1. Neuropeptide Y Family Receptors Traffic via the Bardet-Biedl Syndrome Pathway to Signal in Neuronal Primary Cilia 
Alexander V. Loktev, Peter K. Jackson 
Cell Reports 
Volume 5, Issue 5, Pages (December 2013)
DOI: /j.celrep
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2. Cell Reports 2013 5, 1316-1329DOI: (10.1016/j.celrep.2013.11.011)
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3. Figure 1
Candidate GPCRs Regulating Energy Homeostasis Localize to Primary Cilia in Transfected RPE Cells and Cultured Primary Hypothalamic Neurons
(A) Flowchart of strategies used to identify GPCRs regulating energy homeostasis and localized to neuronal primary cilia. See also Tables S1 and S2.
(B) Candidate ciliary GPCRs were transiently expressed as GFP fusions in RPE cells. Cells were fixed and immunostained for acetylated α-tubulin (Ac-tub) to mark primary cilia and pericentrin (Pcnt) to mark basal bodies.
(C) Cultured primary embryonic rat hypothalamic neurons (DIV13) stained with antibodies against indicated candidate ciliary GPCRs, neuronal primary cilia marker ACIII, and neuropeptide Y (Npy). Example of Npy5r localization to neuronal projections as well as cilia is presented in Figure S1A. Scale bars represent 10 μm. White arrows mark primary cilia enlarged in insets. Hoechst dye was used to label nuclei (DNA) in this and all other figures.
Cell Reports 2013 5, DOI: ( /j.celrep )
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4. Figure 2
GPCRs Controlling Energy Homeostasis, Including the Neuropeptide Y Family Receptor NPY2R, Localize to Neuronal Cilia In Vivo
(A) Coronal brain sections WT mouse stained with antibodies for indicated GPCRs and ACIII. ME, median eminence; ACN, arcuate nucleus; OT, olfactory tubercle; DMH and VMH, dorso- and ventromedial hypothalamus. Chanel-separated images of NPY5R staining in ACN is presented in Figures S1B and S1C.
(B and C) ACN section stained for NPY2R, ACIII, prepro NPY (ppNPY), and POMC, respectively. Scale bars represent 10 μm, unless otherwise indicated. White arrows mark primary cilia enlarged in insets.
Cell Reports 2013 5, DOI: ( /j.celrep )
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5. Figure 3 The NPY2R Receptor Harbors Ciliary Targeting Sequences
(A) Sequence alignment of IC3s of Pgr15l, NPY2R, and GPR83 compared to closely related nonciliary GPCR NPY1R. Boxed amino acids in bold were mutated in NPY2R (mut1 and mut2) and tested for ciliary targeting. NPY1R mut1 and mut2 were tested for gain of ciliary targeting.
(B) RPE cells were transfected with WT or mutated NPY2R and NPY1R GFP-fusion constructs (as in A) and immunostained for Ac-tub and Pcnt. The lower right panel shows ciliary targeting of chimeric NPY1R harboring IC4 from NPY2R (see also Figure S2). Percentage of cilia in GFP-positive cells with ciliary GPCR is shown for each mutant. The effect of CTS mutagenesis of the closely related GPR83 is presented in Figure S3. Scale bars represent 10 μm.
(C) RPE cell line stably expressing NPY2R-GFP was transfected with indicated siRNAs. Percentage of ciliated cells and percentage of cilia positive for NPY2R-GFP were calculated and plotted. Error bars represent ± SEM. ∗∗∗p < compared to control (ANOVA) (n > 300, from three independent experiments).
Cell Reports 2013 5, DOI: ( /j.celrep )
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6. Figure 4
NPY2R Is Missing from Neuronal Primary Cilia in BBS-Defective Mice
(A) Bbip10−/− mice (generated as shown in Figure S4) develop obesity and hyperphagia similar to other BBS mouse models. Photograph of a representative female mice at 12 weeks of age.
(B) Body weight changes of female Bbip10−/− mice over time (n = 6–8).
