Rod and Cone Arrestins Differentially Mobilize Signaling Proteins to Microtubules

Abstract

Purpose:: Rod arrestin binds microtubules (MTs) and this interaction plays an important role in its localization in photoreceptor cells. Here we determined the conformation of MT-bound arrestin, its affinity, and the functional consequences of MT binding of rod and cone arrestins.

Methods:: To determine MT binding sites on arrestin, we used purified proteins for binding assays and site-directed spin labeling electron paramagnetic resonance. In HEK293 cells we explored arrestin-dependent localization of signaling proteins to the cytoskeleton.

Results:: Using purified arrestins and MTs polymerized in vitro from pure tubulin, we confirmed that rod and cone arrestins directly bind microtubules. Deletion of the arrestin C-tail or its detachment from the body of the molecule by a triple alanine substitution increases arrestin flexibility and dramatically enhances its binding to MTs. Progressive deletions in the inter-domain hinge that severely impede rhodopsin binding enhance MT binding of rod arrestin. We found that the MT- and receptor-binding sites on arrestin overlap, making these interactions mutually exclusive. Arrestins mobilize various signaling proteins to the receptors; therefore we tested whether rod and cone arrestins localize ERK1/2 and Mdm2 to MTs. The proportion of ERK1/2 in the cytoskeleton is significantly increased in cells expressing rod (but not cone) arrestin, indicating that rod arrestin brings ERK to the MTs. The total level of active phospho-ERK in the cell is dramatically reduced by its mobilization to MTs. We found that the endogenous Mdm2 present in the cytoskeletal fraction is also significantly increased in cells expressing rod but not cone arrestin. Rod arrestin dramatically increases the ubiquitination of numerous proteins in the cytoskeletal fraction, whereas cone arrestin does not.

Conclusions:: The same surface in rod arrestin interacts with rhodopsin and microtubules, but the conformations of MT- and receptor-bound arrestin are different. Rod (but not cone) arrestin localizes signaling proteins to the microtubules. Arrestin-dependent mobilization of ERK1/2 to MTs decreases the total level of active ERK in the cell by removing it from compartments where it can be activated. Arrestin redirects the activity of Mdm2 towards MT-associated proteins, increasing their ubiquitination. These findings bring to light a distinct contrast between rod and cone arrestin.

Keywords: cytoskeleton • photoreceptors • signal transduction