Mutations In The C–terminus Enhancing Arrestin Binding To Tubulin Affect Its Light–dependent Translocation In Mouse Rod Photoreceptors In Vivo.

Abstract

Abstract: : Purpose: To test whether mutations in the arrestin C–terminus affect its light–dependent translocation in vivo. Methods: Transgenic mice expressing two structurally diverse mutant forms of rod arrestin, truncated arrestin(1–377) (TA) and triple mutant F375A,V376A,F377A (A3A), both of which demonstrate increased affinity for tubulin in biochemical assays, were created. Both transgenes were bred into an arrestin knockout background. The expression of transgenic and wild type (WT) arrestin in whole retinas was measured by quantitative Western blot. Transgenic lines expressing TA and A3A at the level of 25% and 70%, respectively, of arrestin levels in WT mice were used. Arrestin distribution in photoreceptor cells in dark– and light–adapted animals expressing three forms of arrestin (WT, A3A, TA) was compared using quantitative immunofluorescence/ image analysis, and by biochemical fractionation followed by Western blot. Results: In agreement with previous reports, WT arrestin was found predominantly in the outer segments in light–adapted animals, whereas in the dark it distributed throughout the rod cells, with highest immunoreactivity in the synaptic area. In light–adapted retinas both mutants also concentrated in the outer segments. However, in dark–adapted animals a significantly higher proportion of mutant arrestins remained in the outer segments. The distribution of mutant arrestins in the other regions of photoreceptor cells was more even than that of WT, with less conspicuous concentration in the synaptic layer. In both light– and dark–adapted retinas less than 10% of WT arrestin was found in the detergent–insoluble fraction. In contrast, a substantially higher proportion of both mutant forms was detected in the detergent–insoluble fraction enriched in tubulin. Conclusions: We have earlier demonstrated that the deletion of arrestin C–terminus (in TA) or the disruption of its anchoring interaction with the arrestin N–domain by mutagenesis (in A3A) enhances its interaction with tubulin and targets the mutants to the detergent–resistant fraction. Our in vivo data indicate that enhanced tubulin interaction impedes arrestin migration from rod outer segments in the dark, suggesting that tubulin is either involved in arrestin translocation, or that "docking" to tubulin regulates arrestin movement.

Keywords: protein structure/function • photoreceptors • cytoskeleton