Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet-Biedl syndrome proteins

Abstract

Primary cilia are nearly ubiquitous cellular appendages that provide important sensory and signaling functions. Ciliary dysfunction underlies numerous human diseases, collectively termed ciliopathies. Primary cilia have distinct functions on different cell types and these functions are defined by the signaling proteins that localize to the ciliary membrane. Neurons throughout the mammalian brain possess primary cilia upon which certain G protein-coupled receptors localize. Yet, the precise signaling proteins present on the vast majority of neuronal cilia are unknown. Here, we report that dopamine receptor 1 (D1) localizes to cilia on mouse central neurons, thereby implicating neuronal cilia in dopamine signaling. Interestingly, ciliary localization of D1 is dynamic, and the receptor rapidly translocates to and from cilia in response to environmental cues. Notably, the translocation of D1 from cilia requires proteins mutated in the ciliopathy Bardet-Biedl syndrome (BBS), and we find that one of the BBS proteins, Bbs5, specifically interacts with D1.

Fig. 1a–i

Dopamine receptor 1 (D1) localizes to cilia, and ciliary localization is increased in the brains of Bbs4 ^−/− mice. a–c Representative image of transiently transfected inner medullary collecting duct (IMCD) cells expressing D1 fused at the C-terminus to EGFP. a Acetylated α-tubulin (AcTub; red) marks the cilia; b EGFP fluorescence (green) shows expression of the D1 receptor; c merged images. Representative images of the basolateral amygdala in adult WT (d–f) and Bbs4 ^−/− (g–i) mice (n = 3 animals for each genotype) showing labeling for type III adenylyl cyclase (ACIII; red) and D1 (green). Nuclei are stained with DRAQ5 (blue). The appearance and distribution of ACIII-positive cilia is similar between WT (d) and Bbs4 ^−/− (g) sections. The identical fields showing labeling for D1 (green) reveal a lack of D1-positive cilia in the WT (e) section but abundant D1-positive cilia in the Bbs4 ^−/− (h) section. Merged images showing no D1 labeling of cilia in the WT (f) section and colocalization of ACIII and D1 to cilia in the Bbs4 ^−/− (i) section. Scale bars represent 10 μm

Fig. 2a–c

D1 ciliary localization is increased in amygdala-enriched neuronal cultures. Co-immunolabeling of day 7 amygdala neurons from WT (a) and Bbs4 ^−/− (b) mice with antibodies to ACIII (red) and D1 (green) shows the presence of numerous ACIII-positive cilia in both genotypes, a subset of which are also positive for D1 (arrows). Nuclei are stained with DRAQ5 (blue). Scale bars represent 20 µm. c Percentage of ACIII-positive cilia in WT and Bbs4 ^−/− cultures (n = 3 animals for each genotype) that are positive for D1. D1 localizes to 7.7 ± 1.7% (n = 324) of ACIII-positive cilia in WT cultures and 25.4 ± 2.9% (n = 289) of ACIII-positive cilia in Bbs4 ^−/− cultures. Note the percentage of D1-positive cilia is significantly higher in Bbs4 ^−/− cultures compared to WT cultures. Values are expressed as mean ± SEM. **Significantly different from WT percentage (p < 0.01)

Fig. 3

In amygdala neuronal cultures, agonist treatment causes a decrease in D1 ciliary localization in WT but not Bbs4 ^−/− cultures. Percentage of ACIII-positive cilia in WT and Bbs4 ^−/− cultures (n = 3–4 animals for each genotype and condition) that are positive for D1. In unrefed WT cultures (left side of graph), D1 localizes to 8.5 ± 1.6% (n = 216) of ACIII-positive cilia in cultures treated with vehicle and 4.5 ± 1.8% (n = 297) of ACIII-positive cilia in cultures treated with the D1 agonist SKF-81297. In unrefed Bbs4 ^−/− cultures, D1 localizes to 31.7 ± 8% (n = 157) and 25.3 ± 6.3% (n = 140) of ACIII-positive cilia in cultures treated with vehicle and agonist, respectively. In refed WT cultures (right side of graph), D1 localizes to 17.9 ± 3.4% (n = 185) of ACIII-positive cilia in cultures treated with vehicle and 6.2 ± 2.4% (n = 189) of ACIII-positive cilia in cultures treated with agonist. Note the percentage of D1-positive cilia is significantly higher in refed vehicle-treated WT cultures compared to both unrefed vehicle-treated WT cultures (p < 0.05) and refed agonist-treated WT cultures (p < 0.05). In refed Bbs4 ^−/− cultures, D1 localizes to 36.8 ± 1.4% (n = 165) and 36.1 ± 4.3% (n = 144) of ACIII-positive cilia in cultures treated with vehicle and agonist, respectively. Note the percentage of D1-positive cilia in Bbs4 ^−/− cultures is significantly greater than WT cultures under all conditions and does not change significantly in response to refeeding or agonist treatment. Values are expressed as mean ± SEM. *Significantly different from WT percentage (p < 0.05). **Significantly different from WT percentage (p < 0.01)

Fig. 4a, b

D1 ciliary localization is increased in WT amygdala neuronal cultures in response to media containing B-27 supplement or forskolin treatment. a Percentage of ACIII-positive cilia in WT cultures (n = 3 animals for each condition) that are positive for D1. D1 localizes to 6.2 ± 1.0% (n = 322) of ACIII-positive cilia and 12.5 ± 0.8% (n = 327) of ACIII-positive cilia in cultures refed medium without B-27 and with B-27, respectively. Note the percentage of D1-positive cilia is significantly higher in WT cultures refed medium containing B-27 compared to medium lacking B-27. Values are expressed as mean ± SEM. **Significantly different from vehicle-treated percentage (p < 0.01). b Percentage of ACIII-positive cilia in WT cultures (n = 3 animals for each condition) that are positive for D1. D1 localizes to 5.1 ± 0.3% (n = 310) of ACIII-positive cilia in cultures treated with vehicle and 10.8 ± 0.2% (n = 269) of ACIII-positive cilia in cultures treated with forskolin. Note the percentage of D1-positive cilia is significantly higher in forskolin-treated WT cultures compared to vehicle-treated WT cultures. Values are expressed as mean ± SEM. ***Significantly different from vehicle-treated percentage (p < 0.0001)

Fig. 5a, b

D1 and Bardet-Biedl syndrome 5 (Bbs5) proteins interact. a Results of yeast matings between cells expressing the i3 loop of mouse D1, p53 (positive control), or lamin C (negative control) in the bait vector (pGBKT7) and cells expressing each of the seven BBSome subunits or large T antigen (T-Ag) in the prey vector pGADT7. Note that only D1 i3 loop plus Bbs5 mating and the positive control p53 plus T-Ag mating show yeast growth (indicated as +) on selective media. b HA-tagged Bbs5 (Bbs5-HA) was co-expressed with myc-tagged D1 (D1-myc) or myc-tagged glyceraldehyde-3-phosphate dehydrogenase (Gapdh-myc) in HEK293T cells. Cell extracts were immunoprecipitated (IP) with an anti-myc antibody. Immunoprecipitates were analyzed by Western blotting (IB) with an anti-HA antibody (upper). Note that Bbs5 is immunoprecipitated with D1 but not Gapdh. The input, confirming expression of each protein, is also shown (middle and bottom)