Growth hormone receptor/binding protein--clinical implications.
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Animals↗
Biomedical subjects
M C Postel-Vinay
Publications and source records attributed to M C Postel-Vinay.
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↗
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↗
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↗
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↗
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↗
The proline-rich region of the GH receptor is essential for JAK2 phosphorylation, activation of cell proliferation, and gene transcription.
Mutational analysis of the proximal transmembrane region of the cytoplasmic domain of the GH receptor (GHR) allowed us to characterize box 1, a proline-rich sequence of eight amino acids, which has been shown to be critical for signal transduction of many cytokine receptors. Mutants of the box 1 region of the rat GHR were studied for their ability to initiate the phosphorylation of JAK2 and the proliferation of stably transfected BAF B03 cells and also the activation of Spi 2.1 gene transcription in transiently transfected Chinese hamster ovary (CHO) cells. Convergence of effects of the box 1 mutants on JAK 2 phosphorylation, cell proliferation, and gene transcription was found. Our results suggest that no single amino acid in the box 1 sequence is essential for signaling and that the last two prolines (PXP motif) and the hydrophobic residues are necessary for integrity of box 1. Box 1 represents a structural determinant, potentially able to provide an interaction between JAK2 and the receptor; this interaction could be direct or indirect via an adaptor protein.
Amino Acid Sequence↗
Growth hormone receptor: structure and signal transduction.
The growth hormone receptor (GHR) belongs to the superfamily of transmembrane proteins that includes the prolactin receptor and a number of cytokine receptors. Two forms exist for the GHR: the full-length membrane-bound human receptor is a protein of 620 amino acids with a single transmembrane region; and the GH binding protein (GHBP) is a short soluble from corresponding to the extracellular domain of the full-length receptor. In rodents, GHBP is encoded by a specific mRNA of 1.2-1.5 kb, whereas in man and other species GHBP is believed to result from proteolytic cleavage of the membrane receptor. Growth hormone binding protein prolongs the half-life of GH but other functions for GHBP remain to be demonstrated. Recombinant GHBP complexed to human GH shows a 2:1 stoichiometric crystal structure. Growth hormone-induced dimerization of the cell surface GHR appears to be a prerequisite for biological activity of the hormone. JAK2 has been identified as a tyrosine kinase associated with GHR and other receptors of the superfamily. Binding of GH to its receptor results in dimerization of the GHR, phosphorylation of JAK2 and of the GHR. Other substrates for JAK2 have to be identified. Transcription factors belonging to the STAT (signal transducers and activators of transcriptions) family are involved in the transcriptional effects of GH. The activity of mutants of the GHR has been measured in functional tests to identify sequences of the cytoplasmic domain of the receptor that are important for signal transduction. A proline-rich sequence, called Box I, conserved among members of the receptor family has been shown to be crucial for GH effects on gene transcription. MAP kinase activity and cell proliferation. The C-terminal region of the GHR is required for tyrosine phosphorylation of the receptor and for a hormonal effect on gene transcription, whereas only 46 membrane proximal amino acids of the cytoplasmic domain are necessary for activation of JAK2 and transduction of the GH proliferative signal. Much work remains to be done to identify other protein kinases and signalling molecules involved in the mechanism of action of GH.
Amino Acids↗
Expression of the prolactin receptor gene during the breeding and non-breeding seasons in red deer (Cervus elaphus): evidence for the expression of two forms in the testis.
The red deer is a seasonally breeding mammal with a circannual cycle of prolactin secretion which reaches its peak during the non-breeding season. This study investigated expression of the prolactin receptor gene in red deer tissues collected in the breeding and non-breeding seasons. A 562 bp fragment of the extracellular domain of the red deer prolactin receptor cDNA was amplified from red deer liver poly(A)+ RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed from the human sequence. Northern blots were prepared using 10-20 micrograms poly(A)+ RNA. The blots were hybridized to the 562 bp cDNA labelled by random priming with alpha 32P-dCTP. A main transcript of 3.5 kb was expressed in liver, heart, kidney and testis throughout the year and in epididymis during the breeding season only. In the testis an additional major transcript of 1.7 kb was present during the breeding and non-breeding seasons. Competitive binding assays using 125I-ovine prolactin (125I-oPRL) were performed on microsomal membrane fractions prepared from liver. Scatchard analyses confirmed the presence of a single class of lactogen-binding receptor with a mean Ka of 0.87 +/- 0.12 x 10(9) M-1 and a Bmax of 73.6 +/- 9.8 fmol/mg protein (n = 5). Cross-linking of 125I-oPRL to liver microsomes with 0.5 mM disuccinimidyl suberate followed by SDS-PAGE revealed a major band of molecular mass 56 kDa which was displaced by ovine prolactin, suggesting a specific lactogen-binding entity of 33 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
[Growth hormone receptor. Structure and signal transduction].
