrestin 2. Together, our results implicate β-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for β-arrestin in the mediation of Sstr3 ciliary signaling.

FIG 1

Somatostatin treatment causes a decrease in endogenous Sstr3 ciliary localization and localization of β-arrestin to neuronal cilia. (A and B) Representative images of hippocampal neurons from WT mice, after 7 days in culture, labeled with antibodies to Sstr3 (green) and AC3 (red). Neurons were either untreated (UT) (A) or treated with 10 μM SST for 40 min (B). SST treatment causes a reduction in Sstr3-positive cilia. (Insets) Increased magnification of individual channels in the boxed regions to aid visualization. Bars, 10 μm. (C) Percentages of AC3-positive cilia in WT cultures (n = 3) that were positive for Sstr3. Sstr3 localized to 49.5% ± 4.6% of AC3-positive cilia (n = 130), 34% ± 5.8% of AC3-positive cilia (n = 144), and 22.8% ± 8.3% of AC3-positive cilia (n = 146) in cultures treated with 10 μM SST for 0, 20, and 40 min, respectively. The percentage of Sstr3-positive cilia was significantly decreased after 40 min of SST treatment. Values are expressed as the mean ± SEM. *, significantly different results (P < 0.05). (D to I) Representative images of hippocampal neurons from WT mice, after 7 days in culture, labeled with antibodies to βarr1/2 (green) and Sstr3 (red). Neurons were treated with vehicle (Veh) (D to F) or 10 μM SST (G to I) for 20 min. β-Arrestin was not detected within cilia in vehicle-treated neurons but colocalized with Sstr3 throughout the cilium after SST treatment. (Insets) Higher-magnification images of the cilia. Nuclei were stained with DRAQ5. Bars, 10 μm (main images) and 5 μm (insets). (J) Percentages of Sstr3-positive cilia in WT cultures (n = 3) that were positive for βarr1/2. βarr1/2 localized to 0% of Sstr3-positive cilia (n = 73) in vehicle-treated cultures and 55.6% ± 4.1% of Sstr3-positive cilia (n = 127) in cultures treated with SST for 20 min. Values are expressed as the mean ± SEM.

FIG 2

Somatostatin-mediated ciliary localization of β-arrestin 2 in IMCD cells. (A to L) Representative images of IMCD cells transfected with βarr1 fused to GFP (βarr1-GFP; green) (A to F) or βarr2 fused to GFP (βarr2-GFP; green) and myc-tagged Sstr3 (Sstr3-myc; red) (G to L). Cells were treated with vehicle (Veh) (A to C and G to I) or 10 μM SST (D to F and J to L) for 20 min, fixed, and labeled with an antibody to myc. Sstr3-myc is targeted to cilia. βarr1-GFP was not detected in cilia in vehicle- or SST-treated cells. βarr2-GFP was not detected in cilia in vehicle-treated cells but localized to cilia in SST-treated cells. (Insets) Side views of the cilia (the insets in panels A to C correspond to the dashed boxes). Nuclei were stained with DRAQ5. Bars, 10 μm. (M) Percentages of Sstr3-expressing IMCD cell cilia that were positive for βarr1-GFP or βarr2-GFP after 20 min of vehicle or 10 μM SST treatment. In vehicle-treated cultures, βarr1-GFP localized to 0% of Sstr3-positive cilia (n = 43) and βarr2-GFP localized to 0% of Sstr3-positive cilia (n = 52). In SST-treated cultures, βarr1-GFP localized to 0% of Sstr3-positive cilia (n = 51) and βarr2-GFP localized to 87.2% ± 3.4% of Sstr3-positive cilia (n = 54). Values are expressed as the mean ± SEM.

