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M.A. Swiatek–De Lange, M. Beer, M. Ueffing; Analysis of Light–Dependent Dynamics of Protein Complexses in the Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3734. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
A large group of genetically inherited blinding diseases is associated with mutations in genes expressed in photoreceptors. In the light–percepting molecule rhodopsin, more than a hundred different mutations may cause the retinal degenerative disease retinitis pigmentosa. Such mutations not only result in impairment of rhodopsin function but also disruption of functional protein networks downstream of this GPCR and consequently to most severe physiological consequences. We have combined bioanalytical and molecular methods to describe functional rhodopsin interactions and to study their light–dependent dynamics.
Photoreceptor outer segments were isolated from light and dark adapted porcine retinae. OS were divided in a membranous and a cytosolic fraction, and native protein complexes were isolated by means on sucrose density gradient centrifugation or BN–PAGE. The light dependent assembly and dynamics of protein complexes were further analyzed using affinity methods such as immunoprecipitation or active pull–down. Individual proteins were analysed by 1– and 2DE–analyses in combination with MALDI–TOF mass spectrometry and immunoblot methods.
The proteomic analysis of sucrose–density gradient isolated membrane–bound protein complexes from photoreceptor outer segments allowed us to identify several candidates as novel rhodopsin interaction partners. These candidates include small GTPases from the Rho and Rab families. Additionally, we have identified the CRMP–2 protein as a novel Rac and Rho–interaction partner in photoreceptors. Detailed analyses of membrane–associated, Rac–GTP–bound protein complexes allowed us to identify 25 further proteins.
We have detected protein interactions involved in phototransduction and photoreceptor development. Moreover, we have identified protein interactions and signaling networks in photoreceptors that are conserved between different neuronal cell types, thus likely to play a pivotal role in cell physiology. The identified protein complexes show a light and dark regulated dynamic. RhoA and Rac1 participate in protein transport and structural organisation of cells, while the CRMP family is one of main regulators of polarity development in neuronal cells. This links light perception through rhodopsin with signaling networks involved in structural integrity and polarity of photoreceptors and suggests an interdependence of visual perception and proper cellular structure.
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