Amplification of phosphodiesterase activation is greatly reduced by rhodopsin phosphorylation.
In the vertebrate rod outer segment (ROS), the light-dependent activation of a GTP-binding protein (G-protein) and phosphodiesterase (PDE) is quenched by a process that requires ATP [Liebman, P.A., & Pugh, E.N. (1979) Vision Res. 19, 375-380]. The ATP-dependent quenching mechanism apparently requires the phosphorylation of photoactivated rhodopsin (Rho*); however, a 48-kilodalton protein (48K protein) has also been proposed to participate in the inactivation process. Purified species of phosphorylated rhodopsin containing 0, 2, or greater than or equal to 4 (high) phosphates per rhodopsin (PO4/Rho) were reconstituted into phosphatidylcholine (PC) vesicles and reassociated with a hypotonic extract from isotonically washed disk membranes that were depleted of 48K protein; PDE activation, in response to bleaching from 0.01% to 15% of the rhodopsin present, was measured. PDE activity was reduced by at least 30% at high fractional rhodopsin bleaches and by greater than 80% at low fractional rhodopsin bleaches in high PO4/Rho samples when compared to the activity measured in O PO4/Rho controls. A phosphorylation level of 2 PO4/Rho produced PDE activities that were intermediate between O PO4/Rho and high PO4/Rho samples at low bleaches, but were identical with the O PO4/Rho samples at high rhodopsin bleaches. Rhodopsin phosphorylation is thus capable of producing a graded inhibition of light-stimulated PDE activation over a limited range of (near physiological) bleach levels. This effect become less pronounced as the bleach levels approach those that saturate PDE activation. These results are consistent with increasing levels of phosphorylation, producing a reduction of the binding affinity of G-protein for Rho*.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D008027:Light; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000201:enzymology*; D012168:Retinal Pigments / Q000378:metabolism*; D012243:Rhodopsin / Q000302:isolation & purification / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D012168:Retinal Pigments; D000255:Adenosine Triphosphate; D012243:Rhodopsin; D010727:Phosphoric Diester Hydrolases; D019204:GTP-Binding Proteins