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Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial-mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer.Oncogene advance online publication, 2 October 2017; doi:10.1038/onc.2017.352.

A substantial body of evidence supports a role for the growth hormone (GH)-IGF-1 axis in cancer incidence and progression. This includes epidemiological evidence relating elevated plasma IGF-1 to cancer incidence as well as a lack of cancers in GH/IGF-1 deficiency. Rodent models lacking GH or its receptor are strikingly resistant to the induction of a wide range of cancers, and treatment with the GH antagonist pegvisomant slows tumor progression. While GH receptor expression is elevated in many cancers, autocrine GH is present in several types, and overexpression of autocrine GH can induce cell transformation. While the mechanism of autocrine action is not clear, it does involve both STAT5 and STAT3 activation, and probably nuclear translocation of the GH receptor. Development of a more potent GH receptor antagonist or secretion inhibitor is warranted for cancer therapy.

Growth hormone (GH) is a major regulator of postnatal growth and metabolism. There are extensive clinical applications for GH or its antagonists, including treatments for dwarfism, cancer and metabolic wasting. Owing to this, there is considerable interest in the mechanisms of GH receptor (GHR) activation. It is conventionally thought that GH induces dimerization of two GHR monomers, which initiates intracellular signaling cascades. However, recent studies have provided evidence for a ligand-induced conformational change within constitutively dimerized GHRs being responsible for activating signaling pathways. This review will relate the new model of GHR activation to the activation of related cytokine receptors and discuss the implication of this new model for the design of small GH mimetics and antagonists for therapeutic use.

GH receptor (GHR) mediates the anabolic and metabolic effects of GH. We previously characterized a monoclonal antibody (anti-GHR(ext-mAb)) that reacts with subdomain 2 of the rabbit GHR extracellular domain (ECD) and is a conformation-specific inhibitor of GH signaling in cells bearing rabbit or human GHR. Notably, this antibody has little effect on GH binding and also inhibits inducible metalloproteolysis of the GHR that occurs in the perimembranous ECD stem region. In the current study, we demonstrate that anti-GHR(ext-mAb) inhibits GH-dependent cellular proliferation and also inhibits hepatic GH signaling in vivo in mice that adenovirally express rabbit GHR, as assessed with our noninvasive bioluminescence hepatic signaling assay. A separate monoclonal antibody (anti-GHR(mAb 18.24)) is a sister clone of anti-GHR(ext-mAb). Here, we demonstrate that anti-GHR(mAb 18.24) also inhibits rabbit and human GHR signaling and inducible receptor proteolysis. Further, we use a random PCR-generated mutagenic expression system to map the three-dimensional epitopes in the rabbit GHR ECD for both anti-GHR(ext-mAb) and anti-GHR(mAb 18.24). We find that each of the two antibodies has similar, but nonidentical, discontinuous epitopes that include regions of subdomain 2 encompassing the dimerization interface. These results have fundamental implications for understanding the role of the dimerization interface and subdomain 2 in GHR activation and regulated GHR metalloproteolysis and may inform development of therapeutics that target GHR.

Although used as a therapeutic for 50 years, it is only recently that the application of molecular techniques has provided a basis for understanding growth hormone’s (GH) clinical actions. This article reviews progress in our current knowledge of the molecular mechanism of growth hormone (GH) receptor activation based on a number of physicochemical techniques, and documents insights gained into the means used by the activated GH receptor to control the expression of genes regulating growth and metabolism. These findings are related to disorders of short stature, and the therapeutic consequences are summarized.

Once thought to be present only in liver, muscle and adipose tissue, the GH receptor is now known to be ubiquitously distributed, in accord with the many pleiotropic actions of GH. These include the regulation of metabolism, postnatal growth, cognition, immune, cardiac and renal systems and gut function. GH exerts these actions primarily through alterations in gene expression, initiated by activation of its membrane receptor and the resultant activation of the associated JAK2 (Janus kinase 2) and Src family kinases. Receptor activation involves hormone initiated movements within a receptor homodimer, rather than simple receptor dimerization. We have shown that binding of the hormone realigns the orientation of the two receptors both by relative rotation and by closer apposition just above the cell membrane. This is a consequence of the asymmetric placement of the binding sites on the hormone. Binding results in a conversion of parallel receptor transmembrane domains into a rotated crossover orientation, which produces separation of the lower part of the transmembrane helices. Because the JAK2 is bound to the Box1 motif proximal to the inner membrane, receptor activation results in separation of the two associated JAK2s, and in particular the removal of the inhibitory pseudokinase domain from the kinase domain of the other JAK2 (and vice versa). This brings the two kinase domains into position for trans-activation and initiates tyrosine phosphorylation of the receptor cytoplasmic domain and other substrates such as STAT5, the key transcription factor mediating most genomic actions of GH.There are a limited number of genomic actions initiated by the Src kinase family member which also associates with the upper cytoplasmic domain of the receptor, including important immune regulatory actions to dampen exuberant innate immune activation of cells involved in transplant rejection. These findings offer insights for developing specific receptor antagonists which may be valuable in cancer therapy.

Ghrelin, a gut hormone originating from the post-translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS-R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin-O-acyltransferase (GOAT), conferring selective binding to the GHS-R1a receptor via acylated ghrelin. Complete loss of preproghrelin-derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl-ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat(-/-) mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat(-/-) mice compared to age-matched wild-type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH-dependent liver genes. Of interest, circulating levels of insulin-like growth factor (IGF)-1 were elevated in goat(-/-) mice. This rise in circulating levels of IGF-1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF-1 release in goat(-/-) mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of altered GH secretory patterning remains unclear, we propose that this may contribute to sustained IGF-1 release and growth in goat(-/-) mice.

Class I cytokine receptors regulate a wide range of clinically relevant cellular processes. This family of receptors contains around 30 members including receptors for erythropoietin (EPO), prolactin (PRL), growth hormone (GH), thrombopoietin (TPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), interleukin-3 (IL-3), IL-5, and IL-6.1 The growth hormone receptor has been the archetype for the class I cytokine receptor family, as it was the first to be both cloned and have its extracellular domain crystal structure solved. The original paradigm for the mechanism of activation was based on biophysical and structural studies over 20 y ago showing that growth hormone binding to the extracellular domain of the receptor occurred in a sequential fashion, first binding to a high affinity site on a single receptor (site 1) and then binding to a lower affinity site (site 2) on a second receptor.2 The subsequent publication of the crystal structure showed a single hormone bound to 2 receptor domains at the site 1 and site 2 positions, and also revealed the lower fibronectin III domain of the receptor locked together (site 3 or dimerization domain).3 The conclusion from these studies was that hormone binding induced dimerization of the receptor and that this would bring the intracellular signaling domains together to allow association and activation of the associated janus kinases (JAKs). However, despite great progress in elucidating the structural details of ligand binding to the extracellular domain of receptors for several members of this family, the means by which these receptors activate their associated JAKs to initiate signaling had remained elusive. The original ligand-mediated activation model was undermined by several lines of evidence, particularly by studies showing that the GH receptor and other related receptors such as the PRL, EPO, and TPO receptors exist predominantly as dimers prior to ligand binding.4 The question remained as to how ligand binding was able to transmit its signal through the transmembrane domain and to the intracellular domain of the pre-dimerized receptors to activate the associated kinases. Our group embarked on studies to unravel this mechanism, and recently were able to present the first complete mechanistic model of JAK2 activation by an archetypal class I cytokine receptor, the growth hormone receptor.5 We confirmed that the GH receptor exists as a preformed dimer in vivo by using confocal microscopy anisotropy analysis at the surface of live mammalian cells. We then utilized a model system where we could lock receptors in different activation states. We did this by removing the extracellular domain of the receptor and replacing it with the dimerizing leucine zipper from the JUN transcription factor which results in a constitutively active receptor. We interrogated the role of the 12 residue linker that joins the transmembrane domain to the extracellular domain and found that as the zipper was brought closer to the membrane, the activation signal strength increased. We then monitored the relative positioning of the JAK2 binding region of the intracellular domains of the receptor by placing FRET reporters 37 amino acids below the Box1 motif in order to retain JAK2 binding to the receptor. By comparing the FRET signal from the leucine zipper construct with the weakest activation signal (containing the full 12 amino acid linker) to the construct with the strongest signal (no linker) we found that an increase in receptor signaling correlated with an increased distance between the JAK2 binding regions of the receptor, i.e., the intracellular domains were separating upon activation. We then confirmed that GH addition to its receptor induced a separation of the JAK2 binding regions by using a GH receptor construct with FRET reporters placed at the same intracellular position as before. Since the activation of a pre-dimerized receptor must be transmitting a signal through the transmembrane domain, we identified the interaction face of the transmembrane domains of the receptor in the absence of GH by cysteine crosslinking analysis and molecular modeling. Further in silico modeling showed that the transmembrane domains are in a parallel interaction in the inactive state and that transition to the active state involves a rotation resulting in a left-handed crossover dimer. This brings the residues at the extracellular juxtamembrane in close proximity, while C-termini of the transmembrane domains separate, explaining the repositioning of the intracellular domains that we observed with FRET upon activation. Previous models of JAK2 activation did not identify a separation of the JAK2 binding Box1 motifs, therefore we proposed a new model of JAK2 activation where in the inactive state of the predimerized receptor the pseudokinase domain from a JAK2 molecule that is bound to one receptor, interacts with and inhibits the kinase domain of the JAK2 bound to the other receptor. Upon activation, the intracellular domains of the receptor where the JAK2 FERM domain is bound separate, which brings the kinase domains together, facilitating trans-activation (Fig. 1). We were able to use FRET, molecular modeling as well as interaction and JAK2 activation assays to support this model.5 The key remaining question is: does such a mechanism generalize to other class I cytokine receptors? Figure 1. Activation model of homodimeric class I cytokine receptors shown for the archetypal GH receptor. Adapted from Brooks et al.5

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

The growth hormone receptor (GHR) is a critical regulator of postnatal growth and metabolism. However, the GHR signaling domains and pathways that regulate these processes in vivo are not defined. We report the first knock-in mouse models with deletions of specific domains of the receptor that are required for its in vivo actions. Mice expressing truncations at residue m569 (plus Y539/545-F) and at residue m391 displayed a progressive impairment of postnatal growth with receptor truncation. Moreover, after 4 months of age, marked male obesity was observed in both mutant 569 and mutant 391 and was associated with hyperglycemia. Both mutants activated hepatic JAK2 and ERK2, whereas STAT5 phosphorylation was substantially decreased for mutant 569 and absent from mutant 391, correlating with loss of IGF-1 expression and reduction in growth. Microarray analysis of these and GHR(-/-) mice demonstrated that particular signaling domains are responsible for the regulation of different target genes and revealed novel actions of growth hormone. These mice represent the first step in delineating the domains of the GHR regulating body growth and composition and the transcripts associated with these domains.

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp104 and Trp169 in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys165), which in addition to His169, restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.

Monoclonal antibody (MAb) 263 is a widely used monoclonal antibody that recognizes the extracellular domain (ECD) of the GH receptor. It has been shown to act as a GH agonist both in vitro and in vivo, and we report here that it must be divalent to exert its effect on the full-length receptor. To understand the mechanism of its agonist action, we have determined the precise epitope for this antibody using a novel random PCR mutagenesis approach together with expression screening in yeast. A library of 5200 clones of rabbit GH receptor ECD mutants were screened both with MAb 263 and with an anticarboxy-tag antibody to verify complete ECD expression. Sequencing for clones that expressed complete ECD but were not MAb 263 positive identified 20 epitope residues distributed in a discontinuous manner throughout the ECD. The major part of the epitope, as revealed after mapping onto the crystal structure model of the ECD molecule, was located on the side and upper portion of domain 1, particularly within the D–E strand disulfide loop 79–96. Molecular dynamics docking of an antibody of the same isotype as MAb 263 was used to dock the bivalent antibody to the 1528-A 2 epitope and to visualize the likely consequences of MAb binding. The minimized model enables the antibody to grasp two receptors in a pincer-like movement from opposite sides, facilitating alignment of the receptor dimerization domains in a manner similar to, but not identical with, GH. (Molecular Endocrinology 17: 2240–2250, 2003)

OBJECTIVE Because there is discordance between different immunoassay values for serum hGH, and because clinical state may not correlate with immunoreactive hGH, we have developed an assay to accurately measure serum hGH somatogenic bioactivity. The results of this assay were compared with the Elegance two-site ELISA assay across 135 patient samples in a variety of clinical states. DESIGN The somatogenic assay was based on stable expression of hGH receptor in the murine BaF line, allowing these cells to proliferate in response to hGH. To eliminate interference by other growth factors in serum, we created a specific antagonist of the hGH receptor (similar to Trovert or Pegvisomant) which allowed us to obtain a true measure of hGH somatogenic activity by subtraction of the activity in the presence of the antagonist. The assay was carried out in microtiter plates over 24 h, with oxidation of a chromogenic tetrazolium salt (MTT) as the endpoint. PATIENTS These encompassed a number of different clinical conditions related to short stature, including idiopathic short stature, neurosecretory dysfunction and renal failure, as well as obese patients on dietary restriction and normal volunteers. MEASUREMENTS In addition to the colourimetric (MTT) response to hGH, we measured free hGH by stripping out GHBP-bound hGH using beads coupled to a monoclonal antibody to the GHBP (GH binding protein). All samples were measured in both bioassay and ELISA assay. RESULTS This bioassay was sensitive (5 mU/l or 2 mug/l) and precise, and not subject to interference by the GHBP. There was a good correlation (r = 0.95) between bioactivity and immunoactivity across clinical states. There was, however, an increased bioactivity during secretory peaks (over 25 mU/l), which has been reported previously for the Nb2 bioassay. Free hGH did not correlate with clinical state. CONCLUSIONS Because the results of the Elegance ELISA and the bioassay correlate well, even though there is greater bioactivity at higher hormone concentrations, it is evident that an appropriate immunoassay is able to act as a reliable indicator for clinical assessment. In those rare cases where bio-inactive GH exists, our bioassay should provide an appropriate means to demonstrate this.

