The growth and metabolic actions of growth hormone (GH) are believed to be mediated through the GH receptor (GHR) by JAK2 activation. The GHR exists as a constitutive homodimer, with signal transduction by ligand-induced realignment of receptor subunits. Based on the crystal structures, we identify a conformational change in the F'G' loop of the lower cytokine module, which results from binding of hGH but not G120R hGH antagonist. Mutations disabling this conformational change cause impairment of ERK but not JAK2 and STAT5 activation by the GHR in FDC-P1 cells. This results from the use of two associated tyrosine kinases by the GHR, with JAK2 activating STAT5, and Lyn activating ERK1/2. We provide evidence that Lyn signals through phospholipase C gamma, leading to activation of Ras. Accordingly, mice with mutations in the JAK2 association motif respond to GH with activation of hepatic Src and ERK1/2, but not JAK2/STAT5. We suggest that F'G' loop movement alters the signalling choice between JAK2 and a Src family kinase by regulating TMD realignment. Our findings could explain debilitated ERK but not STAT5 signalling in some GH-resistant dwarfs and suggest pathway-specific cytokine agonists.
1. The growth hormone (GH) receptor was the first of the class 1 cytokine receptors to be cloned. It shares a number of structural characteristics with other family members and common signalling mechanisms based on common usage of the Janus kinase 2 (JAK2). 2. Growth hormone receptor activation is initiated by GH-induced homodimerization of receptor molecules. This has enabled the creation of specific hormone antagonists that block receptor dimerization. 3. The details of the transcription factors used by the activated receptor are being revealed as a result of promoter analyses and electrophoretic mobility gelshift analysis. 4. Growth hormone receptors are widespread and their discovery in certain tissues has led to the assignment of new physiological roles for GH. Some of these involve local or paracrine roles for GH, as befits its cytokine status. 5. Four examples of such novel roles are discussed. These are: (i) the brain GH axis; (ii) GH and the vitamin B12 axis; (iii) GH in early pre-implantation development; and (iv) GH in development of the tooth. 6. We propose that the view that GH acts through the intermediacy of insulin-like growth factor-1 is simplistic; rather, GH acts to induce an array of growth factors and their receptors and the composition of this array varies with tissue type and, probably, stage of development.
Signal transduction by the growth hormone receptor (GHR) occurs through growth hormone (GH)-induced dimerization of two GHRs to form a trimeric complex. It is thought that dimerization alone is sufficient for signaling, since monoclonal antibodies (mAbs) against the extracellular domain of the GHR elicit proliferation of FDC-P1 cells transfected with a chimeric receptor comprising the extracellular domain of the GHR and the fibronectin and cytoplasmic domains of the murine granulocyte colony-stimulating factor receptor. We have screened 14 GHR mAbs for proliferative activity against characterized FDC-P1 and BaF-B03 cell lines stably expressing the full-length human, rabbit, or rat GHR, or the chimeric human GHR/granulocyte colonystimulating factor receptor, and for transactivation of the c-fos promoter and STAT activation. With the chimeric receptor, eight mAbs were able to elicit proliferation, although there was no correlation between inhibition of hormone binding and agonist activity. In contrast, no mAbs were able to act as agonists with the full-length GHR FDC-P1 cell lines, although nine competed with GH for binding. A weak proliferative response was observed in the BaF-B03 cell lines with two of the mAbs (263 and 1C9), and the addition of anti-mouse F(ab) 2 resulted in increased signaling in the hGHR BaF-B03 cell line to a plateau of 28 ؎ 4% of the GH maximum for mAb 263. These data could indicate considerable stringency in the ability of mAbs to correctly dimerize the full-length GHR. However, the ability of mAb 263 to stimulate a mutant hGHR altered in the F-G loop of domain 2 was nearly abolished, concurrent with an increased affinity of this mAb for the receptor. Since the F-G loop undergoes a conformational change on GH binding and is necessary for full proliferative signaling, we propose that in addition to promoting receptor dimerization, mAb 263 may induce specific changes in receptor conformation similar to GH, which are required for the biological response. Growth hormone (GH)1 regulates a wide range of processes including somatic growth, metabolism, and synthesis of specific proteins (1). It does this by triggering multiple second messenger pathways in response to ligand binding to the GHR. The first signaling step after ligand binding is thought to be hormone-induced dimerization of identical receptor subunits. It has been shown that formation of the trimeric complex (GH⅐(GHR) 2 ) is a sequential process involving the binding of the hormone, first to receptor 1 and then to receptor 2, on opposite sides of the four-␣-helical bundle hormone. Receptor binding sites on the hormone are referred to as sites 1 and 2, respectively (2, 3). Evidence supporting a role for hormoneinduced dimerization in signaling was first provided by Fuh et al. (4) using a cell line expressing the extracellular binding domain of the GHR fused to the extracellular fibronectin and cytoplasmic domains of the murine granulocyte colony-stimulating factor (mG-CSF) receptor. These workers reported that a hGH analogue mutated in...
