RGR-opsin maintains cellular homeostasis by ameliorating the proteotoxicity of its exon-skipping splice variant

One of the alternative splicing variants of retinal G protein-coupled receptor (RGR), RGR-d, is recognized as a misfolded protein. With or without inhibition of the proteasome system, RGR-d has a cytotoxic effect on retinal pigment epithelium (RPE) cells. RGR-d mutant mice exhibit age-related macular degeneration-like pathologic changes. However, humans with RGR-d likely express both wild-type and splice variant forms. In this study, we investigate the protective influence of the co-expression of RGR and RGR-d protein in RPE cells.

ARPE-19 cells overexpressing both RGR and RGR-d were treated with or without MG132. The protein-protein interaction, ubiquitination, endoplasmic reticulum (ER) stress, autophagy flux, and subcellular localization were explored. Aged RGR-d mice were subjected to ocular examination.

We confirm that RGR binds RGR-d in RPE cells and decreases the polyubiquitin of RGR-d. Formation of the RGR/RGR-d complex could assist RGR-d in correctly folding on the endoplasmic reticulum (ER), and ameliorate ER stress and autophagy inhibition that were induced by misfolded RGR-d protein. The complexes highly colocalize with Golgi apparatus and maintain its typical morphology, avoiding degradation by lysosomes. We observed that RGR-d mice exhibited AMD-like subretinal hyper-reflective deposits on fundus examination.

We have identified a fundamental mechanism in which RGR-opsin binds its splice variant, RGR-d, to form a protective complex that alleviates cytotoxicity to RPE. This discovery is significant as it reveals a previously unknown endogenous defense against proteotoxicity in the retina. Targeting the RGR-d may represent a novel and highly promising intervention for the protein-misfolding pathology of dry age-related macular degeneration.