MASS-SPECTROMETRIC IDENTIFICATION OF PHOSPHORYLATION SITES IN BLEACHED BOVINE RHODOPSIN

Deactivation of the visual cascade is initiated by the phosphorylation of rhodopsin. We report here identification of the two major sites of phosphorylation in bleached bovine rhodopsin using tandem mass spectr ometry in conjunction with synthetic phosphopeptide standards. Both bl eached and unbleached rod outer segments were cleaved with endoprotein ase Asp-N to release the C-terminal fragment, residues 330-348, contai ning seven potential sites of phosphorylation. High-performance liquid chromatographic separation of soluble cleavage products from both unb leached and bleached rod outer segments gave a peak which was identifi ed by tandem mass spectrometry and comparison to synthetic standards a s monophosphorylated (serine 338) DDEASTTVSKTETSQVAPA. Present only in the chromatogram of bleached ROS were two peaks identified as monopho sphorylated (serine 343) and diphosphorylated (serines 338 and 343) de rivatives of DDEASTTVSKTETSQVAPA. These results identify serines 338 a nd 343 as the major sites of phosphorylation within the C-terminal reg ion of bleached bovine rhodopsin and constitute the first example of m ass spectrometric characterization of phosphorylation sites in a G-pro tein coupled receptor.