The Expression Of Phosphorylation–independent "super–arrestins" Protects Rod Outer Segments In Rhodopsin Kinase Knockout Mice.

Abstract

Abstract: : Purpose: To test whether the expression of arrestin mutants that do not require rhodopsin phosphorylation for binding compensates for the lack of rhodopsin kinase in vivo. Methods: Transgenic mice expressing two phosphorylation–independent forms of rod arrestin, truncated arrestin(1–377) (TA) and triple mutant F375A,V376A,F377A (A3A) were created. Both transgenes were bred into arrestin knockout (AKO) and arrestin and rhodopsin kinase double knockout (DKO) background, and raised in 12:12 light:dark cycle. The expression of transgenic and wild type (WT) arrestin in whole retinas was measured by quantitative Western blot. Suction electrodes were used to record flash responses from individual intact transgenic rods. Results: The survival of rod photoreceptors and rod outer segments in three transgenic lines expressing A3A at 2%, 70%, and 150% of WT arrestin level, and three lines expressing TA at 0.5%, 2%, and 25% of WT level were compared to WT, arrestin+/–, AKO, and DKO mice at ages from five through 33 weeks. The expression of either "super–arrestin" (TA or A3A) at 2% of WT level and higher preserved normal retinal morphology on AKO background up to 33 weeks. In contrast, AKO littermates demonstrated progressive loss of outer segments, most of which disappear by 8–10 weeks, without loss of photoreceptor cells, as judged by the thickness of outer nuclear layer. Interestingly, very high (150%) expression of A3A on AKO background triggered progressive loss of photoreceptors, although the remaining cells retained their outer segments. In sharp contrast to AKO, rods expressing 70% and 150% A3A super–arrestin on AKO background demonstrated rapid shut–off of the flash response. Most importantly, this high expression of A3A super–arrestin on DKO background prevented light–induced loss of outer segments and photoreceptor cells observed in transgene–negative DKO littermates. Conclusions: Very low to moderate expression levels of phosphorylation–independent arrestin mutants fully compensated for the lack of WT arrestin in rods. Higher levels of "super–arrestin" expression also compensated for the absence of rhodopsin kinase, apparently by quenching the signaling without rhodopsin phosphorylation effectively enough to prevent light–induced damage to photoreceptor cells.

Keywords: signal transduction • transgenics/knock–outs • photoreceptors