Binding protein for human growth hormone: effects of age and weight.

In human serum, a specific binding protein with high affinity for human growth hormone (GHBP) is found which is identical to the extracellular portion of the hepatic GH receptor. GHBP is assessed by incubating serum samples with [125I]-GH, followed by separation of bound and free radioactivity using gel chromatography. In newborns and children younger than 2 months, GHBP was practically absent and no 'big-big' GH could be found. GHBP values increased rapidly during the first 2 years of life, followed by a slower increase during childhood and puberty. No difference was found between male and female subjects. Apart from age, standardized weight (SDS = z score) had a major positive effect on GHBP concentration. Interestingly, SDS height correlated negatively with GHBP when weight and age were controlled for. These data may relate to two clinical findings: (1) the developmental switch between GH-independent intrauterine and GH-dependent postnatal growth mechanisms, and (2) the accelerated growth velocity encountered in adipose children.     

Immunological identity of human and porcine growth hormones. Application to determination of growth hormone in porcine serum.

In two radioimmunoassay systems with iodinated human growth hormone as tracer and anti-human growth hormone or anti-porcine growth hormone as binding site, the standard curves for human and for porcine growth hormone were parallel. In both systems the porcine growth hormone preparation had to be added in about the same excess as compared to the human hormone. It was concluded that the human and the porcine hormones behave in an identical way and that the values obtained in radioimmunoassay for human growth hormone represent the amount of immunologically active molecules in the porcine growth hormone preparation. As parallel standard curves were obtained with porcine serum, the concentration of growth hormone is porcine serum may be determined by radioimmunoassay for human growth hormone.     

High-affinity serum growth-hormone-binding protein, absent in Laron-type dwarfism, is diminished in heterozygous parents

Human serum high-affinity growth-hormone-binding protein (GHBP), as determined by incubation with 125I-GH followed by chromatography on AcA 44 gel minicolumns, is lacking in patients with Laron-type dwarfism (LTD). We found that the specific binding of 125I-GH to high-affinity GHBP in normal human serum (m +/- SD) was 11.5 +/- 1.8% in 10 children 2-3 years old, 15.3 +/- 2.2% in 10 children 5-8 years old, and 19.3 +/- 2.9% in 15 adults 20-40 years old. It was 0.3% in a 2-year-old child with LTD, and 10.6 +/- 11.3% in his parents. It was 0.1% in another child with LTD, 7 years old, and 14.4 and 14.8% in his parents. The mean value in the heterozygous parents (12.8 +/- 2.1%) was significantly lower (p less than 0.001) than control values. A void volume peak (VVP) of radioactivity, corresponding to the so-called low-affinity GHBP which eluted at the void volume in chromatographs of normal sera remained unchanged with sera of patients with LTD or of their parents and appeared even after incubations of the tracer without serum. This study (1) shows that high-affinity GHBP is diminished in heterozygotes with LTD; (2) confirms that high-affinity GHBP and VVP are independently regulated, and (3) suggests that a part of the VVP may not be related to GH binding to some serum components.     

The quantitation of human growth hormone by a radioreceptor assay using an established human cell line

Membrane receptors on cultured human lymphocytes (IM-9) have been shown to bind human growth hormone (hGH) in a specific manner. The aim of the present study was to develop an in vitro assay of hGH based on this binding. The assay should fulfil established pharmacopoeial requirements for quantitation of hormones. The binding of [125I]hGH was studied as a function of time, temperature, cell density, tracer concentration and the concentration of unlabelled hGH and other related hormones. Also, the dissociation of bound hGH and the chemical stability of hGH in the incubation medium were studied. From these studies, the conditions for an appropriate radioreceptor assay were determined. Briefly, 1.5-3.0 X 10(7) cells ml-1 were incubated with 5-20 X 10(-12) M [125I]hGH and three different concentrations of unlabelled hGH chosen from the linear part of the [125I]hGH displacement curve. The results were analyzed according to general pharmacopoeial principles. The mean values for growth hormone activity tested by radioreceptor assay were within the fiducial limits (P = 0.05) of the corresponding activity determined by the hypophysectomized rat body-weight gain assay. The in vitro assay was found to be more precise and less resource demanding than the in vivo bioassay of hGH. It is concluded that the in vitro bioassay described here is well suited as a screening method for potency determination of hGH preparations.     

