Rhodopsin has recently been recognized as G-protein activating receptor, designed such that the absorption of a photon activates rhodopsin, initiating a sequence of transduction events which lead to the hyperpolarization of the ROS plasma membrane. These events include an amplified activation of G-protein by photoactivated rhodopin, with subsequent activation of a cGMP specific PDE, resulting in closure of cGMP gated sodium channels.
The specific aims of the current application are to further characterize the molecular mechanism of regulation of this excitatory cascade by rhodopsin phosphorylation and its subsequent interaction with 48k protein. The energetic coupling of the two defined functional domains of rhodopsin, the retinal binding site and the G-protein interaction site, will be studied to better understand the intramolecular information flow within receptor molecules. The role of the unusual lipid microenvironment of the disk membrane in controlling the functional activation of rhodopsin will be examined. The effect of calcium on the level of light stimulated phosphodiesterase activity and its implication relative to light adaptation will be studied further. The above goals will be accomplished by characterizing a variety of physical and functional properties of purified preparations of rhodopsin molecules, which have been modified by proteolysis, having variable levels of phosphorylation, and amine group modification, reconstituted into lipid bilayers of defined lipid composition. The binding and activation of G-protein and the resulting PDE activation levels, the thermostability, the metaI to metaII equilibrium are among the properties to be studied. The goals of this proposal are to determine the ability of changes in lipid composition, due either to lipid peroxidation or metabolic defects in lipid metabolism, to effect visual pigment function. In addition, the mechanism whereby phosphorylation, which is a major regulatory mechanism in biological control, exerts its regulatory effect will be characterized at the molecular level. The additional role of the 48k protein in this regulatory process will also be determined. The regulatory mechanisms to be defined in the studies proposed in this application will be of significance in both the field of vision biochemistry and the broader areas of receptor function and metabolic regulation.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY000548-18
Application #
3255428
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1977-07-01
Project End
1993-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
18
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Virginia
Department
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Mitchell, D C; Straume, M; Litman, B J (1992) Role of sn-1-saturated,sn-2-polyunsaturated phospholipids in control of membrane receptor conformational equilibrium: effects of cholesterol and acyl chain unsaturation on the metarhodopsin I in equilibrium with metarhodopsin II equilibrium. Biochemistry 31:662-70
Mitchell, D C; Kibelbek, J; Litman, B J (1992) Effect of phosphorylation on receptor conformation: the metarhodopsin I in equilibrium with metarhodopsin II equilibrium in multiply phosphorylated rhodopsin. Biochemistry 31:8107-11
Mitchell, D C; Kibelbek, J; Litman, B J (1991) Rhodopsin in dimyristoylphosphatidylcholine-reconstituted bilayers forms metarhodopsin II and activates Gt. Biochemistry 30:37-42
Kibelbek, J; Mitchell, D C; Beach, J M et al. (1991) Functional equivalence of metarhodopsin II and the Gt-activating form of photolyzed bovine rhodopsin. Biochemistry 30:6761-8
Litman, B J; Lewis, E N; Levin, I W (1991) Packing characteristics of highly unsaturated bilayer lipids: Raman spectroscopic studies of multilamellar phosphatidylcholine dispersions. Biochemistry 30:313-9
Mitchell, D C; Straume, M; Miller, J L et al. (1990) Modulation of metarhodopsin formation by cholesterol-induced ordering of bilayer lipids. Biochemistry 29:9143-9
Straume, M; Mitchell, D C; Miller, J L et al. (1990) Interconversion of metarhodopsins I and II: a branched photointermediate decay model. Biochemistry 29:9135-42
Miller, J L; Hubbard, C M; Litman, B J et al. (1989) Inhibition of transducin activation and guanosine triphosphatase activity by aluminum ion. J Biol Chem 264:243-50
Straume, M; Litman, B J (1988) Equilibrium and dynamic bilayer structural properties of unsaturated acyl chain phosphatidylcholine-cholesterol-rhodopsin recombinant vesicles and rod outer segment disk membranes as determined from higher order analysis of fluorescence anisotropy decay. Biochemistry 27:7723-33
Miller, J L; Litman, B J; Dratz, E A (1987) Binding and activation of rod outer segment phosphodiesterase and guanosine triphosphate binding protein by disc membranes: influence of reassociation method and divalent cations. Biochim Biophys Acta 898:81-9

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