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. D1RGFP/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.