Cone Phototransduction Shutoff by Phosphorylation and Cone Arrestin Binding

Abstract

Abstract: : Purpose: Shutoff of the rod visual transduction cascade by rod arrestin binding to light-activated, phosphorylated rhodopsin has been well studied; however, deactivation of the cone phototransduction cascade is still not delineated. We sought to explore the potential mechanisms underlying the quenching of the phototransduction cascade in cone photoreceptors. Method: Retinas were dissected from dark-adapted C57 adult mice under infrared light and incubated for 30 min in phosphate-free Krebs buffer containing 1.2 mCi/ml of [₃₂P]H₃PO₄ in the dark. After washing, retinas were either left in the dark or exposed to room light for 10 min before homogenization. Newly created peptide antibodies against mouse M- and S-cone opsins were tested by immunoblot and immunohistochemical analysis and used to immunoprecipitate cone opsins and their associated proteins from the dark and light retinal homogenates. Embryonic day 19 chicken retinal outer segments (OS) were isolated and used for in vitro cone opsin phosphorylation with recombinant GRK1 or 7 and for arrestin binding assays. [₃₅S]-Labeled in vitro-translated arrestins were used in the binding assays. Results: Immunoblot and immunohistochemical analysis demonstrate that both the M- and S-cone opsin antibodies are specific for their respective mouse cone opsin. Immunoprecipitation with either antibody selectively pulled down phosphorylated bands of the appropriate cone opsin monomer, dimer and trimer in light-exposed retinas but not in retinas kept in the dark. Furthermore, only in the light-exposed retinas was cone arrestin co-immunoprecipitated with phosphorylated cone opsins. In vitro experiments with OS membranes confirmed the light-dependent phosphorylation of cone opsins by GRK1/7 and both light- and phosphorylation-dependent binding of cone arrestin to cone opsins. Conclusion: Our results demonstrate, for the first time, that cone arrestin binds to photo-activated, phosphorylated cone opsins both in vivo and in vitro, implying an important signal shutoff function of cone arrestin in the cone phototransduction cascade.

Keywords: 580 signal transduction • 517 photoreceptors • 515 phosphorylation