Michael J. Waters

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

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.

Localization and Ontogeny of Growth-Hormone Receptor Gene-Expression in the Central-Nervous-System

There is literature evidence that both growth hormone (GH) and its mediator, insulin-like growth factor 1 (IGF-1), are able to act upon neuronal and glial cells in the brain. We report here the location of the GH receptor in the brain of the rat and rabbit. Receptor distribution was determined by immunohistochemistry with GH receptor/binding protein (BP) specific monoclonal antibodies and by in situ hybridization with a [35S]riboprobe. GH receptor/BP immunoreactivity in the rat was most prominent in the neonate and declined with postnatal age. Receptor immunoreactivity was generalised with variation in immunoreactivity in regional areas. In the rat, strongest immunoreactivity was seen in layers 2, 3, 5 and especially layer 6 of the cerebral cortex, in neurones of the thalamus and hypothalamus, in Purkinje cells of the cerebellum, in neurones of the trapezoid body of the brainstem, and in retinal ganglion cells. Glial cells, notably astrocytes were also strongly reactive, along with ependyma of the choroid plexus, ventricular lining and pia mater. In the neonatal rabbit, strongest immunoreactivity was evident in layers 2 and 3 of the cerebral cortex, in pyramidal cells of the hippocampus, and in neurones of the inferior and superior colliculi, brain stem reticular formation, dorsal thalamus and hypothalamus. A similar distribution of GH receptor mRNA was seen by in situ hybridization. The ontogeny of GH receptor/BP mRNA in whole rat brain was quantified by solution hybridization-RNAse protection assay. Contrary to its ontogeny in the liver (Endocrinology, 113 (1983) 1325-1329) receptor mRNA decreased with postnatal age.(ABSTRACT TRUNCATED AT 250 WORDS)

The growth hormone receptor: mechanism of activation and clinical implications

Growth hormone is widely used clinically to promote growth and anabolism and for other purposes. Its actions are mediated via the growth hormone receptor, both directly by tyrosine kinase activation and indirectly by induction of insulin-like growth factor 1 (IGF-1). Insensitivity to growth hormone (Laron syndrome) can result from mutations in the growth hormone receptor and can be treated with IGF-1. This treatment is, however, not fully effective owing to the loss of the direct actions of growth hormone and altered availability of exogenous IGF-1. Excessive activation of the growth hormone receptor by circulating growth hormone results in gigantism and acromegaly, whereas cell transformation and cancer can occur in response to autocrine activation of the receptor. Advances in understanding the mechanism of receptor activation have led to a model in which the growth hormone receptor exists as a constitutive dimer. Binding of the hormone realigns the subunits by rotation and closer apposition, resulting in juxtaposition of the catalytic domains of the associated tyrosine-protein kinase JAK2 below the cell membrane. This change results in activation of JAK2 by transphosphorylation, then phosphorylation of receptor tyrosines in the cytoplasmic domain, which enables binding of adaptor proteins, as well as direct phosphorylation of target proteins. This model is discussed in the light of salient information from closely related class 1 cytokine receptors, such as the erythropoietin, prolactin and thrombopoietin receptors.

