Activation of bovine rod outer segment phospholipase C by arrestin.
Phospholipase C (PLC) enzyme activity in rod outer segment (ROS) membranes bleached in the presence of ATP and GTP was assayed using exogenously added [3H]phosphatidylinositol 4,5-bisphosphate vesicles as substrate. The addition of the soluble ROS protein arrestin (also known as S-antigen or 48K protein) to ROS membranes activated PLC 2-3.4-fold. This activation was dose-dependent, and maximal activation was observed at an arrestin concentration of congruent to 110-220 nM. PLC activation by arrestin was dependent on ROS protein concentration and free Ca2+. Soluble PLC (s-PLC) enzyme activity present in hypotonic extracts of bleached ROS was also activated 2-4-fold by arrestin. Maximum activation of s-PLC by arrestin was observed at free Ca2+ of 80 nM. Arrestin activation of s-PLC was not affected by urea-treated and extensively washed ROS membranes, suggesting that rhodopsin was not required for the observed effect of arrestin on s-PLC. The results are indicative of a direct interaction of arrestin with s-PLC, resulting in the activation of the latter. Based on these results and the documented binding of arrestin to bleached and phosphorylated rhodopsin, a model for the light activation of PLC in ROS is proposed.
Ghalayini
A J
AJ
Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030.
Anderson
R E
RE
D000818:Animals; D000941:Antigens / Q000302:isolation & purification / Q000502:physiology*; D019393:Arrestin; D002417:Cattle; D003624:Darkness; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005136:Eye Proteins / Q000302:isolation & purification / Q000502:physiology*; D007700:Kinetics; D008027:Light; D008565:Membrane Proteins / Q000502:physiology*; D008954:Models, Biological; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D010738:Type C Phospholipases / Q000378:metabolism*
D000941:Antigens; D019393:Arrestin; D005136:Eye Proteins; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010738:Type C Phospholipases