EXPRESSION AND MUTAGENESIS OF MOUSE ROD PHOTORECEPTOR CGMP PHOSPHODIESTERASE

Using recombinant baculovirus vectors, the three sub. units of mouse rod photoreceptor cGMP phosphodiesterase (PDE) (alphabetagamma2) have been expressed in insect cells. The recombinant alpha,beta subunits accumulate to 5 mg/liter culture, but most (98%) of the expressed polypeptides 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 holoenzyme associated with ROS membranes under isotonic, but not hypotonic, conditions. The K(m) of the soluble holoenzyme was 11-16 muM both for coexpressed alphabeta subunits and for alphabetagamma subunits, similar 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 beta subunits substantially decreased the protein expression level, abolished post-translational isoprenylation, and prevented subunit binding to the rod outer segment (ROS) membranes. The mutant holoenzyme, however, showed a cGMP hydrolytic activity comparable with that of the normal recombinant enzyme. These results suggest that both alpha and beta subunits are required for the formation of a functional enzyme and that isoprenylation of the subunits is essential for membrane association and stability of PDE.