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M C Postel-Vinay

Publications and source records attributed to M C Postel-Vinay.

At least 19 recordsLinked to original sources
PubMed1997

Monkey growth hormone (GH) receptor gene expression. Evidence for two mechanisms for the generation of the GH binding protein.

The growth hormone receptor (GHR) cDNA was cloned from the liver of Rhesus macaque using polymerase chain reaction. As deduced from the nucleotide sequence, the mature GHR is a protein of 620 amino acids which presents 94.1% identity with the human receptor. The monkey GHR (mkGHR) expressed in 293 cells presented the expected specificity for a primate GHR and was able to transduce a transcriptional effect of GH. Human GH was able to activate tyrosine phosphorylation of both the tyrosine kinase JAK2 and the receptor in 293 cells co-transfected with mkGHR and JAK2 cDNAs. The GH binding protein (GHBP), the soluble short form of the GHR, was also present in monkey serum. Expression of the GHR cDNA in eucaryotic cells indicated that the GHBP can be produced by proteolytic cleavage of the membrane receptor. Northern blot analysis of GHR gene expression in different tissues allowed us to identify three different transcripts of 5.0 and 2.8 kilobase pairs and a smaller one of 1.7 kilobase pairs which could encode a GHBP. Rapid amplification of cDNA extremities (3'-RACE-polymerase chain reaction) was used to identify a cDNA encoding a protein in which the transmembrane and cytoplasmic domains of the receptor are substituted by a short sequence of 9 amino acids. This transcript was present in various tissues and could encode a GHBP as well, suggesting for the first time that two different mechanisms can coexist for the generation of the GHBP: proteolytic cleavage of the membrane receptor and a specific mRNA produced by alternative splicing.

Alternative Splicing↗
PubMed1997

Regulation of human growth hormone receptor gene transcription by human growth hormone binding protein.

The hypothesis that growth hormone binding protein (GHBP) has an effect on its own on the regulation of the GH-receptor/GHBP transcription was tested. Three different forms of human GHBP (recombinant non-glycosylated GHBP, recombinant glycosylated GHBP and GHBP purified and extracted from serum) were added in different concentrations determined by LIFA [0 pmol/l; 50 pmol/l (low level), 200 pmol/l (average level) and 500 pmol/l (high level in circulation)] to a human hepatoma cell line (HuH7 cells) cultured in a serum free hormonally-defined medium. Following the incubation with GHBP for 0, 1 and 2 h, GH-receptor expression was quantitatively assessed by using polymerase chain reaction amplification. Treatment with a GHBP concentration of 50 pmol/l resulted in a significant increase of GH-receptor mRNA molecules given as number of molecules x 10(6)/microg total RNA. In contrast, the concentration of 500 pmol/l presented a significant decrease of GH-receptor mRNA molecules, whereas 200 pmol/l GHBP produced a GH-receptor gene expression which was in between the values of the experiments with 50 and 500 pmol/l of GHBP added. Furthermore, the three different forms of human GHBP used provided similar data and, therefore, did not effect in any variation of GH-receptor expression. In addition, nuclear run-on experiments confirmed the changes in GH-receptor expression; and cycloheximide (10 microg/ml) did not alter the transcription indicating that the up and down regulating effects of GHBP on the GH-receptor/GHBP gene transcription was dependent, at least partly, on pre-existing factors and does not require protein synthesis. In conclusion, we present data showing that GHBP on its own has an effect on GH-receptor gene expression.

Animals↗
PubMed1997

Growth hormone stimulates the proliferation of activated mouse T lymphocytes.