FIG 3

Somatostatin-mediated localization of β-arrestin 2 to neuronal cilia. (A to L) Representative images of hippocampal neurons from WT mice, after 7 days in culture, transfected with βarr1 fused to GFP (βarr1-GFP; green) (A to F) or βarr2 fused to GFP (βarr2-GFP; green) and myc-tagged Sstr3 (Sstr3-myc; red) (G to L). Neurons were treated with vehicle (Veh) (A to C and G to I) or 10 μM SST (D to F and J to L) for 20 min, fixed, and labeled with an antibody to myc. Sstr3-myc is targeted to cilia. βarr1-GFP was not detected in cilia in vehicle- or SST-treated neurons. βarr2-GFP localized to cilia in SST-treated neurons. (Insets) Higher-magnification images of the cilia. Nuclei were stained with DRAQ5. Bars, 10 μm (main images) and 5 μm (insets). (M) Percentages of Sstr3-positive neuronal cilia that were positive for βarr1-GFP or βarr2-GFP after 20 min of vehicle or 10 μM SST treatment. In vehicle-treated cultures. βarr1-GFP localized to 0% of Sstr3-positive cilia (n = 25) and βarr2-GFP localized to 0% of Sstr3-positive cilia (n = 25). In SST-treated cultures βarr1-GFP localized to 0% of Sstr3-positive cilia (n = 25) and βarr2-GFP localized to 100% of Sstr3-positive cilia (n = 25).

FIG 4

Live cell confocal imaging of somatostatin-mediated β-arrestin 2 ciliary localization. Each image is a maximum-intensity projection of a z-stack that encompassed the entire cell body and cilium. (A) Representative images of a WT hippocampal neuron after 7 days in culture transfected with βarr1 fused to GFP (βarr1-GFP; green) and Sstr3 fused to DsRed (Sstr3-DsRed; red). (Top) The green channel; (bottom) both the green and red channels. The elapsed time (t; in minutes) of 10 μM SST treatment is indicated in the upper right corner of each panel. Sstr3-DsRed was targeted to the cilium, but βarr1-GFP was not detected in cilia before (time zero [t0]) or after 10 min (t10) of SST treatment. (B) Representative images of a WT hippocampal neuron after 7 days in a culture transfected with βarr2 fused to GFP (βarr2-GFP; green) and Sstr3-DsRed (red). Panels and labels are the same as those described in the legend to panel A. Before SST treatment (time zero), βarr2-GFP was enriched at the base of the cilium but was not detected within the cilium. βarr2-GFP ciliary localization was apparent after 4 min (t4) of SST treatment and was robust after 10 min (t10). Bars, 10 μm.

FIG 5

Mutation of agonist binding and phosphorylation sites in Sstr3 abolishes β-arrestin 2 ciliary localization in IMCD cells and neurons. (A) IMCD cells were transfected with βarr2 fused to GFP and myc-tagged WT Sstr3 or Sstr3(D124E). Cells were treated with vehicle (Veh) or 10 μM SST for 20 min, fixed, and labeled with an antibody to myc, and βarr2-GFP ciliary localization was quantified. In vehicle-treated cultures, βarr2-GFP localized to 1.2% ± 1.1% of WT (n = 76) and 0% of Sstr3(D124E)-positive cilia (n = 85). In SST-treated cultures βarr2-GFP localized to 92.4% ± 1.7% of WT (n = 85) and 1% ± 1% of Sstr3(D124E)-positive cilia (n = 106). (B) IMCD cells were transfected with βarr2 fused to GFP and myc-tagged WT or mutant Sstr3. Treatments and processing were the same as those described in the legend to panel A. In vehicle-treated cultures, βarr2-GFP localized to 0% of WT or mutant Sstr3-positive cilia (n = 31 to 49). In SST-treated cultures, βarr2-GFP localized to 90% ± 5.1% of WT Sstr3-positive cilia (n = 37), 79.2% ± 4.2% of cilia positive for the Sstr3 triple serine mutant (n = 24), 0% of cilia positive for the Sstr3 quadruple mutant (n = 29), and 0% of cilia positive for the Sstr3(T357A) mutant (n = 25). (C to F) Representative images of hippocampal neurons from WT mice after 7 days in a culture transfected with βarr2 fused to GFP (βarr2-GFP; green) and WT Sstr3 (red) (C and D) or Sstr3(T357A) (red) (E and F). Neurons were treated with vehicle (Veh) (C and E) or 10 μM SST (D and F) for 20 min and then fixed. In vehicle-treated cells, βarr2-GFP was not detected within the cilium. In SST-treated cells, βarr2-GFP localized to WT Sstr3-positive cilia but not Sstr3(T357A)-positive cilia. (Insets) Higher-magnification images of the cilia. Nuclei were stained with DRAQ5. Bars, 10 μm (main images) and 5 μm (insets). (G) Percentages of WT and mutant Sstr3-positive neuronal cilia that were positive for βarr2-GFP (n = 20 to 30 cilia for each condition and construct). In cultures treated with vehicle for 20 min, βarr2-GFP localized to 0% of WT or mutant Sstr3-positive cilia. In cultures treated with SST for 20 min, βarr2-GFP localized to 100% of WT Sstr3-positive cilia and 0% of Sstr3(T357A)-positive cilia. Values are expressed as the mean ± SEM.

