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Igor Peshenko, Elena Olshevskaya, Alexander Dizhoor; Differential Analysis of Retinal Guanylyl Cyclase GUCY2D (RetGC1) Activation and Binding with GCAP1 and RD3 Using In Vitro Activity Assay and Cell-based Binding Assay. Invest. Ophthalmol. Vis. Sci. 2013;54(15):704.
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Regulation of retinal membrane guanylyl cyclase, GUCY2D (RetGC1), plays important role in rod and cone physiology and becomes the cause of retinal diseases when altered by mutations in GUCY2D, GCAPs and RD3. With a variety of mutations causing different forms of congenital blindness, it becomes important to distinguish between the two general mechanisms through which disease-causing mutations affect GUCY2D activity and/or regulation - suppressing the catalytic activity of the enzyme versus those affecting cyclase binding with its regulatory proteins.
Point mutations affecting GUCY2D activity and linked to Leber Congenital Amaurosis 1 (LCA1) [1, 2] were introduced in GUCY2D. GUCY2D was co-expressed in HEK 293 cells with fluorescently tagged GCAP1 or RD3 and their association was tested in cyto using confocal microscopy. GUCY2D activation by GCAP1 expressed in E. coli was measured in vitro.
Mutations that suppressed RetGC activation by GCAP1 produced different effects on the ability of the mutant GUCY2D to bind GCAP1-GFP in HEK 293 cells: some of them suppressed GCAP1 GFP binding while the others had no effect on the binding and evidently caused the loss of GUCY2D activity in vitro through affecting its catalytic activity per se. In some cases, they also differentially affected binding of GCAP1, a positive regulator, and of RD3, a negative regulator of the cyclase activity . Mutations in GCAP1 located in EF-hand 1 and in proximity to the interface between the two lobes of the GCAP molecule were able to completely block GCAP1 binding to guanylyl cyclase.
Comparative analysis of GUCY2D activity and its ability to bind GCAP1-GFP in a cell-based assay provides a useful tool for distinguishing between the direct suppression of GUCY2D activity versus that of GCAP1 or RD3 binding. The binding sites for RD3 and GCAP1 in RetGC1 are likely overlapped but not identical and can become differentially affected by disease-related mutations. References:  S. Jacobson et al. (2012) Hum Mol Genet., in press;  E. Stone (2007) Am J Ophthalmol 144:791-811;  I.Peshenko et al. (2011) Biochemistry 50:9511-9519
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