Abstract
Purpose: :
To determine the role of arrestin1 cleavage products in photoreceptor cell death.
Methods: :
Arrestin1 cleavage by caspases was evaluated in vitro and in cultured cells. The cell lysates were analyzed for arrestin1 cleavage products, and their distribution in the cell was determined by fractionation.
Results: :
Prolonged exposure of mice to intense light induces apoptosis of photoreceptor cells, accompanied by the cleavage of endogenous arrestin1. To determine whether arrestin1 is cleaved by caspases, we tested the sensitivity of purified arrestin1 to recombinant caspases in the presence and absence of P-Rh*. We found that both bovine and mouse arrestin1 are cleaved in the presence of P-Rh*, but not in the absence of the receptor. The fragments generated from both proteins were comparable in size. Interestingly, in the presence of active initiator caspases 8 and 10, mouse, bovine, and human arrestin1 were cleaved at different aspartic acid residues. Mouse and human arrestin1 produce a much larger cleavage product than bovine. Thus, any caspase cleaves P-Rh*-bound arrestin1, but only initiator caspases 8 and 10 cleave free arrestin1. Bovine arrestin1 was also cleaved in apoptotic Rat-1 cells. The cleavage sites were identified as 364D, 397D, and 403D by mutagenesis of individual aspartic acid residues in the C-terminus of the protein. Previously we found that, in contrast to full-length protein, the caspase-generated arrestin2 fragment localizes to the mitochondria, and induces the release of cytochrome C in apoptotic cells. Similarly, caspase-generated bovine arrestin1 fragment also preferentially localizes to the mitochondria under these conditions. Interestingly, arrestin4 was not cleaved by caspases either in vitro or in vivo. The role of arrestin1 cleavage in cell survival will be analyzed by viral expression of arrestin1 fragments and caspase-resistant mutants in photoreceptor cells.
Conclusions: :
Arrestin1 cleavage by caspases likely plays a role in apoptotic death of photoreceptor cells.
Keywords: apoptosis/cell death • photoreceptors • protein structure/function