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.
Showing the most recent 10 out of 11 publications
