Involvement of a growth hormone receptor-associated tyrosine kinase in signal transduction by growth hormone.

Although growth hormone (GH) has been demonstrated to be an important regulator of body growth, cellular differentiation and metabolism, the mechanisms of intracellular signal transduction initiated by GH are unclear. This study investigates whether the cloned liver GH receptor (GHR) forms a complex with a cellular tyrosine kinase and if so, whether activation of this kinase is important in GH signaling and actions. To determine whether the cloned liver GHR associates with a tyrosine kinase and can be tyrosyl phosphorylated by this kinase, the ability of $\sp{125}$I-GH-GHR complexes from cells expressing the cloned GHR to bind to anti-phosphotyrosine antibody and the ability of GH-GHR complexes purified by anti-GH antibody to incorporate $\sp{32}$P-phosphoryl residues in an in vitro assay were examined. Results indicate that the cloned liver GHR co-purifies with tyrosine kinase activity and can be tyrosyl phosphorylated in vivo and in vitro when expressed in COS-7, mouse-L, CHO and RIN5-AH cells. A 121-kDa phosphoprotein (pp121) has been identified in this study as a candidate for the GHR-associated tyrosine kinase. pp121, which does not appear to bind to GH, is present in kinase-active anti-GH antibody immunoprecipitates from CHO cells expressing the cloned liver GH receptor, suggesting that pp121 forms a complex with GHR. Both GHR and pp121 are phosphorylated on tyrosyl residues in an in vitro kinase assay and in intact cells incubated with GH. Regions of the GHR necessary for association and/or activation of the GHR-associated tyrosine kinase and tyrosyl phosphorylation of the GHR were determined using two GHR truncation mutants. When expressed in CHO and RIN5-AH cells, GHR$\sb{1-454}$ which lacks the C-terminal half of the cytoplasmic domain but not GHR$\sb{1-294}$ which lacks all but 5 amino acids of the cytoplasmic domain retains the ability to copurify with tyrosine kinase activity and pp121 and to be phosphorylated in vivo and in vitro. This finding indicates that the N-terminal half of the cytoplasmic domain of the GHR is required for association and/or activation of the GHR-associated tyrosine kinase and contains phosphorylation site(s) for the kinase. Additionally, GH increases MAP kinase activity and induces c-fos gene expression in CHO cells expressing GHR$\sb{1-454}.$ These results strongly suggest that activation of the GHR-associated tyrosine kinase is important in GH signaling and GH actions. (Abstract shortened by UMI.).PhDPhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103510/1/9319651.pdfDescription of 9319651.pdf : Restricted to UM users only