FIG 6

The ciliary localization of FKBP-βarr2-GFP is greater in response to somatostatin treatment than rapamycin treatment. (A) Schematic of rapamycin-induced dimerization in the ciliary compartment. FKBP-GFP diffuses into and out of the cilium. In the absence of rapamycin, the level of FKBP-GFP in the cilium is low. In the presence of rapamycin, FKBP dimerizes with FRB fused to Sstr3, which is enriched in the ciliary membrane, thereby trapping FKBP-GFP in the ciliary compartment. (B) IMCD cells were transfected with Sstr3 fused to DsRed and FRB (Sstr3-DsRed-FRB) and FKBP fused to GFP (FKBP-GFP) or βarr2-GFP (FKBP-βarr2-GFP). Cells were treated with DMSO (vehicle [Veh]), 100 nM rapamycin (Rap), or 10 μM SST for 10 min and fixed, and the GFP ciliary localization was quantified. In DMSO-treated cultures, FKBP-GFP and FKBP-βarr2-GFP localized to 0% of Sstr3-positive cilia (n = 33 to 38). In rapamycin-treated cultures, FKBP-GFP localized to 71.6% ± 4.8% of Sstr3-positive cilia (n = 41) and FKBP-βarr2-GFP localized to 24.8% ± 5.3% of Sstr3-positive cilia (n = 44). In SST-treated cultures, FKBP-GFP localized to 3.7% ± 3.7% of Sstr3-positive cilia (n = 33) and FKBP-βarr2-GFP localized to 87.8% ± 4% of Sstr3-positive cilia (n = 45). The percentage of FKBP-βarr2-GFP ciliary colocalization was significantly greater after SST treatment than rapamycin treatment. Values are expressed as the mean ± SEM. ***, significantly different results (P < 0.001).

FIG 7

Agonist-mediated decreases in endogenous Sstr3 neuronal ciliary localization require βarr2. Hippocampal neurons from WT and βarr2-KO mice after 7 days in culture were treated with vehicle (Veh) or 10 μM SST for 40 min, fixed, and labeled with antibodies to Sstr3 and AC3 (n = 3 experiments per genotype). In vehicle- and SST-treated WT cultures, Sstr3 localized to 60.4% ± 2.4% of AC3-positive cilia (n = 369) and 41.6% ± 2.3% of AC3-positive cilia (n = 388), respectively. In vehicle- and SST-treated βarr2-KO cultures, Sstr3 localized to 50.8% ± 3.4% of AC3-positive cilia (n = 333) and 53% ± 3.4% of AC3-positive cilia (n = 324), respectively. The percentage of Sstr3-positive cilia was significantly decreased after 40 min of SST treatment in WT neuronal cultures but did not change in βarr2-KO neuronal cultures. The percentages of Sstr3-positive cilia in vehicle-treated WT and βarr2-KO neuronal cultures were not significantly different. Values are expressed as the mean ± SEM. **, significantly different results (P < 0.005).