Identification and characterization of the cell surface receptor for Rod-derived Cone Viability Factor

Abstract

Purpose: The Rod-derived Cone Viability Factor (RdCVF) is produced by alternative splicing of the nucleoredoxin-like 1 gene (Nxnl1) which is homologous to thioredoxin genes. The intron retention and the presence of an in frame stop codon produces a truncated thioredoxin without any enzymatic activity. This suggests that the effect of RdCVF is mediated by binding to a cell surface receptor on cones

Methods: Binding of ¹²⁵I-RdCVF was performed of cone-enriched cultures from chicken embryos and the RdCVF interacting proteins on membrane fractions were identified by far-western blotting followed by MS analysis. Binding of the RdCVF-receptor was validated using alkaline phosphatase fusion proteins and its involvement by gene silencing. The RdCVF-receptor associated proteins were identified by co-immunoprecipitation followed by MS analysis and validated by FRET. The downstream signaling was studied using cone-enriched cultures from chicken embryos and validated in the mouse retina. The role of the RdCVF-receptor in generating cone protection was studied in the rd1 mouse using AAV delivery of RdCVF mutant protein which was unable to bind the RdCVF-receptor. The presence of the RdCVF-receptor in the fovea of primates was observed by immunohistochemistry and western blotting

Results: Using proteomics, we identified a single pass transmembrane protein that binds to RdCVF and is required for the trophic effect of RdCVF on cones. The RdCVF-receptor is associated at the surface of the cone cells to another membrane protein that is mediating the effect of RdCVF. When delivered into the rd1 mouse using an AAV vector, a mutant RdCVF unable to bind to the RdCVF-receptor is not protecting the cones contrarily to wild-type RdCVF. The RdCVF-Receptor is located onto cone photoreceptors in the primates

Conclusions: The identification and the characterization of the mode of action of the cell surface receptor of RdCVF opens novel avenues of therapeutics research for inherited retinal degenerations