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Vsevolod Gurevich, Sergey Vishnivetskiy, Qiuyan Chen, Maria Palazzo, Evan Brooks, Christian Altenbach, Tina Iverson, Wayne Hubbell; Independent manipulation of binding selectivity and self-association of arrestin-1. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2460.
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© ARVO (1962-2015); The Authors (2016-present)
To test whether binding selectivity and self-association of arrestin-1 can be independently manipulated for research and therapeutic use
Versions of arrestin-1 with activating mutations enhancing the binding to unphosphorylated light-activated rhodopsin (Rh*) and substitutions suppressing self-association were constructed, expressed, and their binding selectivity, stability, and oligomerization were tested
Arrestin-1 preferentially binds active phosphorylated rhodopsin (P-Rh*). An enhanced mutant with increased binding to Rh* partially compensates for the lack of rhodopsin phosphorylation in vivo. We show that reengineering of the receptor-binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. Mammalian arrestin-1 readily self-associates at physiological concentrations. To elucidate the biological role of this phenomenon, wild type arrestin-1 in living animals must be replaced with a non-oligomerizing mutant retaining all other functions. Constitutively monomeric forms of arrestin-1 with wild type selectivity for P-Rh* are sufficiently stable for in vivo expression. The same mutations eliminate self-association of enhanced forms, while preserving high Rh* binding and stability
Individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Stable forms of arrestin-1 with high Rh* binding can be constructed with or without the ability to self-associate. These results pave the way for testing of the biological role of arrestin-1 self-association and elucidation of full potential of compensational gene therapy of gain-of-function GPCR mutations. NIH grants EY011500, GM077561, GM081756 (VVG), EY05216 and the Jules Stein Professorship Endowment (WLH), GM079419, GM095633 (TII)
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