May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Positional Effects of the Phosphorylated C-terminus of Rhodopsin on Arrestin Activation
Author Affiliations & Notes
  • J. McDowell
    Ophthalmology, University of Florida, Gainesville, Florida
  • J. Standfuss
    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
  • A. Arendt
    Ophthalmology, University of Florida, Gainesville, Florida
  • G. Schertler
    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
  • W. C. Smith
    Ophthalmology, University of Florida, Gainesville, Florida
  • Footnotes
    Commercial Relationships  J. McDowell, None; J. Standfuss, None; A. Arendt, None; G. Schertler, None; W.C. Smith, None.
  • Footnotes
    Support  NIH grants EY 006225, EY 14864, EY 8571, Kirchgessner Foundation (WCS), a grant from RPB (UF), Marie Curie fellowship MEIF-CT-2006-039171 (JS) and Human Frontiers (GS)
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 154. doi:https://doi.org/
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      J. McDowell, J. Standfuss, A. Arendt, G. Schertler, W. C. Smith; Positional Effects of the Phosphorylated C-terminus of Rhodopsin on Arrestin Activation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):154. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : To crosslink the fully phosphorylated region of bovine rhodopsin comprising residues 330-348 to arrestin at various positions to determine if the activation of arrestin depends on the position where the crosslink occurs and to determine if activation leads to a shift in the oligomeric equilibrium.

Methods: : An analog of the fully phosphorylated carboxyl terminus of bovine rhodopsin comprising residues 330-348 (AcK-7PP) was synthesized with an acetylated amino side chain of lysine residue 339. The peptide was modified at its free amino terminus with the bifunctional reagent N-(a-maleimidoacetoxy) succinimide ester that places a sulfhydryl reactive group on the free amino group. The modified peptide was then combined with arrestin and three cysteine mutants of arrestin, K15C/C63A/C143A, I16C/C63A/C143A, and A381C/C63A/C143A. SDS-PAGE and western blotting were performed to determine if the peptide cross-linked to arrestin. Binding to rhodopsin was assessed by a centrifugal pull-down assay. The monomer/dimer ratio of arrestins was tested using blue native electrophoresis.

Results: : As shown earlier in native arrestin, AcK-7PP cross-links only to Cys-143. Cross-linking to this native cysteine leads to a state of arrestin that by limited proteolysis appears active, but the cross-linked arrestin does not bind to either light-activated rhodopsin (R*) nor to light-activated phosphorylated rhodopsin (PR*). Cross-linking AcK-7PP to arrestin at K15C or I16C yields proteins that are activated and bind to both R* and PR*. Conversely, cross-linking the AcK-7PP to arrestin at A381C yields a protein that is little affected. It does not bind significantly to R*, but binds well to PR*. Blue native gels showed that AcK-7PP cross-linked to I16C resulted in a significant increase in the amount of monomer compared to either native arrestin or uncross-linked I16C.

Conclusions: : 1. Cross-linking AcK-7PP to arrestin mutants yields three different states of the protein depending on the position of the crosslink: a) arrestin that is activated and binds to R* as well as PR*; b) arrestin that is not affected and binds to PR* but not R*; and c) arrestin that binds to neither R* nor PR*, but looks activated by limited proteolysis. 2. Crosslinking AcK-7PP to arrestin at I16C shifts the oligomeric equilibrium to more monomer as assessed by blue native gels, suggesting that activation enhances monomer concentration.

Keywords: protein structure/function • signal transduction • phosphorylation 
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