Gao T, Marcelli M, McPhaul M J
Department of Internal Medicine, The University of Texas Southwestern Medical Center at Dallas, 75235-8857, USA.
J Steroid Biochem Mol Biol. 1996 Sep;59(1):9-20. doi: 10.1016/s0960-0760(96)00097-0.
A series of cDNAs containing deletions within the open-reading frame of the human androgen receptor (AR) were constructed and transiently expressed in CV1 cells to investigate the effects of these alterations on the level of expression of the protein and on its capacity to activate a model reporter gene (MMTV-luciferase). The levels of AR expression were assayed using immunoblots made using an antibody directed at an epitope (amino acids 1-21) preserved in all of the deletions. Treatment of the transfected cells with androgen increased the level of normal or mutant AR approximately five-fold in all constructs in which the hormone-binding domain was intact. This finding indicates that an intact hormone-binding domain is necessary and sufficient for the androgen-dependent increase in AR levels. Contraction of expansion or the glutamine repeat or deletion of the glycine repeat in the amino terminus diminished the capacity of the mutant ARs to activate the MMTV luciferase gene. The presence of a large-scale deletion within the amino terminus (amino acid residues 96-483), abolished receptor function, and two smaller deletions (bounded by residues 80-93 and 245-485) within the amino terminus substantially impaired receptor function. As previously described, deletion of the hormone-binding domain (amino acids 708-917) resulted in a constitutively active receptor. Unexpectedly, the large-scale deletion within the amino terminus (amino acids 96-483), in combination with deletion of the carboxy terminus also produced a constitutively active receptor that was almost as active as ligand-activated normal AR. None of the alterations in AR function could be explained by changes in the level of AR expression and the function of some mutant receptors was even more defective when the relative levels of mutant ARs expressed was considered. These findings imply that interaction of the sequences within the amino- and carboxy-terminal portions of the AR, or proteins that interact with these segments, is critical for regulation of transcription by the AR.
构建了一系列在人雄激素受体（AR）开放阅读框内含有缺失的cDNA，并在CV1细胞中瞬时表达，以研究这些改变对蛋白质表达水平及其激活模型报告基因（MMTV-荧光素酶）能力的影响。使用针对在所有缺失中均保留的表位（氨基酸1-21）的抗体进行免疫印迹分析AR表达水平。用雄激素处理转染细胞后，在激素结合域完整的所有构建体中，正常或突变AR的水平增加了约五倍。这一发现表明，完整的激素结合域对于雄激素依赖性AR水平的增加是必要且充分的。氨基末端谷氨酰胺重复序列的收缩或扩展或甘氨酸重复序列的缺失会降低突变AR激活MMTV荧光素酶基因的能力。氨基末端内的大规模缺失（氨基酸残基96-483）消除了受体功能，氨基末端内的两个较小缺失（由残基80-93和245-485界定）严重损害了受体功能。如先前所述，激素结合域（氨基酸708-917）的缺失导致组成型活性受体。出乎意料的是，氨基末端内的大规模缺失（氨基酸96-483）与羧基末端的缺失相结合，也产生了一种组成型活性受体，其活性几乎与配体激活的正常AR一样高。AR功能的改变均不能通过AR表达水平的变化来解释，并且当考虑突变AR的相对表达水平时，一些突变受体的功能甚至更有缺陷。这些发现表明，AR氨基末端和羧基末端部分内的序列或与这些片段相互作用的蛋白质之间的相互作用对于AR调节转录至关重要。
