Brinkmann A O, Kuiper G G, Ris-Stalpers C, van Rooij H C, Romalo G, Trifiro M, Mulder E, Pinsky L, Schweikert H U, Trapman J
Department of Biochemistry II, Erasmus University, Rotterdam, The Netherlands.
J Steroid Biochem Mol Biol. 1991;40(1-3):349-52. doi: 10.1016/0960-0760(91)90201-f.
The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2-8, we identified a G----T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the activation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G----C) results in an aspartic acid----histidine substitution (with negligible androgen binding), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A----G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional errors in the human androgen receptor have an enormous impact on phenotype and fertility.
人类雄激素受体是类固醇激素受体超家族的成员。该蛋白的正常功能是正常男性性分化和发育的先决条件。人类雄激素受体cDNA的克隆以及相应基因基因组结构的阐明，使我们能够在具有雄激素不敏感临床表现的受试者和人前列腺癌细胞系（LNCaP）中研究雄激素受体。通过对外显子2 - 8进行PCR扩增、亚克隆和测序，我们在一名具有完全形式雄激素不敏感的受试者的雄激素受体基因中鉴定出一个G→T突变，该突变使外显子4/内含子4边界处的剪接供体位点失活。此突变导致外显子4中一个隐蔽的剪接供体位点激活，从而使类固醇结合域缺失41个氨基酸。在另外两个独立出现的病例中，我们在位于外显子4的密码子686（GAC；天冬氨酸）中鉴定出两个不同的核苷酸改变。一个突变（G→C）导致天冬氨酸→组氨酸替代（雄激素结合可忽略不计），而另一个突变（G→A）导致天冬氨酸→天冬酰胺替代（雄激素结合正常，但雄激素受体复合物快速解离）。对人LNCaP细胞（前列腺淋巴结癌）中雄激素受体的序列分析显示，外显子8的密码子868处有一个点突变（A→G），导致苏氨酸被丙氨酸替代。该突变是LNCaP细胞雄激素受体类固醇结合特异性改变的原因。在COS和HeLa细胞中瞬时表达突变受体后，确定了所观察到的突变在蛋白质表达、特异性配体结合和转录激活方面的功能后果。这些发现表明，人类雄激素受体中的功能错误对表型和生育能力有巨大影响。
