Interaction of RPGR with RAB8A. (A) Bovine retinal extract was prepared in the presence of the non-hydrolyzable GDP substrate and subjected to immunoprecipitation (IP) using anti-RAB8A, anti-RPGR antibodies or normal IgG and pre-immune (Pre-Imm) serum. Twenty percent of the protein used for IP was loaded in input lane. Vertical line represents different RPGR isoforms expressed in bovine retina, as described earlier (38,42). (B) A known-bait and known-prey yeast two-hybrid analysis. Upper panel represents the growth of yeast transformed with bait (RPGR^1–15) and prey (RAB8A) constructs. Interaction between p53 (bait) and T antigen (T-Ag; prey) was used as a positive control, whereas laminin (LAM) and T-Ag association served as a negative control in this experiment. The lower panel represents β-galactosidase (blue) test of the growth assay shown in the upper panel. (C) Coomassie blue staining of the gel showing the purified WT GST-RPGR^1–15 or GST proteins utilized in GST pulldown assay. Molecular mass markers are in kilo Daltons (kDa). (D and E) GST pulldown assay: 10 µg of purified GST-RPGR^1–15 fusion protein or GST alone was incubated with ³⁵S-labeled in vitro-translated RAB8A (C) or RAB11 (D) (or with indicated mutants). The proteins were collected by binding to Glutathione-Sepharose™ beads and analyzed by SDS–PAGE followed by autoradiography. Lanes are indicated. (F) Ciliated MDCK cells were stained with antibodies to RPGR (purple) and RAB8A (green). Immunostained cells were analyzed with OLYMPUS FV 500. White arrow in ‘Merge’ image shows co-localization of RPGR and RAB8A at the cilium. Nuclei (N) are stained with Hoechst (blue).

Modulation of RAB8A activity by RPGR. (A) Comparison of MANT-GTP incorporation into His-RAB8A as a function of time when incubated with RPGR or Rabin8. The activity of Rabin8 over RAB8A was used as positive control. AFU, arbitrary fluorescent units. (B) Fold change of RAB8A GDP/GTP exchange for each of the RPGR mutants relative to WT. Error bars represent mean ± SD from three independent experiments. Fold change was calculated as the ratio of RAB8A MANT-GTP incorporation after incubation with WT or mutant RPGR proteins with RAB8A MANT-GTP incorporation without incubation with RPGR (RAB8A intrinsic activity). Fold change by each mutant was compared with that depicted by WT RPGR from three independent experiments; level of significance was calculated with two-tailed t-test. *P < 0.05 and **P < 0.005.

Interaction of RPGR mutants with RAB8A. (A) To evaluate the interaction of RPGR mutants with RAB8A-T22N, we expressed and purified GST-RPGR^1–15 WT and mutant proteins. Upper panel represents the Coomassie blue-stained gel showing that equivalent amount of individual protein used in the GST pulldown assay. The proteins were incubated with in vitro-translated ³⁵S RAB8A-T22N, followed by pulldown using Glutathione-Sepharose beads. ³⁵S RAB8A-T22N-positive signal was detected by autoradiography with phosphorimager (Storm 840 GE Healthcare) (lower panel). (B) Quantification of GST pulldown was analyzed from Coomassie blue-stained gels and the autoradiograms, using ImageJ software. Area and intensity of RAB8A signal was measured and plotted against the amount of the respective mutant protein used in the pulldown assay. Graph represents each ratio normalized against the ratio of the binding of WT-RPGR with ³⁵S RAB8A-T22N. Error bars represent mean ± SD from five independent experiments. The level of significance was calculated with two-tailed t-test. *P < 0.001.

RPGR regulates ciliary localization of RAB8A. (A) Generation of RPGR-knockdown cells: lysates from stable transfected hTERT-RPE1 cells with control (scrambled) shRNA or RPGR shRNA were analyzed using anti-RPGR antibody or actin. Arrows represent distinct RPGR isoforms. (B) This panel represents cilia (red; acetylated α-tubulin) formation in control and RPGR-shRNA cells. Nuclei (blue) are stained with Hoechst. (C) Histograms show the distribution of cilia length in RPGR-shRNA-expressing cells. (D) There is increase in the number of cells with shorter cilia (1.5–3.9 µm) when treated with RPGR-shRNA compared with controls. Distribution of cilia length was calculated from three independent experiments with at least 150 cells analyzed in each experiment. Error bars represent standard deviation. (E) Knockdown of RPGR interferes with ciliary localization of RAB8A (green) to primary cilia (acetylated α-tubulin; red). Yellow in the Merge image indicates RAB8A localization at primary cilia. (F) Histogram showing decrease in number of cells with RAB8A at the cilium. We analyzed a total of 180 cells in each group and the experiment was repeated three times. (G) Knockdown of RPGR does not affect the localization of IFT88/Polaris (purple; left panel) to the cilium (red; Ac. α-tubulin; left panel) or of CEP290 (purple; right panel) to basal bodies (red; γ-tubulin; right panel).