This project focuses on the basic molecular mechanisms of transmembrane signal transduction via G-protein coupled receptors. Understanding of how these plasma membrane receptors interact with G-proteins is crucial for a broad range of cellular effects triggered by hormones, neurotransmitters, odorants and light. Practical applications of this knowledge will be important for the development of more specific therapeutics that target G-protein coupled receptors and G-proteins, because it's estimated that more than 40 percent of drugs in use work on G- protein coupled receptors. How exactly receptors activate G- proteins remains unclear, because of the universal technical difficulties in obtaining the high-resolution structural information in receptor/G-protein complexes. This proposal aims at resolving a major question about catalytic signal relay in membrane receptor/G-protein complexes, namely, the molecular role for the G-protein betagamma-subunit complex in the receptor catalyzed activation of the alpha-subunit. We propose to test a hypothesis that the betagamma-subunit complex is actively employed by rhodopsin in order to control the nucleotide-binding site on the alpha-subunit. We have prepared a set of mutant G- proteins in which a rhodopsin interaction domain, the C-terminus of transducin gamma-subunit, is targeted by Alanine replacements, deletions, and sequence reversal mutations. We have shown that some of the mutations severely affect coupling with the light- activated rhodopsin. We will survey extensively the functional properties of these mutants by examining various individual steps of rhodopsin-transducin interactions and transducin activation. Recently, we have developed the model mimetic peptides derived from the surface domains of G-proteins in order to study the dynamics of rhodopsin-transducin interface by NMR spectroscopy. We will study the effect of the inactivating mutations and the lack of farnesylation on the structural features of the C- terminal domain of the gamma-subunit in the light activated rhodopsin-bound state. We will determine whether the conformational switch in the gamma-subunit is affected by mutations. Functional studies of transducin mutants and structural data obtained in parallel with model mimetic peptides will yield valuable information on the role of the betagamma- subunit complex in rhodopsin-catalyzed activation of transducin.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063203-04
Application #
6879535
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Flicker, Paula F
Project Start
2002-04-01
Project End
2007-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
4
Fiscal Year
2005
Total Cost
$183,750
Indirect Cost
Name
Saint Louis University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
050220722
City
Saint Louis
State
MO
Country
United States
Zip Code
63103
Lobysheva, E; Taylor, C M; Marshall, G R et al. (2018) Tauroursodeoxycholic acid binds to the G-protein site on light activated rhodopsin. Exp Eye Res 170:51-57
Lomonosova, Elena; Kolesnikov, Alexander V; Kefalov, Vladimir J et al. (2012) Signaling states of rhodopsin in rod disk membranes lacking transducin ??-complex. Invest Ophthalmol Vis Sci 53:1225-33
Kolesnikov, Alexander V; Rikimaru, Loryn; Hennig, Anne K et al. (2011) G-protein betagamma-complex is crucial for efficient signal amplification in vision. J Neurosci 31:8067-77
Taylor, Christina M; Rockweiler, Nicole B; Liu, Cassie et al. (2010) Using ligand-based virtual screening to allosterically stabilize the activated state of a GPCR. Chem Biol Drug Des 75:325-32
Van Eps, Ned; Anderson, Lori L; Kisselev, Oleg G et al. (2010) Electron paramagnetic resonance studies of functionally active, nitroxide spin-labeled peptide analogues of the C-terminus of a G-protein alpha subunit. Biochemistry 49:6877-86
Kisselev, Oleg G; Downs, Maureen A (2006) Rhodopsin-interacting surface of the transducin gamma subunit. Biochemistry 45:9386-92
Downs, Maureen A; Arimoto, Rieko; Marshall, Garland R et al. (2006) G-protein alpha and beta-gamma subunits interact with conformationally distinct signaling states of rhodopsin. Vision Res 46:4442-8
Kisselev, Oleg G (2005) Focus on molecules: rhodopsin. Exp Eye Res 81:366-7
Kisselev, Oleg G; Downs, Maureen A; McDowell, J Hugh et al. (2004) Conformational changes in the phosphorylated C-terminal domain of rhodopsin during rhodopsin arrestin interactions. J Biol Chem 279:51203-7
Kisselev, Oleg G; Downs, Maureen A (2003) Rhodopsin controls a conformational switch on the transducin gamma subunit. Structure 11:367-73