The long-term goal of this research is to determine the mechanisms underlying eukaryotic gene regulation. Human progesterone receptors (PR), members of the nuclear receptor superfamily of ligand-activated transcription factors, serve as a model system. PR co-exist as two functionally distinct isoforms: an 83 kD A-receptor and a 99 kD B-receptor. The two isoforms are identical except that the B-receptor has an additional 164 amino acids at its N-terminus. Functional differences seen between the two proteins may be due to differential interactions with transcriptional coactivating proteins such as AIB1 (Amplified in Breast Cancer-1). Our studies indicate that residues unique to the B-receptor allosterically modulate recruitment of AIB1 to the promoter complex, thus modulating isoform-specific function. Further, these interactions are coupled to dramatic increases in structural stability due to large-scale disorder to order folding transitions. The goal of this proposal is to carry out a biophysical and structural dissection of the interactions between PR isoforms and the coactivating protein, AIB1, on a PR-regulated promoter. Aim 1a: We will resolve the microscopic free energy contributions associated with isoform-specificAIB1 recruitment using quantitative DNase footprint titration. Aim 1b: We will determine the molecular forces responsible for recruitment by assessing the physical and chemical contributions to receptor-coactivator assembly. Aim 2a: The detailed structural makeup of any intact receptor, coactivator, or receptor-coactivator complex is unknown. We will identify receptor-coactivator-promoter complexes amenable to high-resolution structural approaches using limited proteolysis, mass spectrometry, and analytical ultracentrifugation. Aim 2b: Identified complexes will be assessed structurally using NMR spectroscopy and crystallographic approaches. Together, these studies will contribute to the long-term objective of directly connecting structure and energetics in order to generate quantitatively predictive models of function. This proposal is focused on understanding the fundamental mechanisms by which human proteins (progesterone receptors) regulate gene expression. Elucidation of these mechanisms should eventually allow for the development of drugs that can modulate receptor function. These drugs should then have an impact on hormone dependent diseases such as breast or endometrial cancer.

R21DK071652

2007-07-01

2010-06-30