title: Mapping of the cytoplasmic domain of the human growth hormone receptor required for the activation of Jak2 and Stat proteins.
abstract: 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.
authors.affiliation: Division of Cytokine Biology, Center for Biologics Evaluation and Research, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: R H
authors.initials: RH
authors.lastname: Hackett
authors.forename: Y D
authors.initials: YD
authors.lastname: Wang
authors.forename: A C
authors.initials: AC
authors.lastname: Larner
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D053614:Janus Kinase 2; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D011986:Receptors, Somatotropin / Q000235:genetics*; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D011986:Receptors, Somatotropin; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D014443:Tyrosine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2