Abstract
Abstract: :
Purpose: Arrestin translocates from the inner segment of rod photoreceptors (RIS) to the outer segment of rod photoreceptors (ROS) in response to light. In the absence of transducin and phosphorylated rhodopsin, arrestin is still able to translocate from the RIS to the ROS in response to light [1]. To determine if arrestin translocation could be stimulated through a light–independent pathway, we investigated the translocation of arrestin in response to phorbol 12,13–diacetate (PDA), which is an activator of protein kinase C (PKC). Methods: Arrestin–GFP transgenic tadpoles and wild–type tadpoles were dark–adapted for at least twelve hours. The tadpoles were treated with 1uM PDA in 0.1x TPR (Tadpole Ringers solution) for 5,10, 15, 20, and 30 min in the dark. Additional tadpoles treated with PDA for 30min were also light adapted for 1hr. After each time point, the tadpoles were fixed in 73% MeOH, 3.7% formaldehyde, and 23.3% deionized water. Arrestin–GFP tadpole eyes were cryosectioned and endogenous fluorescence of GFP was visualized using confocal microscopy. Wild–type tadpole eyes were also cryosectioned, immunostained with anti–Xenopus arrestin antibody and visualized using confocal microscopy. Results: Translocation of arrestin from the RIS to the ROS was detected after treating the tadpoles for 5min with 1uM PDA. Maximum translocation was seen at 10min of exposure to PDA. Longer exposure to the PDA (i.e.>15min) resulted in arrestin relocalizing to the RIS. Interestingly, arrestin is able to re–translocate to the outer segments in response to light even after 30min exposure to PDA. Conclusions: Our results show that arrestin translocation in rod photoreceptors can be stimulated by a phorbol ester. This observation suggests an alternative signaling cascade involving PKC that may trigger protein translocation in rod photoreceptors. 1. Mendez A., Lem J., Simon M., Chen J. (2003) J Neurosci, 23: 3124–3129