Abstract
Abstract: :
Purposes: To establish whether light–activated rhodopsin (R*) leads to phosphorylation of dark rhodopsin (R) in intact rod photoreceptor cells. In vitro experiments using isolated rod outer segments suggest that low levels of R* can lead to phosphorylation of dark rhodopsin (Binder BM, et al. J. Biol. Chem. 1996; 271). This has been termed "high gain" phosphorylation, reflecting the high stoichiometry of incorporated phosphate beyond the level expected if only R* were phosphorylated. Whether this process occurs in vivo is uncertain due to difficulties in distinguishing R* from R biochemically. Methods: We generated transgenic mice that expressed mouse cone S–opsin in the rhodopsin–/– and rhodopsin +/– backgrounds. Ectopically expressed S–opsin localized to the rod outer segment, whether in the presence or absence of endogenous rhodopsin. S–opsin is a spectrally distinct visual pigment with an absorption maxima near 360 nm. The large separation in spectral sensitivity between S–opsin and rhodopsin at long wavelengths (>515 nm) allows for specific stimulation of rhodopsin using yellow light (>515 nm). Transgenic S–opsin is functional in rod cells, as indicated by electrophysiological measurements (electroretinogram and suction electrode recordings). Intact retinas were isolated and exposed to 360 nm or >515 nm light, and phosphorylation of rhodopsin and S–opsin was monitored using LC–MS (modified from Kennedy MJ, et al. Neuron 2001; 31). Results: We found that S–opsin, when expressed in the absence of rhodopsin, was phosphorylated following exposure to 360 nm light, but was not phosphorylated after exposure to >515 nm light. However, when S–opsin was co–expressed with rhodopsin, it became phosphorylated at multiple sites when intact retinas were exposed to >515 nm light. Conclusions: Our results support the presence of high gain phosphorylation in vivo. This may occur if activated rhodopsin kinase phosphorylates nearby R via transphosphorylation. High gain phosphorylation may play a role in some aspects of light adaptation, since phosphorylated rhodopsin exhibits lowered efficiency in activating transducin.
Keywords: phosphorylation • opsins • transgenics/knock–outs