A modulatory role for GH on immune function has been suggested, but hormonal effects have been difficult to demonstrate with isolated cells. We have recently shown that GH receptors are present in murine hematopoietic tissues, with a lower receptor number in T lymphocytes than in B cells or macrophages. The binding of bovine GH (bGH) to murine splenocytes is increased after T cell activation with either concanavalin A or anti-CD3 antibody. In the present study, we show that bGH is able to stimulate the proliferation of activated murine T cells. Splenocytes were stimulated with either Con A or anti-CD3 antibody; addition of the mitogen resulted in increased [3H]thymidine uptake. When added together with the mitogen to the culture medium, bGH was able to further stimulate thymidine uptake. A bell-shaped dose-response curve was observed. bGH was able to increase cell proliferation by 2.5-fold over the effect of anti-CD3 alone. The amplitude of the bGH response was greater in unfractionated splenocytes than in purified T lymphocytes or thymocytes. Splenocytes were also stimulated by lipopolysaccharide, a B cell-specific mitogen; no change in the level of bGH binding was observed during activation of B cells, and no effect of bGH on the proliferative response of splenocytes to lipopolysaccharide was detected. The GH proliferative effect on T lymphocytes is probably direct and not through locally produced insulin-like growth factor I, because insulin-like growth factor I did not affect the cell proliferation when added at concentrations ranging from 10(-9)-10(-7) M. Ovine PRL was also able to stimulate [3H]thymidine uptake in splenocytes and thymocytes, and a synergistic effect was observed when bGH and ovine PRL were added together at 10(-8) M. Our findings support the biological significance of the GH receptors identified in murine T lymphocytes and confirm the role of GH in the regulation of immune functions.

Animals↗
PubMed1997

Phenotype: genotype relationships in growth hormone insensitivity syndrome.

GH insensitivity syndrome (GHIS) is associated with many different mutations of the GH receptor (GHR) gene. We examined the phenotypic and biochemical features in 82 GHIS patients from 23 countries, each fulfilling diagnostic criteria of GHIS. There were 45 males and 37 females [mean age, 8.25 yr; mean height, -6.09 SD score, and mean insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), -7.99 SD score]. Sixty-three were GH-binding protein (GHBP) negative; 19 were GHBP positive (> 10% binding). The mean height in GHBP-negative subjects was -6.5 SD score, and that in GHBP-positive patients was -4.9 SD score (P = < 0.001). Clinical and biochemical heterogeneity was demonstrated by the wide range of height (-2.2 to -10.4 SD score) and IGFBP-3 (-1.4 to -14.7 SD score) values, which were positively correlated (r2 = 0.45; P = < 0.001). This contrasted with the lack of correlation between mean parental height SD score and height SD score (r2 = 0.01). Fifteen different GH receptor gene mutations were identified in 27 patients. All had homozygous defects, except 1 who had a compound heterozygous defect. The mutations were 5 nonsense, 2 frame shift, 4 splice, 4 missense, and 1 compound heterozygote. There was no relationship between mutation type or exon of the GHR gene involved and height or IGFBP-3 SD score. In conclusion, GHIS is associated with wide variation in the severity of clinical and biochemical phenotypes. This variation cannot clearly be accounted for by defects in the GHR gene. Other genetic and/or environmental factors must, therefore, contribute to phenotype in GHIS.

Adolescent↗
PubMed1997

A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein.

The GH receptor (GHR) is a member of the cytokine receptor family. Short isoforms resulting from alternative splicing have been reported for a number of proteins in this family. RT-PCR experiments, in human liver and cultured IM-9 cells, using primers in exon 7 and 10 of the GHR, revealed three bands reflecting alternative splicing of GHR mRNA: the predicted product at 453 bp and two other products at 427 and 383 bp. The 427-bp product (GHR1-279) utilized an alternative 3'-acceptor splice site 26 bp downstream in exon 9; the predicted C-terminal residues are six frameshifted exon 9 codons ending in an inframe stop codon. The 383-bp product (GHR1-277) resulted from skipping of exon 9; the predicted C-terminal residues are three frame-shifted exon 10 codons ending in an in-frame stop codon. RNase protection experiments confirmed the presence of the GHR1-279 variant in IM-9 cells and human liver. The proportion of alternative splice to full length was 1-10% for GHR1-279 and less than 1% for GHR1-277. The function of GHR1-279 was examined after subcloning in an expression vector and transient transfection in 293 cells. Scatchard analysis of competition curves for [125l]-hGH bound to cells transfected either with GHR full length (GHRfl) or GHR1-279 revealed a 2-fold reduced affinity and 6-fold increased number of binding sites for GHR1-279. The increased expression of GHR1-279 was confirmed by cross-linking studies. The media of cells transfected with GHR1-279 contained 20-fold more GH-binding protein (GHBP) than that found in the media of cells transfected with the full-length receptor. Immunoprecipitation and Western blotting experiments, using a combination of antibodies directed against extracellular and intracellular GHR epitopes, demonstrated that GHRfl and GHR1-279 can form heterodimers and that the two forms also generate a 60-kDa GHBP similar in size to the GHBP in human serum. Functional tests using a reporter gene, containing Stat5-binding elements, confirmed that while the variant form was inactive by itself, it could inhibit the function of the full-length receptor. We have demonstrated the presence of a splice variant of the GHR in human liver encoding a short form of the receptor similar in size to a protein previously identified in human liver and choroid plexus. Expression studies in 293 cells support the hypothesis that while the expression of the splice variant accounts for only a small proportion of the total GHR transcript, it produces a short isoform that modulates the function of the full-length receptor, inhibits signaling, and generates large amounts of GHBP. The differential expression of GHR receptor short forms may regulate the production of GHBP, and truncated receptors may act as transport proteins or negative regulators of GHR signaling.

