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Zachary Wright, Ryan Alpino, Ratnesh Singh, Visvanathan Ramamurthy; Arl3 regulates trafficking of prenylated phototransduction proteins to the rod outer segment.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2583. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Efficient protein trafficking is important for the function and survival of photoreceptor neurons due to the high rate of protein turnover and polarized distribution of proteins. Recent in vitro studies have implicated a small GTPase, ADP-Ribosylation Factor-Like 3 (Arl3), as a regulator specifically in the transport of lipid-modified proteins, such as phosphodiesterase 6 (Pde6), to the photoreceptor outer segment. The purpose of this research is to identify the role for Arl3 in rod photoreceptor cell protein trafficking.
Transgenic mice expressing a tagged-dominant active form of mouse Arl3 (DA-Arl3) under a rhodopsin (rod-specific) promoter was compared to transgenic negative littermate controls or transgenic Arl3-wildtype (WT-Arl3) for experiments conducted in this study. Electroretinography (ERG) was used to determine rod and cone photoreceptor cell function (N=4). Immunocytochemistry and western blot were utilized for protein localization and levels, respectively (N=4). Finally, co-immunoprecitpation followed by mass spectrometry was used to determine the in vivo interacting partners of Arl3.
Dominant active (DA)-Arl3 animals exhibited extensive rod cell death after post natal day 30 (P30) and degeneration was complete by P70. At P30 before the onset of cell death, rod responses were significantly reduced. Furthermore, trafficking or stability of the lipidated proteins Pde6, rod transducin, and rhodopsin kinase (Grk1) were defective in DA-Arl3 retinae. More specifically, immunolocalization showed accumulation of assembled Pde6 subunits and eventually rod transducin on vesicular structures within the inner segment while Grk1 was depleted in rod OSs. In addition, rod Pde6 and Grk1 were greatly reduced at P30 prior to degeneration in DA-Arl3 animals. Finally, co-immunoprecipitation of transgenic retinal isolates and western blot showed specific interaction of Pde6δ with the DA-Arl3 and not Arl3-WT in vivo.
This study establishes the need for GTPase activity of Arl3 in rod photoreceptor cell function and survival. Loss of appropriate Arl3 regulation leads to defective trafficking of essential lipidated proteins to the OS. Specifically, the stability of Grk1 and trafficking of Pde6 is severely affected prior to photoreceptor degeneration.
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