Phosphorylation of Cone Opsins and Binding to Cone Arrestin During Phototransduction in Cone Photoreceptors of Mouse Retina

Abstract

Abstract: : Purpose: The shutoff of the rod visual transduction cascade by GRK1 phosphorylation of light-activated rhodopsin followed by rod arrestin (SAG) binding has been well established; however, deactivation of the cone phototransduction pathway is still not delineated due, in part, to the low abundance of cone photoreceptors in the mammalian retina. We sought to address the potential mechanisms underlying the quenching of the cone phototransduction cascade using animal models rich in cones and/or lacking GRK1. Methods: Nrl-/-Grk1-/- double knockout (KO) mice were created by breeding the Nrl-/- mice (Mears, A.J. et al., 2001. Nature Genet. 29:447-452) with the Grk1-/- mice (Chen, C.-K. et al., 1999. Proc Natl Acad Sci 96:3718-3722). Retinas were dissected from dark-adapted mice under infrared light. For the phosphorylation and cone arrestin (CAR) binding/immunoprecipitation experiments, retinas were 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 bright sunlight (88,000 Lux) for 10 min before processing. Phosphorylation, isoelectric focusing (IEF), immunoblotting, immunohistochemistry and immunoprecipitation were used to examine the retinas. Results: In situ phosphorylation and immunoprecipitation experiments in the Nrl-/- and Nrl-/-Grk1-/- mouse retinas demonstrate that both S and M opsins are phosphorylated at multiple sites following light exposure and that GRK1 is the responsible kinase. Immunofluorescent labeling of mouse retinal sections with GRK1 and mCAR antibodies reveals expression of both proteins in all S and M cones in both wild type and Nrl-/- mouse retinas. Immunoblotting and immunoprecipitation studies reveal specific binding of mCAR to light-activated, GRK1-phosphorylated cone opsins. Intriguingly, immunoreactive SAG is co-expressed with mCAR in all photoreceptors that are functionally cones in the Nrl-/- mouse retina; however, it is not associated with either S or M opsin either in the light or in the dark. Conclusions: Our results demonstrate, for the first time in the mammalian retina, that cone opsin phosphorylation and CAR binding are key components involved in the shutoff mechanisms of the cone phototransduction cascade following light-activation.

Keywords: opsins • phosphorylation • signal transduction