Mechanism of Activation of Protein Kinase JAK2 by the Growth Hormone Receptor

Introduction Class I cytokines regulate key processes such as growth, lactation, hematopoiesis, and immune function and contribute to oncogenesis. Although the extracellular domain structures of their receptors are well characterized, little is known about how the receptors activate their associated JAK (Janus kinase) protein kinases. We provide a mechanistic description for this process, focusing on the growth hormone (GH) receptor and its associated JAK2. Receptor-JAK2 activation process. (Top) Cartoons of the GH receptor basal state (state 1, left) and the active state (state 2, right) with (Bottom) transmembrane helix alignments for these states derived by modeling. GHR, GH receptor. Rationale We tested whether the receptor exists as a dimer in the inactive state by homo-FRET [fluorescence resonance energy transfer (FRET) between the proteins labeled with the same fluorophore] and other means. Then, to define receptor movements resulting from activation, we attached FRET reporters to the receptor below the cell membrane and correlated their movement with receptor activation, measured as increased cell proliferation. We controlled the position of the transmembrane helices with leucine zippers and mutagenesis, and we again monitored FRET and receptor activation. We used cysteine cross-linking data to define the faces of the transmembrane helices in contact in the basal state and verified this with molecular dynamics, which allowed us to model the activation process. We also used FRET reporters to monitor the movement of JAK2, and we matched this with molecular dynamics docking of the crystal structures of the kinase and its pseudokinase domains to derive a model for activation, which we then verified experimentally. Results We found that the GH receptor exists predominantly as a dimer in vivo, held together by its transmembrane helices. These helices are parallel in the basal state, and binding of the hormone converts them into a left-hand crossover state that induces separation of helices at the lower transmembrane boundary (hence, Box1 separation). This movement is triggered by increased proximity of the juxtamembrane sequences, a consequence of locking together of the lower module of the extracellular domain on hormone binding. This movement is triggered by increased proximity of the juxtamembrane sequences , a Both this locking and the helix state transition require rotation of the receptors, but the key outcome is separation of the Box1 sequences. Because these sequences are bound to the JAK2 FERM (4.1, ezrin, radixin, moesin) domains, this separation results in removal of the pseudokinase inhibitory domain of one JAK2, which is blocking the kinase domain of the other JAK2, and vice versa. This brings the two kinase domains into productive apposition, triggering JAK2 activation. We verified this mechanism by kinase-pseudokinase domain swap, by changes in JAK2 FRET signal on activation, by showing association of pseudokinase-kinase domain pairs, and by docking of the crystal structures. An animation of our complete model of GH receptor activation is provided at http://web-services.imb.uq.edu.au/waters/hgh.html. Conclusion The proposed mechanism will be useful in understanding the many actions of GH, which include altered growth, metabolism, and bone turnover. We expect that it may extend to other members of this important receptor family. The mechanism provides a molecular basis for understanding the oncogenic JAK2 mutations responsible for polycythemia vera and certain other hematologic disorders and may thus be of value in the design of small-molecule inhibitors of clinical applicability. Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well generalize to other class I cytokine receptors. A molecular mechanism for transmembrane signaling by the growth hormone receptor is elucidated. [Also see Perspective by Wells and Kossiakoff] The Hormones Message The receptor for growth hormone is a well-studied representative of a family of cytokine receptors through which binding of hormone molecules at the cell surface is converted into a biochemical signal within the cell. Brooks et al. (10.1126/science.1249783; see the Perspective by Wells and Kossiakoff) used a combination of crystal structures, biophysical measurements, cell biology experiments with modified receptors, and molecular dynamics and modeling to decipher how the receptor actually transmits the information that a hormone molecule is bound. The results suggest that the receptors exist in inactive dimeric complexes in which two associated JAK2 protein kinase molecules interact in an inhibitory manner. Binding of growth hormone causes a structural change in the receptor that results in movement of the receptor intracellular domains apart from one another. This relieves the inhibition of the JAK2 molecules and allows them to activate one another, thus initiating the cellular response to the hormone.

Cellular expression of growth hormone and prolactin receptors in human breast disorders

Growth hormone (GH) and prolactin (PRL) exert their regulatory functions in the mammary gland by acting on specific receptors. Using isotopic in situ hybridization and immunohistochemistry, we have localized the expression of hGH receptor (hGHR) and hPRL receptor (hPRLR) in a panel of human breast disorders. Surgical specimens from adult females included normal breast, inflamatory lesions (mastitis) benign proliferative breast disease (fibroadenoma, papilloma, adenosis, epitheliosis), intraductal carcinoma or lobular carcinoma in situ, and invasive ductal, lobular or medullary carcinoma. Cases of male breast enlargement (gynecomastia) were also studied. In situhybridization analysis demonstrated the co‐expression of hGHR and hPRLR mRNA in all samples tested. Epithelial cells of both normal and tumor tissues were labelled. Quantitative estimation of receptor mRNA levels was regionally measured in areas corresponding to tumor cells and adipose cells from the same section. It demonstrated large individual variation and no correlation emerged according to the histological type of lesion. Receptor immunoreactivity was detected both in the cytoplasm and nuclei or in the cytoplasm alone. Scattered stromal cells were found positive in some cases, but the labeling intensity was always weaker than for neoplastic epithelial cells. Our results demonstrate the expression of the hGHR and hPRLR genes and their translation in epithelial cells of normal, proliferative and neoplastic lesions of the breast. They also demonstrate that stromal components express GHR and PRLR genes. Thus the putative role of hGH or hPRL in the progression of proliferative mammary disorders is not due to grossly altered levels of receptor expression. Int. J. Cancer (Pred. Oncol.) 79:202–211, 1998.© 1998 Wiley‐Liss, Inc.

