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Maxim Sokolov, Marycharmain Belcastro, Hongman Song; The Effect of Phosducin on the Trafficking of Transducin to the Rod Outer Segment. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4407.
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Over-stimulation of the rod phototransduction cascade by light promotes dissociation of the heterotrimeric G protein, transducin (T), into the Tα and Tβγ subunits that are loosely anchored to the membrane, and diffuse from the outer segment disc to other cellular compartments. The reversal of this process in the dark would thus require re-association of the separated Tα and Tβγ subunits with each other and the membrane. We proposed that this process is regulated by phosducin (Pdc), an abundant rod phosphoprotein that binds free Tβγ when de-phosphorylated. This hypothesis was tested in vivo using transgenic mice.
Mice expressing normal levels of Pdc-FLAG and PdcS54/71/A-FLAG, lacking its principal phosphorylation sites, on the Pdc-null background were analyzed. Translocation of rod transducin was induced in isoflurane-anesthetized mice using a mild light-conditioning protocol, after which mice were allowed to wake up and recover in the dark while free-running. The fractions of transducin and arrestin present in the outer segments at different times of dark recovery were quantified using Western blotting of serial tangential sections of the retina.
In the control mice, expressing Pdc-FLAG, transducin subunits returned to the rod outer segments with a half-time of 24 min for Tα and 29 min for Tβγ. The trafficking of Tα, however, was profoundly slowed down in PdcS54/71/A-FLAG mice, where it had a half-time of 95 min, while the trafficking of Tβγ was mostly unaffected. This effect of Pdc was specific for transducin, and had no effect on the translocation of arrestin.
Here we demonstrate that the return of transducin to the rod outer segments under physiological conditions occurs at a much faster rate than previously thought. Our data support the notion that the re-association of Tα with the membrane requires interaction with Tβγ, because phosphorylation of Pdc that releases Tβγ from the Pdc/ Tβγ complex significantly accelerates this process. The potential mechanism of the trafficking of transducin will be discussed.
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