The D152H mutation found in growth hormone insensitivity syndrome impairs expression and function of human growth hormone receptor but is silent in rat receptor

In two patients with growth hormone (GH) insensitivity syndrome (Laron syndrome), in whom the GH receptor is able to bind the hormone, the D152H mutation was identified, and lack of dimerization was proposed to explain GH resistance in these patients. To examine further the consequences of the substitution of conserved aspartate 152 on the function of the GH receptor (GHR), we reproduced the mutation in vitro on the full length GH receptor cDNA from man and rat. Effects of the mutation on expression and activity of the GHR were analyzed in 293 cells transfected with wild-type and mutant GHR cDNAs. Mutant human receptor protein was expressed at a lower level than wild-type receptor and its activity was reduced: GH-dependent signal transducer and activator of transcription 5 (Stat5)-mediated transactivation of a reporter gene was lower in 293 cells transfected with mutant GHR cDNA than in transfected cells expressing a comparable level of wild-type GHR. The membrane-bound form of the mutant and of the wild-type human GHR were able to homodimerize, as suggested by the size of the complexes detected in cross-linking experiments with 125I-human (h) GH, and also by the activity in the functional test. With the soluble GHR resulting from proteolysis of the wild-type membrane form, no dimeric complexes could be detected. However, when a soluble receptor lacking the transmembrane and cytoplasmic domains of the receptor was expressed, wild-type and not mutant GH binding protein (GHBP) was able to form dimers in the presence of hGH. The amino acid substitution has no effect on either expression or function of the rat receptor. Structural modeling of D152H soluble human and rat GHR (GHBP) supports the species-specific functional consequences of the mutation. Evaluation of the functional importance of the mutation strongly suggests that impairment in expression and activity of the mutant receptor, rather than complete lack of dimerization, explains the GH resistance of the patients.

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361 GROWTH HORMONE RECEPTOR MUTANT PIGS PRODUCED BY USING THE CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR) AND CRISPR-ASSOCIATED SYSTEMS IN IN VITRO-PRODUCED ZYGOTES

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab361 ◽

2015 ◽

Vol 27 (1) ◽

pp. 269 ◽

Cited By ~ 2

Author(s):

M. Kurome ◽

M. Dahlhoff ◽

S. Bultmann ◽

S. Krebs ◽

H. Blum ◽

...

Keyword(s):

Growth Hormone ◽

Hormone Receptor ◽

Growth Hormone Receptor ◽

Large Animal Model ◽

Large Animal ◽

Single Mutation ◽

Wild Type ◽

Ghr Gene ◽

Normal Fertilization

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technology is considered as an efficient strategy for generating gene edited large animals, such as pigs. Compared to somatic cell nuclear transfer, this new technology offers a relatively simple way to generate mutant pigs by direct injection of RNA into the cytoplasm of zygotes. Moreover, the use of in vitro produced zygotes would provide a highly effective and practical method for the production of porcine disease models for biomedical research. Here we examined the production efficiency of growth hormone receptor (GHR) mutant pigs by the combination of the CRISPR/Cas system and in vitro produced zygotes. In vitro maturation (IVM) of oocytes was performed as described previously (Kurome et al., Meth. Mol. Biol., in press). In all experiments, the same batch of frozen sperm was used. After IVM, around 20 oocytes with expanded cumulus cells were incubated with 5 × 104 spermatozoa in a 100-μL drop of porcine fertilization medium for 7 h. In vitro-produced embryos were assessed by the ratio of normal fertilization (eggs with 2 pronuclei) and blastocyst formation at Day 7. The Cas9 mRNA and a single guide RNA, recognising a short sequence of 20 base pairs in exon 3 of the GHR gene, were injected directly into the cytoplasm of the embryos 8.5 to 9.5 h after IVF. Injected embryos were transferred laparoscopically to recipient pigs, and 86.4% (57/66) of sperm-penetrated oocytes (66/96) exhibited normal fertilization. Incidence of polyspermy was relatively low (9/66, 13.6%). Developmental ability of in vitro-produced embryos to the blastocyst stage was 17.4% (24/138). In total, 426 RNA-injected embryos were transferred into 2 recipients, one of which became pregnant and gave birth to 8 piglets. All piglets were clinically healthy and developed normally. In 3 out of 8 piglets (37.5%), mutations were introduced. Next-generation sequencing revealed that all of them were mosaics: one with a single mutation (22% wild-type/78% mutant) and 2 piglets with 2 different mutations (80% wild-type/2% mutant_1/18% mutant_2 and 94% wild-type/4% mutant_1/2% mutant_2). Four out of 5 mutations caused a frameshift in the GHR gene. Our study reports for the first time generation of GHR mutant pigs by the use of the CRISPR/Cas system in in vitro-produced zygotes. Because all GHR mutant offspring were mosaic, Cas9 activation probably occurred after the 1-cell stage under our experimental conditions. The founder animal with the highest proportion of mutant GHR alleles will be used for breeding to establish a large animal model for Laron syndrome.This work is supported by the German Research Council (TR-CRC 127).

