Mapping of a Cytoplasmic Domain of the Human Growth Hormone Receptor That Regulates Rates of Inactivation of Jak2 and Stat Proteins

Abstract

It has been previously demonstrated that growth hormone (GH)-stimulated tyrosine phosphorylation of Jak2 and Stat5a and Stat5b occurs in FDP-C1 cells expressing either the entire GH receptor or truncations of the cytoplasmic domain expressing only the membrane-proximal 80 amino acids. However, other receptor domains that might modulate rates of GH activation and inactivation of this cascade have not been examined. Here we have defined a region in the human GH receptor between amino acids 520 and 540 in the cytoplasmic domain that is required for attenuation of GH-activated Jak/Stat signaling. Immunoprecipitations with antibodies to Jak2 indicate that the protein tyrosine phosphatase SHP-1 is associated with this kinase in cells exposed to GH. To address the possibility that SHP-1 could function as a negative regulator of GH signaling, liver extracts from motheaten mice deficient in SHP-1 or unaffected littermates were analyzed for activation of Stats and Jak2. Extracts from motheaten mice displayed prolonged activation of the Stat proteins as measured by their ability to interact with DNA and prolonged tyrosine phosphorylation of Jak2. These results delineate a novel domain in the GH receptor that regulates the inactivation of the Jak/Stat pathway and appears to be modulated by SHP-1. Growth hormone (GH)1 exerts its pleiotropic actions on a variety of tissues including fat, bone, soft tissue, and liver. One of the earliest events that occurs after GH binds to its cell surface receptor is the tyrosine phosphorylation of several cellular proteins, including the SH2 domain-containing transcription factors termed signal transducers and activators of transcription or Stats (1-4). Tyrosine phosphorylated Stat proteins bind enhancers that are present in genes whose transcription is rapidly induced by the treatment of cells with GH and other cytokines. One of these enhancers is the gamma response region (GRR) present in the promoter of the Fc␥RI receptor gene. This enhancer, which is required for IFN␥-activated transcription of the Fc␥RI receptor gene, has a sequence similar to those of enhancers that are required for the activation of cellular genes by a variety of other cytokines. GRR binding activity can be measured in many cells in response to growth hormone treatment, and it serves as an assay for the tyrosine phosphorylation of Stat proteins (1, 5). Most of the cytokine receptors interact with members of the Jak family of tyrosine kinases, and Jak activation closely parallels and in many cases is required for Stat protein phosphorylation on tyrosine. Tyrosine phosphorylated Jak2 has been shown to associate with the GHR after the addition of ligand, which allows Stat1, Stat3, Stat5a, and Stat5b to also be phosphorylated (6).The receptors for growth hormone and other members of this cytokine receptor superfamily have several conserved features including cysteine residues within their extracellular domains and two intracellular subdomains (termed box 1 and box 2) adjacent to the transmembrane region. To elucidate the...

The Signal Transduction of the Growth Hormone Receptor Is Regulated by the Ubiquitin/Proteasome System and Continues After Endocytosis

“…The recruitment of SHP-1 is accompanied by dephosphorylation of JAK2 and subsequent termination of erythropoietin-induced cellular proliferation (33,34). A similar role for SHP-1 in mediating the down-regulation of JAK2 following stimulation of cells with GH has been proposed (11). Our results with GHR-(1-399) and GHR-(1-369) indicate that partial deletion of the C-terminal GHR tail leads to a prolonged JAK2 phosphorylation presumably because of loss of a negative regulator binding site and its consequent activation.…”

“…Part of the dephosphorylation of the GHR has been previously attributed to the activation of the tyrosine phosphatase SHP-1 (11). This enzyme was found to interact with JAK2, and GH stimulates the catalytic activity of SHP-1 (11). Another candidate could be SHP-2 because it associates with the GHR and binding to JAK2 has also been reported.…”

“…The molecular mechanism of JAK deactivation is still poorly understood. Part of the dephosphorylation of the GHR has been previously attributed to the activation of the tyrosine phosphatase SHP-1 (11). This enzyme was found to interact with JAK2, and GH stimulates the catalytic activity of SHP-1 (11).…”

The role of STAT proteins in growth hormone signaling

“…SHP-1 has been demonstrated to negatively regulate JAK/STAT signaling mediated by the various cytokine receptors in hematopoietic cells (David et al, 1996;Haque et al, 1998;Klingmuller et al, 1995;Yi et al, 1993). SHP-1 directly associates with JAK2 (Jiao et al, 1996) and SHP-1 has been suggested to play a role in the dephosphorylation of JAK2 in liver in response to GH (Hackett et al, 1997). Both SHP-1 and SHP-2 have been suggested as potential phosphatases for Stat5 Yu et al, 2000).…”

Two Distinct Domains Within the N-Terminal Region of Janus Kinase 1 Interact with Cytokine Receptors

“…Box 1 is defined as a proline-rich motif (4), whereas box 2 is characterized as a cluster of hydrophobic amino acids (2). Most single subunit cytokine receptors, such as erythropoietin, growth hormone, and prolactin, as well as some heterodimeric receptors, such as IFN-␥ and GM-CSF/IL-3 and -5 interact with Jak2 through the highly conserved box 1 motif (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”