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Adiv A Johnson, Yong S Lee, Lihua Y Marmorstein, Alan D Marmorstein; Disease-causing mutations associated with four bestrophinopathies exhibit disparate effects on the localization, but not the oligomerization, of Bestrophin-1. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2971.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in BEST1, encoding Bestrophin-1 (Best1), cause five clinically distinct retinal degenerative diseases, including adult-onset vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), retinitis pigmentosa (RP), and autosomal dominant vitreoretinochoroidopathy (ADVIRC). Best1 is localized to the basolateral plasma membrane of the RPE, where it forms homo-oligomeric anion channels and regulates intracellular Ca2+ signaling. To gain insight into the pathogenesis of these diseases, we screened 28 Best1 mutants associated with AVMD, ARB, RP, and ADVIRC for defects in localization and oligomerization.
Using confocal microscopy and immunofluorescence, we assessed Best1 localization after expressing YFP-tagged Best1 in highly confluent, polarized MDCK cells via adenovirus-mediated gene transfer. Oligomerization was evaluated by live-cell, confocal fluorescence resonance energy transfer (FRET) between WT Best1-CFP- and WT or mutant Best1-YFP. FRET data were confirmed by reciprocal co-immunoprecipitation as well as co-localization experiments between WT Best1-c-myc- and WT or mutant Best1-YFP.
Many, but not all, AVMD- and ARB-associated mutants were mislocalized to intracellular compartments. In contrast, all RP- and ADVIRC-associated mutants were properly localized to the basolateral plasma membrane. When mislocalized AVMD and ARB mutants were co-expressed with WT Best1, all mutants predominantly co-localized with WT Best1 in intracellular compartments. All 28 mutants exhibited comparable FRET efficiencies to and reciprocally co-immunoprecipitated with WT Best1, indicative of unimpaired oligomerization.
All 28 mutants associated with AVMD, ARB, RP, and ADVIRC formed oligomers with WT Best1, suggesting that oligomeric defects are not associated with the bestrophinopathies. Our data also show that, although other pathogenic mechanisms besides mislocalization are involved for RP and ADVIRC, mislocalization alone is insufficient to distinguish between different disease phenotypes. Moreover, several recessive ARB mutants mislocalized WT Best1 when co-expressed together. That a recessive mutant would mislocalize WT Best1, yet not be pathogenic, indicates that mislocalization on its own cannot cause disease, and that the absence of Best1 at the plasma membrane is well tolerated.
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