Pituitary growth hormone (GH) stimulates postnatal growth and metabolism. The role of GH and its receptor (GHR) during prenatal development, however, is still controversial. As shown by reverse transcription polymerase chain reaction (RT-PCR), bovine in vitro fertilization embryos synthesized the transcript of GHR from Day 2 of embryonic life onwards. Real time RT-PCR revealed that synthesis of GHR mRNA was increased 5.9-fold in 6-day-old embryos compared with 2-day-old embryos. Using in situ hybridization, the mRNA encoding GHR was predominantly localized to the inner cell mass of blastocysts. The GHR protein was first visualized 3 days after fertilization. GH-specific transcripts were first detected in embryos on Day 8 of in vitro culture. As shown by transmission electron microscopy, GH treatment resulted in elimination of glycogen storage in 6- to 8-dayold embryos and an increase in exocytosis of lipid vesicles. These results suggest that a functional GHR able to modulate carbohydrate and lipid metabolism is synthesized during preimplantation development of the bovine embryo and that this GHR may be subject to activation by embryonic GH after Day 8. conceptus, developmental biology, early development, growth hormone, IVF/ART

GH actions are dependent on receptor dimerization. The GH receptor antagonist, B2036-PEG, has been developed for treating acromegaly. B2036 has mutations in site 1 to enhance receptor binding and in site 2 to block receptor dimerization. Pegylation (B2036-PEG) increases half-life and lowers immunogenicity, but high concentrations are required to control insulin-like growth factor-I levels. We examined antagonist structure and function and the impact of pegylation on biological efficacy. Unpegylated B2036 had a 4.5-fold greater affinity for GH binding protein (GHBP) than GH but similar affinity for membrane receptor. Pegylation substantially reduced membrane binding affinity and receptor antagonism, as assessed by a transcription assay, by 39- and 20-fold, respectively. GHBP reduced antagonist activity of unpegylated B2036 but did not effect antagonism by B2036-PEG. B2036 down-regulated receptors, and membrane binding sites doubled in the presence of dimerization-blocking antibodies, suggesting that B2036 binds to a receptor dimer. It is concluded that the high concentration requirement of B2036-PEG for clinical efficacy relates to pegylation, which decreases binding to membrane receptor but has the advantages of reduced clearance, immunogenicity, and interactions with GHBP. Our studies suggest that B2036 binds to a receptor dimer and induces internalization but not signaling.

Insulin modulates the biological actions of GH, but little is known about its effect on human hepatic GH receptors (GHRs). Using the human hepatoma cell line HuH7 as a model, we investigated insulin regulation of total, intracellular, and cell surface GHRs and receptor biosynthesis and turnover. Insulin up-regulated total and intracellular GHRs in a concentration-dependent manner. It increased surface GHRs in a biphasic manner, with a peak response at 10 nmol/L, and modulated GH-induced Janus kinase-2 phosphorylation in parallel with expression of surface GHRs. The abundance of GHR messenger ribonucleic acid and protein, as assessed by RT-PCR and Western analysis, respectively, markedly increased with insulin treatment. To examine whether insulin regulates GHRs at the posttranslational level, its effects on receptor surface translocation and internalization were investigated. Insulin suppressed surface translocation in a concentration-dependent manner, whereas internalization was unaffected. Moreover, insulin actions on total GHRs and surface translocation were inhibited by PD98059 and wortmannin, respectively. In conclusion, insulin regulates hepatic GHR biosynthesis and surface translocation in a reciprocal manner, with surface receptor availability the net result of the divergent effects. The divergent actions of insulin appear to be mediated by the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways, respectively.

Binding of growth hormone (GH) to its receptor (GHR) is a well-studied example of molecular recognition between a cytokine and its receptor. Extensive mutagenesis studies and several crystal structures have defined the key interactive amino acid residues that are involved in binding and subsequent receptor dimerization. This review encompasses each of the three molecular recognition events involved in GHR activation, namely binding of GH to its two receptors and the interactions that occur between these receptors, Particular attention is given to species and ligand specificity of hormone binding and to the molecular recognition events involved in receptor activation, including the possibility that a conformational change in the receptor is required. Copyright (C) 1999 John Wiley & Sons, Ltd.

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 B-12 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 colony-stimulating 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)2resulted 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.

We previously have demonstrated that insulin and insulin-like growth factor-I (IGF-I) down-regulate growth hormone (GH) binding in osteoblasts by reducing the number of surface GH receptors (GHRs). The present study was undertaken to investigate the mechanism of GHR down-regulation. Treatment with 5 nM insulin or IGF-I for 18 hr significantly decreased surface GH binding to 26.4 ± 2.9% and 23.0 ± 2.7% of control (mean ± SE; P < 0.05), respectively. No corresponding reductions in the mRNA level and total cellular content of GHR were found, nor was the rate of receptor internalization affected. The effects on GHR translocation were assessed by measuring the reappearance of GH binding of whole cells after trypsinization to remove the surface receptors. GH binding of control cultures significantly increased ( P < 0.05) over 2 hr after trypsinization, whereas no recovery of binding activity was detected in insulin and IGF-I-treated cultures, indicating that GHR translocation was impaired. Studies on the time course of GHR down-regulation revealed that surface GH binding was reduced significantly by 3-hr treatment ( P ≤ 0.0005), whereas GHR translocation was completely abolished by 75–90 min with insulin and IGF-I. The inhibition of receptor translocation by insulin, but not IGF-I, was attenuated by wortmannin. In conclusion, insulin and IGF-I down-regulated GH binding in osteoblasts by acutely impairing GHR translocation, with their effects exerted through distinct postreceptor signaling pathways.

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 A structure of the human growth hormone (hGH).hGH-binding protein (hGHBP)2 complex that Souza and co-workers (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 (non-primate 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 non-primate hormone to bind to and activate the human GH receptor.