Alternative Splicing↗
PubMed1996

Growth hormone-binding protein in the goat: characterization, evolution under exogenous growth hormone treatment, and correlation with liver growth hormone receptor levels.

This report describes the identification and characterization of a specific, high-affinity growth hormone-binding protein (GHBP) in lactating goat serum. Serum samples were incubated with [125I]human GH as ligand and in the absence or in the presence of bovine GH as competitor. GH-GHBP complex formation was performed by high-performance liquid chromatography, and the radioactivity was recorded on-line with a Berthold LB detector connected to a computer. The results showed that a serum protein was able to bind specifically to human GH and bovine GH but not to ovine prolactin. Scatchard plots indicated an affinity constant of 4.5 x 10(8) M-1 and a maximum binding capacity of 4.8 x 10(-10) mol/l. In addition, we conducted a 4-wk study to determine the effects of recombinant bovine GH administration on milk production in lactating goats. The effects of recombinant bovine GH treatment on milk production and on the regulation of GHBP and hepatic GH receptor levels were studied. As expected, recombinant bovine GH injected daily increased yields of milk, fat, protein (40, 61, and 40%, respectively), and circulating insulin-like growth factor 1 concentrations compared with controls. During the pretreatment and treatment periods, the control goats exhibited a constant amount of GHBP in serum. No consistent effect of GH treatment on GHBP level was observed. The binding of [125I]bovine GH to hepatic microsomal membranes of GH-treated goats was significantly decreased compared with that of control goats. After MgCl2 desaturation of membranes, the results demonstrated that the down-regulation of GH hepatic receptors, observed for the treated goat group, was induced by receptor occupancy without modification of binding affinity. The GH receptor gene expression, analyzed by slot blot and hybridization with an [alpha-32P]GH receptor cDNA probe, was not modified by the GH treatment. In lactating goats, the galactopoietic effect of exogenous GH involved a hepatic receptor occupancy. The individual concentration of GHBP in serum cannot explain the individual variations of responses to GH treatment in goats.

Animals↗
PubMed1996

Growth hormone receptor signalling.

The growth hormone (GH) receptor belongs to the superfamily of transmembrane proteins that includes the prolactin (PRL) receptor and a number of cytokine receptors. Two forms exist for the GH receptor: the membrane-bound form is a protein of 620 amino acid residues with a unique transmembrane domain; the GH-binding protein (GHBP), which is a soluble short form, is identical to the extracellular domain of the membrane receptor. In man and many other species, GHBP is believed to result from proteolytic cleavage of the membrane receptor; in human tissues, only one mRNA form of 4.5 kb encoding the full-length receptor has been detected. In rodents, GHBP is encoded by a specific mRNA of 1.2kb. Binding of GH to its receptor results in dimerization of the receptor, phosphorylation of the tyrosine kinase JAK2 and of the receptor, followed by a cascade of protein phosphorylations. Transcription factors belonging to the signal transducers and activators of transcription (STAT) family are involved in the effects of GH on the transcription of genes such as c-fos, serine protease inhibitor Spi 2.1 and beta-casein. GH is able to activate several STAT proteins including STAT1, 3 and 5. The JAK-STAT pathway is a main pathway for GH effects on gene transcription. Other signalling molecules are involved in GH action through different pathways: GH is able to activate mitogen activated protein (MAP) kinases; the hormone can utilize insulin receptor substrate-1 (IRS-1) and induces the association of phosphatidylinositol 3-kinase with IRS-1. Two main functional regions have been defined in the cytoplasmic domain of the GH receptor by testing the activity of mutant forms of the receptor in several systems: Box 1, a proline-rich sequence in the membrane proximal part, is necessary for all GH effects and is probably the region of association with JAK2; the C-terminal region is required for the induction of specific genes. Other molecules involved in the mechanisms of action of GH remain to be identified. As the same signalling pathways are used by many ligands, explanations for the specificity of the cellular effects have to be determined.

