Human Growth Hormone Receptor Research Articles

Alternative splicing of exon 3 of the human growth hormone receptor is the result of an unusual genetic polymorphism.

Two isoforms of the human growth hormone receptor (hGHR), which differ in the presence (hGHRwt) or absence (hGHRd3) of exon 3, are expressed in the placenta. Specifically, three expression patterns are observed: only hGHRwt, only hGHRd3, or an approximately 1:1 combination of both isoforms. We investigated several potential regulatory mechanisms which might account for the expression of the hGHR isoforms. The frequency of hGHRd3 expression did not change when placentas from differing stages of gestation were examined, suggesting splicing was not developmentally regulated. However, when hGHR isoform expression patterns were examined in each component of a given placenta, it was evident that alternative splicing of exon 3 is individual-specific. Surprisingly, the individual-specific regulation of hGHR isoforms appears to be the result of a polymorphism in the hGHR gene. We analyzed hGHRwt and hGHRd3 expression in Hutterite pedigrees, and our results are consistent with a simple Mendelian inheritance of two differing alleles in which exon 3 is spliced in an "all-or-none" fashion. We conclude the alternative splicing of exon 3 in hGHR transcripts is the result of an unusual polymorphism which significantly alters splicing of the hGHR transcript and that the relatively high frequency (approximately 10%) of homozygous hGHRd3 expression suggests the possibility it may play a role in polygenic determined events.

Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein.

The mechanism underlying the generation of soluble growth hormone binding protein (GHBP) probably differs among species. In rats and mice, it involves an alternatively spliced mRNA, whereas in rabbits, it involves limited proteolysis of the membrane-bound growth hormone receptor (GHR). In humans, this latter mechanism is favored, as no transcript coding for a soluble GHR has been detected so far. To test this hypothesis, we analyzed COS-7 cells transiently expressing the full-length human (h) GHR and observed specific GH-binding activity in the cell supernatants. Concomitantly, an alternatively spliced form in the cytoplasmic domain of GHR, hGHR-tr, was isolated from several human tissues. hGHR-tr is identical in sequence to hGHR, except for a 26-bp deletion leading to a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of the receptor protein. When compared with hGHR, hGHR-tr showed a significantly increased capacity to generate a soluble GHBP. Interestingly, this alternative transcript is also expressed in liver from rabbits, mice, and rats, suggesting that, in these four species, proteolysis of the corresponding truncated transmembrane GHR is a common mechanism leading to GHBP generation. These findings support the hypothesis that GHBP may at least partly result from alternative splicing of the region encoding the intracellular domain and that the absence of a cytoplasmic domain may be involved in increased release of GHBP.

Real-time Kinetic Measurements of the Interactions between Lactogenic Hormones and Prolactin-Receptor Extracellular Domains from Several Species Support the Model of Hormone-induced Transient Receptor Dimerization

Interactions of recombinant soluble prolactin receptors-extracellular domains (PRLR-ECDs) from rabbit, rat, and cow and human growth hormone receptor ECD with immobilized human growth hormone, several prolactins, and bovine placental lactogen were studied utilizing surface plasmon resonance. This method enables real-time kinetic measurements of the interactions and calculations of kinetic constants and of the stoichiometry of interaction, even in cases where only transient interactions occur. In contrast to gel filtration or crystallographic studies, where in most cases the interaction of PRLR-ECDs with various lactogenic hormones indicated formation of 1:1 complexes, our surface plasmon resonance experiments indicated in all cases the transient formation of a 2:1 complex. In most of the interactions the 2:1 complex was very unstable and underwent rapid dissociation to a 1:1 complex. This situation was particularly characteristic of homologous interactions involving hormone and receptor from the same species and was mainly attributed to increased dissociation constants. We suggest that as in the case of growth hormone PRLR activation occurs via hormone-induced transient homodimerization of the receptor, lasting only a few seconds, and that this may be sufficient to initiate the biological signal. Once the signal is initiated, the receptor dimer is no longer required. Its rapid dissociation to a 1:1 complex or to its components may even be advantageous in that it permits activation of additional receptors.

Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A.

The functional mimicry of a protein by an unrelated small molecule has been a formidable challenge. Now, however, the biological activity of a 166-residue hematopoietic growth hormone, erythropoietin (EPO), with its class 1 cytokine receptor has been mimicked by a 20-residue cyclic peptide unrelated in sequence to the natural ligand. The crystal structure at 2.8 A resolution of a complex of this agonist peptide with the extracellular domain of EPO receptor reveals that a peptide dimer induces an almost perfect twofold dimerization of the receptor. The dimer assembly differs from that of the human growth hormone (hGH) receptor complex and suggests that more than one mode of dimerization may be able to induce signal transduction and cell proliferation. The EPO receptor binding site, defined by peptide interaction, corresponds to the smaller functional epitope identified for hGH receptor. Similarly, the EPO mimetic peptide ligand can be considered as a minimal hormone, and suggests the design of nonpeptidic small molecule mimetics for EPO and other cytokines may indeed be achievable.

Molecular model of interleukin 12 that highlights amino acid sequence homologies with adhesion domains and gastrointestinal peptides

A three-dimensional (3-D) model of both subunits of interleukin 12 (IL-12) has been created through molecular modeling. Initial assignment of coordinates in the model of the p40 subunit was based on established amino acid sequence homology between the second and third domains of p40 and the human growth hormone receptor (GHR) and new observations of similarity between the first domain of p40 and the N-terminal domain of CD4. Human growth hormone (GH) served as the reference protein for the p35 chain. Furthermore, thorough analysis of the amino acid sequence of IL-12 revealed two distinct regions of the p40 subunit that display homology with other proteins. The first region (in domain two) contains the sequence RGD, which is found in adhesion proteins (such as fibronectin), and the nearby sequence VTCG, which occurs in a diverse set of molecules, including thrombospondin, properdin, and circumsporozoite proteins of Plasmodium. The second region of homology spans the third domain of p40 and shows marked similarity with the gastrointestinal peptides, such as secretin and glucagon and their preprohormones. We conclude (1) that the regions of homology define functionally important segments of p40 that are fully exposed at the protein surface, and (2) that the third domain of p40 (and its equivalent in the cytokine receptor family) is derived from the same ancestral genes as the gastrointestinal peptides.

Novel Assays Based on Human Growth Hormone Receptor as Alternatives to the Rat Weight Gain Bioassay for Recombinant Human Growth Hormone

Abstract.Two methods, High-Performance Receptor Binding Chromatography (HPRBC) and Cell Proliferation (CP), have been developed as alternatives to the classical hypophysectomized rat weight gain bioassay for the determination of potency for recombinant human growth hormone (rhGH). In the HPRBC assay, rhGH is combined with an excess of the soluble extracellular domain of the recombinant human growth hormone receptor (referred to as ‘receptor’ in the discussion of the HPRBC assay). Nondenaturing size-exclusion chromatography is used to analyze the resulting complex, which forms in a 2:1 receptor to rhGH ratio. The 2:1 complex is assayed at a concentration near the K d(0.4 nM), providing high specificity for rhGH and detection of rhGH variants with reduced activity. In the CP assay, a mouse myeloid leukaemia cell line (FDC—P1) transfected with the full-length receptor is exposed to varying levels of rhGH for 16–20 h. The incorporation of 3H-thymidine into DNA is used as an index of cell proliferation. The results show that the HPRBC assay provides significantly improved precision with a relative standard deviation (RSD) of ≤5% vs.an RSD of 23% for the rat bioassay. The CP assay has RSDs of 4–16% . Analysis of rhGH variants and mutants shows that the potencies measured by both the HPRBC and CP assays are in general agreement with the rat weight gain bioassay. Both of the HPRBC and CP assays are sufficiently rugged for operating in a Good Manufacturing Practices (GMP) routine batch release testing environment. In vitroalternatives such as the HPRBC and CP assays build a foundation for replacing the hypophysectomized rat weight gain bioassay by correlating receptor dimerization, binding specificity and signal transduction with the biological activity of rhGH.

