Mechanism of action of STAMP - a new comodulator of glucocorticoid receptors

点击翻译按钮获取中文摘要

The three aims of this project are 1) to define the interaction surfaces that are used in the interactions of GR, TIF2, and STAMP, 2) to identify the domains of each factor that are needed for the observed modulation of EC50, Amax, and percent partial agonist activity (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485), and 3) to search for other activities of STAMP. Aims 1 and 2 are being assessed by quantifying the ability of different deletions in GR, TIF2, and STAMP, both separately and in combination, to form a ternary complex and to retain the modulatory activity for EC50, percent partial agonist activity, and Amax. Co-IP/re-co-IP experiments demonstrate that GR, TIF2, and STAMP are all present in the same ternary complex. Furthermore, the use of truncated proteins revealed that the smallest fragments of each protein that still gives a ternary complex displays the same EC50, percent partial agonist activity, and Amax for induction of an exogenous reporter gene as does the GR fragment alone. Thus, the added core fragments of TIF2 and STAMP are devoid of modulatory activity with the core GR fragment. This means that the modulatory activities of the full length ternary complex are due to some of the regions that have been deleted from the core fragments. Adding back successive portions of each core protein fragment revealed that specific protein domains encode selected activities. This argues that the determinants of EC50, percent partial agonist activity, and Amax are separable and can proceed via different pathways. These data constitute the strongest support to date of our previous hypothesis that studies of EC50 and/or percent partial agonist activity will yield mechanistic information that is inaccessible from the conventional studies of just Amax. Aim 3 asks whether STAMP mediates any other actions. In fact, we find that changing the level of STAMP (by overexpression of transfected plasmid or by reducing the endogenous protein with siRNAs) alters the growth behavior of cells in a cell-selective and steroid-independent manner. Screening of a variety of cancers uncovered a strong positive correlation between the presence of ovarian cancers and elevated STAMP mRNA levels. Given the lack of early detection methods for ovarian cancer, and the poor survival rate of patients with later stage ovarian cancers, it appears that screening for increased STAMP mRNA could be a useful diagnostic test for early stage ovarian cancers. In summary, we have gained new, hitherto unrealized, molecular information about the modulation of the EC50, percent partial agonist activity, and Amax in steroid receptor-regulated gene induction by modulatory factors. These mechanisms suggest the possibility of a continuum of responses for each parameter, thereby identifying new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. At the same time, STAMP is found to have significant effects on the growth of many cells and may be intimately related to the appearance of ovarian cancers. 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.