Mapping of the cytoplasmic domain of the human growth hormone receptor required for the activation of Jak2 and Stat proteins.

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.