Purchase this article with an account.
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.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
This PDF is available to Subscribers Only