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W Clay Smith, Susan N Bolch, Donald Dugger, Reshmi M Mathew, Gina M D'Ambrosio, J Hugh McDowell; The Complex of Proteins Associated with BBS5 in Photoreceptor Outer Segments. Invest. Ophthalmol. Vis. Sci. 2014;55(13):417.
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We have previously shown that arrestin1 and arrestin4 associate with BBS5 in a light-dependent manner that is regulated by the phosphorylation of BBS5 by protein kinase C. The goal of this current project is to identify other proteins that are associated with this complex of BBS5 and arrestin in rod outer segments.
BBS5 was isolated from rod outer segments using immunoprecipitation with an anti-BBS5 monoclonal antibody cross-linked to Protein G-coated magnetic beads. The isolated complex of proteins was identified by tandem mass spectrometry. Non-specific interactions were discarded based on pull downs with an irrelevant antibody. Potential interaction partners were validated by immunohistochemistry and direct interaction studies using reconstitution assays.
The predominant proteins associated with BBS5 in outer segments were elements of the cytoskeleton, including both tubulin and actin. Also identified in this complex were arrestin1, heterotrimeric G-protein subunits, cGMP-phosphodiesterase holoenzyme, and several chaperone/heat-shock proteins.
Our findings indicate that the BBS5-arrestin interaction in photoreceptor outer segments occurs as part of a larger macromolecular complex associated with the cytoskeleton. This complex includes scaffolding, signaling, and chaperone molecules, suggestive of a functionally organized signaling complex.
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