The species specificity of growth hormone requires the cooperative interaction of two motifs

Primate growth hormones (GH) activate both primate and non-primate somatotrophic receptors (GH receptors), but non-primate GHs do not activate primate GH receptors. Previous studies argued the interaction of Asp(171) of human GH and Arg(43) of the receptor produced an attractive ionic interaction. In non-primate GHs, His(170) replaces the homologous Asp(171), producing a repulsive interaction with Arg(43) of the primate receptor which was believed to reduce the attraction of non-primate GH for the human GH receptor, thus providing species specificity. In this report, H170D bovine GH had activity and affinity for human GH receptors approaching those of human GH. In contrast, replacing Asp(171) of human GH with His did not significantly reduce somatotrophic activity, indicating that species specificity is not wholly explained by this residue's interaction with Arg(43) of the receptor. Deletion of either Phe(44) (a residue present only in primate GHs) or residues 32-46 (20-kDa form of human GH) each only marginally reduced somatotrophic activities. But the combination of the D171H mutation with either DeltaPhe(44) or Delta32-46 in human GH reduced binding and activity in a greater than additive fashion, indicated a functional interaction between these distant structural features. In bovine GH addition of phenylalanine at position 44 increased the somatotrophic activity and receptor affinity in cells containing the human GH receptor. The combination of the H170D mutation and the addition of phenylalanine at position 44 created a bovine GH with activity indistinguishable from wild-type human GH. Based on evidence from both bovine and human GHs, the cooperative interaction of these two distant motifs determined the species specificity and indicated that structural plasticity was a critical feature necessary for the species specificity of somatotrophic activity.     

Cell surface receptors for insulin and human growth hormone. Effect of microtubule and microfilament modifiers.

The receptors for the polypeptide hormones, insulin and growth hormone, are located on the cell surface. Since the cytoplasmic microtubules and microfilaments are involved in the mobility and distribution of surface receptors for immunoglobulins and lectins, we investigated the role of these structures in the binding of insulin and human growth hormone to their receptors on cultured human lymphocytes (IM-9). Cells preincubated with microfilament modifiers, cytochalasin A, B, and D (10 mug/ml), had decreased binding of insulin (30%) and human growth hormone (60%) under steady state conditions, which was not reversed by removing the cytochalasins from the medium and was due entirely to a reduced number of receptor sites on the cell surfact. The lost receptors were not detected in the medium, suggesting a redistribution within the cell. The cytochalasins failed to alter the affinity of the hormones for their receptors or the negative cooperativity of the insulin receptor. The anti-microtubule agents (vincristine, vinblastine, colchicine) had no effect on the binding of insulin and growth hormone to their receptors. Deuterium oxide, a stabilizer of microtubules and other proteins, decreased the affinity (40%) of insulin for its receptors under steady state conditions and accelerated moderately the spontaneous dissociation of 125I-insulin from its receptors. Since cytochalasin decreases the number of available insulin and human growth hormone receptor sites, cytochalasin-sensitive microfilamentous structures appear to modulate the exposure of cell surface hormone receptors, while microtubules do not seem to be involved.     

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.     

Use of calcium dependence as a means to study the interaction between growth hormones and their binding proteins in rabbit liver.

