Abstract
Purpose :
RD3 protects photoreceptors from rapid degeneration by preventing aberrant activation of retinal guanylyl cyclase (RetGC) by calcium-sensor proteins, GCAPs, in the inner segment. RD3 also helps deliver RetGC to the outer segments to enable phototransduction. We studied how RD3 structure defines its ability to prevent photoreceptor degeneration and to enable RetGC delivery to the outer segment.
Methods :
RD3 was mutagenized to identify the location of the key residues required for its high-affinity inhibitory binding to RetGC versus those that are critical for the RD3-assisted delivery of RetGC to the rod outer segments (ROS). The mutants, tested for their abilities to inhibit RetGC in vitro and to bind RetGC in cyto, were expressed in homozygous rd3 mice (Rd3-/-) under control of rhodopsin promoter, followed by testing RetGC activity and localization. Changes in retinal morphology were monitored by spectral domain OCT and the rod function was assessed using dark-adapted ERG.
Results :
The ability of RD3 to bind and inhibit RetGC required two narrow clusters of amino acid residues located in the RD3 N-terminal alpha-helical bundle, whereas its long C-terminal portion did not contribute to the inhibitory binding. RD3 truncated after residue 148 retained high-affinity binding to RetGC and when expressed in Rd3-/- rods protected them from rapid degeneration. The thickness of outer nuclear layer in Rd3-/-Δ148RD3+ aged 4 months was 46±3 µm versus 13±5 µm in Rd3-/- littermates (p<0.0001). Despite their much better preserved retinal morphology, Rd3-/-Δ148+ failed to accumulate RetGC in ROS and improve rod ERG. Extending the length of the C-terminal region past residue 170 did not change RD3 affinity for RetGC, yet restored RetGC accumulation in ROS and improved rod ERG. Wild type and truncated RD3 remained almost exclusively in the inner segment, whereas RetGC and GCAPs accumulated in ROS.
Conclusions :
To prevent photoreceptor degeneration, RD3 binds and inhibits RetGC in the inner segment using its N-terminal helical domain. The C-terminal region of RD3 contains a “guidance signal”, directing the RetGC:RD3 complex delivery to ROS. Upon approaching the outer segment, RD3 is displaced from RetGC by GCAPs and remains in the inner segment.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.