Control of rhodopsin multiple phosphorylation.
The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor at multiple sites near the C-terminus by rhodopsin kinase and binding of a regulatory protein, arrestin. In the present study, the phosphorylation sites were examined in a partially reconstituted system under several experimental conditions. Initial phosphorylation sites were found to be 338Ser, 343Ser, and 334Ser based on analysis by mass spectrometry of proteolytic peptides from the C-terminus. The extent of phosphorylation was found to be limited by two mechanisms: (1) binding of arrestin to phosphorylated rhodopsin (one to three phosphate groups) appeared to prevent further phosphorylation (arrestin has also been observed to promote the initial phosphorylation of rhodopsin at 338Ser in rod outer segment homogenates); and (2) reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol prevented phosphorylation at more than three sites. We propose that previous observations of higher levels of rhodopsin phosphorylation may be the result of the removal of endogenous arrestin, or of exceeding the capacity of retinol dehydrogenase activity by intense bleaches (e.g., by exhausting endogenous NADPH).
Ohguro
H
H
Department of Ophthalmology, University of Washington School of Medicine, Seattle 98195-0001.
Johnson
R S
RS
Ericsson
L H
LH
Walsh
K A
KA
Palczewski
K
K
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000378:metabolism; D019393:Arrestin; D002417:Cattle; D005136:Eye Proteins / Q000378:metabolism; D051606:G-Protein-Coupled Receptor Kinase 1; D008969:Molecular Sequence Data; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D000941:Antigens; D019393:Arrestin; D005136:Eye Proteins; D047428:Protein Kinase Inhibitors; D012694:Serine; D012243:Rhodopsin; D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1