Vertebrate photoreceptors are able to function under a wide variety of illumination conditions. It is likely that multiple mechanisms are involved in the process by which these sensory neurons can adapt to different levels of light. In this application we suggest that at least part of this difference in adaption and sensitivity to light is mediated by the regulated attachment of several of the different phototransduction cascade enzymes to the disk membranes. One area likely to be a major contributor to this regulation, but also one of the least understood, is/are the role(s) played a small 17 kDa polypeptide first identified as a subunit of the light-activated phosphodiesterases (PDE6s). In this application we propose 4 different sets of experiments aimed at elucidating the functions of the PDE6 delta subunit. First we will determine if the delta subunit of the rod PDE6 controls the attachment of PDE6 to the ROS membrane by binding to the C-terminal tail of PDE6 in a manner that requires isoprenylation and methylation. This will be approached by studying the effects of differentially isoprenylated C-terminal PDE peptides on the binding of the delta subunit to PDE6. Second, we will test the idea that the 17 kDa delta subunit of PDE6 regulates PDE6 activity in the light activated visual transduction cascade by altering its binding to the disk membrane. This will be approached by studying the actions of the rod PDE6 delta subunits and peptides from the C-terminal tail of PDE6 in permeablized ROS membranes and also by electrophysiological methods. Third, we propose that the binding of PDE6 alpha and beta subunits to the PDE6 delta subunit is regulated by a light sensitive methylation and/or phosphorylation of the alpha and beta subunits. These studies will utilize standard methods for identification of covalent modification as a regulatory control mechanism for cellular functions. Finally, we want to test the idea that the PDE6 delta subunit will also interact with several other enzymes in the visual transduction cascade and that their functions too may be regulated by similar mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY008197-12
Application #
6363123
Study Section
Visual Sciences C Study Section (VISC)
Program Officer
Mariani, Andrew P
Project Start
1989-08-01
Project End
2003-02-28
Budget Start
2001-03-01
Budget End
2003-02-28
Support Year
12
Fiscal Year
2001
Total Cost
$215,504
Indirect Cost
Name
University of Washington
Department
Pharmacology
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Cook, T A; Ghomashchi, F; Gelb, M H et al. (2001) The delta subunit of type 6 phosphodiesterase reduces light-induced cGMP hydrolysis in rod outer segments. J Biol Chem 276:5248-55
Cook, T A; Ghomashchi, F; Gelb, M H et al. (2000) Binding of the delta subunit to rod phosphodiesterase catalytic subunits requires methylated, prenylated C-termini of the catalytic subunits. Biochemistry 39:13516-23
Mou, H; Grazio 3rd, H J; Cook, T A et al. (1999) cGMP binding to noncatalytic sites on mammalian rod photoreceptor phosphodiesterase is regulated by binding of its gamma and delta subunits. J Biol Chem 274:18813-20
Li, N; Florio, S K; Pettenati, M J et al. (1998) Characterization of human and mouse rod cGMP phosphodiesterase delta subunit (PDE6D) and chromosomal localization of the human gene. Genomics 49:76-82
Florio, S K; Prusti, R K; Beavo, J A (1996) Solubilization of membrane-bound rod phosphodiesterase by the rod phosphodiesterase recombinant delta subunit. J Biol Chem 271:24036-47
Beavo, J A (1995) Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev 75:725-48
Yan, C; Bentley, J K; Sonnenburg, W K et al. (1994) Differential expression of the 61 kDa and 63 kDa calmodulin-dependent phosphodiesterases in the mouse brain. J Neurosci 14:973-84
Sonnenburg, W K; Beavo, J A (1994) Cyclic GMP and regulation of cyclic nucleotide hydrolysis. Adv Pharmacol 26:87-114
Stroop, S D; Beavo, J A (1992) Sequence homology and structure--function studies of the bovine cyclic-GMP-stimulated and retinal phosphodiesterases. Adv Second Messenger Phosphoprotein Res 25:55-71
Bentley, J K; Kadlecek, A; Sherbert, C H et al. (1992) Molecular cloning of cDNA encoding a ""63""-kDa calmodulin-stimulated phosphodiesterase from bovine brain. J Biol Chem 267:18676-82

Showing the most recent 10 out of 17 publications