Mechanisms Regulating Subcellular Distribution of the Thyroid Hormone Receptor

College Of William And Mary, Williamsburg VA

Investigators

Abstract

The nucleus forms a discrete compartment in eukaryotic cells. This allows gene expression to be regulated by altering the nucleocytoplasmic distribution of transcription factors in response to external stimuli. This project explores various aspects of the molecular mechanisms regulating nuclear localization and subcellular distribution of the thyroid hormone receptor (TR), a transcription factor, which either activates or represses the expression of its target genes in response to thyroid hormone. Thyroid hormone is essential for normal growth and development as well as for the regulation of a variety of metabolic pathways. Recent studies of TR subcellular trafficking yielded a number of intriguing results: 1) novel alternative pathways exist for TR import in Xenopus oocytes: nuclear entry occurs by passive diffusion or by a signal-mediated process; 2) TR undergoes nucleocytoplasmic shuttling in Xenopus oocytes and mammalian cells, revealing an additional checkpoint in control of gene expression by TR; 3) there is an energy-requiring step in the nuclear retention/export phase of shuttling; 4) a phosphorylated form of TR is present in the nucleus of Xenopus oocytes but not in the cytoplasm, suggesting a role for phosphorylation in TR nuclear localization; and 5) three dominant negative variants of TR, the DNA binding mutant C122A, the transactivation mutant G121A, and the oncoprotein v-ErbA, have distinct subcellular distribution patterns compared with TR. Such dominant negative TR variants provide important, novel tools for tracking the subcellular pathway of TR. In the proposed studies, the following questions will be addressed: 1) Does TR follow both passive diffusion and signal-mediated nuclear import pathways in mammalian cells? Permeabilized cell in vitro nuclear import assays, which allow for controlled reconstitution of nuclear import, will be used to determine the general mechanism of import of TR monomers, homodimers, and heterodimers. 2) Do dominant negative variants of TR that are defective in ligand binding or transactivation associate with specific subcellular compartments? Does interaction with these variants alter the subcellular distribution of TR? These questions will be investigated using green, yellow, and red fluorescent protein (GFP, YFP, and DsRed)-receptor fusions co-expressed in mammalian cells. Colocalization to cytoplasmic compartments and subnuclear domains will be visualized by staining with compartment-specific probes. 3) Does phosphorylation regulate nucleocytoplasmic shuttling of TR? Site-directed mutagenesis studies will be used to investigate the effect of phosphorylation on nuclear localization of TR. Shuttling characteristics of phosphorylation site mutants will be analyzed by Xenopus oocyte microinjection and mammalian cell heterokaryon assays. In summary, these studies will not only increase understanding of the normal cellular response to thyroid hormone, but should also provide important insight into modulation of gene expression through both compartmentalization and dominant negative transcription factors. The proposed research makes use of techniques that, although sophisticated, are quickly mastered by competent undergraduate and graduate students, and will provide the invaluable opportunity for a diversity of students to gain hands-on experience with the scientific process.