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A. Kanda, J.S. Friedman, K.M. Nishiguchi, T.P. Dryja, A. Swaroop; Retinopathy Mutations Alter Phosphorylation and Transcriptional Regulatory Function of the Basic Motif–Leucine Zipper Protein Nrl . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4899.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the human NRL gene are associated with autosomal dominant retinitis pigmentosa and clumped pigmentary retinal degeneration. This study was undertaken to determine the effect of various mutations in NRL on its function, with a goal to delineate molecular mechanism(s) that underlie disease pathogenesis.
Mutations in NRL were produced by site–directed mutagenesis of the pcDNA4c expression construct. Wild type (WT) NRL and mutants were transfected into established cell cultures to examine protein expression, phosphorylation, and nuclear localization. We used gel–shift and rhodopsin promoter activity assays to compare the DNA–binding and transcription activation function of the WT and mutant NRL proteins.
We generated 17 missense and frame shift mutations (N14K, S50T, S50L, S50P, P51T, P51L, P51S, E63K, P67S, A76V, G122E, H125Q, L160P, L160fs, R218fs, S225N, and L235F) in the NRL protein. As expected, WT NRL exhibited multiple phosphorylated isoforms by immunoblot analysis and localized to the nucleus. Twelve of the NRL mutants showed an immunoblot protein profile different from the WT. Alkaline phosphatase treatment of extracts from NRL–transfected cells abolished all but one NRL isoform, similar to that corresponding to S50 NRL mutant. Only one mutant (L160fs) was mislocalized to the cytoplasm. Many NRL mutants exhibited altered transcriptional activation and DNA–binding properties.
Our results suggest that many mutants affect the function and stability of the NRL protein. These findings provide a better understanding of the cellular defects caused by NRL mutations and will help design further experiments to delineate the mechanisms of disease pathogenesis.
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