The growth hormone receptor (GHR) belongs to the family of the prolactin and cytokine receptors. The full length receptor in a 620 amino acid protein with a unique transmembrane domain. The GH binding protein (GHBP) corresponds to the extracellular domain of the membrane GHR. In all human tissues tested, one form of 4.5 kb for the GHR mRNA was detected, suggesting that GHBP is generated through proteolytic cleavage of the membrane receptor. The three dimensional crystollographic structure of GHBP-hGH complex has identified a homodimer made of two receptor molecules and one molecule of hGH. Hormone-induced receptor dimerisation appears to be crucial for signal transduction. Functional tests using the GH effect on transcription of genes, such as SP12.1 and beta lactoglobulin, have been developed to define the sequences of the receptor which are important for signaling. A proline-rich juxtamembranous sequence, called Box 1, is important for GH effects on gene transcription, on MAP kinase activity, on cell proliferation, and on JAK2 activation. JAK2 has been identified to be a GHR-associated tyrosine kinase. The first 46 amino acids of the cytoplasmic domain are necessary for JAK2 and MAP kinase activation whereas a C-Ter sequence is necessary for the transcriptional effect. Substrates for JAK2, other than the receptor itself, have to be identified. Good candidates are the transcription factors STAT.
Humans↗
Allosteric effects of monoclonal antibodies on human growth hormone.
We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors. The effect of MAbAE5,AC8 and F11 on hGH binding was measured by determining the formation of 125I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding 125I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes. Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case performed 125I-MAb:hGH complexes were added to microsomes. Data showed that 125I-MAb AE5:hGH complexes bound better to the various receptors than 125I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to 125I-MAb AC8 or 125I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32-46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding. Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Allosteric Regulation↗
[Growth hormone receptor and dwarfism].
Laron syndrome is a rare autosomal recessive disease characterized by extreme resistance to growth hormone (GH). A GH receptor defect, which was hypothesized as the cause of the disease, was demonstrated after the cloning of the GH receptor cDNA. Several abnormalities of the GH receptor gene have been identified in the patients, demonstrating the genetic heterogeneity of the disease. Most of the identified mutations belong to the extracellular domain of the receptor, in the hormone-binding region, and this finding is consistent with the absence of GH binding activity found in the patients' plasma. Other mutations will be found, particularly in patients with Laron syndrome associated with a normal high affinity GH binding protein; in these cases, the defect could be at the dimerization level or at a later step in signal transduction. Laron syndrome represents a valuable model to identify structural and functional important regions of the receptor involved in GH action. In other situations of growth failure, such as in the Pygmies, a defect in the regulation of the GH receptor gene expression has been proposed.
Chromosome Deletion↗
Cytoplasmic sequences of the growth hormone receptor necessary for signal transduction.
To study structure-function relationships of the growth hormone (GH) receptor (GHR), two functional systems have been developed. CHO cells were transiently cotransfected with the cDNA encoding the full-length rat GHR and with a construct consisting of the 5' flanking region of one of two GH-dependent genes encoding ovine beta-lactoglobulin or serine protease inhibitor 2.1 (Spi 2.1, formerly Spi.1; the corresponding rat gene has recently been redesignated Spin2a) coupled to the bacterial reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfected cells were grown in the absence and presence of human GH and dexamethasone for the Spi 2.1 gene construct. GH was able to activate each promoter (with approximately 4-fold induction of CAT activity) in a dose-dependent manner. For both tests, the maximal effect was observed at 20 nM human GH. These tests have been used to identify functional domains of the GHR. Two truncated (T) GHRs, lacking most or part of the cytoplasmic domain [called T276 (ending at residue 276) and T436 (ending at residue 436)], were unable to stimulate CAT activity. The GHR contains a proline-rich region, called "Box I," conserved in the cytokine/GH/prolactin receptor family. Alanine substitutions for the four prolines of GHR Box I were introduced. Single proline-to-alanine mutations did not affect the functional activity of the GHR. However, modification of the four prolines together or deletion of the Box I (15 amino acids between positions 279 and 293) resulted in the complete absence of GH stimulation. Thus, the proline-rich region, shown to be important for other members of this receptor superfamily, is also critical for GH signal transduction.
Amino Acid Sequence↗
Expression of two isoforms of the human growth hormone receptor in normal liver and hepatocarcinoma.
In man, two isoforms of growth hormone receptor (GHR) have been reported. The first, hGHR corresponds to the original form described by Leung et al. (J. Biol. Chem. 264 (1987) 9905-9910). The second, hGHRd3, missing 22 N-terminal amino acids is encoded by an mRNA derived from alternative splicing and deletion of the third exon. This latter form has been isolated from placenta and its tissue distribution, as well its function warrant further examination. We studied the relative expression of the two isoforms of GHR in liver at various stages of development of differentiation. Several samples from fetal livers and hepatocarcinomas were analysed and we observed that hGHR and hGHRd3 can be expressed solely or coordinately. Furthermore, we investigated the possibility that alternative splicing of exon 3 could be related to the specific use of different promoters. Our data show that both isoforms have identical 5'-untranslated regions in normal liver or in hepatoma cell lines. Finally, these experiments strongly suggest that hGHRd3 is efficiently translated as a functional receptor, with the soluble form of GHR being recovered in the cell media. The results of the present study support the concept that both isoforms are expressed in human liver, with exon 3 alternative splicing being neither tissue specific nor related to hepatic differentiation.
