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.
| Tan, E; Wang, Q; Quiambao, A B et al. (2001) The relationship between opsin overexpression and photoreceptor degeneration. Invest Ophthalmol Vis Sci 42:589-600 |
| Al-Ubaidi, M R; White, T W; Ripps, H et al. (2000) Functional properties, developmental regulation, and chromosomal localization of murine connexin36, a gap-junctional protein expressed preferentially in retina and brain. J Neurosci Res 59:813-26 |
| O'Brien, J; Bruzzone, R; White, T W et al. (1998) Cloning and expression of two related connexins from the perch retina define a distinct subgroup of the connexin family. J Neurosci 18:7625-37 |