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Distinct mechanisms of induction of hepatic growth hormone resistance by endogenous IL-6, TNF-α, and IL-1β

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00652.2013 ◽

2014 ◽

Vol 307 (2) ◽

pp. E186-E198 ◽

Cited By ~ 23

Author(s):

Yueshui Zhao ◽

Xiaoqiu Xiao ◽

Stuart J. Frank ◽

Herbert Y. Lin ◽

Yin Xia

Keyword(s):

Growth Hormone ◽

Target Gene ◽

Cytokine Signaling ◽

Suppressor Of Cytokine Signaling ◽

Gh Receptor ◽

Tnf Α ◽

Igf I ◽

Socs3 Expression ◽

Gh Resistance

During inflammation, the liver becomes resistant to growth hormone (GH) actions, leading to downregulation of the GH target gene IGF-I and activation of catabolism. Proinflammatory cytokines IL-6, TNF-α, and IL-1β are critically involved in the pathogenesis of hepatic GH resistance. However, the mechanisms used by endogenous IL-6, TNF-α, and IL-1β to inhibit the hepatic GH-IGF-I pathway during inflammation are not fully understood. Here, we show that TNF-α and IL-1β inhibited GH receptor (GHR) expression but had minor effects on the downstream suppressor of cytokine signaling (SOCS)3, while IL-6 induced SOCS3 expression but had no effect on GHR expression in Huh-7 cells. Consistent with the in vitro observations, neutralization of TNF-α and IL-1β in mouse models of inflammation did not significantly alter SOCS3 expression stimulated by inflammation but restored GHR and IGF-I expression suppressed by inflammation. Neutralization of IL-6 did not alter inflammation-suppressed GHR expression but drastically reduced the inflammation-stimulated SOCS3 expression and restored IGF-I expression. Interestingly, when the GH-IGF-I pathway was turned off by maximal inhibition of GHR expression, IL-6 and SOCS3 were no longer able to regulate IGF-I expression. Taken together, our results suggest that TNF-α/IL-1β and IL-6 use distinct mechanisms to induce hepatic GH resistance, with TNF-α and IL-1β acting primarily on GHR and IL-6 acting primarily on SOCS3. IL-6 action may be superseded by factors such as TNF-α and IL-1β that inhibit GHR expression.

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The human growth hormone receptor of cultured human lymphocytes. Structural characteristics and glycosylation properties

Biochemical Journal ◽

10.1042/bj2380379 ◽

1986 ◽

Vol 238 (2) ◽

pp. 379-386 ◽

Cited By ~ 31

Author(s):

K Asakawa ◽

J A Hedo ◽

A McElduff ◽

D G Rouiller ◽

M J Waters ◽

...

Keyword(s):

Growth Hormone ◽

Human Growth Hormone ◽

Hormone Receptor ◽

Gel Electrophoresis ◽

Growth Hormone Receptor ◽

Structural Characteristics ◽

Human Growth ◽

Receptor Complex ◽

Gh Receptor ◽

Binding Subunit

The structural characteristics and glycoprotein nature of the human growth hormone (hGH) receptor in cultured lymphocytes (IM-9 cell line) were studied with the use of a bifunctional reagent (disuccinimidyl suberate) to couple 125I-hGH covalently to intact cells. After cross-linking, the hormone-receptor complexes were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A single band of Mr 140,000 was identified under reducing conditions. The labelling of this band was blocked by unlabelled hGH but not by insulin, ovine prolactin, bovine or ovine growth hormones. The Mr 140,000 band was immunoprecipitated by either anti-hGH antibody or by a monoclonal antibody against rat liver growth hormone receptor. In the absence of reductant two major bands of Mr 270,000 and 140,000 were found. On two-dimensional gel electrophoresis, with the first dimension in the absence of reductant and the second in its presence, the Mr 270,000 complex generated the Mr 140,000 band. The nature of the oligosaccharide chains of the receptor was studied by treatment with different glycosidases. The electrophoretic mobility of the Mr 140,000 receptor complex was markedly increased after digestion with endoglycosidase F but showed no or little change after digestion with endoglycosidase H. The Mr 140,000 band was also sensitive to neuraminidase treatment. In addition the 125I-hGH-receptor complex was adsorbed by immobilized wheat germ agglutinin and to a smaller extent by immobilized concanavalin A, lentil lectin, ricin I and ricin II. In conclusion, taking into account that hGH is a Mr 22,000 polypeptide, the binding subunit of the GH receptor in human IM-9 lymphocytes has an Mr of approx. 120,000. The native receptor may exist as a homodimer of the binding subunit formed by disulphide bonds. Furthermore, the GH receptor subunit contains asparagine N-linked type of oligosaccharide chains. Most, if not all, of these chains are of the complex type and appear to be sialylated whereas no high-mannose type chains are detectable in the mature form of the receptor.

