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A novel approach to investigate the subcellular distribution of nuclear receptors in vivo

By Marko Matic, Sarah Nakhel, Anne M. Lehnert, Patsie Polly, Stephen J. Clarke and Graham R. Robertson

Abstract

Subcellular compartmentalisation and the intracellular movement of nuclear receptors are major regulatory steps in executing their transcriptional function. Though significant progress has been made in understanding these regulatory processes in cultured mammalian cells, such results have rarely been confirmed within cells of a living mammal. This article describes a simple, time-efficient approach to study the nuclear versus cytoplasmic accumulation of nuclear receptors and the regions of nuclear receptor proteins that govern subcellular trafficking within hepatocytes of live mice. Pregnane X receptor, a xenobiotic-activated member of the nuclear receptor family, was used to exemplify the approach. Using dual-labeled wild-type and mutant PXR expression constructs, we outline their in vivo delivery, simultaneous cellular expression, visualization and categorical classification within hepatocytes of live mice. Using this approach, we identified three mutants that had an altered subcellular distribution in the presence and absence of a PXR ligand. This novel in vivo method complements the current cell culture-based experimental systems in protein subcellular localisation studies

Topics: Methods
Publisher: The Nuclear Receptor Signaling Atlas
OAI identifier: oai:pubmedcentral.nih.gov:2686083
Provided by: PubMed Central

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