Lochnan H A, Buteau H, Richards S, Edery M, Kelly P A
INSERM U344-Endocrinologie Moléculaire, Faculté de Médecine Necker, Paris, France.
Mol Cell Endocrinol. 1995 Oct 30;114(1-2):91-9. doi: 10.1016/0303-7207(95)03645-n.
Prolactin receptors (PRLR) have been identified in a number of human tissues and cell lines, although little is known about the human receptor protein. The cloning of the human PRLR cDNA has enabled further characterization of the receptor protein in transfected cells. Since the human cDNA is expressed at lower levels than the rat cDNA, we have constructed a hybrid cDNA (pECE r5'hPRLR) containing nucleotides of the 5' untranslated region and signal peptide of the rat PRLR and the protein coding and 3' untranslated portion of the human receptor. Expression of the hybrid receptor was increased more than two-fold compared to the human receptor as detected by specific binding of 125I-human growth hormone (GH) to transfected COS-7 cells. The relative molecular mass of the receptor was 93,000 Da, as determined by chemical cross-linking studies. Transcriptional assays were used to show the human PRLR was able to activate two milk protein genes; ovine beta-lactoglobulin and rat beta-casein. Transfected cells expressing the human PRLR receptor, treated with human GH or prolactin (PRL), induced a dose-dependent increase in transcriptional activation of the beta-casein/luciferase fusion gene. Glycosylated, and non-glycosylated human PRL, and ovine PRL were equally effective in activating the beta-casein promoter. Human placental lactogen and bovine PRL could also induce a greater than 10-fold induction, whereas insulin did not significantly stimulate the beta-casein promoter. The results show that the human PRLR can activate both beta-lactoglobulin and beta-casein milk gene promoters and that these reporter genes can be used to evaluate the functional activity of agonists and antagonists of the human PRLR.
催乳素受体（PRLR）已在多种人体组织和细胞系中被鉴定出来，尽管对人类受体蛋白了解甚少。人PRLR cDNA的克隆使得在转染细胞中对受体蛋白进行进一步表征成为可能。由于人cDNA的表达水平低于大鼠cDNA，我们构建了一种杂合cDNA（pECE r5'hPRLR），它包含大鼠PRLR的5'非翻译区和信号肽的核苷酸以及人类受体的蛋白质编码区和3'非翻译部分。通过125I-人生长激素（GH）与转染的COS-7细胞的特异性结合检测发现，与人类受体相比，杂合受体的表达增加了两倍多。通过化学交联研究确定，该受体的相对分子质量为93,000 Da。转录分析表明人PRLR能够激活两个乳蛋白基因；绵羊β-乳球蛋白和大鼠β-酪蛋白。用人GH或催乳素（PRL）处理表达人PRLR受体的转染细胞，可诱导β-酪蛋白/荧光素酶融合基因的转录激活呈剂量依赖性增加。糖基化和非糖基化的人PRL以及绵羊PRL在激活β-酪蛋白启动子方面同样有效。人胎盘催乳素和牛PRL也能诱导超过10倍的诱导，而胰岛素对β-酪蛋白启动子没有明显的刺激作用。结果表明，人PRLR可以激活β-乳球蛋白和β-酪蛋白乳基因启动子，并且这些报告基因可用于评估人PRLR激动剂和拮抗剂的功能活性。