Amino Acid Sequence↗
PubMed1996

Growth hormone- and prolactin-binding proteins: soluble forms of receptors.

Receptors for growth hormone (GH) and prolactin (PRL) belong to the GH/PRL/cytokine receptor family, characterized by a unique transmembrane domain and absence of intrinsic tyrosine kinase. The GH receptor (GHR) is a protein of 620 amino acids; the extracellular domain of 246 amino acids is made of two subdomains, one being the domain of interaction with the ligand, the second one being the region of association with another receptor resulting in a homodimer. In addition to the membrane-bound receptor, a soluble form, called the GH-binding protein (GHBP), has been identified in the serum and corresponds to the extracellular domain of the full-length receptor. Two mechanisms of generation for the GHBP exist. In rodents, a 1.2-kb mRNA encodes the GHBP and its hydrophilic C-terminal sequence. In man and many species, no specific mRNA for the GHBP is detected: only one form of mRNA of 4.5 kb encoding the membrane GHR is found by Northern blot analysis. GHBP probably results from proteolytic cleavage of the membrane receptor. Plasma GHBP has a high binding affinity for the hormone comparable to that of the liver GHR. Half-life is longer for GH bound to the binding protein than for free GH. GH-GHBP complex represents a hormone reservoir. Other functions for GHBP remain to be clarified. Plasma levels of GHBP probably reflect the concentration of liver GHRs. Levels of liver GHR and plasma GHBP have been shown to change in parallel. GHBP measurements help in understanding situations of GH resistance. Many factors play a role in the regulation of the plasma GHBP which has been shown to change with age and nutritional status. GH, insulin and sex steroids also influence plasma GHBP levels. No PRL-binding protein has been detected in serum. In rabbit milk a soluble PRL receptor has been identified. The mechanism of its generation and its exact function has to be clarified.

Animals↗
PubMed1996

Expression of growth hormone receptors in murine lymphoid cells analyzed by flow cytofluorometry.

We have analyzed the expression of GH receptors (GHR) in murine lymphoid organs using flow cytofluorometry with biotinylated bovine GH and specific fluorescein isothiocyanate-labeled monoclonal antibodies defining distinct lymphoid cell populations. GHR were widely expressed in al murine hematopoietic tissues and in fetuses, newborns, and 3- and 7-week-old animals. In the bone marrow, all hematopoietic lineages expressed variable levels of GH receptors, whereas in the thymus, this expression was mainly seen in CD4-, CD8-, CD4+CD8+, and CD8+ subpopulations. At the periphery, 50% of splenocytes and peripheral blood lymphocytes and 20% of lymph node cells were GHR positive, with a wider receptor expression on B cells and macrophages (approximately 50%) than on T cells (approximately 20%), where the labeling was seen on both CD4+ and CD8+ cell subsets. Interestingly, the proportion of GHR-bearing CD4+ and CD8+ splenocytes significantly increased after T cell activation with Concanavalin A or anti-CD3. Finally, we demonstrated that all peripheral T cells expressing GHR also expressed PRL receptors. Our study provides a molecular basis to study the factors controlling GHR expression and to better understand the influence of GH in the regulation of immune function.

Animals↗
PubMed1996

A homozygous splice site mutation affecting the intracellular domain of the growth hormone (GH) receptor resulting in Laron syndrome with elevated GH-binding protein.

