Characterisation of novel missense mutations in the GH receptor gene causing severe growth retardation.

Two Swedish brothers, 2.5 and 4 years of age, were found to fulfil all the clinical and laboratory characteristics of Laron's syndrome. They were shown to have unique missense mutations in the GH receptor gene. Both of their parents were of normal height, but they both separately carried one of the identified gene alterations. A molecular model of the first receptor alteration suggests that a collapse in three-dimensional receptor structure most likely contributed to the GH insensitivity in these patients.

Cell transfection as a tool to study growth hormone action.

The isolation of growth hormone receptor (GHR) cDNA clones has made possible the transfection of GHRs into cultured cells. Our aim in this minireview is to show how the application of such approaches have benefited GHR research. GH stimulation of cells expressing GHR cDNAs can cause an alteration of cellular function that mimic those of the endogenous GHR. GHR cDNA transfected cells also offer a system where the mechanism of GH action can be studied. Such a system has been used to demonstrate that the GHR itself becomes tyrosine phosphorylated and that further phosphorylation of downstream proteins is important in GH action. The GH signals are transmitted to the nucleus and GH regulated genes have now begun to be characterized. The ability to use cell transfection for mechanistic studies of GH action will be instrumental to define domains within the receptor that are of functional importance and to determine pathways whereby GH signals are conveyed within the cell.

Growth hormone (GH) regulation of a rat serine protease inhibitor fusion gene in cells transfected with GH receptor cDNA.

The mechanism by which GH transmits a signal to the nucleus via its membrane-bound receptor is unknown. To study this process, Buffalo rat liver (BRL), rat hepatoma (FAO), human hepatoma (HepG2) and Chinese hamster ovary (CHO) cell lines were transfected with GH receptor cDNA, and stable clones expressing GH receptor mRNA and protein were selected. From previous in vivo studies it is known that GH regulates the expression of the rat hepatic serine protease inhibitor (SPI) 2.1 gene at the transcriptional level. However, in all the cell lines tested, SPI gene expression was less than 0.2% of that measured in rat liver, and GH did not affect the expression of the endogenous SPI gene in GH receptor-expressing cells. A 45 bp GH-responsive element (GHRE) has previously been defined in the SPI 2.1 gene. A construct containing six repeats of this GHRE was assembled with the thymidine kinase promoter and a chloramphenicol acetyl transferase (CAT) reporter gene. Transient transfection of this reporter gene resulted in GH stimulation of CAT activity in all GH receptor-transfected cell lines. A 33-fold induction was measured in the GH receptor-expressing BRL cells. Induction of CAT activity was observed after 8 h of GH treatment in the BRL-GHR638 cell line. Stable BRL cell lines expressing GH receptors with carboxy-terminal truncations (GHR380 and GHR454) did not show increased CAT activity on GH stimulation. This suggests that more than half of the intracellular domain of the GH receptor is required to activate transcription of the SPI 2.1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel in vitro model for studying signal transduction and gene regulation via the growth hormone receptor.

Buffalo rat liver cells were stably transfected with an expression vector containing rat GH (rGH) receptor cDNA. Transfected cells expressed rGH receptor mRNA and specifically bound GH with high affinity. When transfected cells were stimulated with GH, levels of lipoprotein lipase (LPL) mRNA were increased in a time- and dose-dependent fashion, while glyceraldehyde-3-phosphate-dehydrogenase mRNA levels were unaffected. No GH binding or LPL mRNA could be detected in untransfected cells. Treatment of transfected cells with actinomycin D inhibited the GH-stimulated increase in LPL mRNA, indicating that GH acts at a transcriptional level. When protein synthesis was inhibited using cycloheximide, basal levels of LPL mRNA were increased, and there was no GH stimulation. This suggests that LPL gene expression is constantly repressed by a labile protein. Chloramphenicol acetyltransferase constructs containing the human LPL promoter could be regulated by GH. In conclusion, stimulation of the rGH receptor in stably transfected Buffalo rat liver cells results in specific induction of LPL gene expression. This provides a novel model to study the mechanism of GH action, particularly in relation to gene regulation.

Growth hormone receptor regulation in growth hormone-deficient dwarf rats.

In the rat, many actions of GH depend upon the sexually dimorphic pattern of exposure to GH. Hepatic human GH (hGH) receptor binding differs between the sexes and is sensitive to GH deficiency, but this has mostly been studied in acutely hypophysectomized rats, which lack all pituitary hormones. We have used a strain of GH-deficient dwarf (Dw) rats to determine whether chronic GH deficiency alters the normal developmental pattern and sexually dimorphic expression of hepatic GH receptors. Adult female Dw rats had lower levels of 125I-labelled hGH binding (reflecting predominantly lactogenic receptors) than their normal counterparts whereas there was no difference between adult Dw and normal males; binding capacity increased from 25 days of age, becoming sexually dimorphic from 40 days to adulthood in both strains (% specific binding/mg protein: normal males 1.6 +/- 0.3, normal females 13.2 +/- 1.1, Dw males 2.1 +/- 0.4, Dw females 10.0 +/- 0.6). In contrast, hepatic 125I-labelled bovine GH (bGH) binding (somatogenic receptors) was much lower, and similar in both Dw and normal animals. A sex difference in 125I-labelled bGH binding was only seen in adult animals, and was considerably less marked in Dw rats compared with normal animals (normal males 1.3 +/- 0.1, normal females 2.5 +/- 0.2, Dw males 1.9 +/- 0.2, Dw females 2.4 +/- 0.2%/mg protein). Continuous hGH infusion stimulated growth in female Dw rats, and raised somatogenic and lactogenic GH binding (3.2 +/- 0.4 and 19.6 +/- 2.5%/mg protein) compared with sham-infused controls (2.4 +/- 0.2 and 7.9 +/- 0.6%/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone (GH) induction of tyrosine phosphorylation and activation of mitogen-activated protein kinases in cells transfected with rat GH receptor cDNA.

