The approach of this project is to use mass spectral identification of co-purified proteins to identify factors that can bind to STAMP and may therefore be relevant for STAMP modulation of the EC50 and partial agonist activity in GR-regulated gene expression. This method is also expected to identify proteins that are involved in other, yet unidentified actions of STAMP. This study is being conducted in collaboration with Dr. Sanford Markey (NIMH, NIH). Due to the very low levels of endogenous STAMP (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485), we have overexpressed an exogenous Flag-tagged STAMP, which we demonstrated retains full biological activities. Transient transfections gave high levels of FlagSTAMP and co-purified proteins. However, many of these proteins were suspected 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. Therefore, 293 human embryonic kidney cells with stably transfected FlagSTAMP were isolated and characterized in hopes of obtaining lower and more physiological levels of STAMP but still elevated enough to permit purification from a reasonable number of cells. The level of FlagSTAMP in these cells is much lower than in the transiently transfected cells. Whole cell extracts revealed a different assortment of associated proteins by mass spectrum analysis. STAMP is concentrated in the nucleus, along with GRs, upon addition of a glucocorticoid agonist (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485). Therefore, we are currently purifying STAMP from the nuclei of glucocorticoid-treated cells with stably transfected STAMP and comparing the array of co-purified proteins to those from the whole cell extracts glucocorticoid. Three to five of the most abundant andor interesting co-purified proteins will be examined in co-IP screens with endogenous proteins before moving on to whole cell assays to determine possible biological activities of the putative STAMP-associated proteins.? ? These studies should identify new proteins that participate in, or modify the activity of, STAMP modulation of the EC50 and partial agonist activity in GR-regulated gene expression. 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. Other currently unknown activities of STAMP may also be unearthed.
| Simons Jr, S Stoney (2008) What goes on behind closed doors: physiological versus pharmacological steroid hormone actions. Bioessays 30:744-56 |
