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U. Wolfrum, A. Gießl, A. Pulvermüller, P. Trojan, K.P. Hofmann; Centrins as potential regulators of light–induced transducin translocation . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3639.
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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 triggered by light. Our previous studies showed that the Ca2+–binding protein centrin1 (Cen 1) interacts with ß–subunit of transducin in the connecting cilium which is the only intracellular bridge between both segments. Here we analyzed the role of the 3 other centrin isoforms (Cen 2 – 4) in the intersegmental exchange of transducin via the cilium. Furthermore, the functional relevance of the phosphorylation of centrin isoforms was examined. Methods: RT–PCR, Western–blots, immunofluorescence and immunoelectron microscopy, overlay assays, immunoprecipitation, GST–pull down experiments, size exclusion chromatography, light scattering binding assays and in vitro and ex vivo phohosphorylation studies were applied. Results: RT–PCR– and Western–blot analyses demonstrate expressions of all 4 centrin isoforms in retinal photoreceptor cells of all mammals investigated. Our protein–protein interacting assays reveal that all centrin isoforms bind to ß–transducin in a strict Ca2+–dependent way. In vitro and ex vivo phosphorylation assays indicate that the centrins Cen 1, 2 and 4 are phosphorylated in a light–dependent manner. Treatments with kinase inhibitors reveal the casein–kinase II as a candidate for these light–dependent centrin phosphorylations. Conclusions: Our data strength the hypothesis that the exchange of transducin through the photoreceptor cilium is modulated by light–induced changes of the intracellular Ca2+–concentration via the assembly of complexes between transducin and centrins. In photoreceptor cells, light–dependent functions of centrins are not only regulated by Ca2+, but also by specific phosphorylation. This phosphorylation may also influence centrins affinity to transducin. In general, the assembly of the visual G–protein with centrins is a novel aspect of the supply of signaling proteins in photoreceptor cells, and a potential link between molecular translocations and signal transduction.
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