Abstract
Purpose: :
Phosducin is a major photoreceptor–specific phosphoprotein that interacts with the beta/gamma subunit complex of heterotrimeric G proteins in its dephosphorylated state. Exposure of photoreceptors to light results in massive de–phosphorylation of phosducin, suggesting that it acts as a light–dependent regulator of G proteins. To address this putative function, we have analyzed the phosphorylation status of phosducin in the different cellular compartments of rods exposed to various luminances.
Methods: :
Dark–adapted rats were sacrificed by focused microwave irradiation to preserve in vivo protein phosphorylation. Phosducin was immunoprecipitated in the retinal extracts, resolved by SDS PAGE, and the phosducin band was excised and digested in–gel. The resulting peptides were eluted and analyzed by LC/MS to identify phosphorylated residues. Obtained data was used to generate phospho–site–specific antibodies against phosducin, which were utilized to monitor its phosphorylation status in the different cellular compartment of rods, under various conditions of illumination, by Western blotting and immunofluorescence microscopy.
Results: :
Two principal phosphorylation sites, serine 54 and serine 73, were identified on phosducin in vivo, however several other potential phosphorylation sites were also detected. In the dark–adapted photoreceptors, phosphorylation of serine 54 and serine 73 occurs in a compartment–specific manner. Phosphorylation of phosducin at serine 54 predominantly occurs at the border between the outer and inner segments of the dark–adapted rods, whereas phosphorylation at serine 73 extends further into the inner segment. Interestingly, serine 73 and 54 phosphorylation appeared to be significantly reduced in the synaptic terminus of the rods, indicating the special status of this compartment. Both sites readily underwent de–phosphorylation in the photoreceptors exposed to light.
Conclusions: :
Our data suggest that phosphorylation of serine 54 and serine 73 residues of phosducin regulates different functions of this protein in different cellular compartments of photoreceptors. The role of light–dependent phosducin phosphorylation will be discussed.
Keywords: photoreceptors • protein structure/function • phosphorylation