Heterodimerization between GR and a New Accessory Factor

已发现类固醇激素（包括肾上腺糖皮质激素）受体的分子机制具有迷人的多样性，这些激素调节许多重要的代谢、发育、免疫和炎症过程。 在类固醇作用的经典模型中，糖皮质激素与靶细胞中的糖皮质激素受体（GR）结合。 GR然后作为同源二聚体与细胞核中的DNA位点相互作用，并诱导附近基因的转录。 我们已经发现了一种根本不同的作用模式，其中GR单体与第二种DNA结合蛋白异二聚化。 虽然机制涉及拴系三元复合物众所周知，这是第一次描述，据我们所知，任何成员的核受体超家族形成一个异源二聚体与无关的蛋白质，与双方的合作伙伴结合的DNA。 这种机制控制着非洲爪蟾中纤维蛋白原γ亚基基因的激活，纤维蛋白原是主要的凝血蛋白。 异源二聚化伴侣，爪蟾糖皮质激素受体辅助因子（XGRAF），结合到一个DNA序列，以前不知道作为一个蛋白质结合位点。 初步数据表明，XGRAF属于基本的螺旋-环-螺旋类转录因子。 这项研究的总体目标是了解GR和XGRAF如何共同激活转录以响应激素。 实验策略是回答三个基本问题：A）XGRAF的身份是什么？ B）GR如何与XGRAF物理交互？ C）XGRAF和GR的作用背后的DNA：蛋白质相互作用的完整补充是什么？ 这些问题将通过以下具体目标来解决：1）通过质谱和蛋白质组学的强大方法来鉴定XGRAF。 2)分离XGRAF cDNA克隆。 3)确定XGRAF：GR协同结合DNA所需的GR中的特定氨基酸。 4)确定GR和XGRAF是否在不存在DNA的情况下异源二聚化。 5)鉴定对诱导转录至关重要的核心GR和XGRAF结合位点侧翼的所有核苷酸。 这一机制对激素作用领域具有重要意义，因为它揭示了非受体蛋白调节类固醇反应的新途径。 此外，由于受体结构在与XGRAF相互作用的GR区域中高度保守，因此类似的调节模式可能适用于其他动物中的GR以及其他核受体。 这项研究对健康和疾病具有重要意义，因为糖皮质激素广泛用于许多常见的免疫炎症性疾病，如关节炎，哮喘和过敏，但由于这些类固醇几乎作用于所有细胞，因此治疗受到严重影响的阻碍。 靶向治疗是必要的，我们的工作表明，针对特定基因的专门调控元件可以提供一种选择性调节某些可诱导基因表达的方法，同时使其他基因不受影响。

A fascinating diversity of molecular mechanisms has been found for the receptors of steroid hormones, including the adrenal glucocorticoids, which regulate many vital metabolic, developmental, immune, and inflammatory processes. In the classical model of steroid action, glucocorticoids bind to the glucocorticoid receptor (GR) in target cells. GR then interacts as a homodimer with DNA sites in the nucleus and induces transcription of nearby genes. We have discovered a fundamentally different mode of action in which a GR monomer heterodimerizes with a second DNA-binding protein. Although mechanisms involving tethered ternary complexes as well known, this is the first description, to our knowledge, of any member of the nuclear receptor superfamily forming a heterodimer with an unrelated protein, with both partners bound to the DNA. This mechanism controls activation of the gene for the gamma subunit of fibrinogen, the major blood-clotting protein, in Xenopus frogs. The heterodimerization partner, Xenopus glucocorticoid receptor accessory factor (XGRAF), binds to a DNA sequence not previously known as a protein binding site. Preliminary data suggest that XGRAF belongs to the basic helix-loop-helix class of transcription factors. The overall goal of this research is to understand how GR and XGRAF together activate transcription in response to hormone. The experimental strategy is to answer three basic questions: A) What is the identity of XGRAF? B) How does GR physically interact with XGRAF? C) What is the full complement of DNA: protein interactions underlying the action of XGRAF and GR? These questions will be addressed by the following specific aims: 1) Identify XGRAF by powerful methods of mass spectrometry and proteomics. 2) Isolate XGRAF cDNA clones. 3) Identify specific amino acids in GR required for XGRAF:GR cooperative binding to DNA. 4) Determine whether GR and XGRAF heterodimerizes in the absence of DNA. 5) Identify all nucleotides flanking the core GR and XGRAF binding sites that are critical for induction of transcription. This mechanism is of fundamental significance to the field of hormone action since it reveals new pathways by which a steroid response can be regulated by nonreceptor proteins. Furthermore, because receptor structure is highly conserved in the region of GR that interacts with XGRAF, it is likely that similar modes of regulation apply to GR, as well as other nuclear receptors, in other animals. This research is of significance to health and disease because glucocorticoids are widely-used for many common immune inflammatory disorders such as arthritis, asthma, and allergies, but the treatments are hampered by serious effects since these steroids act in virtually all cells. Targeted therapies are needed, and our work shows that specialized regulatory elements for particular genes could provide a way to modulate selectively the expression of some hormone-inducible genes, while leaving other unaffected.