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Igor V. Peshenko, Elena V. Olshevskaya, Seifollah Azadi, Laurie S. Molday, Robert Molday, Alexander M. Dizhoor; RD3 As A New Modulator Of Retinal Photoreceptor Guanylyl Cyclase (RetGC): A Strong Inhibition Of RetGC Activity And The Effects Of Retinal Disease-linked Mutations. Invest. Ophthalmol. Vis. Sci. 2011;52(14):44.
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It was recently found that RD3, a 23-kDa product of RD3 (Clorf36) gene, associates with RetGC and is crucial for the expression of RetGC isozymes in photoreceptors . The purpose of this study was to evaluate the functional role of RD3 in regulation of the RetGC activity and probe for the effects of several mutations in human RD3  reported in patients with visual abnormalities.
Mouse and human RD3 were expressed in E. coli or in HEK293 cells, and the recombinant RD3 was then tested in RetGC activity assay or probed for co-localization and co-immunoprecipitation with RetGC1 using methodology previously described in detail for RetGC interactions in vitro and in living cells [1,3].
Recombinant RD3 purified from E. coli or expressed in HEK293 inhibited two native RetGC isozymes present in mouse photoreceptor outer segments, and the recombinant RetGC1 expressed in HEK293 cells. There were two distinct components in RetGC inhibition by RD3 - the suppression of the basal activity observed in the GCAP1/GCAP2 double knockout rods and a competitive suppression of GCAP-stimulated activity of the native and the recombinant RetGC. A GFP-tagged and non-tagged RD3 compartmentalized with the wild type RetGC1 in living HEK293 cells, similarly to GCAP1-GFP  and some of the mutations in RetGC1 that blocked GCAP1 binding also suppressed RD3 binding to RetGC1. However, a mutation in the cyclase C-terminal portion that did not alter the RetGC1 association with GCAP1 abolished RD3 binding. We have tested the function of six RD3 variants described in human patients . The product of an altered RD3 gene splicing derived from mutation in one LCA12-affected family displayed a complete loss of the inhibitory activity.
RD3 acts as a strong negative regulator and a competitive inhibitor of the GCAP-dependent stimulation of native and recombinant RetGC. This previously unappreciated step in RetGC regulation is potentially involved in maturation of the functional outer segments. Azadi et al. (2010) Proc Natl Acad Sci USA, in press;  Friedman, et al. (2006) Am J Hum Genet 79:1059;  Peshenko et al. (2008) J Biol Chem 283:21747.
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