Arrestin binding to microtubules involves a distinct conformational change

Abstract

Arrestins play a key role in the regulation of signaling by G protein-coupled receptors. We found that all four mammalian arrestins directly bind microtubules (MTs) with Kds of 25–50 uM. In the case of rod arrestin which self-associates into tetramers in solution, both monomer and tetramer bind MTs. Using site-directed mutagenesis and spin labeling/EPR we identified the concave side of the two arrestin domains as the MT-binding surface in rod arrestin and arrestin2. This site significantly overlaps with the receptor-binding site, making arrestin interactions with receptors and MTs mutually exclusive. Similar to the receptor, MT binding induces the release of the arrestin C-tail. The deletion of the C-tail and other mutations destabilizing the basal conformation of arrestin enhance binding to MTs, suggesting that arrestins undergo a conformational change in the process. Deletions in the inter-domain hinge that hinder receptor binding enhance the arrestin-MT interaction, suggesting that the receptor- and MT-bound arrestin conformations are different. The extent of the co-localization of wild type and mutant arrestins with MTs in cells and their co-purification with the cytoskeletal fraction is proportional to their affinity for MTs determined in vitro. Thus, arrestins in living cells exist in at least three distinct conformations: basal (free), receptor-bound, and MT-bound. Funded by NIH grants EY11500, GM63097, AI58024, GM70642, EY05216.