Mechanisms Regulating Subcellular Distribution of the Thyroid Hormone Receptor

College Of William And Mary, Williamsburg VA

Investigators

Abstract

Cells are divided into two major compartments: the nucleus, where genetic information is stored and processed, and the cytoplasm. Trafficking of proteins between compartments is fundamental to the organization and functioning of all living cells. This research explores aspects of the mechanisms regulating traffic control of the thyroid hormone receptor (TR). TR is a transcription factor that turns target genes on or off in response to thyroid hormone. Results from prior NSF support show that: 1) TR enters the cell nucleus by a signal-mediated pathway; 2) Mutant forms of TR often mislocalize in discrete spots or "foci" within the cytoplasm and nucleus; 3) the oncoprotein (cancer-causing) v-ErbA binds to TR and sequesters a significant fraction of TR in the cytoplasm; 4) shuttling of TR between the nucleus and cytoplasm requires an intact cytoskeleton; and 5) compartment-specific modification (phosphorylation) of TR regulates its retention in the nucleus. These findings illustrate how a comprehensive understanding of TR trafficking must take into account not only pathways of nuclear import and export, but the role of the cell architecture in mediating localization, and the targeting of misfolded proteins for degradation. In the next phase of these studies the mechanism of TR nuclear import will be further explored and the role of the cellular architecture in mediating nuclear entry and cytoplasmic sequestration of TR mutants will be investigated. The following questions will be addressed: 1) Does TR follow multiple nuclear import pathways? Does TR harbor a thyroid hormone-dependent nuclear localization sequence (NLS)? Do multiple import receptors (importins) function as nuclear transport receptors for TR? In vitro nuclear import and binding assays and live-cell imaging will be used to map different domains of TR required for interaction with the import machinery. An innovative RNA interference (RNAi) gene-silencing strategy for selective knockdown of expression of different importin genes will be developed. 2) Is the cytoskeleton indirectly or directly required for TR shuttling between the nucleus and cytoplasm and/or turnover? Does TR move through the cytoplasm to the nucleus by diffusion or interaction with the microfilament or microtubule cytoskeleton? Does the cytoskeleton play a role in cytoplasmic anchoring, nuclear transport, and/or targeting of the oncoprotein v-ErbA and other TR mutants to aggresomes for degradation? Interaction of TR, v-ErbA, and TR mutants with the cytoskeleton and aggresomes (cell structures involved in protein degradation) will be assessed by the use of cytoskeletal-disrupting drugs and a combined approach of high resolution live-cell imaging and in vitro binding assays. 3) Does post-translational modification regulate subcellular trafficking of TR? Site-directed mutagenesis will be used to investigate the effect of phosphorylation of specific amino acids on trafficking of TR in model cell systems. Intellectual merit: These studies will increase understanding of the normal cellular response to thyroid hormone, provide important insight into how genes are turned on and off, and enhance general understanding of the dynamic balance between cytoplasmic anchoring, targeting of misfolded proteins for degradation, and directed movement to the nucleus mediated by the cytoskeleton. Broader impacts: Opportunity for a diversity of undergraduates to gain hands-on experience with the scientific process will be provided, including participation in scientific meetings. In the wider context, new techniques will be introduced to the departmental confocal microscope user group, and research activities will be integrated into a lab course taught each Spring. Master's students and the post-doctoral associate will have the opportunity to gain experience in lab management and to develop their teaching skills through training and supervising undergraduates in the lab. A recently established collaboration with a faculty member and undergraduates at Hampton University, an historically minority-serving university, will be continued.