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Elif Yaman, Alaron Lewis, Galina Dvoriantchikova, Vladlen Slepak, Susan E. Brockerhoff; Signal Dependent Redistribution of Heterotrimeric G Proteins in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2011;52(14):39.
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© ARVO (1962-2015); The Authors (2016-present)
Light causes the translocation of signaling proteins between outer and inner photoreceptor compartments. In mammals, rod and cone G proteins, transducins, concentrate in the outer segments (OS) in darkness. Interestingly, upon illumination, rod transducin disperses throughout the entire cell, whereas cone transducin stays in the OS. The purpose of our study is to investigate the molecular basis of this differential localization.
We established a transgenic zebrafish, which expresses human rod Gα in cone photoreceptors. EE-tagged rod transducin Gα expression construct was generated and microinjected into freshly fertilized Danio rerio eggs. These fish were then raised to adulthood and screened for germline transmission of the transgene. The localization of endogenous rod and cone transducins and the EE-tagged human rod Gα in cones was examined under different light conditions by immunofluorescent and biochemical techniques.
Rod transducin was solubilized from the OS membranes by GTPγS and light. In contrast, cone transducin was not eluted from the membrane fraction by GTPγS but could be eluted from membranes by mSIRK, a synthetic peptide that binds to Gβ and displaces Gα. Surprisingly, we found that unlike mammalian Gβγ subunits, zebrafish rod Gβγ can be readily eluted from the membrane even in the absence of GTPγS. Another surprising finding was that both zebrafish rod and cone transducins were represented by more than one band on the western blot and interestingly, these isoforms displayed different patterns of membrane association. Finally, immunoflourescence microscopy showed that under different light conditions, localization of the EE-tagged rod Gα in cones paralleled that of the endogenous cone Gα.
Once expressed in zebrafish cones, human rod transducin Gα behaves like zebrafish cone Gα and remains localized to OS in the light and cannot be eluted from membranes with GTPγS. These findings suggest that the behavior of cone transducin Gα is controlled by cellular interaction/modifications that do not have stringent amino acid sequence determinants. We speculate that the different localization patterns and biochemical properties between zebrafish rod and cone transducin Gα are determined by differential lipidation and interaction between G protein subunits.
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