In Vivo Analysis of Growth Hormone Receptor Signaling Domains and Their Associated Transcripts.

ABSTRACT 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.

An immunohistochemical assessment of cellular proliferation markers in head and neck squamous cell cancers.

Prognostic information is essential for the evaluation, judgement and optimal treatment of patients with squamous cell cancers (SCCs) of the upper aerodigestive tract. Using immunohistochemical and flow cytometric techniques, we have studied the significance of cellular expression of the Ki-67 antigen, epidermal growth factor receptor (EGFR), the transferrin receptor (TFR) and DNA ploidy status in a prospective analysis of patients with SCCs of the head and neck region. All 42 fresh tumour samples (five well differentiated; 28 moderately differentiated; nine poorly differentiated) expressed both EGFR and TFR to varying degrees. Receptor expression was most marked on the peripheral invading margin of cancer cell islands although staining was also demonstrated in a random fashion within cellular islands and consistently along the basal cell layer of overlying stratified squamous epithelium. The percentage of cancer cells that reacted with the Ki-67 monoclonal antibody was assessed as low (less than 10%) in 15 samples (35.8%), intermediate (10-30%) in 19 samples (45.2%) and high (greater than 30%) in eight samples (19.0%). Eleven of 15 samples (73%) with a low percentage reactivity were DNA diploid, whereas seven of eight samples (87.5%) with a high percentage reactivity were DNA aneuploid. Poorly differentiated SCCs were significantly more often aneuploid than were either moderately or well differentiated tumours. Our results suggest that EGFR and TFR are widely distributed on SCCs, especially on proliferating cells at the invading tumour margin. In addition, there is a close spatial correlation between cells expressing EGFR, TFR and those expressing the Ki-67 antigen. Tumours in which the staining intensity for both EGFR and TFR was intense invariably expressed the Ki-67 antigen in a high proportion of cells. Further patient follow-up will be important in determining whether intense EGFR and TFR staining, combined with a high percentage reactivity with Ki-67 antibody and DNA aneuploidy, will ultimately define a subset of head and neck cancer patients with a poor clinical outcome.

Growth Hormone Research Society Workshop Summary: Consensus Guidelines for Recombinant Human Growth Hormone Therapy in Prader-Willi Syndrome

Context: Recombinant human GH (rhGH) therapy in Prader-Willi syndrome (PWS) has been used by the medical community and advocated by parental support groups since its approval in the United States in 2000 and in Europe in 2001. Its use in PWS represents a unique therapeutic challenge that includes treating individuals with cognitive disability, varied therapeutic goals that are not focused exclusively on increased height, and concerns about potential life-threatening adverse events. Objective: The aim of the study was to formulate recommendations for the use of rhGH in children and adult patients with PWS. Evidence: We performed a systematic review of the clinical evidence in the pediatric population, including randomized controlled trials, comparative observational studies, and long-term studies (>3.5 y). Adult studies included randomized controlled trials of rhGH treatment for ≥ 6 months and uncontrolled trials. Safety data were obtained from case reports, clinical trials, and pharmaceutical registries. Methodology: Forty-three international experts and stakeholders followed clinical practice guideline development recommendations outlined by the AGREE Collaboration (www.agreetrust.org). Evidence was synthesized and graded using a comprehensive multicriteria methodology (EVIDEM) (http://bit.ly.PWGHIN). Conclusions: Following a multidisciplinary evaluation, preferably by experts, rhGH treatment should be considered for patients with genetically confirmed PWS in conjunction with dietary, environmental, and lifestyle interventions. Cognitive impairment should not be a barrier to treatment, and informed consent/assent should include benefit/risk information. Exclusion criteria should include severe obesity, uncontrolled diabetes mellitus, untreated severe obstructive sleep apnea, active cancer, or psychosis. Clinical outcome priorities should vary depending upon age and the presence of physical, mental, and social disability, and treatment should be continued for as long as demonstrated benefits outweigh the risks.