The affinity of 22,000-Mr human growth hormone (22 K-hGH) for GH binding proteins in rabbit liver is increased approx. 19-fold by 25 mM-Ca2+. In contrast, ovine growth hormone (oGH) binding is Ca2+-independent up to 10 mM, and decreased by greater Ca2+ concentrations. The 20,000-Mr hGH variant (20K-hGH), lacking residues 32-46, exhibits intermediate behaviour. Without Ca2+ there is a residual 40% of maximum specific binding to liver microsomes, and this increases to 65% with liver cytosolic GH binding proteins. In contrast with 22K-hGH, Scatchard analysis of 20K-hGH binding to liver microsomes produces curvilinear plots in the presence of 25 mM-Ca2+. From these results and inhibition studies with monoclonal antibodies to the GH binding proteins, it is concluded that deletion of the region 32-46 from 22K-hGH has eliminated one component of high-affinity Ca2+-potentiable binding. The Ca2+-mediated increase in Ka for the 22K-hGH-binding protein interaction is consistent with convergence of unit negative charges on the hormone and binding protein towards an intercalated Ca2+ ion. A positive charge in the critical region of nonprimate GHs would render their interactions Ca2+-independent and of lower Ka compared with 22K-hGH. A likely candidate for the negatively charged interactive residue is glutamate-33, since it is unique to human GH and is replaced by a positively charged arginine in non-primate GHs. Its absence in 20K-hGH could explain the altered calcium-dependence of 20K-hGH binding to what is probably the type 2 binding protein [Barnard & Waters (1986) Biochem. J. 237, 885-892]. The Ca2+-dependence of 20K-hGH binding to a subset of GH binding proteins provides both a verification and a mechanistic basis for the proposal [Hughes, Tokuhiro, Simpson & Friesen (1983) Endocrinology (Baltimore) 113, 1904-1906] that 20K-hGH binds with high affinity to only a subset of binding proteins in rabbit liver membranes.     

Conformational comparison in the growth hormone family

1. The method of Kubota et al. [Biochim. biophys. Acta 701, 242-252 (1982)] was applied to several members of the growth hormone family in order to examine their conformational homology. 2. The method neither detects differences between rat, cow, sheep, horse and alpaca hormones, nor between monkey and human hormones. 3. Lack of homology between primate and non-primate growth hormones was found in segments 42-49 and 184-191. The first fragment could be linked to species-specificity.     

Increased site 1 affinity improves biopotency of porcine growth hormone. Evidence against diffusion dependent receptor dimerization

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp104 and Trp169 in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys165), which in addition to His169, restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.     

Regulation of receptor concentration by homologous hormone. Effect of human growth hormone on its receptor in IM-9 lymphocytes.

When cultured human lymphocytes of the IM-9 line were exposed to human growth hormone (hGH) at 37 degrees, washed for 2 hours, and incubated with 125I-hGH, the binding of 125I-hGH was reduced. The magnitude of the reduction in binding was dependent on the concentration of growth hormone present as well as the duration of the exposure. As little as 2 X 10(-11) M (0.5 ng/ml) growth hormone had a discernible effect. Growth hormone at 2 X 10(-10) M (5.0 ng/ml), which is a low resting concentration of hormone in vivo and occupies about 20% of the receptors at steady state at 30 degrees, produced a 50% reduction in binding while 20 mg/ml, which occupies about 50% of the receptors under steady state conditions, produced an 80% loss of receptors. Further increases in growth hormone concentration produced little further effect on receptor loss. Thus, the loss of receptors at a given concentration of growth hormone (up to 20 ng/ml) in the preincubation at 37 degrees was greater than the occupancy produced by that concentration of growth hormone receptors under steady state conditions at 30 degrees. Analysis of the data indicated that the decrease in binding of 125I-hGH was due to a loss of receptors per cell without any change in affinity of receptor for hormone or in cell number. The concentration of insulin receptors on these cells was affected by the insulin concentration in the medium, and the concentration of growth hormone receptors was affected by growth hormone, but neither hormone had any effect on the heterologous receptors. Exposure of the cells to cycloheximide (0.1 mM) produced a progressive but smaller loss of growth hormone receptors, and the effect of cycloheximide was additive to the receptor loss induced by growth hormone, suggesting that cycloheximide inhibited synthesis of receptors while growth hormone accelerated loss of receptors. When growth hormone was removed from the medium, receptor concentrations were restored rapidly; half of the loss was restored by 6 to 8 hours and the full complement of receptors was restored by 24 hours following removal of the hormone. If the growth hormone was removed and replaced with cycloheximide, the return of the receptors was delayed until the cycloheximide was removed. Thus restoration of the receptors appeared to require the synthesis of new proteins. These data indicate that in the IM-9 lymphocytes the concentration of growth hormone receptors is very sensitive to regulation by growth hormone and also add further support to the suggestion that hormones in general actively regulate the concentration of their own receptors.     

