Functional characterization of glucocorticoid receptor sequences required for steroid-induced cell death.
AuthorDieken, Ellen Sue.
AdvisorMiesfeld, Roger L.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractGlucocorticoid induction of cell death (apoptosis) in mouse lymphoma S49 cells has long been studied as a molecular genetic model of steroid hormone action. The research described in this dissertation focuses on understanding the transcriptional control of apoptosis in two steroid resistant ntⁱ S49 mutant cell lines (S49.55r and S49.143r). These studies extend earlier biochemical analysis of the mutant ntⁱ glucocorticoid receptor (ntⁱ GR) to the molecular level by isolating and characterizing GR cDNA from the two ntⁱ S49 cell lines, S49.55r and S49.143r, and the wild type (wt) parental line, S49.A2. This analysis revealed that ntⁱ GR transcripts encode intact steroid and DNA binding domains but lack 404 amino-terminal (N-terminal) residues as a result of aberrant RNA splicing between exons 1 and 3. Results from transient co-transfection experiments into CV1 cells using ntⁱ receptor expression plasmids and a glucocorticoid responsive reporter gene demonstrate that the truncated ntⁱ receptor is capable of inducing transcription to only 10% the level of wt GR. Gene fusions containing portions of both the wt and ntⁱ GR coding sequences were constructed and used to functionally map the ntⁱ receptor mutation. It was found that the loss of the N-terminal domain alone is sufficient to cause the observed defect in ntⁱ transcriptional transactivation. In addition, a complementation assay utilizing stable transfection of wt and mutant GR cDNA constructs into a GR-deficient cell line (7r) was developed. By measuring steroid-sensitivity of various 7r derivatives, it was determined that GR is rate-limiting for S49 apoptosis and moreover, that the GR N-terminus is absolutely required for complementation in this system. These data also indicate that at physiological levels of receptor, the GR N-terminus plays a crucial role in controlling lymphocyte apoptosis, even though this portion of the receptor seems to be dispensable for low-level induction of mouse mammary tumor virus (MMTV) transcription. The smallest functionally defined transactivation region in the GR N-terminus has a net negative charge and has been named enh2, tau1 or acidic activation domain (AAD). Results from experiments designed to test whether similar activating sequences from the herpes simplex virus-1 (HSV-1) VP16 protein can substitute for the GR N-terminus demonstrate that 7r cells expressing VP-GR fusions are indeed steroid-sensitive, suggesting that S49 apoptosis requires transcriptional induction of specific genes.