While there is a considerable amount of evidence that signal transduction by the growth hormone (GH) receptor requires receptor homodimerization, there has been no systematic study of the role of receptor dimerization domain residues in this process. In conjunction with the distances derived from the crystal structure of the hGH-hGH receptor (extracellular domain) complex, we have used a luciferase-based c-fos promoter reporter assay in transiently transfected Chinese hamster ovary (CHO) cells, and stable receptor expressing CHO cell populations to define the dimerization domain residues needed for effective signaling. In addition to alanine substitution, we have used both aspartate and lysine substitutions to allow us to provide evidence for proximity relations through charge complementation. Introduced cysteine substitutions were also used, but unlike the erythropoietin receptor, these were unable to generate constitutively active receptor. We conclude that serine 145, histidine 150, aspartate 152, tyrosine 200, and serine 201, but not leucine 146 or threonine 147 are required for effective signal transduction through the dimerization domain. This information may be valuable in designing small molecule antagonists of GH and other cytokines that block dimerization by binding to the dimerization domain.

Studies in non-dental embryonic tissues have suggested that an interaction between growth hormone and its receptor may play a role in growth and development before the foetal pituitary gland is competent. This study reports the distribution of growth hormone, its receptor and binding protein in developing rat tooth germs from embryonic day 17 to 21 and postnatal day 0 using antibodies specific for each of these proteins. Four foetal rats were processed at each time point (E17, E18, E20/21 and postnatal day 0). Following routine fixation and paraffin embedding, sections were treated with antisera to rat growth hormone, rat growth hormone binding protein and growth hormone receptor. Localization of antibody/antigen complexes was subsequently visualized by addition of biotinylated IgG and reaction with streptavidin peroxidase and diaminobenzidine. Assessment of the level of staining was qualitative and based on a subjective rankings ranging from equivocal to very strong staining. Overall, growth hormone and its binding protein were located both in the cellular elements and throughout the extracellular matrix, whereas the growth hormone receptor showed an exclusively intra-cellular location. All three proteins were detectable in cells of the dental epithelium and mesenchyme at the primordial bud stage (E17) which occurs prior to expression of pituitary growth hormone. At the cap stage of odontogenesis (E18-19), numerous cells in both the dental epithelium and mesenchyme were intensely immunoreactive for growth hormone, its binding protein and receptor. In the succeeding early bell stage (E20-21), most of the mesenchymal cells in the dental pulp were mildly positive for these proteins, while the dental epithelium and adjacent mesenchyme were more immunoreactive. At the late bell stage (postnatal day 0), all three proteins were localized in dental epithelium, differentiating mesenchymal cells the cuspal surface facing the epithelial-mesenchymal interface, preodontoblasts, and odontoblasts forming dentine. From these observations, immunoreactive growth hormone, its receptor and binding protein appear to be expressed in odontogenic cells undergoing histodifferentiation, morphodifferentiation and dentinogenesis in a cell-type and stage-specific pattern throughout embryonic tooth development. This suggests the possibility that growth hormone, or a growth hormone-like protein, plays a paracrine/autocrine role in tooth development in utero.

The crystal structure of the extracellular domain of growth hormone receptor complexed to its ligand, growth hormone, has been known since 1992. However, no information exists for the unliganded form of the receptor. The human growth hormone receptor's extracellular ligand-binding domain, encompassing amino-acid residues 1-238, has been expressed in Escherichia coli, purified by anion ion-exchange chromatography and crystallized in its unliganded state by the hanging-drop vapour-diffusion method in 100 mM HEPES pH 7.0 containing 27.5%(w/v) PEG 5000 monomethyl ether and 200 mM ammonium sulfate as the co-precipitants. The crystals belong to the othorhombic space group C222(1), have unit-cell parameters a = 99.7, b = 112.2, c = 93.2 A and diffract to 2.5 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of any conformation changes that occur upon ligand binding and will provide information to develop potential low-molecular-weight agonists/antagonists to treat clinical diseases in which the growth hormone receptor is implicated.

Growth hormone (GH) is believed to signal by dimerizing its receptor through two binding sites on the hormone. Previous attempts to increase the biopotency of GH by increasing its site 1 affinity have been unsuccessful, which has led to a bias toward engineering site 2 interactions in the quest for creation of super agonists. Here we report that increasing site 1 affinity can markedly increase proliferative bioactivity in FDC-P1 cells expressing full-length GHR. In contrast, we find three site 1 mutants with affinities for site one similar to or greater than wild type GH, which have markedly decreased bioactivity. Through crystal structure analysis of the receptor interactive regions of these GH analogues, we are able to suggest why previous mutagenesis on human GH failed to improve biopotency, and thus provide a new avenue for GH and cytokine agonist design.

A panel of monoclonal antibodies to the growth hormone (GH) receptor/binding protein was used to demonstrate the existence and detail the expression of GH receptors in ductal and alveolar epithelial cells from rat and rabbit mammary glands by immunohistochemistry. Intense immunoreactivity was present in membrane, cytoplasm and some nuclei of epithelial cells during proliferation and lactation. Receptor expression decreased during weaning and was absent or weak in regressive mammary glands. Immunoreactivity was weak in ductal epithelial cells from virgin adult animals. Pronounced expression of GH receptor/binding protein was observed with two monoclonal antibodies and lesser reactivity was seen with others, paralleling their affinities for the receptor. The cytoplasmic presence of this putatively plasma membrane located GH receptor is accounted for by the existence of a soluble form on the GH receptor, namely the growth hormone binding protein derived from the membrane receptor by cleavage. Primary localization of the receptor in proliferating and lactating epithelial cells suggests that the rat and rabbit mammary gland is a GH target tissue. This finding is in contradiction to both classical GH action and the somatomedin hypothesis and challenges the widely held view that GH has no direct influence on mammary growth and function. Copyright

Growth hormone (GH) promotes stem cell activation, cell proliferation, differentiation and survival, either directly or through the induction of IGF-1. GH acts via its cell membrane receptor to initiate a range of signalling pathways, with JAK2 kinase activation of STAT5 being the most important. The transcription factor STAT5 acts to induce expression of the key growth mediator, IGF-1, but also regulates the expression of a host of other genes, some of which are important growth regulators. In addition to its signalling from the cell membrane, the GH receptor translocates to the nucleus in a GH-dependent manner, where it regulates the expression of other cell growth-related genes, and sensitises the cell to the proliferative action of GH.

