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 tr anscription factor Stat1 alpha (1-4). When the promyeloid cell line FD C-P1 is transfected with the human growth hormone receptor, these cell s 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 i n extracts prepared from growth hormone treated cells that binds to th e gamma response region, an enhancer present in the promoter of the hi gh affinity Fc gamma R1 receptor to which cytokine-activated Stat comp lexes bind. When truncations of the cytoplasmic domain of the receptor are expressed in FDC-P1 cells only the membrane-proximal 80 amino aci ds (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 the se SO amino acids. Replacement of the one tyrosine in this region of t he receptor with a phenylalanine does not alter the activation of eith er GHSF or Jak2, suggesting that tyrosine phosphorylation of the recep tor is not required for GH activation of GHSF. Moreover, a cell line e xpressing a receptor with only the 54 membrane-proximal amino acids of the intracellular domain (including box 1) shows constitutively tyros ine phosphorylated Jak2 as well as GHSF binding. With this truncated r eceptor, there is little if any additional GH-induced tyrosine phospho rylation of Jak2 or induced binding to the gamma response region. Thes e results define the importance of the membrane-proximal 80 amino acid s 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 t hat within these domains there may be positive and negative elements t hat regulate Jak2 function.