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Sergey Vishnivetskiy, Lori S Sullivan, Sara J Bowne, Stephen P Daiger, Eugenia Gurevich, Vsevolod V Gurevich; Molecular defects of the disease-causing human arrestin-1 C147F mutant. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3062.
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Recently identified C147F mutation in the human arrestin-1 causes an autosomal dominant retinitis pigmentosa (adRP) in a group of families without mutations in known adRP genes. Identification of the molecular defect in disease-causing human arrestin-1 C147F mutant is the purpose of this study.
The binding of WT human arrestin-1 and several mutants with substitutions in position 147, including C147F that causes dominant retinitis pigmentosa in humans, to phosphorylated and unphosphorylated light-activated rhodopsin was determined. Protein yields in cell-free translation system and its expression in E.coli were evaluated. Thermal stability of WT and mutant human arrestin-1, as well as unfolded protein response in photoreceptor-derived 661W cells were also assessed.
WT human arrestin-1 is selective for phosphorylated light-activated rhodopsin. Substitutions of Cys-147 with smaller side chain residues, Ala or Val, did not substantially affect binding selectivity, whereas residues with bulky side chains in the position 147 (Ile, Leu, and disease-causing Phe) increased the binding to unphosphorylated light activated rhodopsin more than 2-fold. Functional survival of mutant proteins with bulky substitutions at physiological and elevated temperature was also compromised. WT arrestin-1 retains full functionality at 42oC, and >80% even at 50oC, whereas the C147F mutant loses almost half of its P-Rh* binding ability at 42oC and virtually all at 50oC. In contrast to stable WT human arrestin-1, C147F mutant almost completely denatures after 24 h at 37oC. The expression of the WT human arrestin-1 in 661W cells does not increase the level of BIP, whereas the expression of the C147F mutant significantly increased BIP expression. Thus, C147F mutant induced unfolded protein response in cultured cells.
Bulky Phe substitution of Cys-147 in human arrestin-1 likely causes rod degeneration due to reduced stability: denaturing protein likely induces unfolded protein response in rods.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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