Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments.
Each photoexcited rhodopsin (R*) molecule catalyzes binding of GTP to many copies of the guanine nucleotide-binding protein transducin, which, in its GTP-binding form, then activates cGMP phosphodiesterase (PDEase). Subsequent deactivation of this light-activated enzyme cascade involves hydrolysis of the GTP bound to transducin, as well as decay of the activating capacity of R*. We report here that deactivation of PDEase in rod outer segment suspensions is highly enhanced by addition of ATP and purified 48-kDa protein, which is an intrinsic rod outer segment protein that is soluble in the dark but binds to photolyzed rhodopsin that has been phosphorylated by rhodopsin kinase and ATP [Kühn, H., Hall, S.W. & Wilden, U. (1984) FEBS Lett. 176, 473-478]. To analyze the mechanism by which ATP and 48-kDa protein deactivate PDEase, we used an ATP-free system consisting of thoroughly washed disk membranes, whose rhodopsin had been previously phosphorylated and chromophore-regenerated, and to which purified PDEase and transducin were reassociated. Such phosphorylated membranes exhibited a significantly lower (by a factor less than or equal to 5) light-induced PDEase-activating capacity than unphosphorylated controls. Addition of purified 48-kDa protein to phosphorylated membranes further suppressed their PDEase-activating capacity; suppression could be as high as 98% (as compared to unphosphorylated membranes), depending on the amount of 48-kDa protein and the flash intensity. Addition of ATP had little further effect. In contrast, PDEase activation or deactivation with unphosphorylated control membranes was not influenced by 48-kDa protein, even in the presence of ATP, provided rhodopsin kinase was absent. Our data suggest that 48-kDa protein binds to phosphorylated R* and thereby quenches its capacity to activate transducin and PDEase.
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D008027:Light; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D010750:Phosphoproteins / Q000502:physiology*; D010786:Photoreceptor Cells / Q000378:metabolism*; D012168:Retinal Pigments / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism* / Q000528:radiation effects; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D015293:Transducin; D014785:Vision, Ocular
D008565:Membrane Proteins; D010750:Phosphoproteins; D012168:Retinal Pigments; D000255:Adenosine Triphosphate; D012243:Rhodopsin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin