PIGMENT DEPHOSPHORYLATION IN MAMMALIAN ROD AND CONE PHOTORECEPTORS

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ABSTRACT The state of phosphorylation mediates the activity of many G protein-coupled receptors. In photoreceptors, the phosphorylation of the visual pigment has been well characterized. However, an often-overlooked step in the resetting of the visual pigment following its photoactivation is its dephosphorylation. Despite remarkable progress in understanding G protein signaling in general, and phototransduction in particular, the enzyme responsible for catalyzing this reaction in vivo remains unknown. Also unknown is the role of visual pigment dephosphorylation in modulating the kinetics of dark adaptation or the susceptibility of photoreceptors to light damage. We have generated mice with loxP-flanked catalytic α-subunit of PP2A (Ppp2cafl/fl) which will allow us to investigate the role of this enzyme in pigment dephosphorylation and the function and survival of mammalian photoreceptors. Crossing these animals with mice expressing Cre recombinase selectively in rod or cone photoreceptors has allowed us to generate rod- and cone-specific PP2A Cα knockout mice. We will perform experiments to determine whether pigment dephosphorylation is suppressed in these mice. These experiments will establish whether PP2A is the elusive pigment phosphatase in mammalian rods and cones. We will also determine how the deletion of PP2A Cα affects the expression profile, morphology, and survival of photoreceptors. We will also analyze the light responses of PP2A Cα-deficient mouse rods and cones to determine the role of PP2A in modulating phototransduction. By in vivo ERG recordings, we will also determine whether the deletion of PP2A affects the kinetics of photoreceptor pigment regeneration and dark adaptation. These experiments will establish the role of pigment dephosphorylation in regulating the dark adaptation of mammalian rods and cones. The link between pigment phosphorylation and photoreceptor degeneration will also be explored using a combination on mutant mice lacking arrestin, rhodopsin kinase, and PP2A. Finally, we will also test the hypothesis that light-induced phosphorylation of ground state cone visual pigment reduces the overall light sensitivity and represents a novel mechanism for mammalian cone background light adaptation. Collectively, our experiments will establish the molecular mechanism for visual pigment dephosphorylation in mammalian photoreceptors. They will also determine the role of pigment dephosphorylation in controlling the kinetics of mammalian rod and cone dark adaptation, as well as the therapeutic potential of modulating this enzymatic reaction in light- or opsin-induced retinal degeneration.