Abstract
Purpose :
Determine the role of arrestin-1 self-association in vivo.
Methods :
Transgenic mouse lines expressing oligomerization-deficient arrestin-1-(F86A+F198A) at different levels (relative to WT) on arestin-1 -/- background were created: M2 – 56+5%; M4 – 96+6%; M5 – 292+15%; M7 – 210+16%; M9 – 298+30%; M10 – 116+3%. The distribution of the mutant in dark- and light-adapted rods was determined by immunohistochemistry of retina sections and by Western blot in separated rod outer segments (ROS).
Results :
Purified mouse arrestin-1-(F86A,F198A) has dimerization constant of ~540 uM and does not form tetramers (unlike WT mouse arrestin-1 with dimerization and tetramerization constants of ~58 uM and ~63 uM). Calculations based on these constants and expression levels suggest that absolute concentration of monomeric mutant in cell bodies and the inner segments (IS) in the dark would be ~490 uM in M4 and M10, ~900 uM in M7, and reach ~1,280 uM in M5 and M9 lines. Apparently, rods tolerate ~500 uM, but higher concentrations are toxic: rods degenerate in lines M7, M5, and M9. However, it appears that at the normal level of arrestin-1 in WT cell bodies in the dark (~2,000 uM), dimerization with a constant of ~500 uM would suffice to protect rods. Thus, more robust oligomerization of bovine, mouse, and human arrestin-1 serves a different purpose. We detected higher than WT concentration of monomeric arrestin-1 in the upper part of the IS and in the thin sliver of ROS cytoplasm, likely around the microtubule bundle, but not in the intra-discal spaces. This suggests that monomeric arrestin-1 binds rod proteins localized in the upper IS better than WT and indicates that active gating in the connecting cilium does not allow it to equilibrate with ROS.
Conclusions :
Oligomerization of arrestin-1 does not significantly affect its light-dependent translocation, but changes its distribution in rods.
Funding – RO1 EY011500 (VVG).
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.