Methionine oxidation in human growth hormone and human chorionic somatomammotropin. Effects on receptor binding and biological activities

The oxidation of the methionine residues of human growth hormone (hGH) and human chorionic somatomammotropin (hCS) to methionine sulfoxide by hydrogen peroxide has been studied. The kinetics of oxidation of individual methionine residues has been measured by reverse-phase high pressure liquid chromatography tryptic peptide mapping. Met-170 is completely resistant to oxidation in both hormones. The other 3 methionine residues in hCS (Met-64, Met-96, and Met-179) have markedly different reaction rates. Oxidation of the methionine residues does not appear to cause gross conformational changes in either hGH or hCS, as judged by CD and 1H NMR spectroscopy. Oxidation of Met-14 and Met-125 in hGH has little effect on affinity of the hormone for lactogenic receptors or on its potency in the Nb2 rat lymphoma in vitro bioassay for lactogenic hormones. The oxidation of Met-64 and/or Met-179 in hCS reduces profoundly both its affinity for lactogenic receptors and its in vitro biological potency. It is inferred by induction that residues 64 and/or 179 are critical for the binding of both hGH and hCS to lactogenic receptors and the expression of lactogenic biological activity.     

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.     

Immunochemical similarity of the human plasma growth hormone-binding protein and the rabbit liver growth hormone receptor

We probed the (immunochemical) relationship between the recently discovered growth hormone binding protein in human plasma and the growth hormone receptor using monoclonal and polyclonal antibodies raised against rabbit liver growth hormone receptor. The human binding protein was recognized by these antibodies; its immunological crossreactivity compared to the rabbit receptor was 1-2%. These data suggest a) that the binding protein and the receptor are structurally related and b) that rabbit and human growth hormone receptors share some but not all epitopes.     

Determination of the effect of acetylation of specific lysine residues in human growth hormone on its affinity for somatogenic receptors by an affinity selection technique

A technique is described to study the effect of acetylation of individual lysine residues in peptide hormones on the affinity for their receptors, and is illustrated for the case of human growth hormone (hGH) binding to somatogenic receptors. The hGH was partially acetylated with high specific activity [3H]-acetic anhydride and the product ([3H]-Ac-hGH) was incubated with solubilised affinity-purified somatogenic receptors (from male rat liver) in the presence and absence of excess unlabelled hGH. The receptor-bound and unbound labelled hormone were separated by gel filtration and subjected to HPLC tryptic peptide mapping after the addition of cold carrier Ac-hGH. Peaks of [3H] radioactivity were assigned to peptides corresponding to the acetylation of specific lysine residues in the hGH sequence by amino acid analysis and sequencing. Comparison of the relative intensities of corresponding [3H] peaks in the peptide maps of added receptor, bound and unbound [3H]-Ac-hGH, enabled the relative receptor-binding potencies of different acetylated hGH species to be determined. Acetylation of lysine 168 or 172 in hGH greatly decreases its receptor-binding affinity, acetylation of lysine 115 probably causes a minor decrease, whereas acetylation of lysines 38, 70, and the N-terminal amino group have no appreciable effect. Acetylation of lysine 140 causes a significant increase in receptor-binding affinity.     

Receptor binding properties of four-helix-bundle growth factors deduced from electrostatic analysis

Hormones of the hematopoietin class mediate signal transduction by binding to specific transmembrane receptors. Structural data show that the human growth hormone (hGH) forms a complex with a homodimeric receptor and that hGH is a member of a class of hematopoietins possessing an antiparallel 4-α-helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormone-receptor recognition. In order to examine the specificity of hormone-receptor complexation, an analysis was made of the electrostatic potentials of hGH, interleukin-2 (IL-2), interleukin-4 (IL-4), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the hGH and IL-4 receptors. The binding surfaces of hGH and its receptor, and of IL-4 and its receptor, show complementary electrostatic potentials. The potentials of the hGH and its receptor display approximately 2-fold rotational symmetry because the receptor subunits are identical. In contrast, the potentials of GM-CSF and IL-2 lack such symmetry, consistent with their known high affinity for hetero-oligomeric receptors. Analysis of the electrostatic potentials supports a recently proposed hetero-oligomeric model for a high-affinity IL-4 receptor and suggests a possible new receptor binding mode for G-CSF; it also provides valuable information for guiding structural and mutagenesis studies of signal-transducing proteins and their receptors.     

