The broad, long-term objectives of the proposed research are to determine the mechanism used by the visual receptor rhodopsin to bind and activate the G-protein transducin. To achieve this objective, the location and timing of the conformational changes that occur must first be determined. Subsequently, the question of which of these changes is minimally required can be addressed. Rhodopsin is the best characterized and understood G-protein coupled receptor. Thus, studies proposed in this application will provide a deeper understanding, at the structural level, of how these receptors become activated. The proposed research will focus on specific conformational changes observed to occur in the transmembrane helices of rhodopsin upon photoactivation. By employing a range of physical and biochemical approaches to study a series of rhodopsin mutants that have unique cysteine residues at the cytoplasmic ends of the helices, this proposal will address the following three questions: (1) whether conformational changes in rhodopsin can be detected using fluorescence and biochemical techniques; (2) what the relationship is between these conformational changes and the location of transducin binding; and (3) whether the conformational changes are required to enable rhodopsin to activate transducin. The latter question will be investigated using the above techniques with rhodopsin mutants known to be functionally defective (i.e., proteins that cannot bind and activate transducin). Mutants of this type include those found in autosomal dominant retinitis pigmentosa, a form of night blindness. The results from these studies may help to provide a molecular, mechanistic explanation for this disease, and increase our general knowledge about a primary step in the signal transduction mechanism of G-protein coupled receptors.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012095-04
Application #
6363157
Study Section
Visual Sciences C Study Section (VISC)
Program Officer
Mariani, Andrew P
Project Start
1998-03-01
Project End
2002-02-28
Budget Start
2001-03-01
Budget End
2002-02-28
Support Year
4
Fiscal Year
2001
Total Cost
$201,666
Indirect Cost
Name
Oregon Health and Science University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Sommer, Martha E; Smith, W Clay; Farrens, David L (2005) Dynamics of arrestin-rhodopsin interactions: arrestin and retinal release are directly linked events. J Biol Chem 280:6861-71
Fay, Jonathan F; Dunham, Thomas D; Farrens, David L (2005) Cysteine residues in the human cannabinoid receptor: only C257 and C264 are required for a functional receptor, and steric bulk at C386 impairs antagonist SR141716A binding. Biochemistry 44:8757-69
Mansoor, Steven E; Farrens, David L (2004) High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane. Biochemistry 43:9426-38
Janz, Jay M; Farrens, David L (2004) Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus. J Biol Chem 279:29767-73
Janz, Jay M; Farrens, David L (2004) Role of the retinal hydrogen bond network in rhodopsin Schiff base stability and hydrolysis. J Biol Chem 279:55886-94
Janz, Jay M; Farrens, David L (2003) Assessing structural elements that influence Schiff base stability: mutants E113Q and D190N destabilize rhodopsin through different mechanisms. Vision Res 43:2991-3002
Janz, Jay M; Fay, Jonathan F; Farrens, David L (2003) Stability of dark state rhodopsin is mediated by a conserved ion pair in intradiscal loop E-2. J Biol Chem 278:16982-91
Mansoor, Steven E; McHaourab, Hassane S; Farrens, David L (2002) Mapping proximity within proteins using fluorescence spectroscopy. A study of T4 lysozyme showing that tryptophan residues quench bimane fluorescence. Biochemistry 41:2475-84
Farrens, D L; Dunham, T D; Fay, J F et al. (2002) Design, expression, and characterization of a synthetic human cannabinoid receptor and cannabinoid receptor/ G-protein fusion protein. J Pept Res 60:336-47
Janz, J M; Farrens, D L (2001) Engineering a functional blue-wavelength-shifted rhodopsin mutant. Biochemistry 40:7219-27

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