Identification of a region critical for proteolysis of the human growth hormone receptor.

Release of soluble growth hormone binding protein (GHBP) corresponding to the extracellular domain of the GH receptor (GHR) occurs via distinct mechanisms depending on species. In human, proteolysis of full length GHR results in liberation of GHBP into the extracellular medium. A putative protease responsive for GHR cleavage has been identified, however, the residues involved are still unknown. In this study, using the mutational approach to the extracellular domain of the human GHR, we demonstrated that deletion of three residues located close to the transmembrane domain abolishes constitutive GHBP shedding without change in cellular GH binding. Deletion also significantly decreased the phorbol 12-myristate 13-acetate (PMA)-induced release of GHBP and the accumulation of membrane-anchored remnant proteins. Taken together, these results suggest that integrity of the juxtamembrane region of GHR is necessary for its biochemical cleavage and that a common mechanism is involved in constitutive and PMA-induced shedding.     

Characterization of the binding of human growth hormone to microsomal membranes from rat liver.

The binding of 125I-labelled human growth hormone to the 100000g microsomal membrane fraction prepared from the livers of normal female rats was dependent on time, temperature, pH, membrane concentration and concentration of 125I-labelled human growth hormone. At 22 degrees C binding reached a steady state after 16h, with the mean maximal specific binding being 20% of the tracer initially added. Dissociation of 125I-labelled human growth hormone from the membranes, after addition of excess of unlabelled hormone, was relatively slow with a half-time greater than 24h. Only minor degradation of the 125I-labelled human growth hormone was observed during incubation with membranes for 16 or 25h at 22 degrees C. Similarly, no significant change in the ability of membranes to bind human growth hormone was evident after preincubation of the membranes for 16 or 25h. Specificity studies showed that up to 90% of the 125I-labelled human growth hormone bound could be displaced by 1 mug of unlabelled hormone. Ovine prolactin also showed considerable competition for the binding site. Non-primate growth-hormone preparations (ovine, bovine, porcine and rat) and non-related hormones (insulin, thyrotropin, lutropin and follitropin) all showed negligible competition. Scatchard analysis of the binding data was consistent with two classes of binding site with binding affinities of 0.64 X 10(10) +/- 0.2 X 10(10)M-1 and 0.03 X 10(10) +/- 0.007 X 10(10)M-1 and corresponding binding capacities of 98.4 +/- 10 fmol/mg of protein and 314.6 +/- 46.3 fmol/mg of protein. These studies provide data which, in general, are consistent with the criteria required for hormone-receptor interaction. However, proof of the thesis that the human-growth-hormone-binding sites in female rat liver represent physiological receptors must await the demonstration of a correlation between hormone binding and a biological response.     

The effect of growth hormone on the proliferation of human Th cell clones

The effects of human growth hormone (hGH) on human Th cell clones were examined. Both 20K and 22K hGH stimulated the proliferation of Th2 and Th0 cells in the presence of mite antigen, whereas they did not stimulate the proliferation of Thl cells. Because the effect of 20K hGH was almost the same as that of 22KhGH, it was suggested that the action of hGH was not mediated through prolactin receptor but through hGH receptors. The application of growth hormone binding protein (GHBP) inhibited the cell growth of Th1 clones. In Th2 and Th0 cells GHBP inhibited the hGH-stimulated cell proliferation. However, GHBP alone did not affect the proliferation of Th2 and Th0 cells. hGH was detected in the supernatant of Th1 clones in the presence of mite antigen but it was not detected in Th2 clones. hGH was detected in one out of 4 batches of Th0 clones. These data indicated that hGH was secreted from Thl clones, and that Th0 clones possessed characteristics of both Th2 and Th0 clones.     

