Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.
PDE6 (phosphodiesterase-6) is the effector molecule in the vertebrate phototransduction cascade. Progress in understanding the structure and function of PDE6 has been hindered by lack of an expression system of the enzyme. Here we report ectopic expression and analysis of compartmentalization and membrane dynamics of the enhanced green fluorescent protein (EGFP) fusion protein of human cone PDE6C in rods of transgenic Xenopus laevis. EGFP-PDE6C is correctly targeted to the rod outer segments in transgenic Xenopus, where it displayed a characteristic striated pattern of EGFP fluorescence. Immunofluorescence labeling indicated significant and light-independent co-localization of EGFP-PDE6C with the disc rim marker peripherin-2 and endogenous frog PDE6. The diffusion of EGFP-PDE6C on disc membranes investigated with fluorescence recovery after photobleaching was markedly slower than theoretically predicted. The enzymatic characteristics of immunoprecipitated recombinant PDE6C were similar to known properties of the native bovine PDE6C. PDE6C was potently inhibited by the cone- and rod-specific PDE6 gamma-subunits. Thus, transgenic Xenopus laevis is a unique expression system for PDE6 well suited for analysis of the mechanisms of visual diseases linked to PDE6 mutations.
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, USA.
Hakim
H
Muradov
Kimberly K
KK
Boyd
Mohammad
M
Haeri
Vasily
V
Kerov
Barry E
BE
Knox
Nikolai O
NO
Artemyev
D000818:Animals; D002417:Cattle; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6 / Q000378:metabolism*; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007381:Intermediate Filament Proteins / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D064531:Peripherins; D011994:Recombinant Proteins / Q000737:chemistry; D017949:Retinal Cone Photoreceptor Cells / Q000201:enzymology*; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C578995:PRPH protein, Xenopus; C578994:PRPH protein, human; D064531:Peripherins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D049452:Green Fluorescent Proteins; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6