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

我们研究了雄激素在具有典型雌雄二态性的小鼠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相互作用的吸附剂，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，对小鼠乳腺肿瘤病毒DNA具有高亲和力。然而，凝血酶裂解消除GST片段导致结合亲和力降低，表明ARDBD的二聚化对于高亲和力的DNA结合至关重要。用小鼠EGF基因启动子上游5′区DNA片段孵育GST-ARDBD亲和柱。该柱特异性捕获了EGF基因-727- -598b处130 bp的片段。与雄激素依赖性雄激素基因的序列比较，发现这些基因之间存在同源片段。研究睾酮对小鼠颌下腺AR mRNA表达的影响。女性的AR mRNA水平高于男性。阉割导致该水平升高，而雌性小鼠注射睾酮导致该水平降低。结果表明，睾酮可下调AR mRNA水平。我们最后研究了90 kda热休克蛋白（HSP90）二聚化的机制，HSP90是类固醇受体的结合成分。HSP90alpha的二聚体结构由c端191个氨基酸介导，由一个亚基的c端区域（Met628/Ala629-Asp732）和相邻的另一个亚基的多n端区域（Val542-Tyr627/Met628）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.