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Elvir Becirovic, Sybille Böhm, Lisa Maria Riedmayr, Andreas Gießl, Christian Schön, Stylianos Michalakis, Martin Biel; Peripherin-2 and Rom-1 Have Opposing Effects on Rod Outer Segment Targeting of Two Misfolded Peripherin-2 Mutants in Rods. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3564.
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© ARVO (1962-2015); The Authors (2016-present)
The tetraspanins peripherin-2 and Rom-1 can form homo- and heteromeric complexes in photoreceptor outer segments (OS). Mutations in peripherin-2, but not in Rom-1, lead to autosomal dominant retinitis pigmentosa (adRP) and the vast majority of these mutations is located in the D2 loop domain. How different complexes assembled by various combinations of wild type (perWT), mutant peripherin-2 (perMT) or Rom-1, which can be formed in heterozygous patients, contribute to the pathophysiology of adRP remained elusive so far. Here, focusing on the two adRP-linked peripherin-2 D2 loop mutants, perP210L and perC214S, we analyzed the binding characteristics, subunit assembly, subcellular localization, and rod OS targeting of perWT-perMT, and Rom-1-perMT complexes in HEK293 cells and in murine rod photoreceptors.
Binding characteristics were analyzed by co-immunoprecipitation, protease accessibility assay, peptide competition assay, and quantitative FRET measurements. Subunit assembly of the single complexes was addressed by sucrose density gradient centrifugation. Rod OS targeting and subcellular localization of the single complexes was examined on retinal slices from wild type mice injected with AAV-vectors expressing different combinations of perWT, perMT, and Rom-1.
perP210L and perC214S are misfolded and lead to decreased binding to perWT and Rom-1 due to impaired binding kinetics. The perWT-perMT binding was stronger affected when compared to the Rom-1-perMT combination. In contrast to perP210L, perC214S was mislocalized throughout the rod OS in presence of transgenic perWT. Both mutants preferentially formed non-covalent dimers with perWT and Rom-1. However, only perWT-perMT complexes could be targeted to rod OS.
We show that perWT-perMT dimers can be targeted to rod OS. Moreover, we unravel unexpected opposing roles of perWT and Rom-1 in the pathophysiology of peripherin-2 mutants. We postulate a novel function of Rom-1 in rods with high relevance for the design of future gene-based treatments of peripherin-2-linked adRP.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Rod OS targeting of perMT dimeric complexes. The rod photoreceptor localization of different perMT-containing dimers (perWT-perMT, Rom-1-perMT and perMT only) is symbolized by a dashed rectangle. The position of the D2 loop is highlighted in red and the D2 loop misfolding caused by perMT is symbolized by an incision.
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