Oakley R H, Webster J C, Sar M, Parker C R, Cidlowski J A
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Endocrinology. 1997 Nov;138(11):5028-38. doi: 10.1210/endo.138.11.5501.
Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two highly homologous protein isoforms, termed hGR alpha and hGRbeta, that differ at their carboxy-termini. In contrast to the well characterized hGR alpha isoform, which modulates gene expression in a hormone-dependent fashion, the biological significance of hGRbeta has only recently begun to emerge. We and others have shown that the hGRbeta messenger RNA transcript is widely expressed in human tissues and that the hGRbeta protein functions as a dominant negative inhibitor of hGR alpha in transfected cells. Unfortunately, these initial studies did not determine whether the hGRbeta protein was made in vivo. Such analyses are hindered because available anti-hGR antibodies cannot discriminate between the similarly sized hGR alpha and hGRbeta proteins. Therefore, to investigate the expression of the hGRbeta protein, we have produced an antipeptide, hGRbeta-specific antibody termed BShGR. This antibody was made against the unique 15-amino acid peptide at the carboxy-terminus of hGRbeta and recognizes both the native and denatured conformations of hGRbeta, but does not cross-react with hGR alpha. Using BShGR on Western blots and in immunoprecipitation experiments, we detected the hGRbeta protein in a variety of human cell lines and tissues. Immunocytochemistry was then performed with BShGR on HeLa S3 and CEM-C7 cells and on tissue sections prepared from lung, thymus, and liver to assess the cellular and subcellular distribution of hGRbeta. In all immunopositive cells, hGRbeta was found in the nucleus independent of glucocorticoid treatment. Within tissues, the hGRbeta protein was expressed most abundantly in the epithelial cells lining the terminal bronchiole of the lung, forming the outer layer of Hassall's corpuscle in the thymus, and lining the bile duct in the liver. As a potential in vivo inhibitor of hGR alpha activity, expression of hGRbeta may be an important factor regulating target cell responsiveness to glucocorticoids.
人类糖皮质激素受体（hGR）初级转录本的可变剪接产生了两种高度同源的蛋白质异构体，分别称为hGRα和hGRβ，它们的羧基末端不同。与特征明确的以激素依赖方式调节基因表达的hGRα异构体不同，hGRβ的生物学意义直到最近才开始显现。我们和其他人已经表明，hGRβ信使核糖核酸转录本在人体组织中广泛表达，并且hGRβ蛋白在转染细胞中作为hGRα的显性负性抑制剂发挥作用。不幸的是，这些初步研究并未确定hGRβ蛋白是否在体内产生。此类分析受到阻碍，因为现有的抗hGR抗体无法区分大小相似的hGRα和hGRβ蛋白。因此，为了研究hGRβ蛋白的表达，我们制备了一种抗肽，即名为BShGR的hGRβ特异性抗体。该抗体是针对hGRβ羧基末端独特的15个氨基酸肽制备的，可识别hGRβ的天然构象和变性构象，但不与hGRα发生交叉反应。在蛋白质印迹和免疫沉淀实验中使用BShGR，我们在多种人类细胞系和组织中检测到了hGRβ蛋白。然后用BShGR对HeLa S3和CEM - C7细胞以及从肺、胸腺和肝脏制备的组织切片进行免疫细胞化学，以评估hGRβ的细胞和亚细胞分布。在所有免疫阳性细胞中，发现hGRβ存在于细胞核中，与糖皮质激素处理无关。在组织内，hGRβ蛋白在肺终末细支气管内衬的上皮细胞、胸腺中哈氏小体的外层以及肝脏胆管内衬中表达最为丰富。作为hGRα活性的潜在体内抑制剂，hGRβ的表达可能是调节靶细胞对糖皮质激素反应性的一个重要因素。
