We are interested in studying two structural features of steroid receptors that have been implicated to be important in controlling functional activities; formation of stable non-covalent complexes with heat shock proteins (hsps), and covalent modification by phosphorylation. Studies will be conducted with human progesterone receptors (PR) of the T47D breast cancer cell line. Anti-receptor monoclonal antibodies (MAbs) will be used for immunoisolation and direct detection of receptor protein. The present application proposes the following studies; 1) We plan further characterization of receptor-hsp complexes shown in preliminary studies to be composed of steroid binding subunits and one (hsp90) or possibly a second (hsp70) heat shock protein. Different native forms of immunoisolated receptor complexes will be characterized for; protein composition, hydrodynamic properties, chemical nature of hsp-PR binding interactions, effect of heat shock on formation of these complexes and, identification of hsp isoforms bound to receptors. 2) Possible functional activities of receptor-hsp complexes will be investigated. The hormone-dependence of hsp90- association with receptors will be tested in vivo. Alteration of hsp-receptor association through cellular stress will be evaluated for effects on receptor function and the ability of microinjected anti-hsp antibodies to block receptor activity in living cells will be tested. Since hsp70 is an ATP binding protein we plan also to measure and characterize ATP binding to immunoisolated receptor complexes, determine ATP dependence of complex formation and the ability of these complexes to hydrolyze ATP. 3) The role of hormone dependent receptor phosphorylation on receptor binding to the progesterone responsive enhancer element (PRE) of MMTV will be explored. Two approaches will be taken: a) receptors will be phosphatase treated in vitro and changes in DNA binding properties related to removal of phosphate, b) different phosphoforms of receptors bound to PRE will be identified by two-dimensional gel electrophoresis. 4) Several of the above studies will be repeated with receptors bound with the potent progestin antagonist, RU 486. Identification of structural alterations in receptor. RU 486 complexes may provide important additional information on structure-function relationships. The overall goal of this proposal is to attempt to relate specific structural features of steroid receptors that might be responsible for regulating functional activities.
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