Expression of exon 3-retaining and -deleted human growth hormone receptor messenger ribonucleic acid isoforms during development.

Recent investigations have suggested that human GH (hGH) and its receptor may have specific functions during human fetal life. To improve our understanding of the mechanisms of hGH action during gestation, we characterized the ontogenic appearance of hGH receptor messenger ribonucleic acid (mRNA) in multiple human fetal and postnatal tissues. Using RT-PCR assays, followed by Southern hybridization to confirm the specificity of the amplified fragments, we scanned the entire coding region of the hGH receptor mRNA. Transcription of the hGH receptor gene was observed in all fetal tissues studied (liver, kidney, skin, muscle, lung, adrenal, spleen, intestine, central nervous system, pancreas, and placental villi) from the earliest stage that could be examined [7-14.8 weeks fetal age (FA)]. Furthermore, we identified only 2 isoforms of the hGH receptor mRNA-coding region: exon 3 can be retained or deleted. Surprisingly, we found individual-specific, not tissue-specific, expression patterns of these two transcripts when we examined multiple tissues (n = 2-6) from 15 individuals (11.5-33 weeks FA); this individual-specific pattern of expression is maintained in cultured dermal fibroblasts for at least 12 generations (n = 2; 16 and 20 weeks FA). In addition, a cross-sectional study of 78 individuals (9 weeks FA to 43 yr postnatal age) showed that the exon 3-deleted transcript is predominantly expressed in tissues from fetuses of 9-20 weeks FA (P < 0.002). Finally, we showed that the absence of exon 3 from the mRNA is not due to genomic deletion of exon 3 by amplifying exon 3 from genomic DNA of 3 fetuses (13.3-19 weeks FA) expressing only the exon 3-deleted mRNA transcript. We conclude that 1) transcription of the hGH receptor gene occurs in multiple tissues as early as the first trimester of human fetal life; 2) the exon 3-retaining and -deleted transcripts are the only two isoforms of the hGH receptor mRNA-coding region during gestation; and 3) the pattern of expression of these transcripts is individual specific and may be developmentally regulated.

Expression of exon 3-retaining and -deleted human growth hormone receptor messenger ribonucleic acid isoforms during development

Expression of exon 3-retaining and -deleted human growth hormone receptor messenger ribonucleic acid isoforms during development

The Crystal Structure of the Extracellular Domain of Human Tissue Factor Refined to 1.7 Å Resolution

Exposure of blood to cells expressing tissue factor results in formation of a high-affinity complex with factor VIIa, initiating the extrinsic pathway of blood coagulation by the activation of factors IX and X. The structure of the extracellular portion of tissue factor was refined to a crystallographic R-value of 20.4% to a resolution of 1.69 Å against synchrotron data collected from a flash-frozen crystal. The structure consists of two fibronectin type III modules whose hydrophobic cores merge in the domain – domain interface, suggesting that the extracellular portion serves as a relatively rigid template for factor VIIa binding. Analysis of the hydrophobic core of each individual module identifies a cluster of residues forming a packing motif centered on Trp25 which appears to be characteristic for fibronectin type III modules. Comparison of the structure to that of the human growth hormone receptor, which belongs to a different class (class I) of the same cytokine receptor superfamily, shows that the structure of the individual domains is very similar but that the relative domain – domain orientation differs greatly. Even though the WSXWS box characteristic of the class I cytokine receptors is not present in tissue factor, the analogous residues have the identical polyproline helical conformation. Mapping of residues important for biological activity on the structure shows that all these are located on β-strands in a small number of distinct clusters, on the opposite side of the molecule compared to the ligand binding determinants of the growth hormone receptor.

