title: Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase.
abstract: 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.
authors.affiliation: Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: N
authors.initials: N
authors.lastname: Qin
authors.forename: W
authors.initials: W
authors.lastname: Baehr
mesh_terms: 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
chemical_list: D017931:DNA Primers; D046911:Macromolecular Substances; D015106:3',5'-Cyclic-GMP Phosphodiesterases