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J. Li, Jr., W. C. Smith; Elements Regulating Light-Driven Translocation in the C-Terminus of Arrestin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4662.
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© ARVO (1962-2015); The Authors (2016-present)
Arrestin translocates in rod photoreceptors in response to light, relocalizing from the inner segment to the outer segment. In previous studies, we have shown that the strongly acidic C-terminus of arrestin contains an important element that supports light-driven translocation. Here, we show that the amino acid order of the C-terminus of arrestin, and not just the charge composition, is important for translocation, but not for binding to rhodopsin.
Four lines of transgenic Xenopus were prepared by nuclear transplantation: 1) native Xenopus arrestin (xArr) fused to GFP; 2) xArr with the C-terminal 33 amino acids scrambled and fused to GFP; 3) xArr with the C-terminal 33 amino acids replaced with bovine arrestin C-terminus fused to GFP; and 4) xArr with the C-terminal 33 amino acids replaced with a scrambled bovine C-terminus fused to GFP. Eyes from each transgenic strain were dark adapted or light adated, and processed for fluorescence microscopy. Each of the four arrestin proteins was also expressed in yeast, and binding to rhodopsin assessed in a pull-down assay.
Arrestin lacking the C-terminal 33 amino acids showed a cellular distribution different than full-length arrestin, having nearly half of the arrestin localized to the outer segments in dark-adapted animals (compared to virtually none for full-length arrestin). If the C-terminus is replaced by either the C-terminus from bovine or Xenopus arrestin, normal arrestin distribution and translocation is restored. On the other hand, if the C-terminal 33 amino acids are replaced by scrambled bovine or Xenopus C-terminal peptides, then arrestin distribution appears like that of the truncated arrestin and does not show light-dependent translocation. Intriguingly, all arrestins, even those with a scrambled C-terminus (either from bovine or Xenopus), retain their selectivity for light-activated, phospho-rhodopsin and do not show and increased binding to unphosphorylated metarhodopsin.
Our results provide evidence that the C-terminus of arrestin is an important element that promotes light-driven translocation. Importantly, these results reveal that the order of the C-terminal peptide is significant, and not just the charge composition. This observation is significant in light of the fact that the C-terminus shows significant affinity for tubulin through charge/charge interactions rather than sequence-specific binding. Further, the lack of translocation in an arrestin that shows specific and selective binding to R*P suggests that binding to rhodopsin and translocation are independent events, requiring different elements on the arrestin protein.
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