Human growth hormone fragments 1-43 and 44-191: in vitro somatogenic activity and receptor binding characteristics in human and nonprimate systems.

Human GH (hGH) fragments 1-43 and 44-191 have potent in vivo effects on glucose homeostasis in rodents but cannot stimulate body growth. To assess the in vitro bioactivity of these hGH fragments we tested their activity against GH-responsive FDC-P1 cell lines expressing full-length human (h), mouse (m), or rabbit (r) GH receptors (GHR). Binding specificity and affinity was tested using GHR-containing membrane preparations from three species and recombinant hGH binding protein (hGHBP). Recombinant hGH 1-43 and recombinant 44-191 stimulated proliferation of FDC-P1-hGHR cells with half-maximal effect at approximately 2000 and 100 nM, respectively, whereas intact hGH stimulates proliferation of FDC-P1-hGHR cells with ED50 of 0.02-0.03 nM. However, these fragments had minimal effect on cells expressing mGHR or rGHR. Although 44-191 did not stimulate proliferation of FDC-P1-rGHR cells, when added to these cells in the presence of 0.23 nM hGH, it antagonized hGH action in a dose-dependent manner (ED50 at 230 nM). Binding of these GH fragments was compared using membrane preparations from rabbit liver, rabbit and mouse adipose tissue, and recombinant hGHBP. Binding competition curves were consistent, with 44-191 having at least a 10-fold lower affinity for rabbit liver GHR and rabbit adipose GHR than bovine GH and a 61-fold lower affinity for hGHBP than hGH. Binding of hGH 1-43 could not be demonstrated to GHRs in rabbit liver microsomes, adipose microsomes, or to hGHBP. HGH 1-43 did not compete for insulin binding sites in adipose microsomes. In conclusion, hGH 44-191 binds with low affinity to the GHR and at supraphysiologic levels stimulates proliferation of FDC-P1-hGHR cells. At high doses, 44-191 can also antagonize GH action in FDC-P1-rGHR cells, presumably by blocking receptor dimerization. Binding of 1-43 to GHR could not be detected, and the basis for its weak in vitro mitogenic effect remains to be elucidated. The low affinity of the fragments for cloned GHRs and low biopotency in these systems suggests that the metabolic actions of these fragments are unlikely to be mediated by the cloned GHR. This raises the possibility of a separate receptor mediating metabolic effects of these fragments.

Expression of functional growth hormone receptors in cultured human osteoblast-like cells.

Recent clinical studies indicate that GH is important for bone remodeling. Patients with GH deficiency exhibit decreased bone density, and GH substitution increases bone density in these patients. The aim of the present study was to investigate the presence of GH receptors and the effects of GH on cultured human osteoblast-like cells. Primary cultures of human osteoblast-like cells were established from trabecular bone. Northern blot analysis, using a probe recognizing exon 10 of the human GH receptor, revealed a 4.7-kilobase transcript corresponding to the human GH receptor. Cultured osteoblast-like cells expressed, as determined by RNase protection assay, approximately one fourth of the GH receptor messenger RNA levels found in liver. Binding studies using 125I-labeled GH revealed a single class of receptors with approximately 2000 binding sites per cell and an association constant (Ka) of 2.6 x 10(9) M-1. GH stimulation of the cultured cells resulted in increased [3H]thymidine incorporation, suggesting that the GH receptors are functional. In summary, the present study shows that cultured human osteoblast-like cells express functional GH receptors.

Cellular localization of the growth hormone receptor/binding protein in the human anterior pituitary gland.