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A single amino acid substitution in the exoplasmic domain of the human growth hormone (GH) receptor confers familial GH resistance (Laron syndrome) with positive GH-binding activity by abolishing receptor homodimerization.

The EMBO Journal ◽

10.1002/j.1460-2075.1994.tb06392.x ◽

1994 ◽

Vol 13 (6) ◽

pp. 1386-1395 ◽

Cited By ~ 116

Author(s):

P. Duquesnoy ◽

M.L. Sobrier ◽

B. Duriez ◽

F. Dastot ◽

C.R. Buchanan ◽

...

Keyword(s):

Growth Hormone ◽

Amino Acid ◽

Human Growth Hormone ◽

Amino Acid Substitution ◽

Human Growth ◽

Binding Activity ◽

Single Amino Acid ◽

Laron Syndrome ◽

Gh Receptor ◽

Gh Resistance

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Effect of Growth Hormone and Calorie Restriction on the Expression of Antioxidant Enzymes in the Liver and Kidney of Growth Hormone Receptor Knockout Mice

Atlas Journal of Biology ◽

10.5147/ajb.v2i2.23 ◽

2017 ◽

Vol 2 (2) ◽

pp. 136-141

Author(s):

Khalid A Al-Regaiey

Keyword(s):

Growth Hormone ◽

Antioxidant Enzymes ◽

Defense Mechanisms ◽

Growth Hormone Receptor ◽

Oxidative Stress Marker ◽

Gh Receptor ◽

Liver And Kidney ◽

Ad Libitum ◽

Gpx Activity ◽

Gh Resistance

Caloric restriction (CR) can delay aging and prolong life span and these actions may be related to reduced oxidative damage. Mice with disrupted growth hormone (GH) receptor/binding protein knockout (GHRKO) live significantly longer than their normal siblings. Therefore, it is of interest to examine the effects of chronic CR on hepatic and renal antioxidant enzymes as well as lipid peroxidation (LP) as an oxidative stress marker in GHRKO mice. Female GHRKO and normal mice were either fed ad libitum (AL) or subjected to 30% CR starting at 2 months of age and examined at the age of 9 months. In the liver, catalase (CAT) activity was significantly increased in GHRKO-AL as compared to normal control -AL animals. CR reduced CAT activity in both GHRKO and normal phenotypes. Cu/Zn superoxide dismutase (SOD1) activity was also higher in GHRKO-AL as compared to normal-AL mice. However, CR reduced SOD1 activity in GHRKO mutants. Glutathione peroxidase (GPx) activity was significantly decreased in GHRKO-AL mice and further reduced in GHRKO-CR group of animals. CR significantly increased LP in GHRKOs while its activity was not altered in GHRKO-AL group of mice. In the kidney, CAT activity was lower in GHRKO-AL as compared to normal-AL, however CR did not induce any significant effect in both phenotypes. Similarly, SOD1 levels were significantly lower in GHRKO than in normal mice. GPx expression was higher in GHRKO-AL as compared to control-AL. CR reduced GPx activity in GHRKO mice but increased it in controls as compared to their AL counterparts. There was no difference in LP expression between GHRKO-AL and normal-AL mice. However, CR significantly increased its levels in both phenotypes. Although these findings do not support the hypothesis that CR would increase the capacity of ROS defense mechanisms in GHRKO mice by increasing antioxidant enzymes levels, they do agree with some of the reported effects of CR on their expression. We suspect that GH resistance and CR may affect aging by different mechanisms and if CR delays aging in GHRKO animals it is not due to changes in the activity of antioxidant enzymes.

