PMC

CLSs from GPCRs and fibrocystin require Tulp3 for trafficking to cilia. (A) CLS wild-type and mutant sequences used for making CD8-CLS-LAP and CLS-LAP fusions (left), and a diagram representing the CD8-CLS-LAP or CLS-LAP fusions (right). EC, extracellular region; IC, intracellular region; TM, transmembrane domain. (B) Stable RPE lines expressing the indicated CD8-CLS-LAP or CLS-LAP fusions were transfected with 100 nM TULP3 siRNA for 72 h, serum starved for the last 24 h, and processed as in . GFP-positive cilia were counted in three experiments, and total counted cells are >300 in each condition. Data represent means ± SD; *, P < 0.001 with respect to corresponding siRNA controls. Also see Fig. S2.

Proximity biotinylation assays determine CLS–Tulp3 membrane proximity. (A) T-Rex-293 cells were cotransfected with CD8-CLS-BirA* and LAP-TULP3 constructs and processed for a tandem immunopurification (IP) procedure for detecting and quantifying the extent of biotin labeling on LAP-TULP3 as indicated (left). Diagram representing the fusion constructs appears on the right. BirA* will biotinylate primary amine-containing lysine residues in TULP3 fusion, if in close proximity. (B) Western blots of inputs and tandem affinity IPs for the indicated conditions are shown after pretreatment with biotin (50 µM) for 24 h. IB for biotin refers to neutravidin-tagged IRDye 680RD antibody-based detection. Normalized (Norm.) IP values refer to biotin/S tag signal in corresponding S tag secondary IPs, with respect to the control linker biotin-pretreated sample. The number of experiments (N) performed is mentioned beneath each panel. Normalized IP values are reported as means ± SD. (C) T-Rex-293 cells were cotransfected with CD8-CLS-BirA* and the LAP-TULP3 wild type (WT) or PI(4,5)P₂ binding-deficient mutant (TULP3^K268A;R270A), treated with biotin (20 µM) for 12 h, and processed for tandem IP procedure for detecting and quantifying the extent of biotin labeling as in A. Normalized IP values are reported as means ± SD from two experiments; **, P < 0.01; ***, P < 0.001, with respect to respective control linker biotin-pretreated samples. Also see Figs. S3 and S4.

CLSs and TULP3 are chemically cross-linked in a ciliary sequence-specific manner. (A–C) T-Rex-293 cells were cotransfected with CD8-CLS-LAP and 6×Myc-TULP3 full-length (B) or 6×Myc–C-terminal tubby domain (C term, 184–442 aa; C) constructs, and processed for cross-linking, quenching, tandem IP procedure, and decross-linking as indicated in the format in the left panel in A. The right panel in A shows a diagram representing the fusion constructs. Western blots of inputs and tandem affinity IPs for the indicated conditions are shown in B and C. Cross-linking enrichment scores shown beneath each respective panel represents (Myc IP/S tag IP)_DSP – (Myc IP/S tag IP)_NoDSP for each CD8-CLS-LAP–transfected condition normalized with respect to control linker (B) and wild-type CLSs in individual panels in C. The number of lysines in the linker/CLS are as follows: linker GSGAAAAAGSG (0); β₂ andrenergic receptor IC3 (β₂AR IC3; RVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKT; 5); Fibro^WTCLS (12); Fibro^IKPCLS (11); Fibro^7ACLS (9); Gpr161^WTIC3 (4); Gpr161^5AIC3 (2); Gpr161^3AIC3 (2); Mchr1^WTIC3 (1); and Mchr1^5AIC3 (1), as shown in . In addition, the rest of the fusion in CD8-CLS-LAP, including CD8α, the attB recombinase site, S tag, and the PreScission protease site from LAP, has a total of 11 lysines. All experiments were performed twice, except the last panel in C, which was performed three times. (B and C) Cross-linking enrichment scores are reported as means ± SD; *, P < 0.5 with respect to control linker (B); *, P < 0.05; **, P < 0.01; ***, P < 0.001with respect to wild-type CLS in each panel (C).

