Bardet-Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia - PubMed (original) (raw)

Bardet-Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia

Nicolas F Berbari et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Primary cilia are ubiquitous cellular appendages that provide important yet not well understood sensory and signaling functions. Ciliary dysfunction underlies numerous human genetic disorders. However, the precise defects in cilia function and the basis of disease pathophysiology remain unclear. Here, we report that the proteins disrupted in the human ciliary disorder Bardet-Biedl syndrome (BBS) are required for the localization of G protein-coupled receptors to primary cilia on central neurons. We demonstrate a lack of ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) in neurons from mice lacking the Bbs2 or Bbs4 gene. Because Mchr1 is involved in the regulation of feeding behavior and BBS is associated with hyperphagia-induced obesity, our results suggest that altered signaling caused by mislocalization of ciliary signaling proteins underlies the BBS phenotypes. Our results also provide a potential molecular mechanism to link cilia defects with obesity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

BBS mice possess neuronal primary cilia in the brain but lack Sstr3-positive cilia. (A–C) Representative images of the CA3 region of the hippocampus in adult WT (A), _Bbs2_−/− (B), and _Bbs4_−/− (C) mice (n = 9) showing labeling for ACIII (red). Nuclei are stained with DRAQ5 (blue). The appearance and distribution of ACIII-positive cilia are similar among all genotypes. (D–F) The identical fields showing labeling for Sstr3 (green) reveals Sstr3-positive cilia in the WT (D) section but a complete lack of Sstr3-positive cilia in the _Bbs2_−/− (E) or _Bbs4_−/− (F) sections. (G–I) The merged images showing colocalization of ACIII and Sstr3 to cilia in the WT (G) section and no Sstr3 labeling of cilia in the _Bbs2_−/− (H) or _Bbs4_−/− (I) sections. (Scale bars, 10 μm.)

Fig. 2.

Fig. 2.

Sstr3 ciliary localization can be restored in vitro. (A–C) Coimmunolabeling of day 7 hippocampal neurons from WT (A), _Bbs2_−/− (B), and _Bbs4_−/− (C) mice with antibodies to ACIII (green) and βTIII (red) shows the presence of cilia (arrows) in all three genotypes. (D–F) Coimmunolabeling of day 7 hippocampal neurons from WT (D), _Bbs2_−/− (E), and _Bbs4_−/− (F) mice with antibodies to Sstr3 (green) and βTIII (red) shows the presence of cilia (arrow) only on the WT neuron. Note the apparent punctate Sstr3 labeling in the _Bbs2_−/− (E) and _Bbs4_−/− (F) neurons. (G and H) Immunolabeling of day 7 hippocampal neurons from _Bbs2_−/− (G) and _Bbs4_−/− (H) mice for Sstr3 (red) 2 days posttransfection with an expression vector encoding Bbs2 and Bbs4, respectively, and a vector expressing enhanced green fluorescence protein (GFP; green) as a transfection marker. Note that heterologous expression of BBS proteins restores Sstr3 ciliary labeling (arrows). Nuclei are stained with DRAQ5 (blue). (Scale bars, 5 μm.)

Fig. 3.

Fig. 3.

BBS mice lack Mchr1 ciliary labeling in the brain. (A–C) Representative images of the nucleus accumbens in adult WT (A), _Bbs2_−/− (B), and _Bbs4_−/− (C) mice (n = 9) showing labeling for ACIII (red). The appearance and distribution of ACIII-positive cilia are similar among all genotypes. (D–F) The identical fields showing labeling for Mchr1 (green) reveals Mchr1-positive cilia in the WT (D) section but a complete lack of Mchr1-positive cilia in the _Bbs2_−/− (E) or _Bbs4_−/− (F) sections. Note the presence of increased punctate labeling in the _Bbs2_−/− (E) and _Bbs4_−/− (F) sections. (G–I) The merged images showing colocalization of ACIII and Mchr1 to cilia in the WT (G) section and no Mchr1 labeling of cilia in the _Bbs2_−/− (H) or _Bbs4_−/− (I) sections. Nuclei are stained with DRAQ5 (blue). (Scale bars, 10 μm.)

Fig. 4.

Fig. 4.

Mchr1 ciliary localization can be restored in vitro. (A–C) Coimmunolabeling of day 7 nucleus accumbens/olfactory tubercle-enriched neurons from WT (A), _Bbs2_−/− (B), and _Bbs4_−/− (C) mice with antibodies to ACIII (green) and βTIII (red). Cilia (arrows) are present in all three genotypes. (D–F) Coimmunolabeling of day 7 nucleus accumbens/olfactory tubercle-enriched neurons from WT (D), _Bbs2_−/− (E), and _Bbs4_−/− (F) mice with antibodies to Mchr1 (green) and βTIII (red) shows the presence of cilia (arrow) only on the WT neuron. Note the punctate staining in E and F. (G and H) Immunolabeling of day 7 hypothalamic neurons from _Bbs2_−/− (G) and _Bbs4_−/− (H) mice for Mchr1 (red) 2 days posttransfection with an expression vector encoding Bbs2 and Bbs4, respectively, and a vector expressing enhanced green fluorescence protein (GFP; green) as a transfection marker. Note that heterologous expression of BBS proteins restores Mchr1 ciliary labeling (arrows). Nuclei are stained with DRAQ5 (blue). (Scale bars, 5 μm.)

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