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Two newly detected mutations and four new single nucleotide polymorphisms in the promotor region of exon 1 of the human growth hormone receptor gene in adult patients with growth hormone deficiency

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/s-2005-920449 ◽

2005 ◽

Vol 113 (08) ◽

Author(s):

S Meyer ◽

M Arnken ◽

A Keth ◽

PH Kann

Keyword(s):

Growth Hormone ◽

Growth Hormone Receptor ◽

Receptor Gene ◽

Human Growth ◽

Hormone Deficiency ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Exon 1 ◽

Growth Hormone Receptor Gene ◽

Human Growth Hormone Receptor

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Reduced and S-carboxymethylated human growth hormone: a probe for diabetogenic action

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1984.247.5.e639 ◽

1984 ◽

Vol 247 (5) ◽

pp. E639-E644

Author(s):

C. M. Cameron ◽

J. L. Kostyo ◽

J. A. Rillema ◽

S. E. Gennick

Keyword(s):

Growth Hormone ◽

Amino Acid ◽

Human Growth Hormone ◽

Human Growth ◽

Mouse Mammary Gland ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Hypophysectomized Rats

The biological activity profile of reduced and S-carboxymethylated human growth hormone (RCM-hGH) was determined to establish its suitability for study of the diabetogenic property of hGH. RCM-hGH was found to have greatly attenuated in vivo growth-promoting activity in the 9-day weight-gain test in hypophysectomized rats (approximately 1%) and to have a similar low order of in vitro activity in stimulating amino acid incorporation into the protein of the isolated rat diaphragm. RCM-hGH also only had approximately 1% of the in vitro insulin-like activity of the native hormone on isolated adipose tissue from hypophysectomized rats. In contrast, RCM-hGH retained substantial in vivo diabetogenic activity in the ob/ob mouse, appearing to have approximately 50% of the activity of the native hormone. RCM-hGH was also found to retain significant, although attenuated (25%), in vitro lactogenic activity when tested for the ability to stimulate amino acid incorporation into a casein-rich protein fraction in mouse mammary gland explants. Because RCM-hGH exhibits a high degree of diabetogenic activity, although lacking significant anabolic or insulin-like activities, it will be useful as a "monovalent" probe for the study of the molecular mechanism of the diabetogenic action of GH.

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Adiponectin inhibits leptin signalling via multiple mechanisms to exert protective effects against hepatic fibrosis

Biochemical Journal ◽

10.1042/bj20102148 ◽

2011 ◽

Vol 440 (3) ◽

pp. 385-395 ◽

Cited By ~ 55

Author(s):

Jeffrey A. Handy ◽

Ping P. Fu ◽

Pradeep Kumar ◽

Jamie E. Mells ◽

Shvetank Sharma ◽

...

Keyword(s):

Hepatic Fibrosis ◽

Leptin Receptor ◽

Tyrosine Phosphatase ◽

Janus Kinase ◽

Protective Effects ◽

Wild Type ◽

Matrix Deposition ◽

Matrix Metalloproteinase 1 ◽

Expression And Activity

Adiponectin is protective against hepatic fibrosis, whereas leptin promotes fibrosis. In HSCs (hepatic stellate cells), leptin signals via a JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) pathway, producing effects that enhance ECM (extracellular matrix) deposition. SOCS-3 (suppressor of cytokine signalling-3) and PTP1B (protein tyrosine phosphatase 1B) are both negative regulators of JAK/STAT signalling, and recent studies have demonstrated a role for adiponectin in regulating SOCS-3 expression. In the present study we investigate mechanisms whereby adiponectin dampens leptin signalling and prevents excess ECM production. We treated culture-activated rat HSCs with recombinant adiponectin, leptin, both or neither, and also treated adiponectin knockout (Ad−/−) and wild-type mice with leptin and/or carbon tetrachloride (CCl4) or saline. We analyse JAK2 and Ob-Rb (long form of the leptin receptor) phosphorylation, and PTP1B expression and activity. We also explore potential mechanisms through which adiponectin regulates SOCS-3–Ob-Rb association. Adiponectin inhibits leptin-stimulated JAK2 activation and Ob-Rb phosphorylation in HSCs, whereas both were increased in Ad−/− mice. Adiponectin stimulates PTP1B expression and activity in vitro, whereas PTP1B expression was lower in Ad−/−mice than in wild-type mice. Adiponectin also promotes SOCS-3–Ob-R association and blocks leptin-stimulated formation of extracellular TIMP-1 (tissue inhibitor of metalloproteinases-1)–MMP-1 (matrix metalloproteinase-1) complexes in vitro. These results suggest two novel mechanisms whereby adiponectin inhibits hepatic fibrosis: (i) by promoting binding of SOCS-3 to Ob-Rb, and (ii) by stimulating PTP1B expression and activity, thus inhibiting JAK2/STAT3 signalling at multiple points.

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