A comprehensive analysis of sequence variants and putative disease-causing mutations in photoreceptor-specific nuclear receptor NR2E3

Abstract

Purpose: The photoreceptor-specific orphan nuclear receptor NR2E3 is a key regulator of transcriptional events during photoreceptor differentiation in mammalian retina. Mutations in NR2E3 are associated with enhanced S-cone syndrome and related retinal phenotypes that reveal characteristic excess of S-cone function. This study was undertaken to determine biochemical as well as functional consequences of reported sequence variants and disease-causing mutations in NR2E3.

Methods: Twenty-five different mutations in the wild-type NR2E3 expression construct were generated by site-directed mutagenesis and performed nuclear localization, gel-shift, rhodopsin promoter activity assays, and co-immunoprecipitation in cultured mammalian cells.

Results: Of the 25 mutant proteins, 15 mislocalize at least partially to the cytoplasm. Eight of the nine changes in the DNA-binding domain (DBD) and 12 of the 14 mutations in the ligand-binding domain (LBD) of NR2E3 exhibited reduced DNA-binding and transcriptional activation of the rhodopsin promoter. Moreover, these mutations dramatically altered the interaction of NR2E3 with NRL as well as with CRX. Two NR2E3 variants between DBD and LBD showed no effect on any biochemical or functional parameter tested.

Conclusions: These data provide a better understanding of sequence variants, validate disease-causing mutations, and demonstrate the significance of DBD and LBD in mediating NR2E3 function. These studies contribute to molecular mechanisms underlying retinal phenotypes caused by NR2E3 mutations.

Figure 1

A schematic diagram of the human NR2E3 protein. A: Arrows indicate altered NR2E3 residues. Abbreviations: DNA binding domain (DBD), ligand binding domain (LBD), nuclear localization signal (NLS; NM_014249.2). B: NR2E3 protein sequence aligned to orthologs. By using ClustalW, we were able to align the sequence of human NR2E3 protein with those of chimp, rhesus, cow, mouse, opossum, chicken, frog, fugu, zebrafish, tetraodon, honeybee, mosquito, fruitfly, and roundworm. Amino acid residues conserved in all orthologs are indicated by asterisk, and lower identity or similarity is shown using either colon or a dot. Shadows indicate NR2E3 mutations analyzed in this report.

Figure 2

Subcellular localization of WT and mutant NR2E3 proteins in COS-1 cells. COS-1 cells expressing WT or mutant NR2E3 proteins were incubated with anti-Xpress antibody (anti-Xp) and visualized using anti-mouse IgG-Alexa 488 antibody (green). Central panels show nuclei labeled with bisbenzimide (blue). The bottom panels display merged images. Scale bar represents 50 μm.

Figure 3

Effect of NR2E3 mutations on DNA binding and transactivation. A: EMSAs were performed using the [³²P]-labeled Kni x2 probe with untransfected (UnTR) COS-1 cells or WT NR2E3 expressing cell extracts. Specificity of DNA binding is demonstrated by competition with unlabeled Kni x2 oligonucleotide (50×) and nonspecific (NS) oligonucleotide (50×). The arrow indicates the position of a specific DNA–protein complex between NR2E3 and Kni x2 oligonucleotide. B: Binding of mutant NR2E3 proteins to the labeled Kni x2 oligonucleotide was examined by EMSA. Mutant NR2E3 protein amount in cell extracts was normalized to WT-NR2E3 by immunoblot analysis. C: NR2E3 expression constructs were cotransfected into HEK293 cells with bovine rhodopsin-130 luciferase reporter plasmid and with NRL and CRX expression constructs. Fold change is relative to the empty expression vector control. Error bars indicate standard error of mean (SE). D: Luciferase assays were performed after co-transfection of mutant NR2E3 construct with the NRL and/or CRX expression constructs. ANOVA with a post hoc test were performed on each sample compared to WT NR2E3. Significant differences of p<0.05, and p<0.01 are shown as * and **, respectively. Error bars correspond to SEM.

Figure 4

Interaction of WT and mutant NR2E3 proteins with NRL or CRX. A: Xpress-tagged WT or mutant NR2E3 expression plasmids were cotransfected into COS-1 cells with V5-tagged NRL expression construct. After immunoprecipitation with goat anti-V5 antibody, NR2E3 protein was detected with mouse anti-Xpress antibody. B: NR2E3 expression constructs were cotransfected into COS-1 cells with V5-tagged CRX expression construct. NR2E3 was visualized with mouse anti-Xpress antibody. In lower panels, the intensity of the WT NR2E3 immunoreactive band (normalized to input NR2E3) was compared with mutant NR2E3 proteins. ANOVA with a post hoc test were performed on each sample compared to WT NR2E3. Significant differences of p<0.05, and p<0.01 are shown as * and **, respectively.