PC1/2 trafficking to cilia requires Tulp3. (A) mIMCD-K2 cells were sequentially transfected with the indicated 200 nM siRNAs twice for 72 h and serum starved for the last 36 h before fixation and immunostained for PC2 using anti-mouse PC2 rabbit polyclonal serum (from G. Pazour; see the Antibodies section of Materials and methods), acetylated tubulin, and DNA. PC2-positive and -negative cilia are marked by white arrows and arrowheads, respectively. The inset shows PC2 levels in control and Tulp3 siRNA-treated cells by immunoblotting. Data represent means ± SD from three experiments. The total number of cells counted is >800 per condition. Quantification of PC2 using a separate antibody available commercially in Fig. S5 (A and B). (B) mIMCD-K2 cells expressing ^LAPTULP3 were treated as in A. The total counted cells are >350 for each condition. ns, not significant. (C) mIMCD-K2 cells stably expressing ^FlagPC1^HA were sequentially transfected with the indicated 200-nM siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 36 h before fixation and immunostained for Flag (green), HA (red), AcTub (magenta), and DNA. N-terminal Flag tag allowed determination of PC1-expressing cells. Flag-positive cells were counted for HA-positive cilia from three independent experiments, and total counted cells were >1,000 per condition with ∼10% cells being Flag positive. PC1^HA-positive and -negative cilia are marked by white arrows and arrowheads, respectively. The magenta arrow marks a cilium in a cell not expressing Flag. Data represent means ± SD. (D) ARPE cells stably expressing PC2^L703XGFP were transfected with the indicated 100 nM siRNAs for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, pericentrin, and DNA. GFP-positive cilia were counted from two experiments, and the total number of GFP-positive cells counted were >120 per condition. GFP-positive and -negative cilia are marked by white arrows and arrowheads, respectively. Yellow arrows point to perinuclear staining for PC2^L703XGFP. Data represent means ± SD. Ciliary lengths of PC2^L703XGFP-positive cells treated with control and TULP3 siRNA are 8.5 ± 4.6 and 3.2 ± 0.8 µm, respectively. n = 20 each. (E) mIMCD-K2 cells were transfected with indicated siRNAs as in A and immunostained for PC2 (using anti-mouse PC2 rabbit polyclonal serum) or Gpr161, acetylated tubulin, and DNA. Pixel intensities of PC2- and Gpr161-positive cilia are shown to the right. White arrows mark PC2-positive cilia, and yellow arrows point to cilia shown in insets. Total counted cells were >240 and >140 per condition for PC2 and Gpr161 immunofluorescence, respectively. (A and C–E) *, P < 0.001 with respect to corresponding siRNA control (A); *, P < 0.01 with respect to corresponding siRNA control (C and D); *, P < 0.0001 with respect to controls (E). A.U., arbitrary units. Bars: (A and C–E, main images) 5 µm; (E, insets) 1 µm. Also see Fig. S5.

TULP3/TUB-mediated trafficking to ciliary membrane. (A) IMCD3 cells stably expressing ^SNAPGpr161^GFP were sequentially transfected with 200 nM of the indicated siRNAs twice as indicated in the experimental format on the top left (see Materials and methods). Starved cells were blocked with SNAP–surface block for 30 min, washed, and then were treated with SNAP–surface 594 at indicated time points. Cells were fixed and immunostained for GFP (green) and acetylated tubulin (magenta). Cilia positive and negative for SNAP–surface 594 are marked by white arrows and arrowheads, respectively. The yellow arrowhead points to cilia faintly stained for SNAP–surface 594. Total counted cells are >150 for control and >200 for Tulp3 siRNA, respectively. Data represent means ± SD from two or more fields from a single experiment. Bars, 5 µm. (B) NIH 3T3 Flp-In cells stably expressing Gpr161-GFP or Gpr161^V158E-GFP mutants were sequentially transfected with 200 nM of indicated siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixing and immunostaining for GFP, acetylated tubulin, and DNA. Ciliary pixel intensities for GFP are shown. Total counted cells were >60 per condition from two independent transfections with >30 cells counted per coverslip. A.U., arbitrary units. Also see Fig. S5 E. (C) IMCD3 Flp-In cells stably and inducibly expressing ^MycTULP3 N terminus (1–183 aa) were starved in the presence of doxycycline (Dox) for 20 h (4 µg/ml). After washing, cells were treated ± SAG (500 nM) for indicated time points in starvation medium before fixing and immunostaining for Gpr161, Myc, acetylated tubulin, and DNA. Myc-positive and -negative cells were scored for Gpr161-positive cilia. Total counted cells are >55 and >180 for Myc-positive and -negative cells, respectively, for each time point. Data represent means ± SD from three coverslips. Also see Fig. S5 F. (A–C) *, P < 0.001 with respect to corresponding siRNA control at each time point measured (A); *, P < 0.0001 with respect to corresponding controls (B); *, P < 0.05; **, P < 0.01 with respect to corresponding 0 h time point (C). (D) Model for TULP3/TUB-mediated trafficking to ciliary membrane. See Discussion. BB, basal body; FP, fusion protein; NT, TULP3/TUB IFT-A binding N-terminus domain. Also see Fig. S5.

