Calcium‐activated Opsin Phosphatase Activity in Retinal Rod Outer Segments

Abstract

We describe the presence in bovine retinal rod outer segments of a phosphatase which dephosphorylates phosphoopsin with an efficiency similar to that of PP2A, and which is stimulated by submicromolar levels of CaZ (half-maximal activation, 0.4-0.5 pM). This enzyme is designated Ca'+-activated opsin phosphatase (CAOP). CAOP has a molecular mass of 70-75 kDa as determined by gel filtration on Superose 12 and exhibits reversible Ca2+-dependent oligomerization. An unidentified protein of approximately 25 kDa is necessary for full activity of CAOP and for cooperative binding of Ca'+ ( h > 2). CAOP does not require MgZ+ and is inhibited by okadaic acid (median inhibitory concentration > 25 FM), which suggests that it is related to the PP1/2A/2B class of protein phosphatases. Like PP2B, CAOP is inhibited by trifluoperazine (median inhibitory concentration 40 pM), but calmodulin has no effect on CAOP activity, and CAOP is inhibited by mastoparan at much higher concentrations than PP2B. This combination of properties suggests that CAOP is not identical to any characterized protein phosphatase. Since the cytoplasmic concentration of Ca'+ in retinal rod outer segments is reduced upon excitation by light, the existence of a Ca2+-sensitive opsin phosphatase activity suggests that light-dependent Ca'' levels may control rhodopsin dephosphorylation.

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

“…For rhodopsin to recycle for full light sensitivity, arrestin and the phosphate moieties must be removed. Light-dependent Ca 2+ concentration fluctuations have also been shown to affect the dephosphorylation of rhodopsin through the Ca 2+ -activated opsin phosphatase (CAOP) enzyme (72). In Drosophila, the Ca 2+ -dependent rhodopsin protein phosphatase, RDGC, dephosphorylates rhodopsin (73).…”

Phosphorylation of the Ca2+-Binding Protein CaBP4 by Protein Kinase C ζ in Photoreceptors

“…This result also shows that Ca 2ϩ -induced conformational change does not mediate the decreased phosphorylation of CaBP4 in the presence of Ca 2ϩ . More likely, dephosphorylation of CaBP4 in the dark may be mediated by a Ca 2ϩ -dependent phosphatase (Kutuzov and Bennett, 1996;Sherman et al, 1997) and its phosphorylation promoted by lightdependent activation of PKC.…”

Regulation of the Phosphorylation State of Rhodopsin by Dopamine

“…Rod outer segments from dopamine-treated and control retinas were purified as described (Method 2, above), and their cytosolic fractions (0.6 mg/ml) were used to assay phosphatase activity (1 nM rhodopsin; 22°C for 30 min, during which time 32 P release was linear) (7,31). Radioactive products were separated by SDS-PAGE and analyzed by a PhosphorImager.…”

“…In experiments to test whether ROSs from dopamine-treated retinas contained greater phosphatase activity, purified ROS membranes containing 32 P-phosphorylated rhodopsin were incubated with ROS cytosol from control or dopamine-treated retinas (7,31). Fig.…”