Members of the cytokine receptor family have a consensus WSXWS sequence (WS motif) in the extracellular domain. With the interleukin-2, erythropoietin, and prolactin receptors, alteration of the WS sequence disrupts ligand binding and receptor signaling. The structural basis for these effects is unclear. To examine the role of the WS equivalent sequence (Y222GEFS226) in the function of the growth hormone receptor, each residue was mutated to alanine or to the WS consensus sequence. Although we used stable cell lines expressing all of these mutants, we show only three mutants, Y222A, G223A, and S226A, which display lower ligand affinity. Using conformation-specific monoclonal antibodies, we show that Y222A and S226A receptors have structural perturbations, which result in decreased signal transduction. This was shown by a decreased ability of growth hormone to stimulate protein synthesis and to transactivate the c-fos promoter with these mutants. The crystal structure of the ligand-occupied extracellular domain of growth hormone receptor indicates that Tyr222 and Ser226 have important interactions within the second beta-barrel domain, providing a structural basis for our results. The WS segment is not involved in sequence-specific accessory protein interaction, as mutation of residues Gly223, Glu224, and Phe225 does not alter receptor function.

In this study we have demonstrated that the C-terminus of helix 1 of porcine GH (pGH) is a receptor-interactive region, thus extending the current binding site model of GH. This was achieved by introducing charge reversal mutations into this region of pGH, which influenced receptor affinity and Ca2+ dependence of binding. The first mutant (R34E pGH, conversion of Arg 34 to Glu) introduced a putative Ca2+ binding site which is present in human GH (hGH) [Barnard et al. (1989) J. Theor. Biol. 140, 355-367] and sits opposite E220 of receptor subunit 1. This mutant exhibited increased Ca2+ dependence of receptor binding but even at optimal Ca2+ did not display higher than wild-type affinity. Introduction of a second Ca2+ binding site adjacent to the first by a second charge reversal (K30E R34E pGH) further increased Ca2+ dependence of binding and also increased affinity for the rabbit GH receptor (2.4 +/- 0.4)-fold relative to wild-type pGH at optimal Ca2+ Equilibrium dialysis and Scatchard analysis of binding of Ca-45(2+) to PGH and K30E R34E pGH revealed two Ca2+ binding sites on wild-type pGH and an additional two Ca2+ binding sites on the K30E R34E pGH mutant (K-d 0.5-0.8 mM), as predicted. A third partial charge reversal mutant in the fourth helix (H170D) also led to enhanced Ca2+ dependence of binding, supporting our proposal that E34 and D170 are responsible for the Ca2+ dependence of hGH binding to the rabbit GH receptor. Examination of the crystal structure shows that E34 and D170 are in close proximity and would interact repulsively with a cluster of acidic residues on the receptor consisting of E126, E127, and E220 unless neutralized by Ca2+ or an introduced basic residue. Accordingly, charge reversal at the adjacent PGH residue E33 (E33K pGH) led to a Ca2+ independent (3.0 +/- 0.4)-fold increase in affinity of binding. As well as extending the binding site model of GH, these studies provide a mechanistic explanation for the unique Ca2+ dependence of hGH binding to the rabbit GH receptor. They also indicate that charge reversal can be used to design higher affinity GH analogues and could assist in the mapping of interactive regions in ligand-receptor complexes generally.

Summary A panel of monoclonal antibodies to the growth hormone (GH) receptor/binding protein was used to demonstrate the existence and detail the expression of GH receptors in the cerebellum of 2, 10, 28 days old postnatal and adult rats and 10, 20 days old and adult rabbits by immunohistochemistry to define potential targets for endogenous GH action in the cerebellum. Receptors were localized in membrane and cytoplasmic components of neurons and glial cells and expression decreased with age. Intense immunoreactivity was observed in the cytoplasm and dendrites of Purkinje cells and in cells of the cerebellar nuclei. Glial cells also showed receptor expression. Strong immunoreactivity was observed with two monoclonal antibodies and lesser reactivity was seen with others, paralleling their affinities for the receptor. The cytoplasmic presence of this putatively plasma membrane located GH receptor is accounted for by the high receptor content of endoplasmic reticulum and the existence of a soluble form of the GH receptor, namely the GH binding protein (BP) derived from the membrane receptor by cleavage, and receptor localization reported here correlate well with the distribution of insulin-like growth factor 1 (IGF-1 ) mRNA and immunoreactivity in cerebellar Purkinje cells and glial cells. Primary localization of the receptor in the cerebellum is in direct contradiction to both classical GH action and the somatomedin hypothesis and supports and extends the theory of genetically regulated macroneuronal maturation.

Although several reports indicate proliferative and functional effects of human GH (hGH) on peripheral blood lymphocytes (PBL), no information is available about hGH receptor (GHR) expression in PBL subsets. Here, the surface membrane GHR levels were investigated in different human PBL subpopulations using a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody specific for the GHR (mAb263) in dual fluorochrome flow cytometric assays. Strong GHR expression was found in B-cells (CD20+), whereas CD2+ lymphocytes, including T-cells as well as natural killer cells, exhibited considerably lower levels of receptor expression. Similarly, using FITC-labeled recombinant hGH, receptor expression on CD20+ cells was significantly higher than that on CD2+ cells. Abundant expression of GHR in B-lymphocytes was confirmed by reverse transcriptase-polymerase chain reaction analysis of GHR messenger ribonucleic acid from isolated B-cells. Accordingly, the B-cell merits greater consideration as a GH target cell. The use of FITC-labeled mAb263 and hGH is of potential use for the study of GHR levels in patients exhibiting different types of growth disorders. Because of its high specificity for GHR, FITC-labeled mAb263 is also of considerable value for specifically demonstrating the presence of GHR, because hGH may interact with and act through PRL receptor, as shown previously in human neutrophils.

We have previously shown that the growth hormone (GH) receptor-binding protein is associated with the nucleus. We show here both by electron microscopy and nuclear isolation that GH is subject to rapid nuclear translocation. The intracellular fate of intravenously injected 125I-bovine growth hormone (bGH) was examined in the rat hepatocyte by electron microscopic autoradiography. The hormone appeared rapidly at the plasma membrane, then sequentially in lysosomal and multivesicular bodies and/or the nuclear membrane before final translocation to the nuclear matrix. Maximal translocation to the nuclear matrix occurred within 30 min of injection. Nuclear translocation of 125I-hGH was also studied by isolation of nuclei from cells stably transfected with cDNAs encoding the GH receptor, GH-binding protein, and a membrane bound but cytoplasmic domain-deficient receptor. Specific internalization and nuclear translocation of hormone only occurred in cells transfected with the full-length receptor. The translocation was rapid and became saturated within 1 h after addition of hormone to the culture media. SDS-polyacrylamide gel electrophoresis of isolated nuclei showed that GH is transported to the nucleus as the intact molecule. Pretreatment of cells with lysosomotropic agents (chloroquine, ammonium chloride, and bacitracin) decreased hormone degradation and increased nuclear translocation of GH. The nuclear translocation of GH was achieved independent of the cytoskeletal system (microtubular, microfilament, and intermediate filament networks). Thus, GH is subject to rapid receptor-dependent nuclear translocation via the endosomal pathway.

After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca++ concentration. PRL stimulates Ca++ entry and induces secondary Ca++ mobilization. The entry of Ca++ is a result of an increase in K+ conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 microM herbimycin in CHO cells co-transfected with PRL receptor cDNA and the beta lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes.

Laron-type dwarfism is an autosomal recessive disorder characterised by extreme growth retardation and growth hormone (GH) resistance and has been shown in some cases to be associated with mutations in the GH receptor gene. Limited data suggest that in this condition specific liver GH binding is absent. In the majority of reported cases specific GH binding is also absent in serum. However it is not known whether the GH receptor and/or the serum GH binding protein are expressed in this condition. Using the techniques of immunohistochemistry and Northern blotting we have demonstrated that in cultured skin fibroblasts derived from four patients with Laron-type dwarfism the GH receptor gene is transcribed and the GH receptor protein is expressed on the cell surface. Further study of one of these patients, who has not previously been reported, has also revealed low but detectable levels of GH binding protein in serum using a two-site immunradiometric assay which does not depend on GH binding. These results indicate that the growth hormone receptor/binding protein is expressed in Laron-type dwarfism.

Summary OBJECTIVE Studies in rodents have shown GH binding protein (GHBP) levels to be dependent on the mode of GH administration. The aim is to determine whether GHBP levels in man are also modulated by the pattern of GH administration. PATIENTS Six GH deficient subjects participated in a randomized study in which 2 IU GH were administered either as (i) a continuous 24-hour infusion, (ii) two intravenous boluses or (iii) eight intravenous boluses every 3 hours. In a second study, six normal men received a single subcutaneous injection of 0 2 U/kg GH and GHBP activity was measured over 24 hours. Control data were obtained from an untreated group of six age-matched normal men. MEASUREMENTS GHBP activity was measured by immunoprecipitation using a monoclonal antibody that recognizes the human GHBP, and expressed as percentage specific binding of 125I-GH in 50 μl of serum. RESULTS GHBP activity was not significantly different between the GH deficient and normal subjects. GHBP activity did not rise significantly during GH administration with each of the three intravenous patterns of delivery nor were there any significant differences between treatments. in the second study, GHBP activity did not change significantly following subcutaneous GH injection nor did results differ from untreated normal controls. CONCLUSIONS The level of GHBP in man is not dependent on GH secretory status or altered by short-term GH treatment or the mode of administration. These findings stand in contrast to GH treatment effects in rodents and suggest that GH regulation of GHBP may be different between species.

Growth hormone (GH) gene transfer can markedly increase growth in transgenic fish. In the present study we have developed a transcriptional assay to evaluate GH-signal activation (GHSA) in zebrafish embryos. By analyzing the transcription of c-fos and igf1, and the promoter activity of spi2.1, in zebrafish embryos injected with different constructs, we found that overexpression of either GH or growth hormone receptor (GHR) resulted in GHSA, while a synergetic overexpression of GH and GHR gave greater activation. Conversely, overexpression of a C-terminal truncated dominant-negative GHR (ΔC-GHR) efficiently blocked GHSA epistatic to GH overexpression, demonstrating the requirement for a full GHR homodimer in signaling. In view of the importance of signal-competent GHR dimerization by extracellular GH, we introduced into zebrafish embryos a constitutively activated GHR (CA-GHR) construct, which protein products constitutively dimerize the GHR productively by Jun-zippers to activate downstream signaling in vitro. Importantly, overexpression of CA-GHR led to markedly higher level of GHSA than the synergetic overexpression of GH and GHR. CA-GHR transgenic zebrafish were then studied in a growth trial. The transgenic zebrafish showed higher growth rate than the control fish, which was not achievable by GH transgenesis in these zebrafish. Our study demonstrates GH-independent growth by CA-GHR in vivo which bypasses normal IGF-1 feedback control of GH secretion. This provides a novel means of producing growth enhanced transgenic animals based on molecular protein design.

Residues within the first disulphide loop of the GH receptor are highly conserved, and the two cysteines forming this motif are conserved across many cytokine receptors. We have used site-directed mutagenesis and the polymerase chain reaction with splicing by overlap extension to show that these residues are essential for binding of bovine (b)GH and human (h)GH to the rabbit GH receptor. When all residues within this loop were replaced with an equivalent polyalanine sequence, hormone binding was abolished. Conversion of Arg 39 within the loop to aspartate (R39D) decreased affinity for bGH by up to 20-fold. Conversion of Glu 42 to lysine (E42K) also resulted in a fivefold loss of affinity for bGH. However, charge reversals at Lys 37, Glu 44 and the conversion of Leu 43 to an arginine (as in the human receptor) were without a major effect on bGH binding. The lack of effect of the L43R mutation on bGH affinity, despite a significant (threefold) decrease in hGH affinity, argues against a role for Arg 43 as a residue conferring primate GH-binding specificity on the human receptor. Examination of the affinities of poly Ala, R39D and E42K mutants for a variety of hormone-binding-site directed and other monoclonal antibodies (MAbs) to the GH receptor revealed that these mutations were without a major effect on tertiary structure. It is of interest that the epitopes for the hormone-binding inhibitory MAbs 263 and 7 are located within this first loop, since the poly Ala mutation abolished the binding of both MAbs, and the R39D mutation abolished binding of MAb 7. In conclusion, residues within the first disulphide loop of the GH receptor are critical for hormone binding, and are epitopes for the binding of inhibitory MAbs. These findings may be applicable to other cytokine receptors.

Pituitary GH secretion appears largely unnecessary for the attainment of normal birth size in many species, including man. This is believed to be due to an immaturity and/or an absence of GH receptors in many fetal tissues. However, in vitro studies using late first trimester human fetal tissues have demonstrated mitogenic actions of GH on liver and stimulation of insulin biosynthesis in pancreas. To resolve this discrepancy, we have employed immunocytochemistry to identify the presence and distribution of GH receptors in various human fetal tissues. Fetuses of 14-16 weeks gestation were obtained after therapeutic abortion, tissues were fixed, and immunocytochemistry was performed using monoclonal antibodies against purified rat or rabbit GH receptor. The specificity of staining was confirmed by preabsorption of the antibodies with 1) adult rat liver membranes or 2) human fetal liver membranes, both of which possess specific GH-binding sites, or 3) human fetal skeletal muscle membranes, which do not specifically bind GH. Positive staining was seen in a subpopulation of liver parenchymal cells, in the ductal and endocrine tissue of pancreas, in the germinal layer of the epidermis and the deeper dermal layers of skin, and in the tubular epithelium of kidney. No immunopositive staining was seen in skeletal or cardiac muscle, epiphyseal growth plate, lung, intestine, or adrenal. Positive staining was present in the neuronal cell bodies of the cerebral cortex. GH receptor was also detectable as early as 8 weeks gestation in syncytial layers of the placenta and was maintained until term. Results demonstrate the presence of immunoreactive GH receptor/binding protein in some human fetal tissues early in development. In particular, these results would support a role for GH in the growth and function of liver and pancreas.