TULP3 determines localization of multiple rhodopsin family GPCRs to primary cilia. (A) Stable RPE hTERT or IMCD3 Flp-In cell lines expressing the indicated GPCRs C-terminally tagged with GFP were transfected with 100 nM TULP3 si #3 siRNA (for RPE, see Materials and methods) or were sequentially transfected with 200 nM Tulp3 siRNA twice (for IMCD3 expressing P2RY1, see Materials and methods) for 72 h and serum starved for the last 24 h before fixation and then were immunostained for GFP, acetylated tubulin, and DNA. GFP-positive cilia were counted from two experiments, and total counted cells are >200 for each condition. Data represent means ± SD. (B) Same as in A, with RPE hTERT stable lines expressing GPR83 and KISS1R, except the ciliary intensity of GFP was quantified. Total counted cells are >100 for each condition. A.U., arbitrary units. (C) Stable IMCD3 Flp-In cells expressing D1R C-terminally tagged with GFP were sequentially transfected with 200 nM Tulp3 siRNA twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixing and staining for Gpr161, acetylated tubulin, and DNA. D1R^GFP/Gpr161-positive cilia were counted from two experiments, and total counted cells are 300–500 for each condition. Data represent means ± SD. (D) IMCD3 Flp-In cells stably expressing ^LAPTULP3 (LAP; S tag–PreScission-GFP) were sequentially transfected with 200 nM Tulp3 siRNA twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixing and staining for Gpr161, acetylated tubulin, and DNA. Data represent means ± SD from three experiments, and total counted cells are >400 for each condition. (A–D) *, P < 0.01 (A); *, P < 0.001 (B); *, P < 0.05 (C; A–C, with respect to corresponding siRNA controls); and *, P < 0.001 (D). (E) Table summarizing rhodopsin family GPCRs tested for ciliary localization and the role of TULP3/TUB in trafficking. Unlike the long D2R isoform, the D2R short isoform (NCBI RefSeq database accession no. NP_057658) is ciliary (). ND, not determined or ciliary, but limited stable expression in RPE hTERT cells. Other class A GPCRs reported to be ciliary by transient expression or endogenously in neurons or thyrocytes but not detected in cilia upon stable expression in RPE hTERT cells include the neuromedin receptor NMUR1 (), the orphan PGR15L, the pyroglutamylated RFamide peptide (QRFP) receptor QRFPR (), and the trace amine receptor TAAR1 (). Two other cilia-localized seven-transmembrane (7TM) receptors, GPR157 () and Gpr175 (TPRA1; ), that were not tested are either not readily classifiable or have no homology to known GPCRs, respectively (; ; http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=113). Also see Fig. S1.

Differential effects of Tub on ciliary GPCR trafficking. (A) IMCD3 Flp-In cells stably expressing LAP–TUB isoform b () were sequentially transfected with 200 nM of the indicated siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, and DNA. GFP-positive cilia were counted in two experiments, and total counted cells are >400/condition. Data represent means ± SD. Bars, 5 µm. (B) ^MBPTUB isoform b and ^MBPTULP3 immobilized on amylose resin were incubated with PreScission eluates from IFT140^LAP RPE cells ± ^HisTULP3 (1–183 aa; see Materials and methods; ). LAP, S tag–PreScission-GFP. ^MBPTUB- and ^MBPTULP3-bound proteins and corresponding flowthroughs were immunoblotted for S tag (IFT140^{S tag}), maltose-binding protein (MBP), and His-tag as indicated. Data are representative of two experiments. (C and D) Embryonic day 16.5, day in vitro (DIV) 8 hippocampal neurons from wild-type (WT) and Tub mice were immunostained for Gpr161/Gpr19 (green), DyLight594-labeled ACIII (red), and Sstr3 (white; C). Gpr161/Gpr19-positive and -negative cilia are marked by arrows (A and C) and arrowheads (C), respectively. Gpr161/Sstr3 coexpressing cells are marked by yellow arrows. The red dot indicates debris under the coverslip. Data represents means ± SD from cultures of two different embryos belonging to each genotype. Total counted cells are >300 per condition for Gpr161/Sstr3 and >90 per condition for Gpr19. Bars, 5 µm. Ciliary lengths are 3.2 ± 0.1 µm (wild type) and 3.5 ± 0.1 µm (Tub) neurons in experiments with Gpr161 staining and 3.7 ± 0.9 µm (wild type) and 3.5 ± 0.8 µm (Tub) in experiments with Gpr19 staining. Data represent means ± SD. n > 50 each. (E) Embryonic day 16.5 DIV5 hippocampal neurons from wild-type mice were transfected with indicated GFP-tagged N terminus (NT) wild-type or non–IFT-A binding (mut12) TULP3 constructs (), fixed at DIV8, and immunostained for Gpr161 and DyLight594-labeled ACIII. GFP-positive cells were quantified for Gpr161-positive cilia from two different coverslips, and total counted neurons are >24 per condition. (F) Embryonic day 18.5 DIV5 glia from wild-type mice were transfected with constructs as in C, fixed at DIV8, and immunostained for Gpr19 and Arl13b. GFP-positive cells were quantified for Gpr19-positive cilia from cultures from two different mice, and total counted cells are >110 per condition. (A and D–F) *, P < 0.001 with respect to corresponding siRNA control (A); *, P < 0.05 with respect to corresponding wild type; ns, not significant (D); *, P < 0.05 with respect to ^GFPTULP3-NTmut12 transfected cells (E); and *, P < 0.05 with respect to ^GFPTULP3-NTmut12-transfected cells. Also see Fig. S4.