GH-dependent STAT5 signaling plays an important role in hepatic lipid metabolism.

GH deficiency is known to be clinically associated with a high incidence of nonalcoholic fatty liver disease, and this can be reversed by GH administration. Here we investigated the mechanistic basis for this phenomenon using engineered male mice lacking different signaling elements of the GH receptor, hepatic stat5a/b(-/-) mice and a mouse hepatoma line. We found deficient GH-dependent signal transducer and activator of transcription (STAT)-5 signaling correlates with steatosis, and through microarray analysis, quantitative PCR, and chromatin immunoprecipitation, identified putative targets of STAT5 signaling responsible for the steatosis seen on a normal diet. These targets were verified with liver-specific stat5a/b deletion in vivo, and in vitro we show that dominant-negative (DN) STAT5 increases lipid uptake in a mouse hepatoma line. Because loss of STAT5 signaling results in elevated STAT1 and STAT3 activity and intracellular lipid accumulation, we have used DN-STAT5a/b, DN-STAT1, constitutively active (CA)-STAT3, or addition of oleate/palmitate in the hepatoma line to assign which of these apply to individual targets in STAT5 signaling deficiency. These findings and published mouse models of steatosis enable us to propose elevated cd36, pparγ, and pgc1α/β expression as primary instigators of the steatosis along with elevated fatty acid synthase, lipoprotein lipase, and very low-density lipoprotein receptor expression. Decreased fgf21 and insig2 expression may also contribute. In conclusion, despite normal plasma free fatty acids and minimal obesity, absent GH activation leads to steatosis because activated STAT5 prevents hepatic steatosis. These results raise the possibility of low-dose GH treatment for nonalcoholic fatty liver disease.

Exercise increases neural stem cell number in a growth hormone-dependent manner, augmenting the regenerative response in aged mice

The exercise‐induced enhancement of learning and memory, and its ability to slow age‐related cognitive decline in humans led us to investigate whether running stimulates periventricular (PVR) neural stem cells (NSCs) in aging mice, thereby augmenting the regenerative capacity of the brain. To establish a benchmark of normal aging on endogenous NSCs, we harvested the PVR from serial vibratome sections through the lateral ventricles of juvenile (6‐8 weeks), 6‐, 12‐, 18‐, and 24‐month‐old mice, culturing the cells in the neural colony‐forming cell assay. A significant decline in NSC frequency was apparent by 6 months (∼40%), ultimately resulting in a ∼90% reduction by 24 months. Concurrent with this decline was a progressive loss in regenerative capacity, as reflected by an incomplete repopulation of neurosphere‐forming cells following gamma cell irradiation‐induced depletion of the PVR. However, voluntary exercise (i.e., 21 days of running) significantly increased NSC frequency in mice ≥ 18 months of age, augmenting the regeneration of irradiation‐ablated periventricular cells and restoring NSC numbers to youthful levels. Importantly, and consistent with the demonstrated ability of growth hormone (GH) to increase NSC proliferation, and the elevated secretion of GH during exercise, exercise failed to stimulate NSCs in GH receptor‐null mice. These findings now provide a novel basis for understanding the ability of exercise to delay the onset and rate of decline in neurodegenerative conditions not typically associated with the hippocampus and suggest that the GH‐dependent activation of endogenous NSCs may be effective in reversing or preventing age‐related neurodegeneration in humans. STEM CELLS 2009;27:2044–2052