Proteomic Changes in the Photoreceptor Outer Segment upon Intense Light Exposure

Sep. 1, 2010

Acute light-induced photoreceptor degeneration has been studied in experimental animals as a model for photoreceptor cell loss in human retinal degenerative diseases. Light absorption by rhodopsin in rod photoreceptor outer segments (OS) induces oxidative stress and initiates apoptotic cell death. However, the molecular events that induce oxidative stress and initiate the apoptotic cascade remain poorly understood. To better understand the molecular mechanisms of light-induced photoreceptor cell death, we studied the proteomic changes in OS upon intense light exposure by using a proteolytic O18 labeling method. Of 171 proteins identified, the relative abundance of 98 proteins in light-exposed and unexposed OS was determined. The quantities of 11 proteins were found to differ by more than 2-fold between light-exposed OS and those remaining in darkness. Among the 11 proteins, 8 were phototransduction proteins and 7 of these were altered such that the efficiency of phototransduction would be reduced or quenched during light exposure. In contrast, the amount of OS rhodopsin kinase was reduced by 2-fold after light exposure, suggesting attenuation in the mechanism of quenching phototransduction. Liquid chromatography multiple reaction monitoring (LC-MRM) was performed to confirm this reduction in the quantity of rhodopsin kinase. As revealed by immunofluorescence microscopy, this reduction of rhodopsin kinase is not a result of protein translocation from the outer to the inner segment. Collectively, our findings suggest that the absolute quantity of rhodopsin kinase in rod photoreceptors is reduced upon light stimulation and that this reduction may be a contributing factor to light-induced photoreceptor cell death. This report provides new insights into the proteomic changes in the OS upon intense light exposure and creates a foundation for understanding the mechanisms of light-induced photoreceptor cell death.

Figure Molecular steps in activation (a) and deactivation (b) of the phototransduction cascade. The activation cascade is initiated upon photon absorption by rhodopsin. Absorption of a photon by rhodopsin causes a series of conformational changes in the protein leading to an activated state of rhodopsin. The activated rhodopsin binds a heterotrimeric protein transducin and allows for exchange of GDP nucleotide for GTP in the subunit of transducin. The GTP bound subunit dissociates from the subunits of transducin and activates phosphodiesterase that catalyzes hydrolysis of cGMP. In response to a decreased concentration of cGMP, cGMP gated cation channels in the plasma membrane close, resulting in decreased calcium levels which cause the photoreceptor cell to become hyperpolarized (a). Recoverin is normally bound to rhodopsin kinase when the calcium concentration is high. However, during phototransduction, the calcium levels fall, resulting in the release of rhodopsin kinase that leads to initiation of the deactivation cascade of rhodopsin. The activated rhodopsin is first phosphorylated by rhodopsin kinase. The phosphorylated rhodopsin allows arrestin to bind, which stearically interferes with the binding of the subunit of transducin. In addition to deactivating rhodopsin, transducin-mediated signaling could be quenched. The complex of G-protein beta 5 (GBB5), regulator of signaling 9 (RGS9) and regulator of signaling 9 anchor protein (R9AP) binds to the transducin subunit.phosphodiesterase complex. This stimulates the GTPase activity of transducin subunit and results in dissociation of the transducin subunit from phosphodiesterase, therefore, slowing down the hydrolysis of cGMP, allowing the cGMP channels to open (b). Adapted from Burns and Arshavsky.

Results from: Hajkova, D., Imanishi, Y., Palamalai, V., Rao, K.C., Yuan, C., Sheng, Q., Tang, H., Zeng, R., Darrow, R.M., Organisciak, D.T., Miyagi, M. J Proteome Res. 9(2):1173-81, 2010.