Laron syndrome (LS) is a severe autosomal recessive form of GH resistance resulting from molecular defects in the GH receptor (GHR). Affected individuals have extreme short stature and a typical facial phenotype. The point mutations in the GHR gene identified in this condition have until now been confined to the region encoding the extracellular domain of the receptor. We report here the first homozygous point mutation within the intracellular domain of the GHR in two LS cousins distinguishable from classical LS patients only by the presence of elevated GH-binding protein (GHBP) in their serum. A G to C transversion at the vital - 1 position in the splice donor site of exon 8 disrupts normal splicing, resulting in the complete skipping of exon 8, producing a mutant GHR protein lacking transmembrane and intracellular domains. We predict that this mutant protein would not be anchored in the cell membrane and would be measurable in the circulation as GHBP, hence explaining the phenotype of severe GH resistance combined with elevated circulating GHBP.

Base Sequence↗
PubMed1996

Ontogeny of GH receptor and GH-binding protein in the pig.

The present study was undertaken to examine the developmental pattern of GH receptor (GHR) and GHR gene expression in skeletal muscle (longissimus dorsi and trapezius (TR)) and liver from the last third of gestation until 1 year of age in male Large White (LW) and Meishan (MS) pigs. Plasma GH-binding protein (GHBP) levels were also measured. 125I-Labelled bovine GH (bGH) specific binding (not determined in foetal TR) and GHR mRNA were detected in skeletal muscle from 75 days of gestation until the adult stage with no clear age-related changes. By contrast, 125I-labelled bGH specific binding and GHR mRNA were undetectable or barely detectable in foetal liver. After birth, 125I-labelled bGH specific binding (P < 0.001) and GHR mRNA in liver increased with age. The level of bGH binding to liver membranes was higher in MS than in LW pigs at 1, 45, 80 and 120 days of age and did not differ between breeds at the other ages. Specific binding of 125I-labelled human GH (hGH) to plasma GHBP was easily detected as early as 75 days of gestation and increased with age (P < 0.001). The level of hGH binding to plasma GHBP was higher in MS than in LW pigs at 1, 80 and 120 days of age. It can be concluded that (1) the developmental expression of the GHR is tissue-specific, (2) the presence of GHBP in foetuses despite the absence of GHR in liver suggests that other tissues such as skeletal muscle could contribute to the generation of GHBP and (3) the presence of GHR in skeletal muscle as early as 75 days of gestation suggests that GH may play a role in foetal muscle growth.

Animals↗
PubMed1995

Identification of phenylalanine 346 in the rat growth hormone receptor as being critical for ligand-mediated internalization and down-regulation.

The functional significance of growth hormone (GH) receptor (GHR) internalization is unknown; therefore, we have analyzed domains and individual amino acids in the cytoplasmic region of the rat GHR required for ligand-mediated receptor internalization, receptor down-regulation, and transcriptional signaling. When various mutated GHR cDNAs were transfected stably into Chinese hamster ovary cells or transiently into monkey kidney (COS-7) cells, internalization of the GHR was found to be dependent upon a domain located between amino acids 318 and 380. Mutational analysis of aromatic residues in this domain revealed that phenylalanine 346 is required for internalization. Receptor down-regulation in transiently transfected COS-7 cells was also dependent upon the phenylalanine 346 residue of the GHR, since no GH-induced down-regulation was observed in cells expressing the F346A GHR mutant. In contrast, the ability to stimulate transcription of the serine protease inhibitor 2.1 promoter by the GHR was not affected by the phenylalanine 346 to alanine mutation. These results demonstrate that phenylalanine 346 is essential for GHR internalization and down-regulation but not for transcriptional signaling, suggesting that ligand-mediated endocytosis is not a prerequisite for GH-induced gene transcription.

Animals↗
PubMed1995

Expression and regulation of growth hormone (GH) and prolactin (PRL) receptors in a rat insulin producing cell line (INS-1).

We have examined the expression of growth hormone (GH) and prolactin (PRL) receptors, and the binding parameters of human GH (hGH), on the rat insulin producing cell line INS-1. We found that, like normal insulin producing beta-cells, INS-1 cells express both GH and PRL receptors, and the majority of human GH (hGH) binding sites on this cell line are of lactogenic specificity. As calculated from Scatchard plots, about 6600 hGH binding sites with a Ka of 6.2 x 10(8) M-1 are present per cell. Northern blot analysis showed two mRNA species of 4 and 1.6 kb for the GH receptor and, one major species of 10.5 kb for the PRL receptor. The PRL receptor mRNA was up-regulated by bovine GH (bGH), rat PRL (rPRL), in a time- and dose-dependent manner. On the contrary, bGH and rPRL, down-regulated the expression of GH receptor gene. The importance of this differential regulation in in vivo and in vitro studies are discussed.

