The purpose of this project is to characterize the components involved in signaling through the growth hormone and cytokine receptors. Many cytokines regulate gene expression by the tyrosine phosphorylation of a family of DNA binding factors known as Stats (for Signal Transducers and Activators of Transcription). These activated protein complexes bind to enhancers present in the promoters of early response genes such as the high affinity Fcgamma receptor (FcgammaR1). Our studies have shown that the activation the Stats occurs through cytokine and growth factor receptors. Treatment of human peripheral blood monocytes with interleukin-3 (IL-3), interleukin-5 (IL-5), interleukin-10 (IL-10), granulocyte-macrophage colony stimulating factor, growth hormone (GH), prolactin or the interferons (IFNs) differentially activated DNA-binding proteins all of which recognized the same IFN gamma response region (GRR) located in the promoter of the FcgammaRI gene. This year, I have continued these studies using murine cell lines that are stably transfected with full length or mutated growth hormone or prolactin receptors to identify sequences in the cytoplasmic domain of these receptors that are required for the activation and deactivation of the GRR binding complex. This work revealed that only the membrane proximal 80 amino acids of the GH receptor are required for the activation of a GH- stimulated DNA binding complex and the tyrosine phosphorylation of other cell proteins. Immunological studies with these cell lines and antibodies to various components of cytokine signaling cascades showed that the GH- activated DNA binding complex contains STAT5 in these cells and that the Janus kinase Jak2 is one of the major tyrosine phosphorylated proteins upon GH treatment. Currently, I am further delineating the sequence of events and protein associations that occur during growth hormone signaling using coimmunoprecipitations of GH receptor-associated signaling components as well as by transfecting these cell lines with mutated genes for several of the signaling proteins. These studies are providing a broader understanding of the components involved in signaling through the GH receptor and, consequently, the mechanisms of cytokine-stimulated signaling and gene activation.
