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K.A. Martemyanov, G. Keresztes, C.M. Krispel, P. Lishko, N. Calero, J. Lem, C.–K.J. Chen, M.E. Burns, S. Heller, V.Y. Arshavsky; The interaction between the DEP domain of RGS9 and R9AP determines subcellular localization and stability of the RGS9–Gß5 GTPase activating complex in photoreceptors . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2211.
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Purpose: Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the RGS9–Gß5 protein complex. A transmembrane protein R9AP anchors RGS9–Gß5 to photoreceptor disc membranes by interacting with the DEP domain of RGS9. Our goal was to identify the functional consequences of this interaction in vivo. Methods: We generated two genetically modified mice: a transgenic mouse where the RGS9 mutant lacking the DEP domain was expressed in the rods of RGS9 knockout animals (DEP–less) and another mouse where the R9AP gene was knocked out (R9AP KO). The levels of mRNA and proteins were analyzed using Northern and Western blots respectively. The distribution of proteins in the rod photoreceptor compartments was studied using a combination of immunohistochemical methods and the serial tangential sectioning/Western blotting technique. The photoresponses from transgenic rods were recorded using the suction electrode technique. Results: We have found that RGS9 was completely absent from the retinas of R9AP KO mice, whereas the level of RGS9 mRNA was not affected by the R9AP knockout. On the other hand, the RGS9 mutant protein lacking the DEP domain was expressed in the normal amount but was not delivered to rod outer segments. Electrophysiological studies of the mouse photoreceptors revealed that the light responses of both R9AP KO and DEP–less rods were very slow to recover and indistinguishable from those of rods of the RGS9 knockout mice. Conclusions: We conclude that both the stability of RGS9 in the rod cell and its delivery to the rod outer segment is determined by the interaction between the DEP domain of RGS9 and R9AP.
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