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Joshua Sammons, Alecia Gross; Monitoring Kinetic Changes of Proper and Improper Rhodopsin Transport ex vivo. Invest. Ophthalmol. Vis. Sci. 2013;54(15):701.
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© ARVO (1962-2015); The Authors (2016-present)
We have generated knock-in mice expressing human rhodopsin fused to photoactivatable green fluorescent protein (rho-paGFP) with the C-terminal targeting epitope appended to the C-terminus of the fusion protein (rho-paGFP-1D4) as a real-time probe of rhodopsin transport and mediator of proper rod outer segment formation. Rho-paGFP-1D4 mice were bred to mice expressing rhodopsin (WT) or the rhodopsin C-terminal truncation autosomal dominant retinitis pigmentosa mutant, Q344ter. We have characterized the fusion protein, monitored localization under different environments, and performed real time ex vivo trafficking studies.
To test for proper folding, rho-paGFP-1D4 was expressed in COS cells, reconstituted, solubilized, and subjected to UV/Vis spectroscopy. Activation of the G-protein transducin was tested by uptake of GTPγ35S whereas rhodopsin phosphorylation was tested for by using ATPγ32P. Polarized mouse Inner Medullary Collecting Duct (IMCD) cells were transfected with rho-paGFP-1D4 cDNA to observe in vitro localization relative to a primary cilium. Transgenic Xenopus laevis were created expressing rho-paGFP-1D4 under the Xenopus opsin promoter to examine in vivo localization in transgenic animals and knock-in mice were generated expressing rho-paGFP-1D4 to monitor in vivo localization and real time rhodopsin transport.
Rho-paGFP-1D4 folds, activates transducin, and is phosphorylated similarly to WT rhodopsin. Rho-paGFP-1D4 localizes similarly to WT rhodopsin in vitro and in vivo unless the rhodopsin trafficking mutant Q344ter is expressed in the same system. This has allowed us to monitor the differences in proper and improper transport of rhodopsin in live mouse retinas.
Rho-paGFP-1D4 functions and localizes similarly to WT, despite the addition of 258 amino acids to the C-terminus. As such, it is being used as a real time probe of rhodopsin transport when expressed in retinas containing wild type properly-localizing rhodopsin and the mislocalizing mutant Q344ter.
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