title: Engineering visual arrestin-1 with special functional characteristics.
abstract: 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.
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Vishnivetskiy
authors.forename: Qiuyan
authors.initials: Q
authors.lastname: Chen
authors.forename: Maria C
authors.initials: MC
authors.lastname: Palazzo
authors.forename: Evan K
authors.initials: EK
authors.lastname: Brooks
authors.forename: Christian
authors.initials: C
authors.lastname: Altenbach
authors.forename: Tina M
authors.initials: TM
authors.lastname: Iverson
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
mesh_terms: D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005123:Eye / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012243:Rhodopsin / Q000378:metabolism; D055672:Static Electricity; D013696:Temperature; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D050505:Mutant Proteins; D010710:Phosphates; D000071557:beta-Arrestins; D012243:Rhodopsin