The increasing use of GH therapy has led to the description of its target cells in human tissues, but no data are yet available on the localization of the GH receptor in the human pituitary. In the present study, we used immunocytochemistry to detect the presence of GH receptor/binding protein (GHR/BP), and we examined its distribution among the different types of human anterior pituitary cells. Human pituitaries were taken from autopsies and were processed for embedding in paraffin wax. Immunocytochemistry was performed by using monoclonal antibody 263 raised against purified rat and rabbit GHR/BP, which cross-reacts with the human GH receptor. In order to determine the types of cells that display immunoreactivity for GHR/BP, adjacent pituitary sections were used to detect immunoreactivity for GH, PRL, ACTH, TSH, LH, and FSH. Several controls were carried out to verify the specificity of the immunostaining. Receptor immunoreactivity was found in the cytoplasm and nuclei of the somatotrophs, lactotrophs, and gonadotrophs but not in the thyrotrophs or corticotrophs. In order to demonstrate that the detected GHR/BP immunoreactivity was not caused solely by a cellular capture, we also investigated the cellular distribution of GHR gene expression. This was performed by in situ hybridization with use of complementary oligonucleotide radioactive probes encoding distinct domains of the GHR. Several tests were carried out to validate the detection of gene expression. In situ hybridization demonstrated the presence of GHR messenger RNAs in the anterior lobe of human pituitary, and examination of the signal strengthened the cell-specificity of GHR gene expression. These results demonstrate the presence of GHR/BP in discrete human pituitary cells and indicate a paracrine, autocrine, or intracrine role for GH in the pituitary.

Requirement for the Immunoglobulin-like Domain of Granulocyte Colony-stimulating Factor Receptor in Formation of a 2:1 Receptor-Ligand Complex

The extracellular portion of the granulocyte colony-stimulating factor (G-CSF) receptor has a mosaic structure of six domains (each approximately 100 amino acid residues) consisting of an immunoglobulin-like (Ig) domain, a cytokine receptor homologous region subdivided into amino-terminal (BN) and carboxyl-terminal (BC) domains, and three fibronectin type III repeats. In the present study, we expressed the Ig-BN and the BN-BC regions and purified them to homogeneity as monomers using G-CSF affinity column chromatography. Using gel filtration high performance liquid chromatography, we investigated the molecular composition of receptor-ligand complexes formed between G-CSF and purified BN-BC or Ig-BN domains. In contrast to the well characterized example of the human growth hormone (GH) receptor, in which the BN-BC.GH complex shows a 2:1 receptor-ligand complex stoichiometry, the BN-BC domain of the G-CSF receptor formed a 1:1 complex. The isolated Ig-BN domain also formed a 1:1 complex with G-CSF. However, in the presence of both Ig-BN and BN-BC domains, we detected a 1:1:1 Ig-BN.G-CSF.BN-BC complex corresponding to the 2:1 receptor: ligand stoichiometry. These results suggest that 1) the Ig domain and both the BN and the BC domains are required for oligomerization of the G-CSF receptor, 2) G-CSF contains two binding sites for its receptor, and 3) there are two ligand binding sites on the G-CSF receptor, one site on the BN-BC domain and one on the Ig-BN domain.

Mapping of the Cytoplasmic Domain of the Human Growth Hormone Receptor Required for the Activation of Jak2 and Stat Proteins

