The loss of transcriptional inhibition by the photoreceptor-cell specific nuclear receptor (NR2E3) is not a necessary cause of enhanced S-cone syndrome

Abstract

Purpose: To investigate functional consequence on photoreceptor-cell specific nuclear receptor (NR2E3) transcriptional activity of enhanced S-cone syndrome (ESCS) mutations localized in ligand binding domain (LBD).

Methods: Point mutations were introduced into the LBD of full length and Gal4 chimeric NR2E3 receptors and transcriptional activity was investigated by using transient co-transfection assay on corresponding luciferase reporters. Expression and DNA binding properties of transfected mutant and wild-type receptors were tested by Western blotting and gel shift assay.

Results: Our analysis show that two ESCS mutations, missense mutations R385P and M407K, abolished NR2E3 repressive activity in the context of full-length and Gal4 chimeric receptors, while W234S and R311Q mutants retained their repressive activity in both assays. All mutant receptors maintained their stability and DNA binding ability.

Conclusions: These results showed that NR2E3 mutations localized in LBD induce ESCS disease without affecting inhibitory activity as recorded in vitro. This demonstrates the absence of correlation between transcriptional inhibition and ESCS phenotype. This analysis suggests that NR2E3 might have transcriptional activation properties not yet identified.

Figure 1

Transcriptional repression by NR2E3^LBD. COS-1 cells were transiently transfected with Gal4^DBD or Gal4-NR2E3^LBD expression plasmids. Transcriptional activity was measured for the different luciferase Gal4-reporter genes. A: Luciferase activity in the presence of an increasing amont of GAL-NR2E3^LBD. B: Luciferase acitity in the presence of an increasing amount of GAL-RARα^LBD. C: Luciferase activity in the presence of an increasing amount of GAL-RARα^LBD in the presence of its ligand RA. Normalized values are expressed as relative luciferase activity.

Figure 2

Transcriptional effect of NR2E3 mutations on Gal4 chimeric receptor. A: COS-1 cells were transiently transfected with 100 ng of Gal4-NR2E3^LBD wild-type and mutant expression plasmids. Transcriptional activity of a Gal4 responsive reporter gene was measured. B: HeLa cells were transiently transfected with 100 ng of Gal4-NR2E3^LBD wild-type and mutant expression plasmids, and transcriptional activity of a Gal4 responsive reporter gene was measured. Normalized values are expressed as relative luciferase activity. C: Expression of Gal4-NR2E3^LBD wild-type and mutants in COS-1 transfected cells. D: Electrophoretic mobility shift assay of Gal4 full-length, wild-type (WT), and R385P mutated Gal4-NR2E3^LBD using a Gal4 probe. Bound indicates the different shifted bands, and free denotes unbound probe.

Figure 3

Transcriptional effect of NR2E3 mutations on full-length receptor. A: COS-1 cells were transiently transfected with 100 ng of NR2E3 wild-type and mutant expression plasmids, and transcriptional activity of a NR2E3 responsive reporter gene was measured. Normalized values are expressed as relative luciferase activity. B: Expression of NR2E3 wild-type and mutants in COS-1 transfected cells. C: Electrophoretic mobility shift assay of full-length wild-type (WT) and mutated NR2E3 using a Kni x2 probe [6]. Bound indicates shifted bands, and free denotes unbound probe.

Figure 4

Expression of Gal4-NR2E3^LBD. A and B mutant proteins after transfection into COS-1 cells. The bottom panels represents β-actin loading controls. A: Western blotting against the GAL4-NR2E3 fusion proteins. B:L Western blotting against the hemaglutinin antigene (HA) tagged NR2E3 proteins.

Figure 5

Repression of Gal4 activated promoter by NR2E3 wild-type and mutant full-length proteins. COS-1 cells were transfected with various combinations of Gal4 (50 ng) and NR2E3 wild-type and mutant (100 ng) expression plasmids. Transcriptional activity of a NR2E3 responsive reporter gene was measured. Normalized values are expressed as relative luciferase activity.

Figure 6

Molecular model of NR2E3^LBD. Homology modeling of the NR2E3^LBD based on the RAR^LBD crystal structure. Highlighted are several residues that mutated in enhanced S-cone syndrome. The residue R385, shown in green, was predicted to localize in the ligand hydrophobic pocket. Mutation of W234, shown in red, was predicted to modify the ligand pocket conformation.