The long-term objective of this study is to understand the cellular processing of steroid receptors, identifying steps that might be target for intervention in steroid-dependent disease, such as breast cancer, or for potentiation of steroidal therapies. Transcriptional activation by steroid receptors has been studied intensively but processing of receptors prior to their interaction with the transcriptional machinery is less well understood. Recent studies in vitro have suggested several interactions between steroid receptors and components of the protein folding/assembly machinery; in most cases, the relevance of these interactions to steroid receptor function has not been shown. The following hypothesis is to be tested here: A protein component, FKBP54, of progesterone receptor complexes modifies structural features of receptor that are important for hormone binding or hormone-dependent receptor activation. FKBP54 (54 kDa FK506 Binding Protein) is a novel immunophlinin, protein targets for FK506, cyclosporine, and other immunosuppressant drugs; immunophilins are also important components of protein folding/assembly pathways. FKBP54 was first identified in progesterone receptor (PR) complexes. A closely related protein, FKBP52, (p59 or hsp56), was previously identified in PR and other steroid receptor complexes, however, the binding of FKBP54, unlike FKBP52 or hsp90, is highly sensitive to progesterone in vitro, implicating FKBP54 in hormone-related structural changes of PR.
These Specific Aims will address whether FKBP interaction is important to normal PR function: 1. Clone and sequence both chicken and human cDNAs for FKBP54 2. Prepare recombinant FKBP54 for functional analyses and monoclonal antibody production 3. Using a cell-free system for assembly of PR complexes, define in vitro the role of FKBP54 in hormone binding and hormone-dependent dissociation of PR complexes 4. Using cDNA deletion mutants for chick PR and FKPB54, define PR-FKBP54 interaction sites. Learning the role of FKBP54 interaction with PR may add an important facet to our understanding of steroid receptor biology. It might also lead to novel pharmacological alternatives or enhancements in reproductive medicine or treatment of steroid-dependent disease.
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