Our previous approach of using mass spectral identification of co-purified proteins to identify factors that bind to STAMP was thwarted by the very low levels of endogenous STAMP. Transient transfections of Flag/STAMP plasmid did give high levels of overexpressed Flag/STAMP and co-purified proteins. However, most of these proteins appeared to be non-specifically bound and were not among the known associated proteins of TIF2, SRC-1, and GR. Quantitative Western blot analysis of the purified STAMP indicated that only 0.4% of the STAMP was associated with GRs in these samples. Stably transfected 293 human embryonic kidney cells did afford lower and more physiological levels of STAMP and a different assortment of associated proteins by mass spectrum analysis. However, no co-purified protein appeared to be physiologically interesting. In view of the limited results using mass spectral identification of co-purified proteins, we have concentrated on using microarray data from cells with and without STAMP to identify possible binding proteins. As described elsewhere (DK047039-04), we have recently prepared mouse embryo fibroblasts (MEFs) from wild type mice and mice in which the endogenous STAMP gene has been knocked out (KO mice). Microarray analyses have been performed on cells that were induced by glucocorticoid steroid for 8 hr. High quality data were obtained for almost 3,000 genes, for which the level of expression changed by ≥1.5 fold after Dex treatment. Those genes with the largest change in expression between wild type and KO MEF cells are being examined for the involvement of other known cofactors. Our hypothesis is that STAMP interaction with these known cofactors may be responsible for the significant change in glucocorticoid-regulated gene expression in the STAMP KO MEF cells. Those genes for which there are major differences in basal level expression are candidates for mediating the apparent steroid-independent effects of STAMP, such as cell growth and tumor formation. Initially identified cofactors will be further examined for STAMP binding by co-immunoprecipitation and for cofactor activity in gene induction assays using transiently transfected cells. These studies should identify new proteins that not only participate in, or modify the activity of, STAMP modulation of the EC50 and PAA in GR-regulated gene expression but also affect cell growth. These results will increase our understanding of several physiologically relevant transcriptional properties of GR-steroid complexes that permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.
