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U Wolfrum, A Pulvermüller, A Gießl, M Heck, A Schmitt, OP Ernst, K Hofman; Ca2+-Induced Assembly of Centrin/Transducin Complex in Mammalian Photoreceptors . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1958.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Vertebrate photoreceptor cells consist of morphological and functional distinct cellular compartments. The visual G-protein transducin migrates between the inner segment and outer segment in a strict light dependent manner. Here we analyzed the role of the EF-hand Ca2+-binding protein centrin1 in the molecular mechanism of the exchange of transducin between both segments via the connecting cilium. Methods: Immunofluorescence and immunoelectron microscopy, Western-blot overlay assays, immunoprecipitation, size exclusion chromatography, and light scattering binding assays were applied. Results: We demonstrated that transducin forms a Ca2+-dependent complex with the cytoskeletal protein centrin1. Immunoelectron microscopy revealed that transducin and centrin1 co-localize in a specific domain of the photoreceptor connecting cilium. We showed by co-immunoprecipitation, overlay assays, size exclusion chromatography, and kinetic light scattering experiments that Ca2+-activated centrin1 binds with high affinity and specificity to transducin. Furthermore, our set of experiments revealed that the assembly of the centrin/G-protein complex is mediated by the b-subunit of the heterotrimeric complex of transducin. Conclusion: Our data indicate that the exchange of transducin through the photoreceptor cilium is modulated by light-induced changes of the intracellular Ca2+-concentration via the assembly of the centrin/transducin complex. The present Ca2+-dependent assembly of the visual G-protein with centrin is a novel aspect of the supply of signaling proteins in photoreceptor cells, and a potential link between molecular translocations and signal transduction in general.
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