June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Interactions Between Wildtype Rhodopsin and Misfolded Mutant Rhodopsin
Author Affiliations & Notes
  • Megan Gragg
    Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH
    Molecular Medicine, Case Western Reserve University, Cleveland, OH
  • Paul Shin-Hyun Park
    Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH
    Molecular Medicine, Case Western Reserve University, Cleveland, OH
  • Footnotes
    Commercial Relationships Megan Gragg, None; Paul Park, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4657. doi:
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      Megan Gragg, Paul Shin-Hyun Park; Interactions Between Wildtype Rhodopsin and Misfolded Mutant Rhodopsin. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4657.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: Over 100 clinically identified rhodopsin mutations cause autosomal dominant retinitis pigmentosa (adRP). Many of these mutations cause misfolding of the receptor. In these cases, physical interactions between wildtype (WT) rhodopsin and the misfolded mutant rhodopsin may explain the autosomal dominant phenotype. To test this possibility, we examined the interactions between WT rhodopsin and the G188R mutant, a severely misfolded clinically identified adRP rhodopsin mutant.

Methods: To investigate rhodopsin oligomerization or aggregation, we used Förster Resonance Energy Transfer (FRET) to monitor the interactions between fluorescently tagged rhodopsins expressed in tansfected HEK293 cells.

Results: Tagged WT rhodopsins exhibited FRET in HEK293 cells, indicating that rhodopsin oligomerizes in cell membranes. The FRET signal was eliminated by treatment with n-dodecyl β-D-maltoside (DDM), suggesting that mild detergents disrupt rhodopsin oligomers. Tagged G188R mutants also exhibited FRET. However, the FRET signal was not completely disrupted by DDM. Therefore G188R rhodopsins form different complexes than WT rhodopsins. Confocal microscopy of G188R rhodopsins showed that the mutant is mislocalized and retained intracellularly. The FRET between tagged G188R mutants may therefore be indicative of misfolded aggregates. When mutant and WT rhodopsins were coexpressed in HEK293 cells, a FRET signal was also observed that could not be completely disrupted by DDM. The FRET signal, however, was significantly lower than WT or G188R rhodopsins expressed alone.

Conclusions: The detergent sensitivity of the FRET signal between tagged rhodopsins can differentiate between functional oligomers and misfolded aggregates. When WT and G188R rhodopsins are coexpressed, as occurs in some adRP patients, misfolded aggregates containing both receptor forms were detected. The lower FRET signal from these heterocomplexes, however, indicates that heterocomplexes are less likely to form compared to homocomplexes of each receptor kind. Misfolding mutants therefore may not significantly impede the synthesis and trafficking of properly folded rhodopsins. Thus, strategies increasing the expression of properly folded rhodopsin molecules, such as gene therapy approaches, may be a viable option for treating patients with misfolded rhodopsin mutants.


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