SEQUENTIAL PHOSPHORYLATION OF RHODOPSIN AT MULTIPLE SITES

Photolyzed rhodopsin is phosphorylated at multiple serine and threonin e residues during the quenching of phototransduction. Sites of phospho rylation by rhodopsin kinase have been localized to the C-terminal reg ion of rhodopsin, but no information was available on the kinetics and identity of phosphorylated residues. To determine the kinetics of pho sphorylation at specific residues, the phosphorylated C-terminal pepti de of rhodopsin (330DDEASTTVSKTETSQVAPA) obtained by proteolysis of rh odopsin with endoproteinase Asp-N was subjected to further subdigestio n followed by electrospray mass spectrometry. Analysis of monophosphor ylated peptide revealed that the major initial phosphorylation site is 338Ser. The analysis of di- and triphosphorylated peptides indicated that 343Ser or 336Thr residues are subsequent phosphorylation sites. T hese three residues, located in the C-terminal region of rhodopsin, ar e likely to be key phosphorylation sites of rhodopsin during the quenc hing of phototransduction. Identification of the kinetics of phosphory lation will facilitate understanding the functional significance of rh odopsin phosphorylation at multiple sites and the mechanism of rhodops in kinase action.