Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase.
Using recombinant baculovirus vectors, the three subunits of mouse rod photoreceptor cGMP phosphodiesterase (PDE) (alpha beta gamma 2) 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 alpha and beta subunits showed insignificant PDE activity, but coexpression (by coinfection) of alpha beta subunits elevated PDE activity 7-fold and coexpression of alpha beta gamma up to 15-fold. The soluble expressed holoenzyme associated with ROS membranes under isotonic, but not hypotonic, conditions. The Km of the soluble holoenzyme was 11-16 microM both for coexpressed alpha beta subunits and for alpha beta gamma subunits, similar to the Km (6-80 microM) 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.
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Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Baehr
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D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D046911:Macromolecular Substances; D015106:3',5'-Cyclic-GMP Phosphodiesterases