Molecular characterization of the interaction between peripherin-2 and opsins in rod and cone photoreceptors

Episode von Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 06/06 von Ludwig-Maximilians-Universität München · 24. Februar 2016

The tetraspanin peripherin-2 is a glyco-membrane protein exclusively expressed in the outer segments of rod and cone photoreceptors. Mutations in peripherin-2 are associated with retinal disorders characterized by Degeneration of rod or cone cells. Previous unpublished work identified peripherin-2 as a potential novel part of the protein complex comprising the B-subunit of the cyclic nucleotide-gated channel (CNGB1a and the light detector rhodopsin.

In the first part of this study, using a combination of protein biochemical and FRET approaches in transfected HEK293 cells and in virally transduced murine rod outer segments, it could be demonstrated that peripherin-2 simultaneously binds to both, CNGB1a and rhodopsin. The interaction between peripherin-2 and rhodopsin was not described in previous studies. The binding domain mediating the peripherin-2/rhodopsin interaction could be narrowed down to the fourth transmembrane domain (TM4) of peripherin-2. Finally, the data revealed that the G266D point mutation in TM4 of peripherin-2 that is linked to a rod degenerative disease selectively disrupts the peripherin-2/rhodopsin interaction.

To analyze if peripherin-2 also binds to cone opsins in the second part of this study, a similar experimental approach was conducted as used for the investigation of the peripherin-2/rhodopsin interaction. In this context, it was unveiled that peripherin-2 binds to both, short wavelength-and medium wavelength-sensitive cone opsin (S-opsin and M-opsin, respectively) in transfected HEK293 cells and in outer segments of transduced murine cones. Co-immunoprecipitation and quantitative FRET analysis revealed that binding of peripherin-2 to M-opsin was stronger than the peripherin-2/S-opsin interaction. This result was supported by transmission electron microscopy studies using gold particles coupled to

opsin- and peripherin-2-specific antibodies. Finally, quantitative FRET analysis in transfected HEK293 cells and in transduced cone outer segments demonstrated that the V268I Point mutation in TM4 of peripherin-2 associated with a degenerative cone disease significantly attenuates the peripherin-2/M-opsin interaction.

Taken together, this study provides a proof-of-principle for FRET-based analysis of protein-protein interactions in the outer segments of rod and cone photoreceptors. This approach led to the identification of hitherto unknown Protein complexes between peripherin-2 and opsins suggesting a novel physiological role of peripherin-2 in rods and cones. Finally, Analysis of disease-linked point mutations unveiled the molecular determinants of the peripherin-2/opsin interaction. These results might contribute to understanding the differential penetrance of certain point mutations in rods and cones.

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