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和AR，2）定义用于GR、TIF 2和STAMP相互作用的相互作用表面，3）鉴定观察到的EC 50、Amax和部分激动剂活性百分比的调节所需的每个因子的结构域（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，孕酮受体（PR）在哺乳动物双杂交测定中与STAMP相互作用（He和Simons Jr.，2007，Mol. Cell.生物学，27，1467-1485）。 因此，PR诱导特性（Amax、部分激动剂活性百分比和EC 50）类似地被STAMP调节并不意外，尽管在相同条件下调节至比GR略低的幅度。 然而，不同的STAMP序列对于与PR和GR的最大相互作用是重要的，这表明PR和GR对STAMP的反应在其他条件下可能引起更显著的差异。 目的2和3通过单独和组合定量GR、TIF 2和STAMP中不同缺失的能力来评估，以保留EC 50、部分激动剂活性百分比和Amax的调节活性。 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，不同地区的STAMP需要的最佳调制的每个诱导属性与GR。此外，显着部分的GR，TIF 2，和STAMP可以删除，而不会在最终的调制活动的重大变化。 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，最近报道TTLL家族蛋白中的重叠区域具有聚谷氨酰胺酶活性（货车Dijk等人，2007，Mol Cell，26，437-448）。 我们已经证实STAMP（TTLL 5）具有多聚谷氨酰胺酶活性，并且除了微管蛋白之外还识别TIF 2。 然而，TIF 2多聚谷氨酰化的水平非常低，并且与STAMP的调节活性无关。 初步数据表明，STAMP还以细胞选择性方式影响细胞的生长行为。 总之，新的辅因子（STAMP）以加和的，有时是协同的方式与共激活因子TIF 2一起改变PR调节的基因表达的几个特性。 因此，STAMP调节AR，GR和PR的转录特性。作为我们正在进行的研究的结果，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百分比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 and ARs, 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, Amax, and percent partial agonist activity (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, progesterone receptors (PRs) interact with STAMP in a mammalian two-hybrid assay (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485). Therefore, it was not unexpected that PR induction properties (Amax, percent partial agonist activity, and EC50) are similarly modulated by STAMP, albeit to a slightly lower magnitude than GR under the same conditions. However, different STAMP sequences are important for maximal interaction with PR vs. GR., which suggests that more significant differences in the responses of PR and GR to STAMP may be elicited under other conditions. 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, percent partial agonist activity, and Amax. 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 optimal modulation of each induction property with GR. Furthermore, significant portions of GR, TIF2, and STAMP can be deleted without major changes in the final modulatory activities. 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 was recently reported that an overlapping region in TTLL family proteins has polyglutamylase activity (van Dijk et al., 2007, Mol Cell, 26, 437-448). We have confirmed that STAMP (TTLL5) has polyglutamylase activity and recognizes TIF2 in addition to tubulin. The level of TIF2 polyglutamylation is very low, though, and unrelated to the modulatory activity of STAMP. Preliminary data indicate that STAMP also affects the growth behavior of cells in a cell-selective manner. In summary, the new cofactor (STAMP) that acts in an additive, and sometimes synergistic, manner with the coactivator TIF2 to alter several properties of PR-regulated gene expression. Thus STAMP modulates the transcriptional properties of AR, GR, and PR. 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 percent 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.