Animals↗
PubMed1995

Nutritional status and growth hormone-binding protein.

To study the effects of nutrition on growth hormone (GH) receptor status, the plasma GH-binding protein was evaluated under conditions of poor nutrition, anorexia nervosa, celiac disease, and obesity. Nine patients, aged 12-30 years, presented anorexia nervosa and had a mean weight loss of -19% of their initial weight at the time of the study. Ten patients with celiac disease, aged 3-14 years, had a mean height at -4.2 SD, and normal body weight for height. Fourteen severely obese children, aged 3-10 years, had a mean body mass index (BMI) of 25.7 +/- 0.9. GH-binding protein was low in patients with anorexia nervosa (16.8 +/- 1.9% of radioactivity) and in patients with celiac disease (16.1 +/- 2.2%) whereas it was very high in obese children (57.2 +/- 3.3%). A strong correlation was found between GH-binding protein and BMI. GH-binding protein was also correlated with insulin-like growth factor-1 plasma levels. Nutrition is an important regulator of the GH receptor/binding protein. The growth failure presented by undernourished children is associated with partial GH resistance and low GH receptor level. On the contrary, children with obesity and normal growth have a high GH receptor level.

Adolescent↗
PubMed1995

Progressive normalization of growth hormone-binding protein and IGF-I levels in treated growth hormone-deficient children.

The short- and long-term effects of hGH treatment on growth hormone (GH)-binding protein (GHBP) were examined in 18 prepubertal children, aged 1.5-10 y, with isolated idiopathic GH deficiency. The patients were studied before and at regular intervals during 24 mo of hGH therapy (0.6 IU/kg/wk, given daily). Pretreatment GHBP values were low: 14.6 +/- 1.2% of radioactivity (p < 0.0001 versus normal prepubertal children). After the first hGH injection, GHBP levels fell significantly at 6 h (8.2 +/- 1.3% of radioactivity) and then remained at basal level during the first week. Under hGH therapy, an increase in GHBP was observed, but it occurred at different times of treatment, from 1 to 12 mo, and the mean GHBP value became significantly higher than the value before treatment after 12 mo of therapy. An increase in serum IGF-I level was observed as soon as 1 wk of hGH therapy, and after 3 mo, the mean IGF-I value was normal. No correlation was found between the increase in GHBP, IGF-I levels, and the growth velocity at 12 and 24 mo of treatment. These findings support the role of GH in the regulation of GHBP/receptor in man. The time course of the GH effect appears to be progressive and variable.

Carrier Proteins↗
PubMed1995

Quantitative analysis by polymerase chain reaction of growth hormone receptor gene expression in human liver and muscle.

A single form of GH receptor (GHR) messenger RNA (mRNA) of 4.5 kilobases, encoding the full-length GHR, has been found in man. To measure the absolute number of mRNA molecules encoding the GHR in human tissues, we developed a quantitative polymerase chain reaction assay. An internal control RNA was constructed by inserting a 50-basepair fragment of the rat PRL receptor complementary DNA into a portion of the human GHR complementary DNA. The internal control RNA and the target mRNA were amplified together with the same set of primers. Twenty-four cycles of amplification were used to satisfy an exponential phase of amplification. It was possible to detect as few as 500 molecules of target mRNA/micrograms total RNA. In 3 liver samples obtained from normal donors at the time of transplant, the amount of GHR mRNA ranged from 0.5 +/- 0.1 to 1.4 +/- 0.4 x 10(6) molecules/micrograms total RNA. These results were confirmed by slot blot analysis of the same samples. The number of receptor transcripts did not appear to be correlated with the receptor-binding capacity found in the 3 liver samples. In 7 muscle biopsies, GH receptor mRNA varied between 4.0 +/- 0.4 and 34.6 +/- 1.4 x 10(4) molecules/micrograms total RNA. This technique allows direct measurement of GHR gene expression in human tissues and represents a valuable tool, particularly for tissues such as muscle, in which the receptor protein cannot be measured using conventional binding assays.

Base Sequence↗