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Vsevolod V Gurevich, Srimal Aminda Samaranayake, Sergey A Vishnivetskiy; The effect of arrestin-1 self-association on its distribution in rods. Invest. Ophthalmol. Vis. Sci. 2019;60(9):558.
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© ARVO (1962-2015); The Authors (2016-present)
Determine the role of arrestin-1 self-association in vivo.
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).
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.
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.
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