It has been known for more than 4 decades that only primate growth hormones are effective in primate species, but it is only with the availability of the 2.8 Å structure of the human growth hormone (hGH)⅐hGH-binding protein (hGHBP) 2 complex that Souza and coworkers (Souza, S. C., Frick, G. P., Wang, X., Kopchick, J. J., Lobo, R. B., and Goodman, H. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 959 -963) were able to provide evidence that Arg-43 on the primate receptor is responsible. Here we have examined systematically the interaction between Arg-43 (primate receptor) or Leu-43 (non-primate receptors) and their complementary hormone residues Asp-171 (primate GH) and His-170 (nonprimate hormones) in a four-way comparison involving exchanges of histidine and aspartate and exchanges of arginine and leucine. BAF/B03 lines were created and characterized which stably expressed hGH receptor, R43L hGH receptor, rabbit GH receptor, and L43R rabbit GH receptor. These were examined for site 1 affinity, for the ability to bind intact cells, and for proliferative biopotency using hGH, D171H hGH, porcine GH, or H170D porcine GH. We find that the single interaction between Arg-43 and His-170/171 is sufficient to explain virtually all of the primate species specificity, and this is congruent with the crystal structure. Accordingly, for the first time we have been able to engineer a nonprimate hormone to bind to and activate the human GH receptor. The growth hormone receptor (GHR)1 is a member of the hematopoietic cytokine receptor family, sharing common structural and functional features with receptors for prolactin, erythropoietin, granulocyte and granulocyte-macrophage colony-stimulating factors, several interleukins, thrombopoietin, ciliary neurotrophic factor, oncostatin M, and leptin (for review, see Refs. 1 and 2). Of this family, the interaction of GH with its receptor is the best characterized, primarily because of the extensive structure/function studies that have been carried out on both GH and the GHR (for review, see Ref. 1) and because it is the only member for which the crystal structure of the hormone-receptor complex is known (3). Both crystal structure and solution studies support the concept that two identical receptor subunits bind the helix bundle hormone through similar loop determinants on the receptor -sandwich structures. The hormone is captured by receptor 1 through binding to determinants located in a 900-Å 2 patch encompassing helices 1 and 4 and the unstructured loop between helices 1 and 2. Eight key residues account for 85% of the binding energy, with electrostatic interactions governing the approach of hormone to the receptor binding site (4, 5). Electrostatic interactions are also important specificity determinants because 5 of the 7 residues that were modified to enable prolactin to bind to the GH receptor with high affinity involved charged residues (6).GHs from humans and monkeys (primate GHs) are unique in that they are able to bind with and activate non-primate GHRs as well as primate GHR...
Growth hormone initiates signaling by inducing homodimerization of two GH receptors. Here, we have sought to determine whether constitutively active receptor can be created in the absence of the extracellular domain by substituting it with high affinity leucine zippers to create dimers of the growth hormone receptor (GHR) signaling domain. The entire extracellular domain of the GHR was replaced by the hemagglutinintagged zipper sequence of either the c-Fos or c-Jun transcription factor (termed Fos-GHR and Jun-GHR, respectively). Transient transfection of Fos-GHR or Jun-GHR resulted in activation of the serine protease inhibitor 2.1 promoter in Chinese hamster ovary-K1 cells to a level equal to that achieved by fully activated wild type GHR. Furthermore, stable expression of Jun-GHR alone or Fos-GHR and Jun-GHR together in the interleukin 3-dependent BaF-B03 cell line resulted in cell proliferation after interleukin 3 withdrawal at a rate equal to maximally stimulated wild type GHR-expressing cells. Activation of STAT 5b was also observed in Fos-Jun-GHR-expressing cells at a level equal to that in chronically GH-treated GHR-expressing cells. Thus, forced dimerization of the transmembrane and cytoplasmic domains of the GHR in the absence of the extracellular domain can lead to the constitutive activation of known GH signaling end points, supporting the view that proximity of Janus kinase 2 (JAK2) kinases is the essential element in signaling. Such constitutively active GH receptors may have particular utility for transgenic livestock applications. Growth hormone (GH)1 is a potent regulator of somatic growth and a broad spectrum of metabolic processes. Its wide variety of effects on target cells are mediated by a single membrane-spanning receptor (GHR), which is expressed in over 40 different tissues (reviewed in Ref. 1). The GHR is a class 1 homomeric cytokine receptor belonging to the same group as erythropoietin receptor, prolactin receptor, thrombopoietin receptor, and granulocyte colony-stimulating factor receptor (G-CSFR), all of which homodimerize upon ligand binding to form a complex consisting of one ligand and two identical receptor subunits. This contrasts with other class 1 cytokine receptors, which undergo heterodimeric or multimeric associations in response to ligand binding (reviewed in Ref. 2). Early studies showed that GH binds via a high affinity interaction with one GH receptor, followed by a lower affinity interaction with the second identical GHR forming a GH(GHR) 2 trimeric complex (3). Formation of this complex is the first step in signal transduction, which is presumed to bring together and activate two JAK2 molecules, each of which associates with a highly conserved membrane proximal proline rich region in the cytoplasmic domain referred to as box 1 (4). Activation of JAK2 results in activation of signaling molecules, such as STATs, MAP kinases, phosphatidylinositol 3Ј-phosphate kinase, and protein kinase C, and elevates intracellular calcium, leading to the transcriptional regulation of ma...
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