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S.M. Hanson, D.J. Francis, S.A. Vishnivetskiy, K.S. Nair, V.Z. Slepak, W.L. Hubbell, C.S. Klug, V.V. Gurevich; Rhodopsin and Microtubules Compete for Arrestin Binding in Rod Photoreceptors . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1715.
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Purpose: Arrestin and transducin translocate between the compartments of rod photoreceptors upon illumination. In the dark arrestin is located in the microtubule–rich inner segment (IS), while in the light it moves to the outer segment (OS). Recently we found that rod arrestin binds directly to microtubules (MTs) in vitro and in vivo. This interaction is important for localizing arrestin to the IS in the dark. Here we identify the MT–binding site on rod arrestin. Methods: The ability of wild type arrestin and charge reversal mutants to bind MTs was tested in a direct binding assay. A number of cysteine point mutants were created on a cys–less arrestin background, purified, spin–labeled, and their EPR spectra in the presence and absence of MTs were compared. Direct competition between MTs and various functional forms of rhodopsin was also tested. Results: Several charge reversal mutations in arrestin (R18E, K166E, K236E) on the concave side of both arrestin domains inhibit MT binding, whereas K330E enhances it two–fold. Site–directed spin–labeling EPR (SDSL–EPR) at several positions (including 74 and 173) also implicates the concave surface of arrestin in MT binding. Wild type arrestin binds better to every functional form of rhodopsin except dark Rh and opsin than to MTs. Conclusions: We have previously demonstrated that MT binding plays an important role in arrestin translocation by concentrating it in the MT–rich inner segments in the dark. Several arrestin residues implicated in rhodopsin binding were also found to be critical for its binding to microtubules. Significant overlap of arrestin binding sites for microtubules and rhodopsin provides structural basis for their competition. Arrestin binding to rhodopsin and microtubules is mutually exclusive.
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