Engineering visual arrestin-1 with special functional characteristics.

Vishnivetskiy SA, Chen Q, Palazzo MC, Brooks EK, Altenbach C, Iverson TM, Hubbell WL, Gurevich VV
J Biol Chem. 2013 288 (5): 3394-405

PMID: 23250748 · PMCID: PMC3561558 · DOI:10.1074/jbc.M112.445437

Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations.

MeSH Terms (18)

Animals Arrestins beta-Arrestins Eye HEK293 Cells Humans Mice Models, Molecular Mutant Proteins Mutation Phosphates Protein Binding Protein Engineering Protein Stability Protein Structure, Tertiary Rhodopsin Static Electricity Temperature

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