Fig 1.
A screen to identify ciliary GPCRs using NIH3T3 cells.
A) Strategy of the screen to identify ciliary GPCRs. We constructed plasmids expressing 138 non-odorant GPCRs fused with a FLAG or mCherry tags at their C-terminals. These constructs were transfected into NIH3T3 cells. At 24 hrs after transfection, ciliogenesis was induced by serum starvation. At 48 hrs after transfection, subcellular localization of GPCRs was analyzed by immunostaining using an anti-FLAG or anti-mCherry antibody. Cilia were marked with an anti-acetylated α-tubulin antibody. B, C) Localization of FLAG-tagged Sstr3 (B) and MCHR1 (C) to cilia in NIH3T3 cells. GPCRs were stained with an anti-FLAG antibody (red) and cilia were stained with the anti-acetylated α-tubulin antibody (green). Co-localization of FLAG (red) and acetylated α-tubulin (green) signals was observed in cilia. Nuclei were stained with DAPI (blue). Arrowheads indicate cilia. Scale bars, 10 μm (B, C) and 5 μm (insets in B, C).
Fig 2.
Newly identified GPCRs localized to cilia.
A–C) Three GPCRs, PRLHR (A), NMUR1 (B) and NPFFR1 (C), were identified to localize to primary cilia. GPCRs were stained with the anti-FLAG antibody (red) and cilia were stained with the anti-acetylated α-tubulin antibody (green). Co-localization of FLAG (red) and acetylated α-tubulin (green) signals was observed in the cilia. D, E) NMUR2 (D) and Npffr2 (E), highly conserved paralogs of NMUR1 and NPFFR1, respectively, did not localize to cilia. GPCRs were stained with the anti-FLAG antibody (red) and cilia were stained with the anti-acetylated α-tubulin antibody (green). FLAG (red) and acetylated α-tubulin (green) signals were non-overlapped. Nuclei were stained with DAPI (blue). Arrowheads indicate cilia. Scale bars, 10 μm (A-E) and 5 μm (insets in A-E).
Fig 3.
A short isoform of dopamine D2 receptor (DRD2S) efficiently localizes to cilia.
A) Schematic diagrams of DRD2 isoforms, DRD2S and DRD2L. DRD2L differs from DRD2S in the presence of a 29-amino-acid insertion in the third intracellular loop. B–D) Subcellular localization of DRD2 isoforms. FLAG-tagged DRD2L (B) or DRD2S (C) was expressed in the NIH3T3 cells. Cells were stained with the anti-acetylated α-tubulin (green) and anti-FLAG (red) antibodies. DRD2L was mainly accumulated in perinuclear Golgi apparatuses-like structures but was barely localized to the cilia (B). In contrast, DRD2S was clearly enriched in cilia (C). Ratios of localization of DRD2S and DRD2L to cilia in ciliated NIH3T3 cells were quantified (D). The average values from three independent transfection experiments are shown (*p<0.03). Nuclei were stained with DAPI (blue). Arrowheads indicate cilia. Scale bars, 10 μm (B, C) and 5 μm (insets in B, C).
Fig 4.
Prlhr localizes to cilia in the hypothalamic third ventricle.
A–D) Prlhr was localized to the ventricular cilium in the adult mouse brain. Frozen sections containing the third ventricle of the adult mouse brain were immunostained with an anti-Prlhr antibody (green). The Prlhr signal was observed in cilia protruding from cells on the surface of the third ventricle (arrowheads in A, B, D). Preabsorption of the anti-Prlhr antibody with a synthetic Prlhr-peptide antigen abolished the Prlhr staining in the cilia (C). Cilia were coimmunostained with an anti-Arl13b antibody (D, red). Cilia with the Prlhr signal (arrowheads) were not clustered together, unlike the multiple cilia of ependymal cells that lacked the Prlhr signal (arrows). E–H) Ventricular Prlhr-positive cilia (red) were double stained with GFAP (green) which stains a subtype of tanycytes. A partial population of the GFAP-positive tanycytes (green) possesses Prlhr-positive cilia (arrowheads). Nuclei were stained with DAPI (blue). V: third ventricles. Scale bars, 20 μm (the left panel in A; B, C, F, G), 10 μm (the right panel in A; H), 5 μm (D), and 40 μm (E).
