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

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 Previous unpublished work identified peripherin-2 as a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.