In a recent study we identified GH receptor/binding protein in cells of the gastric mucosa. In order to define the role of the GH receptor/binding protein in gastric function, we have investigated the effect of GH on gastric structure and function in the GH-deficient Lewis (dwarf) rat. Bovine GH, 65 micrograms/100 g body wt, was administered twice daily to adult male dwarf rats for 6 days (DW+) while control animals received vehicle only (DW-). Administration of GH produced a significant increase in body wt (P less than 0.001), stomach wt (P less than 0.01), and stomach to body wt ratio (P less than 0.05). GH administration also resulted in increased total gastric DNA, RNA, and protein content but did not produce significant differences in DNA, RNA, or protein content when normalized to stomach wt. Morphometric analysis of the gastric mucosa revealed a significantly (P less than 0.05) increased gastric epithelial height and mucosal surface area along with an increase in the proportion of nuclei with multiple nucleoli (P less than 0.01). The number of gastric mucosal cells in S-phase was determined by immunohistochemical detection of nuclear 5'-bromo-2'-deoxyuridine (BrdU) incorporated during a 2 h pulse of BrdU. GH treatment resulted in a 74% increase (P less than 0.05) in the number of BrdU-labeled nuclei/mm2 mucosa relative to vehicle-injected control animals. A modification of Zimmerman's method for the differential staining of gastric mucosa was used to delineate cell type for morphometric analysis. This showed that the density of differentiated (parietal and chief) cell types was not significantly different between DW- and to DW+ animals. Soluble extracts of gastric mucosa were prepared for estimation of pepsinogen content and [57Co]cyanocobalamin (vitamin B12) binding. GH administration produced no significant change in pepsinogen content per mg protein and did not affect the relative levels of pepsinogen isoenzymes as determined by polyacrylamide gel electrophoresis. GH administration did however result in an 86% increase (P less than 0.01) in [57Co]cyanocobalamin binding per mg protein. The increase in binding was totally displaceable by 1 microgram/ml unlabeled cyanocobalamin but not by 1 microgram/ml cobinamide dicyanide indicating it was the result of increased intrinsic factor rather than R protein. Sephadex S-300 gel filtration of mucosal extracts revealed an elution profile for [57Co]cyanocobalamin identical to that of purified porcine intrinsic factor and different from that of human salivary R protein. In conclusion, we have demonstrated that GH stimulates proliferation and enlargement of the gastric mucosa without significant alteration in cellular composition.(ABSTRACT TRUNCATED AT 400 WORDS)

Although fetal growth is generally considered to be independent of pituitary growth hormone (GH), it is possible that pituitary GH plays a modulatory role in organ development or that a GH-like substance of non pituitary origin may influence fetal growth through the GH receptor. Accordingly, we have used immunohisto-chemisty, northern blot analysis, the reverse transcriptase-polymerase chain reaction and solution hybridization to study the ontogeny of the GH receptor/binding protein (BP) from the 12-day-old embryo (E12) to the E18 rat fetus. GH receptor/BP immunoreactivity was observed in all major organ systems of the E18 rat fetus and was not preferentially associated with any germ layer derivative. A general increase in GH receptor/BP immunoreactivity was evident from E12 to E18, with a marked increase occurring between E16 and E18. Hemangioblastic tissue was, however, strongly or intensely immunoreactive at all stages of development, as was the placenta. Most noteworthy of the other tissues expressing GH receptor/BP immunoreactivity by day 18 were skeletal and smooth muscle, chondroprogenitor cells, epithelial lining cells, neuronal ganglia, ependymal cells and the adrenal cortex. In the placenta, the most prominent immunoreactivity was associated with decidual cells. Total RNA was isolated from E12 to E18 rat fetuses and adult rat liver. Northern hybridization with a 35S-labelled rat GH receptor cRNA probe revealed that 3.9 kb and 1.2 kb transcripts complementary to the rat GH receptor ribopobe are present from at least E16. The existence of GH receptor mRNA at E12 and E14 was demonstrated by the polymerase chain reaction. Solution hybridization analysis demonstrated an exponential increase in the total embryonic GH receptor/BP mRNA during fetal development. However even by E18 levels were only 8% those of adult liver. In conclusion, we have demonstrated the presence and defined the ontogeny of the GH receptor/BP in the rat foetus from mid gestation. These results, when combined with earlier studies of GH action on fetal tissues suggest a réévaluation of the role of GH in prenatal growth.

GH has physiological functions in many tissues, but the cellular targets for direct effects of GH remain ill defined in complex tissues such as the growth plate in which the contribution of direct vs. indirect actions of GH remains controversial. The Janus kinase (Jak)-signal transducer and activator of transcription (STAT)-5 pathway is activated by GH, so we developed a method to visualize nuclear Stat5b and phosphorylated Stat5 in single cells in response to a pulse of GH. Hep2 cells did not show a Stat5 phosphorylation (pY-Stat5) response to GH except in cells transfected to express GH receptors. ATDC5 cells express GH receptors and showed GH-induced pY-Stat5 responses, which varied with their state of chondrocyte differentiation. In vivo, Stat5b+ve nuclei were seen in the resting and prehypertrophic chondrocytes of the growth plate. After a single ip pulse of human GH or mouse GH, but not prolactin, pY-Stat5 responses were visible in cells in the resting zone and groove of Ranvier, 10–45 min later. Prehypertrophic chondrocytes showed no pY-Stat5 response to GH. GH target cells were also identified in other tissues, and a marked variability in spatiotemporal pY-Stat5 responses was evident. Endogenous hepatic pY-Stat5 was detected in mice with intact GH secretion but only during a GH pulse. Fasting and chronic exposure to GH attenuated the pY-Stat5 response to an acute GH injection. In conclusion, pY-Stat5 responses to GH vary in time and space, are sensitive to nutritional status, and may be inhibited by prior GH exposure. In the growth plate, our data provide direct in vivo support for an early role of GH to regulate the fate of immature chondrocytes.

