Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.

BACKGROUND: NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD). The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2)). NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients

Methods to Investigate the Nucleocytoplasmic Shuttling Properties of β-Arrestins

International audienc

A biosensor to monitor dynamic regulation and function of tumour suppressor PTEN in living cells

Tumour suppressor ​PTEN is a phosphatase that negatively regulates the PI3K/AKT pathway. The ability to directly monitor ​PTEN conformation and function in a rapid, sensitive manner is a key step towards developing anti-cancer drugs aimed at enhancing or restoring ​PTEN-dependent pathways. Here we developed an intramolecular bioluminescence resonance energy transfer (BRET)-based biosensor, capable of detecting signal-dependent ​PTEN conformational changes in live cells. The biosensor retains intrinsic properties of ​PTEN, enabling structure–function and kinetic analyses. BRET shifts, indicating conformational change, were detected following mutations that disrupt intramolecular ​PTEN interactions, promoting plasma membrane targeting and also following physiological ​PTEN activation. Using the biosensor as a reporter, we uncovered ​PTEN activation by several G protein-coupled receptors, previously unknown as ​PTEN regulators. Trastuzumab, used to treat ​ERBB2-overexpressing breast cancers also elicited activation-associated ​PTEN conformational rearrangement. We propose the biosensor can be used to identify pathways regulating ​PTEN or molecules that enhance its anti-tumour activity