CONTROL OF RHODOPSIN MULTIPLE PHOSPHORYLATION

The inactivation of photolyzed rhodopsin requires phosphorylation of t he receptor at multiple sites near the C-terminus by rhodopsin kinase and binding of a regulatory protein, arrestin. In the present study, t he phosphorylation sites were examined in a partially reconstituted sy stem under several experimental conditions. Initial phosphorylation si tes were found to be 338Ser, 343Ser, and 334Ser based on analysis by m ass spectrometry of proteolytic peptides from the C-terminus. The exte nt of phosphorylation was found to be limited by two mechanisms: (1) b inding of arrestin to phosphorylated rhodopsin (one to three phosphate groups) appeared to prevent further phosphorylation (arrestin has als o been observed to promote the initial phosphorylation of rhodopsin at 338Ser in rod outer segment homogenates); and (2) reduction of the ph otolyzed chromophore all-trans-retinal to all-trans-retinol prevented phosphorylation at more than three sites. We propose that previous obs ervations of higher levels of rhodopsin phosphorylation may be the res ult of the removal of endogenous arrestin, or of exceeding the capacit y of retinol dehydrogenase activity by intense bleaches (e.g., by exha usting endogenous NADPH).