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表现典型性二型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。与谷胱甘肽S转移酶(GST)融合的ARDBD通过GST-GST相互作用以二聚体的形式存在，与小鼠乳腺肿瘤病毒DNA具有较高的亲和力，但凝血酶切割消除GST部分会导致结合亲和力降低，表明ARDBD的二聚化是高亲和力结合DNA所必需的。GST-ARDBD亲和层析柱与小鼠EGF基因启动子5‘-上游区DNA片段温育。该柱特异性捕获了EGF基因-727--598b处的130个碱基片段。将该序列与雄激素依赖性雄激素基因的序列进行比较，发现这些基因的片段具有同源性。AR基因表达水平女性高于男性。去势会导致水平升高，而雌性小鼠注射睾丸素会导致水平下降。这些结果表明，睾酮下调了AR的mRNA水平。最后，我们研究了类固醇受体结合成分90-kDa热休克蛋白(HSP90)的二聚化机制。HSP90α的二聚体结构由191个氨基酸组成，由一个亚基的C-末端区域(Met628/Ala629-Asp732)和另一个亚基相邻的多个N-末端区域(Val542-Tyr627/Met628)重复相互作用组成。290-732位氨基酸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.