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AJ Ghalayini, MH Elliott; Light-dependent Reciprocal Association of T and Arrestin With Photoreceptor Rod Outer Segment(ROS) Cytoskeleten In Vivo . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2898.
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Purpose: Light-dependent protein translocation of phototransduction components between the inner and outer segments of photoreceptors has been reported by several investigators. We have investigated the biochemical mechanisms involved in protein movement between compartments of photoreceptor cells and the possible role of the cytoskeleton in protein recruitment Methods: Albino rats were dark-adapted (D) or light-adapted(L) for 30 minutes in room light (300 lux), and ROS prepared on a discontinuous sucrose gradient. ROS membranes were solubilized with buffer containing 1% Triton -X-100 and the detergent resistant pellet (Cytoskeleton, Csk) obtained following centrifugation at 27,000xg was subjected to immunoblot analysis with anti-transducin alpha (Tα) or anti-arrestin followed by densitometry. In other studies, ROS were solubilized with both 1% Triton -X-100 and octylglucoside followed by immunoprecipitation with either anti-Tα or anti-caveolin-1. Results:DROS and DCsk were enriched 2-3 fold in Tα over LROS and LCsk, respectively. Conversely, arrestin was enriched 3-5 fold in LROS and LCsk over DROS and DCsk, respectively, indicating a reciprocal, light-dependent association of Tα and arrestin with the cytoskeleton. Other studies revealed that Tα and caveolin co-immunoprecipitate only from DROS. Conclusion:Our data show that detergent-resistant membranes obtained from DROS and LROS play a role in recruiting (binding) both arrestin and Tα in a light-dependent fashion. Moreover, we propose that the scaffolding protein caveolin-1 present in detergent- resistant membranes may play a role in the association of Tα with these membranes. Support:NIH grants EY11504; EY12190;EY13674 and Research to Prevent Blindness, Inc.
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