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S. A. Vishnivetskiy, T. H. Bayburt, T. Morizumi, C.-C. Huang, J. J. Tesmer, O. P. Ernst, S. G. Sligar, V. V. Gurevich; Rhodopsin Monomer is Phosphorylated by Rhodopsin Kinase and Binds Arrestin With High Affinity in the Presence of Negatively Charged Lipids. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1098.
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© ARVO (1962-2015); The Authors (2016-present)
Determine the role of negatively charges lipids in arrestin1-rhodopsin interaction
Arrestin1 binding to monomeric rhodopsin in nanodiscs was measured.
The role of rhodopsin dimerization is hotly debated. We found that monomeric rhodopsin in nanodiscs is phosphorylated by rhodopsin kinase (GRK1) as efficiently as in native membranes. Arrestin1 binds P-Rh* monomer with affinity: Kd app ~ 28nM, very close to ~ 25nM in native disc membranes. The stoichiometry of arrestin1-monomeric P-Rh* interaction is 0.86 mol/mol, similar to 0.99 mol/mol in native membranes with purified arrestin1, and ~0.83:1 in rod photoreceptors of living mice. The efficiency of rhodopsin phosphorylation is the same in phosphatidylcholine (PC) nanodiscs containing 0-50% phosphatidylserine (PS). In contrast, arrestin1 binding to P-Rh* in pure PC nanodiscs is low. The binding increases ~10-fold and plateaus at 30-50% PS. We compared WT arrestin1 and structurally different "enhanced" mutants that bind P-Rh* and Rh* with comparable affinity. Both truncated arrestin1(1-378) that has no C-tail and 3A mutant where the interaction anchoring the C-tail to the body of the molecule is destabilized are much less dependent on PS, demonstrating >50% of maximum binding in pure PC. In contrast, polar core mutants with the same phenotype, R175E and D296R, depend on PS virtually as much as WT arrestin1. Elimination of positive charges on arrestin1 surface does not affect PS dependence of R175E mutant binding to rhodopsin.
Monomeric rhodopsin is normally phosphorylated and binds arrestin. Negatively charged lipids promote interaction by "pushing" the arrestin C-tail away from the receptor-binding surface. This is the first indication of the function of multiple negative charges present in the C-tail of all arrestin subtypes.
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