The purpose of the proposed research is to determine the functional significance, catalytic and structural, of C-terminal palmitoylation and N-terminal glycosylation of vertebrate rhodopsin, the rod visual pigment. Polymerase chain reaction will be utilized to modify, by site-directed mutagenesis, the opsin glycosylation and palmitoylation sites and mutant opsins will be expressed in transgenic mice. The mutant opsins will also contain a three amino acid, bovine-specific C-terminal epitope """"""""tag"""""""" which, in conjunction with an epitope-specific monoclonal anti-bovine opsin antibody, will permit identification and localization of the transgenic """"""""bouse"""""""" opsin against the background of the normal mouse opsin, using conventional Western blotting and immunocytochemical methods. Initial experiments will be performed to determine the level of expression of """"""""bouse"""""""" opsin (not containing modified palmitoylation and glycosylation sites) that does not by itself produce structural and functional alternations. This proposal aims at testing the hypothesis that palmitoylation of rhodopsin is involved in the deactivation of photoactivated rhodopsin. It also aims at testing the hypothesis that impaired N-terminus glycosylation of rhodopsin is sufficient to cause defective morphogenesis of the outer segment. These features will be studied using a combination of biochemical methods, light microscopic autoradiography, and light and electron microscopy and immunocytochemistry. Electroretinography and suction pipette single cell recording will be used to determine the effects of transgene expression on phototransduction and overall electrophysiological competence of the retina.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
1R01EY011376-01A1
Application #
2020048
Study Section
Special Emphasis Panel (ZRG1-VISC (02))
Project Start
1996-12-01
Project End
1999-11-30
Budget Start
1996-12-01
Budget End
1997-11-30
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612