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

该项目的四个目的是1）确定STAMP的调节作用是否限于GR，2）定义用于GR、TIF 2和STAMP相互作用的相互作用表面，3）鉴定除了总基因诱导的变化之外，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，或Vmax（He和Simons Jr.，2007，Mol. cell.生物学，27，1467 - 1485），和4）寻找其他活动的邮票。 关于第一个目的，雄激素受体（AR）在哺乳动物双杂交测定中显示出与STAMP最强的相互作用（He和Simons Jr.，2007，Mol. cell.生物学：27，1467 - 1485）。 因此，我们正在测试STAMP调节瞬时转染CV-1猴肾细胞和LNCaP人前列腺肿瘤细胞中AR调节基因表达的EC50、部分激动剂活性和Vmax的能力。 改变AR转录特性的能力将表明STAMP在前列腺癌的进展和治疗中的作用。 目的2和3通过单独和组合定量GR、TIF 2和STAMP中不同缺失保留EC50、部分激动剂活性和Vmax的调节活性的能力来评估。 ChiP-reChIP（他和小西蒙斯，2007，Mol. cell.生物学，27，1467 - 1485）和co-IP实验支持GR、TIF 2和STAMP经由含有所有三种因子的复合物起作用的模型。 初步数据表明，不同区域的STAMP需要EC50和Vmax的调制。 最后，目标4试图确定STAMP是否介导任何其他行为。 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，其不是STAMP调节活性所必需的（He和Simons Jr.，2007，Mol. cell.生物学，27，1467 - 1485）。 然而，它是不可能的，STAMP，1277个氨基酸的蛋白质，不具有与TTL或调节活性无关的许多区域相关的额外功能。 初步实验表明，我们可能已经发现了一种额外的活性，这是通过确定所需的STAMP区域和靶分子的性质来实现的。 总之，我们已经确定了一种新的辅因子（STAMP），它以加性的，有时是协同的方式与共激活因子TIF 2一起改变GR调节基因表达的两个主要方面的几个特性：诱导和抑制。 由于我们正在进行的研究，我们已经获得了新的分子信息的调制的剂量-反应曲线和总活性的激动剂和部分激动剂活性的抗类固醇辅因子。 这些调节因子允许一个连续的反应，并构成新的治疗目标的差异控制基因表达的类固醇激素在发展，分化，稳态和内分泌治疗。 这些综合研究结果有助于我们在分子水平上定义类固醇激素的作用并了解其在人体生理学中的作用的长期目标。

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.