The photoreceptor rod outer segment cGMP phosphodiesterase (PDE, alphabetagamma2) converts cytoplasmic 3',5'-cGMP to 5'-GMP in a light- dependent manner, acting as a link in the amplification cascade of visual transduction. In mouse, the non-processed alpha subunit consists of 859 and the beta subunit of 856 residues. Alpha and beta have a duplicated domain structure and are, signalled by a CAAX box motif at their C- termini, posttranslationally isoprenylated, a modification that provides the molecular anchor for membrane association. A defect (truncation at residue 347) in the gene encoding the beta subunit is presumed to disable PDE catalytic activity and to cause a retinal degeneration in the rd mouse. Since the alpha subunit appears to be expressed normally (although at a reduced level), we conclude that both alpha and beta subunits are necessary to form a functional enzyme. To prove this hypothesis, we wish in specific aim 1 to express first intact, later mutated beta subunits in rod photoreceptors of a transgenic rd mouse. Expression of the transgene is predicted to provide a full size complement for alpha and gamma to form a functional PDE, thus allowing development of normal rod outer segments. We also propose to introduce a mutation (C853S) in the C-terminal CAAX box of the beta subunit to study the biological consequences of a defect in membrane association. In the second specific aim we wish to functionally express both the alpha and beta subunits in unicellular expression systems (bacteria and insect cells infected with recombinant baculovirus). The goal of this research is to reconstitute an active enzyme in vitro and to determine the factors essential for reconstitution. In the last part of specific aim 2, we propose to introduce specific point mutations in the CAAX box and the proposed catalytic domain to study their effects upon membrane binding, reconstitution, and catalytic activity. This research, using gene expression in bacteria, insect cells, and transgenic mice, will yield detailed information about the structure and function of PDE not available to date.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY008123-04
Application #
3265291
Study Section
Visual Sciences C Study Section (VISC)
Project Start
1989-12-01
Project End
1997-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
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