Novel ARNT-mediated Regulatory Paradigm of AHR Signaling

 描述（由申请人提供）：AHR是一种细胞质受体，其对许多异生物质配体具有亲和力，所述异生物质配体包括卤代芳烃，例如2，3，7，8-四氯二苯并-对-二恶英（TCDD），其是最有效的AHR激活剂之一，并且AHR信号传导介导环境污染物对身体组织和器官的有害影响。配体结合诱导AHR的核转位以及与AHR结合配偶体芳烃受体核转位器（ARNT）的相互作用，允许识别特异性DNA增强子序列。最近，内源性和天然AHR配体，如色氨酸代谢物和膳食化合物，已被确定。这些配体诱导AHR介导的免疫调节作用，例如控制正常T细胞分化和免疫耐受。然而，关于ARNT在AHR免疫信号传导中的作用知之甚少。我们实验室和其他实验室对ARNT在AHR介导的免疫调节中的调节作用的初步研究表明，ARNT是不同免疫信号传导途径（包括AHR和NF-κB信号传导）中不可或缺的辅助因子。ARNT通常在AHR信号传导中被描述为AHR的组成型表达的、非调节的核结合伴侣。然而，我们的数据挑战了这一假设，并表明实际的监管模式更为复杂。例如，ARNT表达为两种同种型，同种型1和3，它们在同种型1中仅存在15个氨基酸的差异。尽管它们的序列相似，我们发现ARNT亚型似乎在AHR信号传导中具有相反的功能。有趣的是，亚型1中额外的15个氨基酸包括一个独特的磷酸化位点，在那里我们观察到TCDD诱导的磷酸化。此外，我们发现ARNT亚型1的磷酸化对于RNA聚合酶II的募集和AHR/ARNT靶基因的反式激活至关重要。我们预测ARNT活性是亚型1的独特磷酸化和特定细胞类型内给定亚型比率的函数，这反过来可能对调节AHR反应的幅度/结果很重要。为了研究我们的假设，我们建议1）定义一个全面的分子框架的AHR调节的ARNT亚型，2）检查AHR活性的作用，由ARNT亚型，在淋巴瘤细胞的生长，和3）表征AHR信号的调节ARNT亚型在体内。最终，这些研究将为基于ARNT的治疗奠定基础，例如剪接调节作为操纵ARNT亚型以控制自身免疫性疾病和癌症中AHR信号传导的手段。

 DESCRIPTION (provided by applicant): AHR is a cytoplasmic receptor that has affinity for numerous xenobiotic ligands, which include halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), one of the most potent AHR activators, and AHR signaling mediates the detrimental effects of environmental contaminants on body tissues and organs. Ligand binding induces nuclear translocation of AHR and interaction with the AHR binding partner, aryl hydrocarbon receptor nuclear translocator (ARNT), allowing recognition of specific DNA enhancer sequences. More recently endogenous and natural AHR ligands, such as tryptophan metabolites and dietary compounds, have been identified. These ligands induce AHR-mediated immunomodulatory effects such as controlling normal T cell differentiation and immune tolerance. However, little is known about the role of ARNT in AHR immune signaling. Initial studies by our laboratory, and others, into the regulatory role of ARNT in AHR-mediated immunomodulation have suggested that ARNT is an integral cofactor in different immune signaling pathways, including AHR and NF-κB signaling. ARNT is often described in AHR signaling as a constitutively expressed, non-regulated, nuclear binding partner for AHR. However, our data challenge this assumption and show that the actual regulatory paradigm is more intricate. For instance, ARNT is expressed as two isoforms, isoform 1 and 3, which differ in only 15 amino acids present in isoform 1. Despite their sequence similarity, we have found that the ARNT isoforms appear to have opposing functions in AHR signaling. Intriguingly, the extra 15 amino acids in isoform 1 include a unique phosphorylation site, where we have observed TCDD-induced phosphorylation. Furthermore, we find that the phosphorylation of ARNT isoform 1 is crucial for the recruitment of RNA polymerase II and transactivation of AHR/ARNT target genes. We predict that ARNT activity is a function of both unique phosphorylation of isoform 1 and the given isoform ratio within a particular cell type, which in turn is likely important for regulating the magnitude/outcome of the AHR response. To investigate our hypothesis we propose to 1) Define a comprehensive molecular framework for AHR regulation by the ARNT isoforms, 2) Examine the role of AHR activity, as governed by the ARNT isoforms, in lymphoma cell growth, and 3) Characterize the regulation of AHR signaling by ARNT isoforms in vivo. Ultimately, these studies will lay the groundwork for ARNT-based therapies such as splice modulation as a means of manipulating the ARNT isoforms to control AHR signaling in autoimmune diseases and cancer.