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, 114 (1), 131-40

Structural and Functional Impairment of Endocytic Pathways by Retinitis Pigmentosa Mutant Rhodopsin-Arrestin Complexes

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Structural and Functional Impairment of Endocytic Pathways by Retinitis Pigmentosa Mutant Rhodopsin-Arrestin Complexes

Jen-Zen Chuang et al. J Clin Invest.

Abstract

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, we propose that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations.

Figures

Figure 1
Figure 1
Confocal images of WT and mutant rhodopsins expressed in HEK cells. (A and B) Fixed HEK cells transfected with WT (A) or R135L (B) rhodopsin were incubated with anti–rhodopsin mAb B6-30 and detected by Alexa 488 secondary antibodies. (CK) HEK cells transfected with R135L were incubated with Alexa 594–Tf for 5 minutes to label early endosomes (CE); incubated with Alexa 594–Tf for 2 minutes followed by a 28-minute chase to label recycling endosomes (FH); or incubated with rhodamine-labeled dextran for 2 hours to label late endosomes/lysosomes (IK). Cells were then fixed and permeabilized for rhodopsin immunostaining (green). Rhodopsin immunoreactivity colocalized with the internalized Tf or dextran is marked by arrows, and merge images are shown in E, H, and K. Scale bars: 10 μm.
Figure 2
Figure 2
Phosphorylation and v-arr association of R135L rhodopsin in HEK cells. (A) Immunoprecipitated rhodopsin, obtained from transfected cells that had been metabolically labeled with [32P] orthophosphate, were subjected to SDS-PAGE and either autoradiographed (left) or immunoblotted (right). (B) Rhodopsin immunoprecipitates obtained from cells transfected with pRK5 vector, rhodopsin and/or GFPv-arr (as indicated) were separated by SDS-PAGE and immunoblotted with either anti-rhodopsin Ab (top) or anti-GFP Ab (bottom) (GFPv-arr: ∼75 kDa). Rhodopsin expressed in tissue culture is heterogeneously glycosylated and prone to forming higher-order aggregates (2, 5), which accounts for the broadened bands on SDS-PAGE. Arrowheads point to the rhodopsin monomer (∼40 kDa), and the brackets indicate the heterogeneous species of oligomerized rhodopsins. The electrophoretic patterns of R135L rhodopsins were distinct from those of WT rhodopsins, perhaps because of the conformation changes caused by the mutation. Some 50-kDa Ig heavy chains were reactive with the secondary antibodies (arrows). Results are representative of three experiments. IB, immunoblot; IP, immunoprecipitate.
Figure 3
Figure 3
Subcellular distribution and trafficking of rhodopsin in HEK cells cotransfected with GFPv-arr. (AI) Cells double transfected with GFPv-arr and either WT rhodopsin (AC), R135L (DF), or P23H (GI) were immunolabeled for rhodopsin (red), and GFPv-arr was directly visualized by its GFP fluorescence. In DF, some cells expressed low levels of R135L and contained both surface and cytosolic GFP signals (open arrows); the vesicular structures near the cell periphery (arrows) and the perinuclear structure (arrowheads) contain both the R135L and the GFPv-arr. In GI, the P23H mutant–induced aggresomes (open arrows) are not enriched for GFPv-arr. (JL) Live, R135L/GFPv-arr–transfected cells were incubated with mAb B6-30 at 4–C, followed by a 1-hour, 37–C chase. The cells were fixed and permeabilized, and the internalized rhodopsin was detected by the labeling of Alexa 594–anti-mouse Ab (red). Internalized surface rhodopsins appeared on GFPv-arr+ vesicle and perinuclear compartments (arrows). (MO) Cells triple-transfected with WT-rhodopsin, RK, and GFPv-arr were fixed, permeabilized, and either immunolabeled with mAb B6-30 followed by Alexa 594–anti-mouse Ab (M) or directly visualized by GFP signals (N). Phosphorylated WT rhodopsin was colocalized with GFPv-arr on the vesicles underneath PM (arrows) and the perinuclear compartments (arrowheads). A GFPv-arr–singly-transfected neighboring cell exhibited cytosolic green fluorescence. Scale bars: 20 μm.
Figure 4
Figure 4
Endogenous v-arr was mislocalized in rodent rods that expressed R135L but not WT or Q344ter rhodopsin. (A) The schematic illustration shows the in vivo electroporation technique. Plasmid is injected into the subretinal space followed by electroporation. Tweezer-type electrodes are placed across the eye, with the anode facing the cornea. (B) A schematic illustration of the bicistronic expression vector pCAG-rhodopsin-IRES-GFP. These vectors permit both rhodopsin and GFP to be translated from a single mRNA and simultaneously expressed in the transfected cells. (CH) Retinas were transfected with either pCAG-WT-IRES-GFP (C and D), pCAG-Q344ter-IRES-GFP (E and F), or pCAG-R135L-IRES-GFP (G and H). These retinas were immunolabeled with the antibodies indicated, followed by Alexa 594–conjugated secondary antibodies. Representative optical images of photoreceptor layer show transfected rhodopsin immunolabeling (C, E, and G). The GFP signals were directly visualized and are shown in the merge images in (D, F, and H). Note that mAb 3A6 effectively detected the WT but not the R135L mutant in transfected cells. (IN) Retina sections prepared from eyes transfected with pCAG-WT-IRES-GFP (I and J), pCAG-Q344ter-IRES-GFP (K and L), and pCAG-R135L-GFP (M and N) were immunolabeled with anti–v-arr antibody (red). Representative confocal images of v-arr labeling (I, K, and M) and merge views with GFP+- transfected cells are shown (J, L, and N). The PM (arrow and arrowhead) and the intracellular vacuole accumulation of v-arr (open arrow) are marked. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 20 μm.
Figure 5
Figure 5
Aberrant endosomal organization caused by R135L/GFPv-arr. Fixed, transfected cells were incubated with mAb’s that recognized early endosome marker EEA1 (A and B), early/recycling endosome marker TfR (C and D), and late endosome/lysosome marker lysosomal-associated membrane protein 1 (LAMP1) (E and F), followed by Alexa 594–conjugated anti-mouse Ab. GFP signals were used to directly visualize the GFPv-arr. The transfected cells are encircled in B, D, and F to show the cell margins. Although EEA1, TfR, and LAMP1 signals were dispersed in nontransfected cells, their signals were predominantly concentrated in the GFP+ perinuclear structures in the R135L/GFPv-arr–transfected cells. Scale bars: 10 μm.
Figure 6
Figure 6
The altered endocytic activity of Tf and LDL in cells that expressed R135L/v-arr. Cells transfected with R135L/GFPv-arr (A and B, E and F, I and J, M and N) and WT rhodopsin/GFPv-arr (C and D, G and H, K and L, O and P) were incubated with Alexa 594–conjugated Tf at 37–C for 5 minutes (A and B, C and D), for 2 hours (E and F, G and H), or for 2 hours plus a 30-minute chase (I and J, K and L) before fixation and visualization. Alternatively, cells were treated with DiI-LDL for 2 minutes followed by a 28-minute chase before the fixation (M and N, O and P). Confocal images of GFPv-arr (green) and internalized Tf (red) or LDL (red) are shown. In J and L, the DAPI nuclear labeling demonstrated that the nontransfected cells (in J and L) and WT/GFPv-arr–transfected cells (in L) have no Tf signals, as the Tf was completely expelled from cells. Scale bars: 20 μm.

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