Incubation of cells with growth hormone (GH) stimulates both tyrosine phosphorylation of the Jak2 tyrosine kinase and, in some cells, the transcription factor Stat1 alpha (1-4). When the promyeloid cell line FDC-P1 is transfected with the human growth hormone receptor, these cells can grow in the presence of GH and in the absence of interleukin-3. Growth hormone treatment of cells expressing the human growth hormone receptor did not activate Stat1 alpha. However, a complex is present in extracts prepared from growth hormone-treated cells that binds to the gamma response region, an enhancer present in the promoter of the high affinity Fc gamma R1 receptor to which cytokine-activated Stat complexes bind. When truncations of the cytoplasmic domain of the receptor are expressed in FDC-P1 cells only the membrane-proximal 80 amino acids (containing box 1 and box 2) are required for activation of both a GH-stimulated binding activity (GHSF) and tyrosine phosphorylation of Jak2. Activation of GHSF can be inhibited in a cell-free system by the addition of a glutathione S-transferase fusion protein containing these 80 amino acids. Replacement of the one tyrosine in this region of the receptor with a phenylalanine does not alter the activation of either GHSF or Jak2, suggesting that tyrosine phosphorylation of the receptor is not required for GH activation of GHSF. Moreover, a cell line expressing a receptor with only the 54 membrane-proximal amino acids of the intracellular domain (including box 1) shows constitutively tyrosine-phosphorylated Jak2 as well as GHSF binding. With this truncated receptor, there is little if any additional GH-induced tyrosine phosphorylation of Jak2 or induced binding to the gamma response region. These results define the importance of the membrane-proximal 80 amino acids of the GH receptor (with the conserved box 1 and box 2 domains) with regard to GH activation of both Jak2 and Stat(s). They also suggest that within these domains there may be positive and negative elements that regulate Jak2 function.

Detection of human growth hormone receptors on IM-9 cells and peripheral blood mononuclear cell subsets by flow cytometry: correlation with growth hormone-binding protein levels

Detection of human growth hormone receptors on IM-9 cells and peripheral blood mononuclear cell subsets by flow cytometry: correlation with growth hormone-binding protein levels

Release of a Soluble Form of Growth Hormone Receptors (Growth Hormone-Binding Proteins) from Human IM-9 Cells by Proteolytic Cleavage1

Soluble forms of growth hormone receptors, growth hormone-binding proteins (GH-BPs), with molecular weights of 60 and 55kDa were found to be constitutively released from human IM-9 cells. The release of the GH-BPs was not inhibited by inhibitors of protein synthesis (cycloheximide) and transport (brefeldin A). Down-regulation by human growth hormone or trypsin pretreatment of surface growth hormone receptors abolished the GH-BP release, suggesting that cell-surface human growth hormone receptors are involved in the GH-BP release. Several inhibitors of serine-, thiol-, and acid-proteases did not affect the GH-BP release. EDTA efficiently blocked the GH-BP release. This inhibition by EDTA was restored by addition of Mg(2+) and Co(2+). These results suggest that human GH-BPs are constitutively released by proteolytic cleavage of cell-surface growth hormone receptors by a metalloprotease.

Detection of human growth hormone receptors on IM-9 cells and peripheral blood mononuclear cell subsets by flow cytometry: correlation with growth hormone-binding protein levels.

We have developed a method using flow cytometry to identify fluorescein-conjugated GH receptors (GHR) on IM-9 lymphocytes and circulating peripheral blood mononuclear cell subsets. Binding to IM-9 cells and peripheral blood mononuclear cells was concentration dependent and could be competitively blocked by the addition of unlabeled human GH, but not by the addition of rat or bovine GH or human insulin or PRL. Using two-color flow cytometric analysis, fluorescein-conjugated human GHR were readily detected on more than 90% of B lymphocytes and monocytes, but only variably on T lymphocytes. B Lymphocytes and monocytes had approximately 6000 GHR/cell. Using two-color flow cytometry, we identified GHR on circulating B lymphocytes in subjects with GH deficiency (n = 9), precocious puberty (n = 6), and Turner syndrome (n = 5) and in seven subjects with miscellaneous disorders, including familial short stature, bone dysplasia, Crohn disease, congenital adrenal hyperplasia, and acromegaly. The percentage of B lymphocytes expressing GHR in subjects with GH deficiency (mean +/- SD, 95 +/- 9%), precocious puberty (91 +/- 15%), and Turner syndrome (84 +/- 15%) was not different from that in normal volunteers (90 +/- 12%; n = 14). In 10 subjects, serum GH-binding protein levels were assayed simultaneously with B lymphocyte GHR. GH-binding protein was normal in all (mean, 1255 pmol/L; range, 773-1809). There was a good correlation between GHR expression on B lymphocytes and GH-binding protein levels (r = 0.75; P = 0.01). We postulate that GHR found on circulating B lymphocytes may contribute to the pool of receptors identified in serum as GH-binding proteins. Two-color flow cytometry appears to be an effective method for the detection of GHR on circulating peripheral blood mononuclear cell subsets. The evaluation of GHR on circulating B lymphocytes may prove to be a useful means of evaluating GH-GHR interactions in subjects with growth disorders.