Serum leptin, insulin-like growth factor-I components and sex-hormone binding globulin. Relationship with sex, age and body composition in healthy population

Leptin plays an important role in the regulation of food intake and thermogenesis, regulates long term energy balance and reproductive function and its concentrations are closely linked to body mass index. Leptin secretion is influenced by many factors and the age-related changes in different hormones might modify circulating leptin concentrations. Sex dimorphism in leptin concentrations has been clearly shown in previous studies and its concentrations were lower in men than in women in all decades of life. Insulin growth factor-I (IGF-I) is a peptide growth factor that is present in all types of physiologic fluids and is also produced by connective tissue cell types and its autocrine/paracrine secretion is nearly always present within tissues. There is a physiological decline of the growth hormone (GH)/IGF-I axis with ageing and in addition, insulin, thyroid hormones and the supply of dietary energy may directly regulate the circulating levels of the IGFs and growth hormone binding protein (GHBP). Furthermore, there is no doubt that GH participates in the regulation of body composition, and with advanced age there is a decrease in muscle and an increase in adiposity associated with a decline in GH and total IGF-I. The biological activities of the IGF ligands are modulated by the family of high affinity GHBP. Sex hormone binding globulin (SHBG) concentrations are thought to be regulated primarily through opposing actions of sex steroids on hepatic SHBG production, with oestrogen stimulating and androgen inhibiting SHBG production, and thyroid hormones are also a potent stimulator of SHBG production concentrations. Some studies support an independent IGFBP3 contribution to SHBG variability and these findings are compatible with the hypothesis that some of the anabolic effects ascribed to the GH/IGF axis may be caused by SHBG-mediated changes in testosterone activity or SHBG/total testosterone index.     

Bushbaby growth hormone is much more similar to nonprimate growth hormones than to rhesus monkey and human growth hormones

Unlike other mammals, Old World primates have five growth hormone-like genes that are highly divergent at the amino acid level from the single growth hormone genes found in nonprimates. Additionally, there is a change in the interaction of growth hormone with its receptor in humans such that human growth hormone functions in nonprimates, whereas nonprimate growth hormone is ineffective in humans. A Southern blotting analysis of the genome of a prosimian, Galago senegalensis, revealed a single growth hormone locus. This single gene was PCR-amplified from genomic DNA and sequenced. It has a rate of nonsynonymous nucleotide substitution less than one fourth that of the human growth hormone gene, while the rates of synonymous substitution in the two species are less different. Human and rhesus monkey growth hormones exhibit variation at a number of amino acid residues that can affect receptor binding. The galago growth hormone is conservative at each of these sites, indicating that this growth hormone is functionally like nonprimate growth hormones. These observations indicate that the amplification and rapid divergence of primate growth hormones occurred after the separation of the higher primate lineage from the galago lineage.     

The study of the metal-binding region in human growth hormone using immobilized metal ion affinity gel-electrophoresis

The zinc(II)-binding affinities of recombinant human growth hormone and two its mutants, 14-33 and 14-95, were studied using Immobilized Metal Ion Affinity Gel-electrophoresis (IMAG). The mutant hormones, composed of polypeptide chain segments of the human and porcine growth hormones, lacked His18, which may be crucial for binding of the intact hormone to the transition metal ions. The mutations did not affect the affinity of human growth hormone to immobilized zinc ions; the structural analysis implied that the human growth hormone contains two IDA-Zn(II) potential sorption sites formed by amino acid residues His21, Asp171, and Glu174 and/or His18 and Glu174.     

Immunological relatedness of human and porcine growth hormones.

