Studies of hormonal signalling have fundamental implications for the molecular basis of organ physiology, embryonic development, cell differentiation, and human disease. The goal of this proposal is to establish a molecular basis for the interaction for steroid and related receptors with proto-oncogene signalling pathways. We have reported that AP-1 is able to block induction by the glucocorticoid and retinoic acid receptor and reciprocally, that glucocorticoids and retinoids are able to inhibit activation of promoters containing AP-1 sites. In this proposal, we will utilize the yeast two hybrid interaction screen to identify nuclear proteins that may mediate AP-1 and glucocorticoid receptor cross-talk. This will be based on a sequential screen isolating interacting clones for one protein and rescreening against the other to identify the unique subsets specific for both. Full-length cDNA clones will be isolated for dually reactive proteins and antibodies to these newly cloned factors will be prepared. These proteins will be subjected to extensive truncation and deletion analysis to map sites of interaction with the receptors utilizing co-immunoprecipitation in vitro as a rapid assay. In addition, functional studies will be initiated using co- transfection based assays. We have recently demonstrated that CBP may represent a common co-factor for both CREB and the glucocorticoid receptor. These studies will be extended to determine whether or not this may also serve as a putative co-factor mediating retinoid signalling. Assays including mobility shifts, microinjection of anti-CBP antibody into cultured cells and co- transfection will be used to characterize these regulatory interactions. Utilizing deletion and truncation mutants, the sites of interaction will be localized. As with AP-1, we have shown that p65/cREL inhibits the activity of the glucocorticoid receptor. We will characterize the functional interactions of p50/p65 (NF-kappaB) with the GR and if successful, we will extend these studies to a similar analysis with the RAR. GR interacting clones from the yeast two hybrid screen will be rescreened with NF-kappaB. Full-length clones for these co-interacting proteins will be isolated, sequenced and antibodies will be prepared. Alternatively, proteins being characterized as associated with NF-kappaB complexes and the activation of these complexes will be directly screened utilizing co- immunoprecipitation, cross-linking, and mobility shift assays for their association with the GR. In summary, the work in this proposal focuses on an alternative route for steroid hormone action mediated by the hormone dependent association of the receptors with factors or co-factors that serve as critical mediators for alternative signalling pathways. The work in this proposal will provide a molecular basis for this hormonal signalling pathway.
