Analysis of the androgen response element of mouse EGF gene by use of the androgen receptor expressed in Echerichia coli

本实验研究了雄激素在具有典型两性异形的小鼠下颌下腺中的作用机制。为了制备与潜在雄激素反应元件特异性结合的吸附剂，在大肠杆菌中表达了人雄激素受体（ARDBD）的DNA结构域。ARDBD与谷胱甘肽S-转移酶（GST）通过GST-GST相互作用以二聚体形式存在，与小鼠乳腺肿瘤病毒DNA具有高亲和力，但凝血酶裂解GST后，其亲和力降低，说明ARDBD的二聚化是其与DNA高亲和力结合的必要条件。将含有GST-ARDBD的亲和柱与来自小鼠EGF基因启动子5 ′上游区的DNA片段孵育。该柱特异性地捕获了EGF基因-727 ~-598b的130 bp片段。与雄激素依赖性雄激素基因序列比较，发现它们之间存在同源性。AR mRNA水平女性高于男性。去势导致水平升高，而对雌性小鼠注射睾酮导致降低。最后，我们研究了类固醇受体结合组分热休克蛋白（90-kDa heat shock protein，HSP 90）二聚化的机制。HSP 90 α的二聚体结构由C-末端191个氨基酸介导，并且由一个亚基的C-末端区域（Met 628/Ala 629-Asp 732）和另一个亚基的相邻的N-末端区域（Val 542-Tyr 627/Met 628）的重复相互作用组成。氨基酸290-732足以与类固醇受体相互作用。

We studied the action mechanism of androgen in the mouse submandibular gland that shows the typical sexual dimorphism. To prepare the adsorbent specifically interacting with potential androgen response element, the DNA-domain of human androgen receptor (ARDBD) was expressed in E.coli. ARDBD fused to glutathione S-transferase (GST) existed as a dimer via GST-GST interaction and had a high affinity for mouse mammary tumor virus DNA.However, elimination of GST mojety by thrombin cleavage caused the reduction of the binding affinity, indicating that the dimerization of ARDBD is essential for the DNA binding of high affinity. The affinity column with GST-ARDBD was incubated with DNA fragments derived from 5'-upstream region of mouse EGF gene promoter. The column specifically trapped the 130 bp fragment located at -727- -598b of EGF gene. Comparison of the sequence with those of androgen-dependent androgen genes revealed the segment homologous among these genes.The effect of testosterone on the expression of AR mRNA in mouse submandibular glands was examined. The AR mRNA level was higher in females than in males. Castration resulted in elevation of the level, while testosterone injection to female mice caused the reduction. Thses results show that the AR mRNA level is down-regulated by testosterone.We finally investigated the mechanism of dimerization of the 90-kDa heat shock protein (HSP90), a binding component of steroid receptors. The dimeric structure of HSP90alpha is mediated by the C-terminal 191 amino acids and consists of duplicate interactions of the C-terminal region (Met628/Ala629-Asp732) of one subunit and the adjacent more N-terminal region (Val542-Tyr627/Met628) of the other subunit. The amino acids 290-732 were sufficient for the interaction with steroid receptors.