EXPRESSION AND MUTAGENESIS OF MOUSE ROD PHOTORECEPTOR CGMP PHOSPHODIESTERASE

Using recombinant baculovirus vectors, the three sub. units of mouse r od photoreceptor cGMP phosphodiesterase (PDE) (alphabetagamma2) have b een expressed in insect cells. The recombinant alpha,beta subunits acc umulate to 5 mg/liter culture, but most (98%) of the expressed polypep tides are insoluble. In the soluble fraction, individually expressed a and 13 subunits showed insignificant PDE activity, but coexpression ( by coinfection) of alphabeta subunits elevated PDE activity 7-fold and coexpression of alphabetagamma up to 15-fold. The soluble expressed h oloenzyme associated with ROS membranes under isotonic, but not hypoto nic, conditions. The K(m) of the soluble holoenzyme was 11-16 muM both for coexpressed alphabeta subunits and for alphabetagamma subunits, s imilar to the K(m) (6-80 muM) of native PDE. Site-directed mutagenesis of cysteine to serine in the C-terminal CAAX box of both alpha and be ta subunits substantially decreased the protein expression level, abol ished post-translational isoprenylation, and prevented subunit binding to the rod outer segment (ROS) membranes. The mutant holoenzyme, howe ver, showed a cGMP hydrolytic activity comparable with that of the nor mal recombinant enzyme. These results suggest that both alpha and beta subunits are required for the formation of a functional enzyme and th at isoprenylation of the subunits is essential for membrane associatio n and stability of PDE.