The immunological relatedness of human and porcine growth hormones is examined by means of labelled human growth hormone and guinea pig antiserum. 1) Labelled human growth hormone is found in the precipitate after reaction with antiserum against porcine growth hormone. Parallel dilution curves are obtained with antisera against human and porcine growth hormones. 2) After addition of antiserum against porcine growth hormone, all the radioactivity is eluted from Sephadex G-100 with the void volume. 3) The addition of an excess of porcine hormone displaces labelled human growth hormone from antibodies against human growth hormone to the same extent as an excess of non-labelled human growth hormone does. 4) The standard radioimmunoprecipitation curves for porcine and human growth hormones obtained in the assay system for the human hormone are parallel in slope, provided that the human hormone and our preparation of the porcine hormone are introduced at a proportion of 1 to 560. 5) In a double diffusion test in agarose gel layers, with human and porcine growth hormones diffusing against guinea pig anti-porcine serum, cross reaction is observed. The conclusion is drawn that with guinea pig antisera, human and porcine growth hormones behave immunologically in a similar fashion. Labelled human growth hormone seems to have only such immunodeterminants as are also found in porcine growth hormone.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Single-step purification of recombinant human growth hormone (hGH) directly from bacterial osmotic shock fluids, for the purpose of (125)I-hGH preparation

A good quality tracer, to be used in the radioimmunoassay of human growth hormone, was prepared by applying the chloramine-T iodination technique to the recombinant product obtained after a single-step high-performance size-exclusion chromatography purification of a bacterial osmotic shock fluid. The labeling reaction presented a yield of about 65% and the purified tracer exhibited an antibody binding of approximately 50% (NIDDK reference antiserum diluted 1:600,000). These values are very similar to those obtained by radioiodinating highly purified clinical-grade recombinant human growth hormone obtained from the same periplasmic extract after the regular six-step purification process. Both tracers provided the same accuracy, when evaluated with the use of commercial-quality control samples in a classical radioimmunoassay methodology, their stability being practically identical: about 18% decrease in antibody binding after 2 months of storage at -20 degrees C. The novel approach permits the utilization of transformed Escherichia coli strains as a source of freshly prepared, radioiodination-grade recombinant proteins, capable of providing better reproducibility and reagent continuity.     

Molecular feature of the nerve growth factor in human serum

High molecular weight binding components which bind [¹²⁵I] mouse β nerve growth factor exist in human serum. The binding of β nerve growth factor to the serum components was inhibited at alkaline condition. After gel filtration of human serum on a Sephadex G-150 column at neutral condition, the nerve growth factor-like immunoreactivity was observed in only one peak, differing from the high molecular weight serum components. However, at alkaline condition two peaks with nerve growth factor-like immunoreactivity appeared; one was almost at the position observed at neutral pH, and the other was a new peak eluted approximately to the column volume. these results suggest that there are at least two nerve growth factor-like molecules in human serum and most of the nerve growth factor in the serum exists in a complex form associated with serum components with high molecular weight.     

Hormones and body size evolution in papionin primates

This study examines the evolution of size differences among papionin primates by measuring hormones that regulate size growth during ontogeny and influence ultimate adult size (insulin‐like growth factor‐I (IGF‐I), insulin‐like growth factor binding protein‐3 (IGFBP‐3), growth hormone binding protein (GHBP), dehydroepiandrosterone sulfate (DHEAS), testosterone, estradiol). The analyses assess longstanding ideas about circulating hormone levels and body size. Importantly, because the consensus papionin molecular phylogeny implies at least two episodes of size increase, this study offers opportunities to determine whether or not similar hormone profiles regulate this apparent evolutionary convergence (i.e., do larger‐bodied papionins have higher levels of growth‐related hormones than smaller‐bodied papionins?). Five hundred and sixty serum samples (from 161 individuals) from 11 papionin species were analyzed using a two‐level approach to address this issue. One used mixed longitudinal samples from two papionin species to test whether, during growth, large‐ and small‐bodied species have higher and lower hormone levels, respectively. The second compared multiple papionin species to assess whether or not hormone levels covary with size in adult animals. Result show that size and hormone levels do not covary consistently across papionins, either during growth or in adulthood. Specifically, some smaller‐bodied papionin species have higher absolute hormone levels than larger‐bodied species. Differences in some hormone levels appear to track phylogeny more closely than body size. In contrast to studies based on single species, we demonstrate that, while the hormones analyzed affect growth, absolute circulating hormone levels either during growth or adulthood may be decoupled from interspecific differences in body size. Am J Phys Anthropol, 2007. © 2006 Wiley‐Liss, Inc.     

