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Y. Cao, K. A. Martemyanov; Transgenic Expression of R7BP in Rod Photoreceptors of R9AP Knockouts Reverses RGS9-1/Gβ5 Loss and Disrupts Its Targeting to the Outer Segments. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3427.
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© ARVO (1962-2015); The Authors (2016-present)
Recovery of vertebrate photoreceptors from light excitation requires an action of GTPase-activating complex RGS9-1/Gβ5/R9AP. In this complex, R9AP subunit controls membrane association and expression level of the entire RGS9-1/Gβ5/R9AP. Knockout of R9AP in mice results in elimination of RGS9-1 and Gβ5 and leads to slow photoresponse recovery. Recently, a new R9AP-like protein called R7 Binding Protein (R7BP) was found. However, unlike R9AP, R7BP is not expressed in the photoreceptors and thus is not found in complex with RGS9-1. Instead, it is expressed in downstream bipolar cells where it interacts with other RGS9-1 like proteins. The goal of this study was to gain an insight into the role of the R7BP/R9AP subunit diversity in mediating RGS9-1/Gβ5 stabilization and targeting by analyzing the consequences of transgenic substitution of R7BP for R9AP.
Rho-R7BP transgenic mice were generated by pronuclear injections with a construct where R7BP cDNA was placed under the control of rhodopsin promoter. Transgenic animals were backcrossed with R9AP knockout mice in order to reduce and/or eliminate R9AP expression. Expression and localization of proteins in transgenic mice were determined by immunohistochemistry and Western blotting. Interactions between RGS9-1 and its binding partners were investigated by immunoprecipitation.
We were able to achieve a substantial overexpression of R7BP in the photoreceptors which did not affect retina morphology. Expression of R7BP in R9AP heterozygous mice resulted in RGS9-1/Gβ5 overexpression. Elimination of RGS9-1/Gβ5 observed in R9AP knockout mice was prevented in Rho-R7BP transgenic mice. Co-immunoprecipitation assays showed that R7BP effectively competed with R9AP for forming complex with RGS9-1. Transgenic R7BP was found to be localized on the plasma membrane and synapses of the rods and excluded from the outer segments. Furthermore, expression of R7BP in transgenic mice, de-localized RGS9-1/Gβ5 complex from the outer segments of photoreceptors.
RGS9-1/Gβ5 complex in photoreceptors relies on its membrane anchor subunit containing targeting signals for reaching its intracellular destination. R7BP much like R9AP can protect RGS9-1 from degradation; however unlike R9AP it lacks targeting signals for the delivery of the complex to the outer segments of photoreceptors.
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