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D. Raman, M.J. Kennedy, J.B. Hurley, V.V. Gurevich; Threshold Mechanism of Arrestin Activation: Two Rhodopsin–Attached Phosphates Are Necessary and Sufficient for High–Affinity Arrestin Binding . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1177.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the number of rhodopsin–attached phosphates that are required for high affinity arrestin binding by using purified forms of rhodopsin with unambiguously defined stoichiometry of phosphorylation . Methods: Rhodopsin in purified bovine rod outer segments was phosphorylated by endogenous rhodopsin kinase and solubilized with 1.5% octyl glucoside. Rhodopsin species with different phosphorylation stoichiometry were separated by chromatofocusing on Mono P FPLC and reconstituted into liposomes. The presence of individual phosphorylated forms of rhodopsin in each fraction was determined quantitatively by mass spectrometry. The binding of radiolabeled arrestin (50 fmol/assay) to rhodopsin (50 ng/assay) containing 0, 1, 2, 3, 4, and 5 phosphates was tested. Results: Rhodopsin fractions containing either unphosphorylated or a mix of unphosphorylated and monophosphorylated (36%+ 64%) light–activated rhodopsin demonstrated very low arrestin binding (<0.2 fmol/assay). Light–activated rhodopsin with two to five attached phosphates at the C–terminus (with the predominant species at 74–95%) bound arrestin at the level of 9–14 fmol/assay. The presence of more than two rhodopsin–attached phosphates marginally increased the arrestin binding. Phosphorylation–independent truncated arrestin mutant (1–378) binds to light–activated rhodopsin regardless of its phosphorylation. This mutant specifically bound to all forms of activated rhodopsin preparations, demonstrating their full functionality. Conclusions: A single rhodopsin–attached phosphate is not sufficient for the high affinity arrestin binding. At least two phosphates per rhodopsin molecule are necessary to induce arrestin transition into high–affinity rhodopsin–binding state. These data agree with in vivo analysis of the number of rhodopsin phosphorylation sites that are necessary for arrestin–dependent rapid shut–off of the photoresponse and with the structure–function studies of the phosphate–binding elements of arrestin that include at least four positively charged residues.
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