This project will study the molecular mechanisms regulating the kinetics of light response in photoreceptor cells. In vertebrates, rhodopsin triggers the light response by stimulating the binding of GTP to the alpha subunit of the G protein transducin (Gt-alpha), which activates its effector enzyme cGMP phosphodiesterase (PDE). GTP hydrolysis by Gt-alpha terminates this active state, leading to recovery from a light stimulus. The rate of Gt's intrinsic GTPase activity is too slow to explain the rapid termination of photoresponse in vivo. Previous research demonstrated that GTPase activity of Gt could be brought to a sub-second time scale by interaction with the PDE-gamma subunit (PDE-gamma) acting synergistically with the photoreceptor-specific RGS protein RGS9. Importantly, when added separately, neither PDE-gamma nor native RGS9 can act as a GAP (GTPase-activating protein). In contrast, the isolated RGS domain of RGS9 is a GAP, indicating that in situ, RGS9 is inhibited. This application is based on a recent and unexpected discovery that RGS9 is bound to the photoreceptor specific G protein beta subunit, Gbeta5L. Preliminary data with non-photoreceptor isoforms, RGS7 and Gbeta5, suggest that Gbeta5L can attenuate RGS9 activity. The working hypothesis driving this project is that Gbeta5L attenuates RGS9-stimulated GTP hydrolysis until Gt-alpha-GTP interacts with PDE-gamma, resulting in stronger signal amplification by the cascade.
Specific aim 1 will investigate the protein complexes involving Gbeta5L and RGS9 in the native extracts of photoreceptors. Using chromatography and immunoprecipitation, these experiments will show whether or not Gbeta5L, Gt-alpha and PDE-gamma can bind to RGS9 simultaneously, and elucidate the role of Gt's GDP/GTP cycle in the formation of these complexes.
Aim 2 will study purified proteins in vitro, and particularly, will determine the effect of Gbeta5L on the GAP activity of RGS9. In addition, studies utilizing surface plasmon resonance (SPR) will characterize protein-protein interactions with respect to their kinetics and regulation.
Aim 3 will study RGS9 and Gbeta5L by mutational analysis, gaining insight into how these molecules work, and providing molecular tools for the future investigation of their physiological role. This project will result in a better understanding of phototransduction at the molecular level. Many retinopathies occur due to disregulation of signal transduction mechanisms in photoreceptors and, therefore, the knowledge gained by this research will help to develop future therapies.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
1R01EY012982-01
Application #
6090420
Study Section
Visual Sciences C Study Section (VISC)
Program Officer
Mariani, Andrew P
Project Start
2000-05-01
Project End
2004-04-30
Budget Start
2000-05-01
Budget End
2001-04-30
Support Year
1
Fiscal Year
2000
Total Cost
$253,296
Indirect Cost
Name
University of Miami School of Medicine
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33146
Levay, Konstantin; Slepak, Vladlen Z (2010) Up- or downregulation of tescalcin in HL-60 cells is associated with their differentiation to either granulocytic or macrophage-like lineage. Exp Cell Res 316:1254-62
Slepak, Vladlen Z (2009) Structure, function, and localization of Gýý5-RGS complexes. Prog Mol Biol Transl Sci 86:157-203
Rosenzweig, Derek H; Nair, K Saidas; Levay, Konstantin et al. (2009) Interaction of retinal guanylate cyclase with the alpha subunit of transducin: potential role in transducin localization. Biochem J 417:803-12
Rosenzweig, Derek H; Nair, K Saidas; Wei, Junhua et al. (2007) Subunit dissociation and diffusion determine the subcellular localization of rod and cone transducins. J Neurosci 27:5484-94
Nair, K Saidas; Hanson, Susan M; Mendez, Ana et al. (2005) Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein-protein interactions. Neuron 46:555-67
Nair, K Saidas; Mendez, Ana; Blumer, Joe B et al. (2005) The presence of a Leu-Gly-Asn repeat-enriched protein (LGN), a putative binding partner of transducin, in ROD photoreceptors. Invest Ophthalmol Vis Sci 46:383-9
Gutierrez-Ford, Christina; Levay, Konstantin; Gomes, Aldrin V et al. (2003) Characterization of tescalcin, a novel EF-hand protein with a single Ca2+-binding site: metal-binding properties, localization in tissues and cells, and effect on calcineurin. Biochemistry 42:14553-65
Witherow, D Scott; Tovey, Steven C; Wang, Qiang et al. (2003) G beta 5.RGS7 inhibits G alpha q-mediated signaling via a direct protein-protein interaction. J Biol Chem 278:21307-13
Balasubramanian, Nagaraj; Slepak, Vladlen Z (2003) Light-mediated activation of Rac-1 in photoreceptor outer segments. Curr Biol 13:1306-10
Nair, K Saidas; Balasubramanian, Nagaraj; Slepak, Vladlen Z (2002) Signal-dependent translocation of transducin, RGS9-1-Gbeta5L complex, and arrestin to detergent-resistant membrane rafts in photoreceptors. Curr Biol 12:421-5

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