Husmann D A, Wilson C M, McPhaul M J, Tilley W D, Wilson J D
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
Endocrinology. 1990 May;126(5):2359-68. doi: 10.1210/endo-126-5-2359.
We have developed polyclonal antibodies to two synthetic peptides corresponding to the amino-(N-)terminal or carboxyl-(C-)terminal segments of the human androgen receptor (hAR) protein, as deduced from the nucleic acid sequence of the androgen receptor cDNA. Immunoreactive antisera were identified by solid phase enzyme-linked immunosorbent assay and purified by peptide affinity chromatography. Specific immunoreactivity with the hAR was confirmed by immunoblotting, using both a fusion protein produced in E. coli that contains the C-terminal 880-amino acid sequence of hAR and the full-length receptor protein produced in COS cells after transfection with a plasmid containing the entire hAR-coding region. Immunohistological evaluation of rat and human prostatic tissue using anti-C-terminal or anti-N-terminal antibodies demonstrated similar patterns of specific staining of the nuclei of epithelial and stromal cells. Castration resulted in a decrease in the amount of nuclear AR detected in the rat prostate after a short time of exposure to anti-C-terminal antibodies (less than 4 h), but did not alter the level of specific staining obtained with anti-N-terminal antibodies. This decrease in nuclear staining using anti-C-terminal antibodies could be reversed by treating castrated animals with dihydrotestosterone. When longer times of exposure to the primary antibodies were used, high levels of nuclear staining were obtained with both types of antibodies in prostate specimens from castrate as well as as intact rats. This immunohistochemical staining pattern contrasts with receptor measurements in rat prostate homogenates that indicate the partition of AR binding into the low salt (cytosolic) fraction in the castrate animal and into the high salt (nuclear) fraction in the intact animal. Our results suggest that the AR is predominantly a nuclear protein even in the absence of ligand and that dihydrotestosterone serves to tighten its association with the nucleus. These data also suggest that the immunoreactivity of anti-C-terminal antibodies is influenced by the presence of dihydrotestosterone, presumably via an alteration in the physical state of the receptor protein.
我们根据雄激素受体cDNA的核酸序列推导，针对人雄激素受体（hAR）蛋白的氨基（N）末端或羧基（C）末端片段对应的两种合成肽制备了多克隆抗体。通过固相酶联免疫吸附测定法鉴定免疫反应性抗血清，并通过肽亲和色谱法进行纯化。使用在大肠杆菌中产生的包含hAR C末端880个氨基酸序列的融合蛋白以及用包含整个hAR编码区的质粒转染后在COS细胞中产生的全长受体蛋白，通过免疫印迹法确认了与hAR的特异性免疫反应性。使用抗C末端或抗N末端抗体对大鼠和人前列腺组织进行免疫组织学评估，结果显示上皮细胞和基质细胞核的特异性染色模式相似。去势导致在短时间（少于4小时）暴露于抗C末端抗体后，大鼠前列腺中检测到的核AR量减少，但未改变抗N末端抗体获得的特异性染色水平。用二氢睾酮处理去势动物可逆转使用抗C末端抗体时核染色的这种减少。当使用更长时间暴露于一抗时，去势大鼠和完整大鼠的前列腺标本中两种类型的抗体均获得了高水平的核染色。这种免疫组织化学染色模式与大鼠前列腺匀浆中的受体测量结果形成对比，后者表明在去势动物中AR结合分配到低盐（胞质）部分，而在完整动物中分配到高盐（核）部分。我们的结果表明，即使在没有配体的情况下，AR主要也是一种核蛋白，并且二氢睾酮有助于加强其与细胞核的结合。这些数据还表明，抗C末端抗体的免疫反应性受二氢睾酮的存在影响，可能是通过受体蛋白物理状态的改变。
