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

该项目的四个目标是：1)确定STAMP的调节作用是否仅限于GR； 2)确定GR、TIF2和STAMP相互作用中使用的相互作用表面；3)确定所观察到的EC50和部分激动剂活性的调节所需的每个因子的结构域，以及总基因诱导或Vmax的变化（He和Simons Jr., 2007, Mol. Cell.）。医学杂志。（27,1467 -1485），以及4)查找STAMP的其他活动。关于第一个目标，雄激素受体（ARs）在哺乳动物双杂交实验中显示出与STAMP最强的相互作用（He and Simons Jr., 2007, Mol. Cell.）。医学杂志。， 27, 1467-1485)。因此，我们正在测试STAMP对瞬时转染CV-1猴肾细胞和LNCaP人前列腺肿瘤细胞中ar调控基因表达的EC50、部分激动剂活性和Vmax的调节能力。改变AR转录特性的能力可能提示STAMP在前列腺癌的进展和治疗中发挥作用。目的2和3是通过量化GR、TIF2和STAMP中不同缺失的能力来评估的，无论是单独的还是联合的，以保持对EC50、部分激动剂活性和Vmax的调节活性。ChiP-reChIP （He and Simons Jr., 2007, Mol. Cell.）医学杂志。研究报告，27,1467 -1485)和co-IP实验支持GR， TIF2和STAMP通过包含所有三个因素的复合物起作用的模型。初步数据表明，调制EC50和Vmax需要不同的STAMP区域。最后，Aim 4试图确定STAMP是否调解任何其他操作。STAMP中唯一可识别的结构域是酪氨酸微管蛋白连接酶样（TTL）结构域，这不是STAMP调节活性所必需的（He and Simons Jr., 2007, Mol. Cell.）。医学杂志。， 27, 1467-1485)。然而，作为一个由1277个氨基酸组成的蛋白质，STAMP不可能不具有与TTL或调节活性无关的许多区域相关的附加功能。初步实验表明，我们可能已经发现了一个额外的活动，这是通过确定所需的STAMP区域和目标分子的性质来追求的。

The four aims of this project are 1) to determine whether the modulatory actions of STAMP are limited to GRs, 2) to define the interaction surfaces that are used in the interactions of GR, TIF2, and STAMP, 3) to identify the domains of each factor that are needed for the observed modulation of EC50 and partial agonist activity in addition to changes in total gene induction, or Vmax (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485), and 4) to search for other activities of STAMP. With regard to the first aim, androgen receptors (ARs) display the strongest interaction with STAMP in a mammalian two-hybrid assay (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485). Therefore, we are testing the ability of STAMP to modulate the EC50, partial agonist activity, and Vmax, of AR-regulated gene expression in transiently transfected CV-1 monkey kidney cells and LNCaP human prostate tumor cells. An ability of modify AR transcriptional properties would suggest a role for STAMP in the progression and treatment of prostate cancer. Aims 2 and 3 are being assessed by quantifying the ability of different deletions in GR, TIF2, and STAMP, both separately and in combination, to retain the modulatory activity for EC50, partial agonist activity, and Vmax. ChiP-reChIP (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485) and co-IP experiments support a model where GR, TIF2, and STAMP act via a complex containing all three factors. Preliminary data indicate that different regions of STAMP are required for the modulation of EC50 and Vmax. Finally, Aim 4 seeks to determine whether STAMP mediates any other actions. The only identifiable domain in STAMP is a tyrosine tubulin ligase-like (TTL) domain, which is not required for STAMP modulatory activity (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485). However, it is unlikely that STAMP, a protein of 1277 amino acids, does not have additional functions associated with the many regions not associated with TTL or modulatory activity. Preliminary experiments suggest that we may have discovered an additional activity, which is being pursued by identifying the region of STAMP required and the nature of the target molecules. In summary, we have identified a new cofactor (STAMP) that acts in an additive, and sometime synergistic, manner with the coactivator TIF2 to alter several properties in both major aspects of GR-regulated gene expression: induction and repression. As a result of our on-going studies, we have gained new molecular information about the modulation of the dose-response curve and total activity of agonists and the partial agonist activity of antisteroids by cofactors. These modulatory factors 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.