Progesterone receptor nucleocytoplasmic trafficking and applications in drug delivery.

Nucleocytoplasmic trafficking of steroid hormone receptors is a conundrum that remains to be solved. The mechanism of nuclear import has been established, whereas the mode of nuclear export is unknown. Steroid receptors are vital to cell functioning and are involved in several signal transduction pathways. These receptors are also promising pharmacological targets as they are implicated in a number of diseases. Identifying the means of nucleocytoplasmic shuttling is important in understanding the molecular basis of functioning of these receptors and their potential use in therapeutics. The research presented here investigated the mechanism of nucleocytoplasmic shuttling of steroid hormone receptors. The knowledge of signal mediated transport was then applied to construct a regulatable protein switch with potential use in the field of gene therapy. The first half of the dissertation describes the search for the export mechanism. A model system using Enhanced Green Fluorescent Protein (EGFP) was established to examine classical nuclear export signals. The most well-known export pathway is mediated by a karyopherin called Crm1 and was the starting point of this investigation. The role of a protein called Calreticulin that binds to the DNA binding domain of these receptors was also tested. In addition, the involvement of heat shock proteins (HSPs) that bind to these steroid receptors at their ligand binding domain was investigated for potential in mediating export. Results could not rule out the possibility of Crm1 and Calreticulin being involved in the transport pathway but showed that the LBD determines the subcellular localization of receptors and also that HSPs may be involved in export. In the second half of this study, signal sequences, regulated by an external stimulus, were used to facilitate or block the localization of a delivered protein to its active compartment. A bidirectional on/off switch was constructed with nuclear export signals, import signals, and a truncated PRLBD, which could be regulated by an external ligand, mifepristone. This work describes a prospective direction for search of proteins responsible for steroid receptor nuclear transport and established a method to regulate protein function by altering its subcellular localization.