Fig 5.
Subcellular localization of Npy2r and BAC-Npy2r-Cre transgenic mouse.
A) The subcellular localization of Npy2r. FLAG-tagged Npy2r was expressed in the NIH3T3 cells. Cells were stained with the anti-acetylated α-tubulin (green) and anti-FLAG (red) antibodies. FLAG-tagged Npy2r signals were observed in NIH3T3 cells (red). Co-localization of Npy2r and acetylated α-tubulin signals was observed in cilia (arrowheads). Nuclei were stained with DAPI (blue). B) Diagram representing the BAC-Npy2r-Cre transgene construct. C–L) Expression of mTFP1 in the brains of R26-CAG-LoxP-mTFP1; BAC-Npy2r-Cre transgenic mice (C, D, G-I, K, L) and control mice (E, F). BAC-Npy2r-Cre transgenic mice were crossed with reporter mice, R26-CAG-LoxP-mTFP1, to detect the expression of the Cre recombinase in BAC-Npy2r-Cre transgenic mice. Dorsal (C, E), ventral (D, F, G) and coronal (H, I) views of the brain are shown. Broad expression of mTFP1 in the brain including the hippocampus, cerebral cortex, and hypothalamus (paraventricular nucleus, arrowheads in G, I) was observed. In the arcuate nucleus, Npy2r (green in J, arrowheads) colocalized with Adcy3 (a ciliary marker, red in J) in cilia. Npy2r-stained cilia (red) were often observed in mTFP1-positive cells (green in K). Preabsorption of the anti-Npy2r antibody with a synthetic Npy2r-peptide antigen abolished the Npy2r staining in the cilia (L). Scale bars, 10 μm (A, J), 5 μm (the inset in A), 5 mm (C–F), 1 mm (G–I), and 20 μm (K, L).
Fig 6.
Ift80 Npy2r-Cre CKO mice exhibited body weight increase.
A) Strategy for the conditional deletion of Ift80. The Ift80 exon 6 was deleted by Cre-mediated recombination, resulting in an Ift80-null allele. B) PCR products of 248 bp or 466 bp were amplified from wild type or floxed Ift80 alleles, respectively. C, D) Cilia in the hypothalamic arcuate nucleus in control and Ift80 Npy2r-Cre CKO brains were immunostained with an anti-Npy2r antibody (C). The number of stained cilia/field (25,600 μm2) is indicated. The number of the anti-Npy2r antibody-stained cilia (arrowheads) significantly decreased in the hypothalamic arcuate nucleus in the Ift80 Npy2r-Cre CKO brain (D) (n = 3; * p<0.03). Nuclei were stained with DAPI (blue). E) The Npy2r mRNA expression levels were measured by Q-PCR in the control and Ift80 Npy2r-Cre CKO mouse hypothalamuses (n = 3). F, G) Cilia in the hypothalamic arcuate nucleus in control and Ift80 Npy2r-Cre CKO brains were immunostained with an anti-Adcy3 antibody (green in F). The number of immunostained cilia/field (25,600 μm2) is indicated. The number of cilia immunostained with the anti-Adcy3 antibody was statistically unaltered between control and Ift80 Npy2r-Cre CKO mice (G) (n = 3). H, I) Ift80 Npy2r-Cre CKO mice showed an increased body weight at 19 weeks. Body weights of 13- (H) or 19-week-old (I) male Ift80 Npy2r-Cre CKO mice were measured. Body weights of Ift80 Npy2r-Cre CKO mice were statistically unaltered at 13 weeks and significantly increased at 19 weeks compared with those of control mice (control, n = 6; CKO, n = 4; *p<0.03). EC: ependymal cells; V: third ventricles. Scale bars, 50 μm (the upper panels in C, F) and 10 μm (the lower panels in C, F).