Hackett R H, Wang Y D, Larner A C
Division of Cytokine Biology, Center for Biologics Evaluation and Research, National Institutes of Health, Bethesda, MD 20892, USA.
J Biol Chem. 1995 Sep 8;270(36):21326-30. doi: 10.1074/jbc.270.36.21326.
Incubation of cells with growth hormone (GH) stimulates both tyrosine phosphorylation of the Jak2 tyrosine kinase and, in some cells, the transcription factor Stat1 alpha (1-4). When the promyeloid cell line FDC-P1 is transfected with the human growth hormone receptor, these cells can grow in the presence of GH and in the absence of interleukin-3. Growth hormone treatment of cells expressing the human growth hormone receptor did not activate Stat1 alpha. However, a complex is present in extracts prepared from growth hormone-treated cells that binds to the gamma response region, an enhancer present in the promoter of the high affinity Fc gamma R1 receptor to which cytokine-activated Stat complexes bind. When truncations of the cytoplasmic domain of the receptor are expressed in FDC-P1 cells only the membrane-proximal 80 amino acids (containing box 1 and box 2) are required for activation of both a GH-stimulated binding activity (GHSF) and tyrosine phosphorylation of Jak2. Activation of GHSF can be inhibited in a cell-free system by the addition of a glutathione S-transferase fusion protein containing these 80 amino acids. Replacement of the one tyrosine in this region of the receptor with a phenylalanine does not alter the activation of either GHSF or Jak2, suggesting that tyrosine phosphorylation of the receptor is not required for GH activation of GHSF. Moreover, a cell line expressing a receptor with only the 54 membrane-proximal amino acids of the intracellular domain (including box 1) shows constitutively tyrosine-phosphorylated Jak2 as well as GHSF binding. With this truncated receptor, there is little if any additional GH-induced tyrosine phosphorylation of Jak2 or induced binding to the gamma response region. These results define the importance of the membrane-proximal 80 amino acids of the GH receptor (with the conserved box 1 and box 2 domains) with regard to GH activation of both Jak2 and Stat(s). They also suggest that within these domains there may be positive and negative elements that regulate Jak2 function.
用生长激素（GH）孵育细胞可刺激Jak2酪氨酸激酶的酪氨酸磷酸化，并且在某些细胞中还可刺激转录因子Stat1α（1 - 4）。当将人生长激素受体转染到早幼粒细胞系FDC - P1中时，这些细胞在有GH存在且无白细胞介素 - 3的情况下能够生长。对表达人生长激素受体的细胞进行生长激素处理不会激活Stat1α。然而，在从生长激素处理的细胞中制备的提取物中存在一种复合物，它能与γ反应区域结合，γ反应区域是高亲和力FcγR1受体启动子中的一种增强子，细胞因子激活的Stat复合物可与之结合。当在FDC - P1细胞中表达受体胞质结构域的截短形式时，对于激活GH刺激的结合活性（GHSF）和Jak2的酪氨酸磷酸化，仅需要膜近端的80个氨基酸（包含框1和框2）。在无细胞系统中，通过添加含有这80个氨基酸的谷胱甘肽S - 转移酶融合蛋白可抑制GHSF的激活。将受体该区域中的一个酪氨酸替换为苯丙氨酸不会改变GHSF或Jak2的激活，这表明受体的酪氨酸磷酸化对于GH激活GHSF不是必需的。此外，一个表达仅具有细胞内结构域54个膜近端氨基酸（包括框1）的受体的细胞系显示Jak2持续酪氨酸磷酸化以及GHSF结合。对于这种截短的受体，几乎没有（如果有的话）额外的GH诱导的Jak2酪氨酸磷酸化或诱导的与γ反应区域的结合。这些结果确定了GH受体膜近端80个氨基酸（具有保守的框1和框2结构域）对于Jak2和Stat的GH激活的重要性。它们还表明在这些结构域内可能存在调节Jak2功能的正负元件。
