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H. Muradov, M. Haeri, K. K. Boyd, B. E. Knox, N. O. Artemyev; Expression and Characterization of Human Cone Pde6 in Xenopus Laevis Rods. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2994.
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© ARVO (1962-2015); The Authors (2016-present)
Progress toward understanding structure and function of retinal phosphodiesterase 6 (PDE6), the central enzyme in vision, is hindered by lack of the protein expression system. Transgenic Xenopus laevis expressing EGFP-fused wild-type and mutant N610S human cone PDE6C were generated to assess the utility of this approach for expression and mutational analysis of PDE6. The N610S model was selected because the corresponding mutation in mouse rod PDE6B results in relatively slow retinal degeneration.
Transgenic X. laevis expressing EGFP-PDE6C and the N610S mutant in rod photoreceptors have been produced with restriction enzyme mediated integration of the transgene under the Xenopus rhodopsin promoter. Expression and localization of EGFP-PDE6 and EGFP-PDE6CN605S in the frog retina were examined by Western blotting and EGFP-fluorescence. The fusion proteins were immunoprecipitated with anti-EGFP antibodies and examined for PDE6 activity and inhibition by the PDE6 γ-subunit (Pγ). Diffusion of EGFP-PDE6C on the disc membrane was investigated using Fluorescence Recovery After Photobleaching (FRAP).
EGFP-PDE6C was correctly targeted to the rod outer segments in the retina of transgenic tadpoles, suggesting intact transport of the protein. The levels of EGFP-PDE6C expression in individual Xenopus tadpoles were ranging from 3% to 15% of the endogenous rod PDE6 level. EGFP-PDE6C was selectively precipitated by anti-GFP antibody without concomitant precipitation of frog rod PDE6. Thus, EGFP-PDE6C does not form heterodimers with the frog PDE6A and PDE6B subunits. The enzymatic characteristics of EGFP-PDE6C (kcat, KM) and the inhibition by the Pγ subunit were consistent with known properties of the native bovine PDE6C. The analysis of the EGFP-PDE6CN610S mutant revealed an impairment of the PDE6C activity inhibition by Pγ. The FRAP analysis indicated a slower than theoretically predicted diffusion of PDE6.
Transgenic Xenopus laevis is an excellent system to study PDE6. Functional EGFP- PDE6 can be expressed in Xenopus rods and isolated in quantities sufficient for biochemical analysis.
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