Abstract
Purpose::
In rods, phosphorylation of phosducin (Pdc), a major protein partner of the heterotrimeric G proteins, is tightly controlled by light. To get insight into Pdc function, we conducted quantitative analysis of Pdc phosphorylation in the retinas of free running animals under physiological levels of illumination.
Methods::
Mice and rats were exposed to various levels of constant illumination, sacrificed and their eyes harvested. Previously identified light-regulated phosphorylation sites of Pdc, Ser54 and Ser73, were monitored using phospho-specific antibodies. The degree and time course of Pdc phosphorylation were determined using immunoprecipitation with phospho-specific antibodies and quantitative immunoblotting. Compartmentalization of phosphorylated Pdc and light-driven translocation of rod transducin were determined by immunoblotting serial tangential sections of the retina.
Results::
In the dark-adapted rods, 45±8% of total Pdc was found to be phosphorylated at Ser73 and appeared to be evenly distributed within the entire rod cytoplasm; however some enrichment in the inner segment ellipsoid was observed. Pdc phosphorylated at Ser54 was strongly compartmentalized and located predominantly in the ellipsoid and the adjacent part of the outer segment, being virtually undetectable in the outer nuclear and plexiform layers. Exposure of animals to dim 8 lux light below the threshold of transducin translocation resulted in near complete de-phosphorylation of both Ser73 and Ser54 within a minute. Shutting off the light restored the phospho-state of both serines after ~3 min. Brighter 1000 lux light triggered translocation of transducin from the rod outer segments and resulted in massive phosphorylation of Pdc in this compartment. Under these conditions, Ser73 phosphorylation levels exceeded those in the dark-adapted retinas, whereas Ser54 was completely de-phosphorylated.
Conclusions::
Our quantitative analysis of Pdc phosphorylation in vivo in response to physiologically relevant changes of illumination reveals differential spatial and temporal phosphorylation of its two principal light-regulated sites, Ser54 and Ser73. A hitherto unknown phenomenon of massive Pdc phosphorylation at Ser73 in the outer segments of rod exposed to bright illumination is described. Our data is consistent with the hypothesis that phosphorylation of Ser54 governs Pdc interaction with transducin and thus regulates light-driven transducin translocation; however, this data suggests a different function for Ser73.
Keywords: photoreceptors • phosphorylation • protein structure/function