Wild-type opsin does not aggregate with a misfolded opsin mutant

Abstract

Rhodopsin is the light receptor in photoreceptor cells that plays a central role in phototransduction and photoreceptor cell health. Mutations in rhodopsin are the leading cause of autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease. A majority of mutations in rhodopsin cause misfolding and aggregation of the apoprotein opsin. The pathogenesis of adRP caused by misfolded opsin is unclear. It has been proposed that physical interactions between wild-type opsin and misfolded opsin mutants may underlie the autosomal dominant phenotype. To test whether or not wild-type opsin can form a complex with misfolded opsin mutants, we examined the interactions between wild-type opsin and opsin with a G188R mutation, a clinically identified mutation causing adRP. Förster resonance energy transfer (FRET) was utilized to monitor the interactions between fluorescently tagged opsins expressed in live cells. The FRET assay employed was able to discriminate between properly folded opsin oligomers and misfolded opsin aggregates. Wild-type opsin predominantly formed oligomers and only a minor population formed aggregates. Conversely, the G188R opsin mutant predominantly formed aggregates. When wild-type opsin and G188R opsin were coexpressed in cells, properly folded wild-type opsin did not aggregate with G188R opsin and was trafficked normally to the plasma membrane. Thus, the autosomal dominant phenotype in adRP caused by misfolded opsin mutants is not predicted to arise from physical interactions between wild-type opsin and misfolded opsin mutants.

Effect of dietary docosahexaenoic acid on rhodopsin content and packing in photoreceptor cell membranes

“…DNA constructs for the expression of opsin tagged with either the yellow fluorescent protein variant SYFP2 or mTurquoise (mTq) were described previously [50, 51]. Procedures for the transfection of HEK293 cells and FRET measurements were also described previously [50, 51]. Briefly, HEK293 cells were cotransfected with different ratios of DNA coding for opsin tagged with SYFP2 or mTq using Lipofectamine 2000 (Invitrogen, Carlsbad, CA).…”

“…(B) FRET curves were generated by measuring the FRET efficiency at different ratios of SYFP- and mTq-tagged opsin (A:D). Plotted is the DM-sensitive FRET efficiency, which corresponds to opsin oligomers [50].…”

Quaternary structures of opsin in live cells revealed by FRET spectrometry

“…The vectors pmRho-SYFP2-1D4 and pmRho-mTq-1D4 were generated as described previously [46, 47]. The full CMV promoter in these vectors was replaced by a truncated CMV promoter to decrease the expression level of rhodopsin.…”

“…To answer fundamental questions about the oligomerization of rhodopsin in live cells, we have tagged the apoprotein opsin with fluorescent proteins and applied a novel spectrometric method – ‘Förster resonance energy transfer (FRET) spectrometry’ [39]. Oligomers of rhodopsin or opsin have previously been detected in transfected cells and lipid vesicles using standard FRET methods [29, 40-47]. Bulk approaches, however, are unable to distinguish between different arrangements and sizes of oligomers formed by the receptors, unless structural information is already known from separate experiments, which is then inserted into suitable theoretical models for FRET in oligomeric complexes.…”

The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy

“…For example, co-expression of WT and P23H rhodopsin in vitro resulted in the formation of inclusions containing the WT protein (Mendes and Cheetham, 2008; Saliba et al, 2002), and co-aggregation of the mutant and WT protein with enhanced proteasome-mediated degradation of the WT protein (Rajan and Kopito, 2005). This property might not be shared by all rod opsin mutants, however, as the G188R mutant also aggregates, but has only small effects on the WT protein and they do not co-aggregate (Gragg et al, 2016). Therefore, mutations in rhodopsin that cause misfolding can lead to enhanced mutant, and WT, rhodopsin degradation; potential ER stress; aggregation and alter photoreceptor homeostasis (Figure 3).…”