The mechanisms involved in transcriptional regulation by growth hormone (GH) remain unknown. We report here that GH receptor immunoreactivity can be demonstrated in the nuclei of GH-responsive rat and rabbit tissues at both the light and electron micrograph level using monoclonal antibodies to the receptor extracellular domain. Nuclear staining is heterogeneous and associated with both chromatin and the nuclear membrane. To confirm these observations, nuclei were isolated from rabbit liver by two methods, one involving extensive nonionic detergent washes. Scatchard analysis of nuclear fractions revealed high affinity somatogenic receptor in nuclear membranes, nucleoplasm, and chromatin fractions. A panel of GH receptor monoclonal antibodies was used to further define these nuclear binding sites as being antigenically identical to microsomal receptor in all but one case. In addition, affinity cross-linking experiments showed the somatogenic binding subunit to have a reduced Mr of 67,000, similar to the Mr of the GH binding protein. We propose that the association of a GH binding protein with the nucleus may provide a means whereby GH can regulate the transcription of specific genes either directly or indirectly through nuclear kinase C activation. This speculation is congruent with the recent demonstration of a GH response element by Yoon et al. (Yoon, J. B., Berry, S. A., Seelig, S., and Towle, H. C. (1990) J. Biol. Chem. 265, 19947-19954).

The presence of GH receptor (GHR) in the cell nucleus correlates with cell division, and targeting the GHR to the nucleus results in constitutive proliferation and transformation because of increased sensitivity to autocrine GH. Here we have sought additional mechanisms that might account for the enhanced proliferation seen with nuclear GHR, commencing with a yeast two-hybrid (Y2H) screen for interactors with the extracellular domain of the GHR [GH-binding protein (GHBP)]. We find that the GHBP is a transcriptional activator in yeast and mammalian cells, and this activity resides in the lower cytokine receptor module. Activity is dependent on S226, the conserved serine of the cytokine receptor consensus WSXWS box. By using parallel GHBP affinity columns and tandem mass spectrometry of tryptic digests of proteins bound to wild-type GHBP and S226A columns, we identified proteins that bind to the transcriptionally active GHBP. These include a nucleoporin and two transcriptional regulators, notably the coactivator activator (CoAA), which is also an RNA binding splicing protein. Binding of CoAA to the GHBP was confirmed by glutathione S-transferase pulldown and coimmunoprecipitation, and shown to be GH dependent in pro-B Ba/F3 cells. Importantly, stable expression of CoAA in Ba/F3 cells resulted in an increased maximum proliferation in response to GH, but not IL-3. Because CoAA overexpression has been identified in many cancers and its stable expression promotes cell proliferation and cell transformation in NIH-3T3 cells, we suggest CoAA contributes to the proliferative actions of nuclear GHR by the hormone-dependent recruitment of this powerful coactivator to the GHR.

The sites of action of GH in the human infant remain unclear; recent evidence in animals suggests direct actions on growth plate and other tissues. We have used a monoclonal antibody recognizing the human GH receptor to visually identify and localize GH receptors in the human infant growth plate. Sternochondral cartilage was obtained at postmortem from infants dying of sudden infant death (n = 20), and either decalcified, fixed, and cut into longitudinal sections or digested with collagenase for monolayer culture of chondrocytes. Sections of cultured chondrocytes were stained immunocytochemically with a monoclonal antibody recognizing human GH receptor (MAb 263), using an avidin-biotin system. Sternochondral cartilage was also obtained at operation from adolescents undergoing sternochondroplasty. In infant tissue, GH receptor was identified in sections in chondrocytes of the proliferative and hypertrophic layers, in perichondrium, in osteocytes in new bone, and in hemopoietic precursor cells in marrow. Cultured chondrocytes showed heterogeneous staining for GH receptor. With prolonged culture from 5-8 days, the pattern of staining changed from individual cells to groups of cells. [125I]Human (h)GH showed specific binding to chondrocyte monolayer (0.6 +/- 0.3%), confirmed visually on emulsion autoradiography. In support of specificity of MAb263, it was able to displace [125I]hGH from monolayers by 35%. Adolescent cultured chondrocytes failed to demonstrate specific binding of [125I]hGH. We conclude that GH receptors are widely distributed in a range of mesenchyme cells in the human infant growth plate, including bone and hemopoietic precursors. The expression of these receptors appears to be maturation dependent in both intact tissue and culture, while they may no longer be expressed after the peak growth phase of puberty.

It has been 75 years since Evans and Long identified a somatic growth-promoting substance in pituitary extracts, yet it is only in the last 20 years that the molecular basis for this action has been established. Three key elements in this elucidation were the cloning of the GH receptor, the identification of Janus kinase (JAK) 2 as the receptor-associated tyrosine kinase, and the delineation of signal transduction and activators of transcription (STAT) 5a/b as the key transcription factor(s) activated by JAK2. The interaction between these three elements results in enhanced postnatal growth and is the subject of this review. We describe a new model for GH receptor activation based on subunit rotation within a constitutive dimer, together with the phenotype and hepatic transcript profile of mice with targeted knockins to the receptor cytoplasmic domain. These support a central role for STAT5a/b in postnatal growth.

The GH receptor (GHR) is essential for normal postnatal growth and development, and the molecular basis of GHR action has been studied intensively. Clinical case studies and more recently mouse models have revealed the extensive phenotype of impaired GH action. We recently reported two new mouse models, possessing cytoplasmic truncations at position 569 (plus Y539/545-F) and 391, which were created to identify functional subdomains within the cytoplasmic signaling domain. In the homozygous state, these animals show progressively impaired postnatal growth coupled with complex changes in gene expression. We describe here an extended phenotype analysis encompassing the heterozygote state to identify whether single copies of these mutant receptors bring about partial or dominant-negative phenotypes. It appears that the retention of the ubiquitin-dependent endocytosis motif in the N-terminal cytoplasmic domain permits turnover of these mutant receptors because no dominant-negative phenotype is seen. Nonetheless, we do observe partial impairment of postnatal growth in heterozygotes supporting limited haploinsufficiency. Reproductive function is impaired in these models in a progressive manner, in parallel with loss of signal transducer and activator of transcription-5 activation ability. In summary, we describe a more comprehensive phenotypic analysis of these mouse models, encompassing overall and longitudinal body growth, reproductive function, and hormonal status in both the heterozygote and homozygote state. Our results suggest that patients expressing single copies of similarly mutated GHRs would not display an obvious clinical phenotype.

Cementum is known to be growth-hormone (GH)-responsive, but to what extent is unclear. This study examines the effects of extremes of GH status on cementogenesis in three lines of genetically modified mice; GH excess (giant), GH antagonist excess (dwarf), and GH receptor-deleted (GHR-KO) (dwarf). Age-matched mandibular molar tissues were processed for light microscope histology. Digital images of sections of first molar teeth were captured for morphometric analysis of lingual root cementum. Cross-sectional area of the cellular cementum was a sensitive guide to GH status, being reduced nearly 10-fold in GHR-KO mice, three-fold in GH antagonist mice, and increased almost two-fold in giant mice (p < 0.001). Cellular cementum length was similarly influenced by GH status, but to a lesser extent. Acellular cementum was generally unaffected. This study reveals cellular cementum to be a highly responsive GH target tissue, which may have therapeutic applications in assisting regeneration of the periodontium.