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M Sokolov, KJ Strissel, VI Govardovskii, VY Arshavsky; Transducin-phosducin Interactions in Rod Inner Segments of the Light-adapted Rat . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1957.
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Purpose: Phosducin is a protein that interacts with various G protein ßγ subunits and is thought to be a negative regulator of G protein signaling. It has been suggested that phosducin down-regulates visual transduction in vertebrate photoreceptors by reducing the concentration of transducin ßγ subunits upon illumination (Lee et al., J. Biol. Chem. 267, 25104, 1992; Willardson et al., Proc. Natl. Acad. Sci. USA 93, 1475, 1996). However, recent papers indicate that rod outer segments (ROS) where the phosducin-transducin interaction is thought to take place contain only a small fraction of the total phosducin pool (Thulin et al., Mol. Vis. 5:40, 1999; Nakano et al., Proc. Natl. Acad. Sci. U.S.A. 98, 4693, 2001). To resolve this controversy, we have applied a novel technique to measure phosducin distribution among different rod compartments in dark- and light-adapted rats. We also compare subcellular distribution of phosducin and transducin. Methods: Phosducin content in whole rat retina and ROS was determined by quantitative Western blotting. Phosducin and transducin distribution among rod subcellular compartments was analyzed by a technique combining serial tangential cryosectioning of the frozen flat-mounted retina with Western blot analysis of proteins in the sections. Results: Whole rat retina was found to contain one molecule of phosducin per 11 molecules of rhodopsin, which is identical to the transducin content. However, ROS of both dark- and light-adapted rats contain only ∼7% of the total phosducin pool. ∼ 60% of phosducin is localized to the ellipsoid and myoid of the rod inner segment and the rest is present within the nuclear and synaptic terminal regions. Unlike phosducin, transducin undergoes massive light-dependent movement from ROS to other rod compartments. We found that a large fraction of the transducin ßγ subunit is bound to phosducin in rod inner segments of the light-adapted retina. Conclusion: Our data indicate that phosducin is predominantly a rod inner segment protein whose subcellular distribution is not affected by light. It interacts with transducin ßγ subunits upon illumination and likely participates in light-dependent transducin re-allocation from ROS into other rod cell compartments.
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