(C) Mean daily food intake of 5-month-old female Bbip10−/− mice (n = 8).
(D) Coronal sections of ACN from 6- to 8-week-old Bbip10−/− and Tubby mice stained for NPY2R and ACIII, showing decrease in NPY2R ciliary localization in mutant mice (n = 4). Error bars represent ± SEM. Scale bars represent 20 μm. ∗p < ∗∗p < 0.01.
Cell Reports 2013 5, DOI: ( /j.celrep )
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7. Figure 5
Localization of NPY2R to Neuronal Cilia Is Essential for the Anorexigenic Effect of the NPY2R Ligand PYY3-36
(A) Food intake over time in fasted Bbip10−/− and Bbip10+/+ mice following injection of PYY3-36 or saline (n = 6 for each group). Mice were tested at 8–9 weeks of age before onset of obesity.
(B) Changes in relative levels of POMC mRNA in hypothalamus of Bbip10−/− mice 2 hr after PYY3-36 injection (n = 3, per group).
(C and D) c-Fos staining of ACN coronal sections from fasted Bbip10−/− and Bbip10+/+ mice (n = 3, per group) 2 hr after injection with PYY3-36 or saline.
(E) Npy2r mRNA expression in hypothalamus is not perturbed in Bbip10 knockout animals. Relative levels of Npy2r mRNA were analyzed in the total RNA samples used in Figure 6B.
(F) Western blot analysis of brain lysates from WT and Bbip10−/− mice showing similar levels NPY2R protein and tubulin acetylation. Error bars represent ± SEM. Scale bars represent 20 μm. ∗p < ∗∗p < 0.01.
Cell Reports 2013 5, DOI: ( /j.celrep )
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8. Figure 6
NPY2R Exits Primary Cilia in Primary Embryonic Rat Hypothalamic Neurons after Binding by Its Specific Cognate Ligand PYY3-36
(A and B) Representative images of cultured primary rat hypothalamic neurons (DIV13) before (A) and after (B), 120 min treatment with 100 nM PYY3-36 stained with antibodies against NPY2R, ACIII, and neuronal marker MAP2. Scale bars represent 10 μm. Arrows mark primary cilia.
(C) Cultured neurons (DIV13) were treated as indicated in the legend for 30, 60, and 90 min and processed as in (A). The percentage of neurons (MAP2 positive) displaying ciliary NPY2R is plotted over time (n > 200 from three independent experiments). Error barsrepresent ± SEM. ∗∗p < 0.01.
Cell Reports 2013 5, DOI: ( /j.celrep )
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9. Figure 7
Primary Cilia Augment cAMP Inhibition upon Activation of NPY2R Receptor by PYY3-36
(A) Diagram of the epac2 cAMP FRET-based assay. RPE cells stably expressing TEpacVV cAMP sensor and NPY2R-mCherry (RPE-Epac-NPY2R) were used to measure FRET, which is inversely proportional to cAMP concentration in cells.
(B) Normalized aggregated FRET measured in RPE-Epac-NPY2R cells during perfusion with buffer, PYY3-36, or BIIE0246 followed by PYY3-36 and compared among cells with or without primary cilia (± cilia) (Movie S1).
(C) Ciliated cells exhibit significantly augmented NPY2R-dependent inhibition of cAMP signaling upon addition of PYY3-36 ligand in epac2 cAMP sensor assay. RPE-Epac-NPY2R were perfused for 30 s with either PYY3-36 or PYY3-36 and then NPY2R antagonist BIIE0246, followed by 30 s in forskolin. FRET was measured during perfusion and compared between cells with or without primary cilia (± cilia) (Movie S2). Examples of cell images are presented in Figure S7. The number of cells measured in each treatment is indicated in the legend (n). Error bars represent ± SEM. ∗∗p < 0.01.
Cell Reports 2013 5, DOI: ( /j.celrep )
Copyright © 2013 The Authors Terms and Conditions