Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase

J Biol Chem. 1994 Feb 4;269(5):3265-71.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3',5'-Cyclic-GMP Phosphodiesterases / biosynthesis*
  • 3',5'-Cyclic-GMP Phosphodiesterases / isolation & purification
  • 3',5'-Cyclic-GMP Phosphodiesterases / metabolism
  • Amino Acid Sequence
  • Animals
  • Baculoviridae / genetics
  • Base Sequence
  • Blotting, Western
  • Cell Line
  • Cell Membrane / metabolism
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression
  • Kinetics
  • Macromolecular Substances
  • Mice
  • Molecular Sequence Data
  • Moths
  • Mutagenesis, Site-Directed
  • Point Mutation*
  • Protein Binding
  • Protein Prenylation
  • Protein Processing, Post-Translational
  • Retinal Rod Photoreceptor Cells / enzymology*
  • Rod Cell Outer Segment / metabolism
  • Transfection


  • DNA Primers
  • Macromolecular Substances
  • 3',5'-Cyclic-GMP Phosphodiesterases