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W. Orisme, W. C. Smith; Arrestin Translocates to the Rod Outer Segments in Response to m-3M3FBS,an Activator of Phospholipase C. Invest. Ophthalmol. Vis. Sci. 2007;48(13):593.
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© ARVO (1962-2015); The Authors (2016-present)
The signaling cascade for arrestin translocation to the rod outer segments (ROS) is not fully understood. We have previously shown arrestin translocation to the ROS can be stimulated by a diacylglycerol analog, phorbol 12, 13-diacetate (PDA). Phospholipase C (PLC) cleaves phosphatidylinositol 4, 5 bis-phosphates (PIP2) to produce inositol trisphosphate and diacylglycerol. Here, we investigate the possible role of PLC in the signaling cascade for arrestin translocation.
Dark-adapted arrestin-GFP (Arr-GFP) transgenic tadpole eyes were treated with 1uM m-3M3FBS (an activator of PLC) for 2.5, 5, 10, 20, and 30 min in the dark. Eyes were also light adapted (LA) for 30min. After each time point, the eyes were fixed in 73% MeOH and 3.7% formaldehyde in deionized water. The eyes were cryosectioned and Arr-GFP was visualized using confocal microscopy. In parallel, wild-type eyes were pretreated with 32P-ATP for 12-15hrs and then treated with 1uM m-3M3FBS for 2.5, 5, 10, and 20min or light adapted for 30min. Retinal extracts were collected from each time point and analyzed by 1D SDS-PAGE.
Arrestin translocated to the base of the ROS within ten minutes of 1uM m-3M3FBS treatment. Arrestin returned back to the RIS with prolonged exposure to m-3M3FBS, behaving similar to PDA treatment. Radiographs of the retinal extracts revealed a ~30kDa protein that significantly increases in phosphorylation at 5min of m-3M3FBS treatment. After 5min of m-3M3FBS, phosphorylation of the band decreased to nearly basal levels.
Our results show that activating PLC with m-3M3FBS can stimulate arrestin translocation to the outer segments in rod photoreceptors in the absence of light. We have also identified a ~30kDa band that may play a role in the signaling cascade for arrestin translocation. These observations suggest that light may trigger arrestin translocation through a signaling cascade that involves PLC.
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