Abstract
Purpose: :
The heterodimerization of rod phosphodiesterase-6 (PDE6) catalytic subunits PDE6A and PDE6B is unique among PDEs. The individual properties of PDE6A and PDE6B and the functional significance of rod PDE6 heterodimerization in phototransduction are unknown. EGFP-fusion proteins of chimeric homodimeric PDE6 enzymes containing the PDE6A or the PDE6B catalytic domains were expressed in transgenic Xenopus laevis to investigate the catalytic properties of PDE6A and PDE6B.
Methods: :
Constructs for PDE6 chimeras contained sequences for thrombin-cleavable EGFP and the N-terminal regulatory GAF domains of human cone PDE6C linked to the C-terminal catalytic domains of PDE6A (PDE6C-A) or PDE6B (PDE6C-B). Transgenic X. laevis expressing EGFP-PDE6C, EGFP-PDE6C-A, and EGFP-PDE6C-B in rods have been produced with restriction enzyme mediated integration of the transgenes under the Xenopus rhodopsin promoter. Expression and localization of the EGFP-fusion proteins in the frog retina were examined by Western blotting and EGFP-fluorescence. The proteins were immunoprecipitated with anti-EGFP antibodies. Soluble PDE6C, PDE6C-A, and PDE6C-B were released with thrombin and examined for PDE6 activity and inhibition by the cone and rod PDE6 γ-subunits (Pγ).
Results: :
Similarly to EGFP-PDE6C, EGFP-PDE6C-A and EGFP-PDE6C-B were targeted to the rod outer segments in the retina of transgenic tadpoles. EGFP-PDE6C-A and PDE6C-B were selectively precipitated by anti-GFP antibody with no co-precipitation of frog rod PDE6A and PDE6B. All three enzymes, PDE6C, PDE6C-A, and PDE6C-B, hydrolyzed cGMP with analogous KM (40-50 µM) and kcat (4000-5000 s-1) values. Likewise, the Ki values for the PDE6C, PDE6C-A, and PDE6C-B inhibition by the cone- and rod-specific Pγ subunits were comparable.
Conclusions: :
Our results suggest that the rod PDE6A and PDE6B subunits are equivalent with respect to catalytic properties and the interaction with Pγ. Furthermore, the PDE6A and PDE6B subunits are functionally similar to PDE6C. The functional significance of the conserved differences between PDE6A, PDE6B, and PDE6C does not appear to lie with the enzymatic properties of PDE6 catalytic subunits.
Keywords: photoreceptors • signal transduction