The broad view f this research is to better understand mechanisms of action of steroid receptors and their role in hormone dependent diseases such as breast cancer. More specifically, this grant will investigate the effects of progestin agonists and antagonists on the structure and function of the ligand binding domain (LBD) of the human progesterone receptor (PR).
Aim #1 will use the baculovirus system to express PR-LBD which is capable of binding steroids in a manner similar to full length PR and will establish the optimal expression vector for high levels of expression for subsequent purification of PR-LBD.
Aim #2 will determine whether PR-LBD undergoes distinct conformational changes induced by agonist and antagonist, as detected by partial proteolysis, differential antibody recognition and hydrophobic phase chromatography.
Aim #3 will study the effects of agonists and antagonists on the ability of PR-LBD to bind heat shock protein 90 (hsp90) and homodimerize, two distinct function of ligand-dependant activation of PR. Binding of hsp90 will be studied by co-immunoprecipitation of labeled PR-LBD with antibodies to hsp90, while homodimerization will be investigated by binding non-fusion PR-LBD to histidine-tagged PR-LBD by nickel-chelation affinity resins in a homogenous and biologically active form suitable for future crystallographic studies. The findings from these experiments will further our understanding of the effects of agonists and antagonists on the function and structure of the PR-LBD and could ultimately aid in the design of more effective antiprogestin for the treatment of hormone dependent diseases.
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| Tetel, M J; Jung, S; Carbajo, P et al. (1997) Hinge and amino-terminal sequences contribute to solution dimerization of human progesterone receptor. Mol Endocrinol 11:1114-28 |