Alternative Splicing↗
Reduced food intake is the main cause of low growth hormone receptor expression in uremic rats.
To examine the respective effects of reduced food intake and of uremia on the growth defect in uremic rats, we have studied the expression of GH receptors in three groups of male rats: Group 1, rats fed ad libitum; Group 2, food-restricted to be pair-fed with uremic rats; Group 3, uremic rats. Animals were studied for a time period of 9 days starting 1 week after surgery (sham operation in rats of Groups 1 and 2, 5/6 nephrectomy in rats of Group 3). The gain in body length and weight of pair-fed controls and of uremic rats was comparable and significantly lower than that of rats fed ad libitum. IGF-1 plasma levels were low in rats of groups 2 and 3. Low food intake (50% that of rats fed ad libitum) resulted in a reduced number of GH receptors in liver membranes and a low plasma level of GH-binding protein (GHBP); GH receptor gene expression in the liver, as analyzed by Northern blots, was not significantly lower in normal food-restricted animals. In uremic rats, the low level of GH binding to liver membranes was comparable to that found in pair-fed controls; but the level of GHBP activity was normal, not different from the values found in rats fed ad libitum. However, expression of the liver GHBP mRNA was reduced in uremic rats. In uremia, the GH receptor dysfunction is not only at a transcriptional level but also at a post-transcriptional level. These findings suggest that uremia, as such, is not primarily responsible for the growth failure.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Growth hormone receptor structure, dimerization and function.
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Animals↗
Improvement of diagnostic criteria in growth hormone insensitivity syndrome: solutions and pitfalls. Pharmacia Study Group on Insulin-like Growth Factor I Treatment in Growth Hormone Insensitivity Syndromes.
A survey to identify children and adolescents with primary growth hormone insensitivity syndrome (GHIS) yielded 38 patients who were positively identified using a scoring system that included five criteria: height, basal growth hormone (GH), GH binding protein, basal insulin-like growth factor I (IGF-I) and the increase of IGF-I after 4 days of GH administration (IGF generation test). Because of an overlap of the accepted and excluded groups with respect to points scored, an attempt was made to improve the scoring system. The new criteria were: height below -3 SDS, basal GH 4 mU/l or above, GH binding below 10%, basal IGF-I and basal IGF binding protein-3 (IGFBP-3) below the 0.1 centile for age, an increase of IGF-I in the IGF generation test less than 15 micrograms/l, and the increase of IGFBP-3 less than 0.4 mg/l. With this scoring system, a clear separation between the accepted and the excluded groups was obtained. IGFBP-3 was included to give the GH-dependent parameters of the IGF system more weight and because the accuracy of IGFBP-3 in the IGF generation tests was greater than the accuracy of IGF-I, when the group of patients with GHIS was compared with a group of patients with GH deficiency. Unexpectedly, the IGF generation test was unable to segregate both cohorts completely. In the GHIS-positive group, a significant correlation was found between basal IGF-I or IGFBP-3 levels corrected for age (SDS) and height SDS (r = 0.49, p < 0.002 and r = 0.61, p < 0.0001, respectively). There was also a significant correlation between the changes of IGF-I or IGFBP-3 in the IGF generation test and height SDS. That is, the patients with a slight response to GH were those with the least growth retardation, suggesting the existence of partial GH insensitivity.
Adolescent↗
The GH receptor and signal transduction.
The primary structure of the growth hormone (GH) receptor in rabbits and humans determined by complementary DNA cloning revealed a single membrane-spanning protein of approximately 620 amino acids. A binding protein (bp) specific for GH has been identified in the serum of a number of species. In rabbits and man, a single 4.5-kb transcript has been identified that encodes the full-length receptor. In rats and mice, however, a smaller transcript produced by alternative splicing has been reported which is specific for the GHbp. Recently, the X-ray crystallographic structure of GH and its receptor have clearly shown the formation of an unusual homodimer, consisting of one molecule of GH and two molecules of hGHbp. Formation of the GH dimer is a necessary prerequisite for biological activity. The transcriptional activity of wild-type and mutant forms of GH receptor has been determined by co-transfecting the promoter of a GH-responsive gene, coupled to CAT along with the receptor cDNA. A 25-amino acid region near the transmembrane domain has been shown to be important for functional activity, although 8 amino acids (known as Box 1), rich in prolines, is essential. Alanine scanning mutagenesis has revealed that individual substitution of each residue is without effect, while the replacement of the last 2 or all 4 of the prolines abolishes activity. Finally, GH has been shown to induce rapid tyrosine phosphorylation of several proteins in cells expressing the receptor, one of which has recently been identified as the kinase JAK2 and another as MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