Identification and Characterization of GH Receptor and Serum GH-binding Protein in Chinese Sturgeon （Acipenser sinensis）

Growth hormone (GH) can stimulate bone and carti-lage cell proliferation and influence carbohydrate and lipidmetabolism. The binding of GH to its specific receptor(GHR) on the surface of target cells will induce dimeriza-tion of GHR, which allows the cytoplasmic region of GHRto interact and trigger downstream signaling and geneexpression [1,2]. GHR belongs to the cytokine receptor superfamily, andis expressed in many tissues such as the liver, muscle,adipose tissue, cartilage, and brain…     

Regulation of hepatic growth hormone receptors by insulin.

Induction of diabetes in the rat with streptozotocin caused a decrease in the specific binding of human growth hormone to liver receptors. The decrease was due to a loss of binding sites, with no change in the affinity constant for growth hormone (5.6 × 10⁹M^?1). A highly significant correlation was seen between serum insulin levels and hepatic growth hormone binding. Specific insulin binding to hepatic receptors was increased in diabetes, with a highly significant negative correlation between serum insulin levels and insulin binding. The loss of growth hormone receptors was reversed by treating diabetic rats with insulin. Since hormones which bind to “lactogenic” binding sites in the liver are reported to regulate somatomedin levels, the insulin dependence of human growth hormone receptors might account for the decrease in serum somatomedin in diabetes.     

Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

The glycoprotein hormones, luteinizing hormone (LH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH), play important roles in overall physiology and reproduction. These hormones are heterodimeric molecules consisting of an identical alpha subunit non-covalently associated with the hormone-specific beta subunit. The inherent structural intricacies possessed by these hormones make them very interesting model systems for structure-function relationship studies of complex dimeric glycoproteins. The structural studies, as well as, the therapeutic applications require large quantities of biologically active hormones free of any contaminants. In this study, we report hyperexpression and purification of biologically active recombinant hLH and hCG expressed using Pichia pastoris expression system. A combination of hydrophobic interaction chromatography and ion exchange chromatography has been used to purify these recombinant hormones to homogeneity. Using a number of biochemical and immunological criteria, the recombinant hormones have been shown to be similar to the natural hormones and were equally biologically active. The preliminary data also suggested that P. pastoris cells express a low molecular weight isoform of hCG that appeared to be less glycosylated. This isoform exhibited lesser affinity for the receptor as compared to hCG, but was found to be fully biologically active.     

Plerocercoid growth factor: a homologue of human growth hormone

Plerocercoids of the tapeworm, Spirometra mansonoides, produce a factor with characteristics similar to those of mammalian growth hormone (GH). Plerocercoid growth factor (PGF) stimulates growth and mimics other actions of GH but does not possess the anti-insulin/diabetogenic activities intrinsic to mammalian growth hormones. Duplication of activities unique to human GH, chemical and physical similarities, plus crossreactivity with strictly specific anti-hGH monoclonal antibodies, underlie the hypothesis that S. mansonoides has obtained and expresses a human gene for GH. In this article, Kirk Phares discusses the similarities between the two hormones.     

An electrostatic model for the interaction between growth hormone and its receptor involving chelation of Ca2+ to the human growth hormone molecule

We previously postulated the local involvement of cations in the complex between human growth hormone and its receptors in the liver. The original electrostatic model involved convergence of unit negative charges on the hormone and receptor, towards an interposed Ca2+ ion. That model was consistent with (a) the Ca2+ dependence of human growth hormone binding, (b) the magnitude of the Ca2+ mediated increase in the affinity of human growth hormone binding, and (c) could also explain the relative affinities of human and non-primate growth hormones for growth hormone receptors. In the present report, the original electrostatic model is revised with the postulate that Ca2+ is chelated to the human growth hormone molecule. The consequences of this postulate are explored mathematically with the result that it becomes necessary to propose an additional unique hindrance determinant (positive residues in helices one and four are good candidates) to account for the lower affinity of non-primate growth hormones relative to human growth hormone. Predictions are made regarding the effect of a particular point mutation (at position 34) on the affinity of hormone binding.