Differential rescue of protein expression and localization of two adRP-linked PRPH2 mutants in rods is mediated by their specific binding characteristics

Abstract

Purpose : Some autosomal-dominant retinitis pigmentosa (adRP)-linked mutations in the photoreceptor outer segment (OS)-specific peripherin-2 (PRPH2) interfere with binding to wild type (WT) PRPH2, leading to protein mislocalization and reduced protein expression in rods. Using recombinant adeno-associated virus (rAAV)-mediated gene delivery in combination with FRET on isolated murine rod OS, this study addressed two questions: i) Is it possible to rescue protein expression or localization of two adRP-linked PRPH2 mutants, P210L and C214S, by a simultaneous co-delivery of WT PRPH2? ii) What is the role of protein-protein interactions between the single mutants and WT PRPH2 in this context?

Methods : WT mice (N=3) were injected on P14 with rAAV particles bearing WT+WT, WT+P210L, or WT+C214S PRPH2 constructs fused to FRET fluorophores citrine or cerulean driven by the human Rhodopsin promoter. Rescue efficiencies were analyzed via fundus photography, confocal and transmission electron microscopy, and Western blotting of injected retinas 3 weeks post injection. FRET was measured 4 weeks post injection on isolated rod OS for the WT+WT (N=17), WT+P210L (N=17), and WT+C214S (N=14) combinations. For binding curves, FRET ratio was plotted against the cerulean/citrine molar ratio. The relative maximal binding affinity given by the maximal FRET ratio (FR_max) and the relative K_d values were calculated using the FR=(FR_max+x)/(K_d+x) formula.

Results : Simultaneous co-delivery of WT PRPH2 led to an almost full rescue of protein expression and localization for the C214S mutant and to a partial rescue of these parameters for the P210L mutant. WT+WT interaction yields a K_d=0.06±0.02 and FR_max=5.66±0.42. The C214S mutant increases the K_d to 0.12±0.04 and reduces the FR_max to 2.15±0.23. The K_d was even stronger increased for the P210L mutant (0.21±0.08) explaining its lower rescue efficiency. Surprisingly, however, the FR_max of P210L was increased up to the highest levels detectable via FRET (11.35±1.31).

Conclusions : We show that differential rescue effects of WT PRPH2 on protein expression and localization of two adRP-linked PRPH2 mutants in photoreceptors is shaped by their differential binding characteristics. We also provide a proof-of principle for rAAV-based ex vivo FRET as a tool to address specific binding properties of proteins in a subcellular compartment.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.