Expression of exon 3-retaining and exon 3-excluding isoforms of the human growth hormone-receptor is regulated in an interindividual, rather than a tissue-specific, manner

Expression of exon 3-retaining and exon 3-excluding isoforms of the human growth hormone-receptor is regulated in an interindividual, rather than a tissue-specific, manner

Distinct regions of c-Mpl cytoplasmic domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation.

c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin. To investigate signal transduction by c-Mpl, a chimeric receptor, composed of the extracellular domain of human growth hormone receptor and the intracellular domain of c-Mpl, was introduced into the interleukin 3-dependent cell line Ba/F3. In response to growth hormone, this chimeric receptor induced growth in the absence of interleukin 3. Deletion analysis of the 123-amino acid intracellular domain indicated that the elements responsible for this effect are present within the 63 amino acids proximal to the transmembrane domain. Mutation of the recently described box 1 motif abrogated the proliferative response. Tyrosine phosphorylation of the tyrosine kinase JAK-2 and activation of STAT proteins were dependent on box 1 and sequences within 63 amino acids of the plasma membrane. STAT proteins activated by thrombopoietin in a megakaryocytic cell line were purified and shown to be STAT1 and STAT3. A separate region located at the C terminus of the c-Mpl intracellular domain was found to be required for induction of Shc phosphorylation and c-fos mRNA accumulation, suggesting involvement of the Ras signal transduction pathway. Thus, at least two distinct regions are involved in signal transduction by the c-Mpl.

Regulation of human growth hormone-binding protein production by human growth hormone in a hepatoma cell line

The mechanism by which growth hormone-binding protein (GH-BP) is generated in humans remains unclear. To address this question, we analysed human GH-receptor/GH-BP gene expression in a human hepatoma cell line (HuH7). Northern hybridisation showed that HuH7 cells contain a single mRNA species hybridising with a probe for the sequences encoding the extracellular domain of the hGH-receptor/GH-BP. These data were confirmed by solution hybridisation methods. Thereafter, the cells were treated with r-hGH at physiological (12.5, 25, 50 ng/ml) and supra-physiological (150, 500 ng/ml) concentrations over the period of 48 h. At intervals, RNase protection assays were performed to determine GH-receptor/GH-BP mRNA levels, nuclear run-on assays were carried out to determine whether changes in mRNA levels represented changes in transcription rate, and a radio-ligand binding assay was performed to measure levels of GH-BP in the medium. We found that the r-hGH-regulated changes in GH-receptor/GH-BP mRNA levels detected with the probe for sequences encoding the extracellular domain of human GH-receptor/GH-BP were identical to those previously detected using a probe for the sequences encoding the transmembrane/ intracellular domain of the human GH-receptor. In addition, we found that r-hGH had a rapid effect on the levels of GH-BP in the culture medium, which differed from its effect on the GH-receptor/GH-BP mRNA levels. Furthermore, lowering of temperature resulted in a decrease of GH-BP released into the medium implying that enzymes may be involved in the releasing mechanism. These data support the idea that GH-receptor and GH-BP are encoded by a single mRNA species in humans. In addition, they suggest that GH-BP levels are not an accurate reflection of GH-receptor/GH-BP mRNA levels, but that GH-BP production is subject to r-hGH-dependent post-transcriptional regulation, perhaps at the level of post-translational cleavage of the full-length GH-receptor protein. The notion that GH-BP measurements might represent GH-receptor status at the functional level must therefore be taken with caution.