Light Dependent Membrane Association of PRL–1, a Phosphatase Expressed in Cone Photoreceptors

Abstract

Abstract: : Purpose: To describe the light dependent subcellular localization of PRL–1 (Phosphatase of Regenerative Liver–1) in cone photoreceptors. Methods: Light– and dark–adapted porcine photoreceptor outer segment (OS) samples were prepared essentially as described by Papermaster. Cultures of the murine cone photoreceptor–derived, 661W, cells, were grown to 85∼90% confluence, starved overnight and either kept dark or exposed to varying durations of light exposure (up to 4 hours, 700 Lux). Cells were harvested and subfractionated into cytosolic (C), membrane (M), nuclear (N) and cytoskeleton (CK) fractions using a ProteoExtract S–PEK Kit. The distribution of PRL–1 was analyzed on Western blots of light– and dark–adapted porcine OS and subcellular fractions of light– and dark–exposed 661W cells labeled with affinity–purified anti–PRL–1 antibodies. Results: PRL–1 localized primarily within light– and dark–adapted porcine OS samples and within the 661W cell C and M fractions, but not the N and CK fractions. Densitometric analysis of PRL–1 immunopositive bands revealed a statistically significant light–associated increase in the amount of PRL–1 in the light–adapted cone OS enriched fractionII as well as light–exposed 661W cell membranes. Conclusions: Our studies have demonstrated PRL–1 protein is localized in photoreceptor OS and of 661W cell membranes, in a light–dependent manner, which is unrelated to its nuclear role in other non–neuron cells. We are testing whether PRL–1 plays a regulatory role through dephosphorylation in a cone signal transduction pathway.

Keywords: photoreceptors • signal transduction • protein structure/function