Perrot-Applanat M, Gualillo O, Buteau H, Edery M, Kelly P A
INSERM U 344, Faculté de Médecine Necker-Enfants Malades, Paris, France.
J Cell Sci. 1997 May;110 ( Pt 9):1123-32. doi: 10.1242/jcs.110.9.1123.
Prolactin (PRL) interacts with a specific, well characterized plasma membrane receptor (PRLR) that is coupled to signal transduction pathways involving Jak2, Fyn, and MAP kinases, and signal transducers and activators of transcription (STAT). Although a few previous studies have indicated nuclear translocation of PRL in IL-2 stimulated T lymphocytes, PRL-dependent Nb2 lymphoma cell lines and 235-1 lactotrophs, the mechanisms of nuclear targeting remain unknown and conflicting results have been reported concerning the putative nuclear translocation of the PRLR. We therefore decided to investigate nuclear translocation of PRLR and PRL in various cell lines transfected with an expression plasmid encoding PRLR, using confocal laser microscopy. We have constructed various cDNAs of the long and short forms of the rat PRLR containing an oligonucleotide encoding a Flag epitope inserted either just before the N-terminal amino acid or in the C-terminal end of the mature receptor (named N-terminal or C-terminal Flag-tagged PRLR). The corresponding receptors function as the PRLR in transfected cells: they are expressed at the plasma membrane and in compartments of the secretory pathway, they bind PRL with normal affinity (Kd= 4x10(-10) M) and have the same capacity to stimulate the transcriptional activity of a milk protein (beta-casein) gene as wild-type PRLR. In addition, the tagged receptors are much more efficiently immunodetected using anti-Flag antibodies, as compared to anti-PRL antibodies (U5 or U6). Immunofluorescence combined with detailed confocal laser microscopy showed that addition of PRL (0 to 12 hours) to COS-7, CHO and NIH-3T3 transfected fibroblasts induces rapid internalization of the receptor (long form), without any translocation to the nucleus. Using PRL-R tagged both in the N-terminal or C-terminal regions of the mature receptor excludes the possibility of a cleaved fragment which could have been subsequently imported into the nucleus. An absence of nuclear translocation of PRLR was also observed in a 293 cell line stably expressing the receptor, and in physiological targets for PRL, i.e. in Nb2 lymphoma cells expressing the Nb2 form of the receptor or in BGME mammary gland epithelial cells upon overexpression of a Flag-tagged PRLR. Similarly, the short form of the PRLR was not detected in nuclei of transfected COS cells upon PRL treatment. Clearly, our results provide evidence that internalization of the plasma membrane PRLR does not lead to nuclear translocation of the receptor, or part of it, in most fibroblasts and epithelial cells at physiological concentrations of PRL. Also, in co-localization experiments, PRL was internalized without nuclear translocation. Activation of STATs transcription factors and MAP kinases, as well as translocation of these proteins to the nucleus following their phosphorylation, probably remains the intracellular mechanism coupling stimulation to nuclear events.
催乳素（PRL）与一种特定的、特征明确的质膜受体（PRLR）相互作用，该受体与涉及Jak2、Fyn和丝裂原活化蛋白激酶（MAP激酶）以及信号转导子和转录激活子（STAT）的信号转导途径偶联。尽管先前的一些研究表明PRL在白细胞介素-2刺激的T淋巴细胞、PRL依赖的Nb2淋巴瘤细胞系和235-1催乳细胞中有核转位现象，但核靶向机制仍不清楚，并且关于PRLR假定的核转位已报道了相互矛盾的结果。因此，我们决定使用共聚焦激光显微镜研究在转染了编码PRLR的表达质粒的各种细胞系中PRLR和PRL的核转位。我们构建了大鼠PRLR长、短形式的各种cDNA，其包含一个编码Flag表位的寡核苷酸，该寡核苷酸插入在成熟受体的N末端氨基酸之前或C末端（分别命名为N末端或C末端Flag标记的PRLR）。相应的受体在转染细胞中作为PRLR发挥作用：它们在质膜和分泌途径的区室中表达，以正常亲和力（Kd = 4×10⁻¹⁰ M）结合PRL，并且与野生型PRLR具有相同的刺激乳蛋白（β-酪蛋白）基因转录活性的能力。此外，与抗PRL抗体（U5或U6）相比，使用抗Flag抗体能更有效地免疫检测标记的受体。免疫荧光结合详细的共聚焦激光显微镜显示，向转染的COS-7、CHO和NIH-3T3成纤维细胞中添加PRL（0至12小时）会诱导受体（长形式）快速内化，而没有任何转位到细胞核。在成熟受体的N末端或C末端区域都标记PRL-R排除了可能被切割并随后导入细胞核的片段的可能性。在稳定表达该受体的293细胞系以及PRL的生理靶细胞中，即在表达受体Nb2形式的Nb2淋巴瘤细胞或过表达Flag标记的PRLR的BGME乳腺上皮细胞中，也观察到PRLR没有核转位。同样，在PRL处理后，转染的COS细胞的细胞核中未检测到PRLR的短形式。显然，我们的结果提供了证据，即在生理浓度的PRL下，大多数成纤维细胞和上皮细胞质膜PRLR的内化不会导致受体或其部分的核转位。此外，在共定位实验中，PRL被内化但没有核转位。STAT转录因子和MAP激酶的激活，以及这些蛋白质在磷酸化后转位到细胞核，可能仍然是将刺激与核事件偶联的细胞内机制。
