Contribution of protein kinase C to the phosphorylation of rhodopsin in intact retinas.
Similar to other G protein-coupled receptors, the visual receptor, rhodopsin, is phosphorylated by both a substrate-regulated kinase, rhodopsin kinase, and a second messenger-regulated kinase, protein kinase C. In the present study, the extent of involvement of protein kinase C in the light-dependent phosphorylation of rhodopsin in intact retinas was assessed using a specific activator (phorbol ester) and specific inhibitor (calphostin C) of protein kinase C. Kinetic analysis of rhodopsin phosphorylation following different illumination conditions revealed that hyperactivation of protein kinase C with phorbol ester resulted in a relative increase in rhodopsin phosphorylation that peaked 10-15 min after the onset of illumination. Following this period, the rate of rhodopsin dephosphorylation was increased in the phorbol ester-treated retinas, so that by about 30 min the amount of phosphorylation was similar to that in control retinas. Treatment of retinas with calphostin C, a potent regulatory domain-directed inhibitor of protein kinase C, resulted in an approximately 50% reduction in the light-dependent phosphorylation of rhodopsin. This inhibitor had no effect on the activity of rhodopsin kinase in vitro. Last, we show that frog rhodopsin is phosphorylated in vitro by protein kinase C from frog rod outer segments, indicating that this kinase could directly modulate rhodopsin in vivo. In conclusion, the present results reveal that the kinetics of rhodopsin phosphorylation/dephosphorylation differ markedly, depending on whether protein kinase C or rhodopsin kinase activity dominates, and that, under the conditions studied, protein kinase C contributes to approximately half of the phosphorylation of rhodopsin in intact frog retinas.
Udovichenko
I P
IP
Department of Pharmacology, University of California at San Diego School of Medicine, La Jolla, California 92093-0983, USA.
Newton
A C
AC
Williams
D S
DS
D000818:Animals; D001001:Anura; D002417:Cattle; D004789:Enzyme Activation; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D007700:Kinetics; D008027:Light; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D012160:Retina / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005136:Eye Proteins; D012243:Rhodopsin; D011494:Protein Kinases; D011493:Protein Kinase C; D051606:G-Protein-Coupled Receptor Kinase 1; D013755:Tetradecanoylphorbol Acetate