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S. A. Vishnivetskiy, X. Song, H. E. Boyd, V. V. Gurevich; Improving Phosphorylation-Independent Arrestin1: Redesign of the Rhodopsin-Binding Surface. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4792.
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To construct phosphorylation-independent arrestin1 mutant that can fully compensate for the defects in rhodopsin phosphorylation in vivo.
Site-directed mutagenesis and direct binding assay were used to create arrestin1 that binds unphosphorylated light-activated Rh* with the same affinity that wild type (WT) arrestin shows for phosphorylated P-Rh*.
Previously we found that triple alanine substitution that detaches the C-tail from the body of the molecule (3A) enhances arrestin binding to Rh* in vitro. Transgenic expression of arrestin1-3A in mice lacking rhodopsin kinase or expressing mutant rhodopsin that does not have phosphorylation sites improves photoreceptor survival and facilitates photoresponse recovery (measured by double-flash ERG) up to three-fold, as compared to animals expressing WT arrestin on the same genetic backgrounds. However, photoresponse recovery in "rescued" animals was slower than in normal WT mice. We found that mutant arrestin-Rh* complex was less stable than WT arrestin-P-Rh* complex. Since receptor-binding surface of WT arrestin fits multi-phosphorylated rhodopsin best, we hypothesized that it must be redesigned for optimal fit to unphosphorylated Rh*. We identified mutations on the rhodopsin-binding concave sides of both arrestin domains that greatly increase the affinity of 3A mutant for Rh*. The levels of binding of several mutants to Rh* in vitro approaches that of WT arrestin to P-Rh*. Combinations of individual mutations improving arrestin affinity for Rh* further increases the binding.
Targeted redesign of rhodopsin-binding surface of the phosphorylation-independent arrestin1 mutant is necessary to ensure better fit for the cytoplasmic tip of unphosphorylated Rh*. Higher affinity of new phosphorylation-independent versions of arrestin1 for Rh* will increase their ability to compensate for the defects in rhodopsin phosphorylation in vivo, enhancing their therapeutic potential in human disorders associated with excessive signaling by mutant forms of rhodopsin.
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