The mechanism of growth hormone (GH) action was studied in Chinese hamster ovary (CHO) cells transfected with GH receptor cDNA. Cytosolic extracts from GH- or phorbol ester (12-O-tetradecanoyl 4 beta-phorbol 13-acetate)-treated cells, transfected with full-length GH receptor cDNA, had an enhanced ability to phosphorylate myelin basic protein. Myelin basic protein, a substrate for mitogen-activated protein (MAP) kinase, was maximally phosphorylated using extracts from cells treated with 50 nM bovine GH for 10 min. In addition, GH treatment resulted in an increased cell proliferation by 30-60%. GH and 12-O-tetradecanoyl 4 beta-phorbol 13-acetate cause tyrosine phosphorylation of two proteins with M(r) of 40,000 and 42,000 that are also recognized by MAP kinase antibodies. These proteins were identified as MAP kinases by analyzing phosphotyrosine immunoprecipitates on Western blots using MAP kinase antibodies. In addition, GH induces mitogenicity, as well as MAP kinase activation, in CHO cells expressing a receptor in which 184 amino acids had been deleted in the carboxyl-terminal part of the intracellular domain. No GH effects were seen in untransfected cells, in CHO cells expressing a truncated GH receptor containing only 5 of 349 amino acids in the intracellular domain, or in cells expressing the soluble GH-binding protein. In conclusion, our data show that GH treatment of CHO cells, reconstituted with GH receptors, initiates a phosphorylation cascade which includes MAP kinase.

Growth hormone (GH) regulation of cytochrome P-450IIC12, insulin-like growth factor-I (IGF-I), and GH receptor messenger RNA expression in primary rat hepatocytes: a hormonal interplay with insulin, IGF-I, and thyroid hormone.

We have studied the GH-dependent expression of cytochrome P-450IIC12 (P-450(15)beta) mRNA and insulin-like growth factor-I (IGF-I) mRNA in primary adult rat hepatocytes. The GH receptor (GHR), being the common denominator for the GH response, was also studied. The respective mRNA levels were measured with specific solution hybridization assays. By investigating the effects of insulin, IGF-I, T3, and corticosterone, alone or in combinations, in the presence or absence of GH we concluded that GH is indeed the inducer of P-450(15)beta mRNA and IGF-I mRNA. However, insulin and IGF-I exerted a 2-fold potentiation of the GH-induced expression of the P-450(15)beta and IGF-I mRNA species. No significant effect of insulin was observed on GHR mRNA expression, but a translational or posttranslational effect on GHR was seen, in that insulin increased the binding of GH to the cells 4-fold. Furthermore, T3 caused a 9-fold increase in the GH-induced expression of IGF-I mRNA. These observations led us to postulate a possible mechanism of hormonal interplay between GH, thyroid hormone, and IGF-I in vivo, i.e. a thyroid hormone potentiation of the GH-induced IGF-I expression, which, in turn, leads to an increased GHR level and thereby a potentiation of the GH-induced expression of P-450(15)beta and, at least transiently, of IGF-I. A transcriptional mechanism of GH action on P-450(15)beta and IGF-I mRNA induction was indicated by the similar half-lives of respective mRNAs in the presence or absence of GH in cell cultures treated with actinomycin-D.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of insulin-like growth factor-I and growth hormone receptor gene expression by diabetes and nutritional state in rat tissues.

Insulin-like growth factor-I (IGF-I) mRNA and GH receptor mRNA levels were analysed in different tissues from rats made diabetic with streptozotocin, fasted rats and rats fed with a protein-reduced diet. Diabetes decreased IGF-I mRNA levels in liver, heart, diaphragm, kidney and aorta, but not in brain. GH receptor mRNA levels were decreased in heart and diaphragm, but not in liver and kidney. Fasting decreased IGF-I mRNA in all tissues studied except brain, and decreased GH receptor mRNA in liver, heart and diaphragm, but not in kidney. A protein-reduced diet decreased hepatic IGF-I mRNA levels but did not significantly affect other tissues, while GH receptor mRNA levels were reduced in liver and diaphragm. In conclusion, both diabetes and limited nutrition affected IGF-I and GH receptor mRNA in different tissues, but the two mRNAs were not co-ordinately regulated in all tissues studied. While reduced GH receptor gene expression may thus be responsible for decreased IGF-I gene expression in some states and tissues, additional regulatory mechanisms may be of importance.

Regulation of rat growth hormone receptor gene expression.

A cDNA encoding the growth hormone (GH) receptor was cloned from rat liver. Both the nucleotide and translated amino acid sequence share greater than 70% similarity with the GH receptors from rabbit and human. An RNA probe was generated from this sequence for use in a solution hybridization assay to quantitate GH receptor mRNA expression in rat tissues. Expression was detected in 9/12 tissues examined, with the highest levels observed in the liver. Expression in liver, kidney, heart and muscle was developmentally regulated, being low at birth and rising to adult levels in 5 weeks. No difference was observed between hepatic expression in males and females, although livers from pregnant rats had elevated levels. Hypophysectomy and GH treatment did not affect hepatic GH receptor mRNA levels.