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).     

Growth hormone receptors in lymphocytes of growth hormone-deficient children

Human growth hormone was injected intravenously into 18 growth hormone-deficient children and growth hormone binding sites in lymphocytes were investigated. Fresh circulating lymphocytes had a low initial value for the binding of growth hormone to solubilized receptors (3.45 +/- 1.46%) but after growth hormone injection, the binding rapidly increased to 14.8 +/- 4.2% at 2 1/2 h and 8.7 +/- 1.8% at 5 h. The sharp increase in binding is due to increase in the number of binding sites. Two control children who received chorionic gonadotropin had no change in lymphocyte growth hormone receptors. The methodological differences between the present study and previous attempts to identify human growth receptors in lymphocytes were (1) lymphocytes were separated and disrupted with Triton X-100 as quickly as possible (to avoid error from receptor leaking out of the cell) and (2) the receptors were assayed at 2 1/2 h after growth hormone administration (previous studies were 12-24 h later). One possible explanation for the data is that growth hormone receptor from liver is taken up by lymphocytes and rapidly released again, thus, contributing to the hormonal receptor economy in humans.     

Receptor-binding and down-regulatory properties of 22000-Mr human growth hormone and its natural 20000-Mr variant on IM-9 human lymphocytes.

Our earlier binding studies of the 22000- and 20000-Mr variants of human growth hormone (somatotropin) to pregnant-rabbit liver and mammary receptors [Closset, Smal, Gomez & Hennen (1983) Biochem. J. 214, 885-892] suggested that the 20000-Mr variant was a lower-affinity analogue of the 22000-Mr molecule. Since the receptor population in these tissues is not fully characterized, we have now investigated the binding of both variants to the well-characterized and highly specific human-growth-hormone receptor of the human lymphocyte IM-9 cell line. The maximum bindability of radioiodinated 22000- and 22000-Mr to IM-9 cells was 60 and 45% respectively. Both hormone variants have essentially the same binding characteristics: slow association (equilibrium reached in 8-10h at 30 degrees C), poor reversibility ('tight binding'), linear Scatchard plot, same specificity as shown by lack of competition by bovine, porcine or equine growth hormones or human growth hormone-(32-46)-(missing in the 20000-Mr variant),-(1-134)- and -(141-191)-peptides. Both unlabelled hormones inhibit binding of both tracers completely, with the 20000-Mr variant being only half as potent as the 22000-Mr one. The apparent affinity is 2.8 X 10(9)M-1 for the 22000-Mr variant and 1.6 X 10(9)M-1 for the 20000-Mr variant. This decreased affinity of the 20000-Mr variant appears to be due to a lower association rate constant. Concentrations (5 ng/ml) of the two variants that occupy about 15% of the total sites induce a marked down-regulation of the receptors after 18h incubation, but the 20000-Mr variant (50% decrease) has a smaller effect than the 22000-Mr variant (75% decrease). Thus the only consequence of the residues-32-46 deletion in the 20000-Mr variant is a lower association rate and affinity for the IM-9 lymphocyte human-growth-hormone receptor. The close binding characteristics of the two forms suggest that the known differences in their insulin-like effects cannot be explained by differences in the nature of their interaction with the human-growth-hormone receptor.     

Alpha and beta thyroid hormone receptors bind immediately adjacent to the rat growth hormone gene TATA box in a negatively hormone-responsive promoter region

We report that alpha and beta type rat thyroid hormone receptors bind specifically and with high affinity to the 10-base pair sequence immediately 3' of the rat growth hormone TATA box (positions -25 to -16) in a region of the rat growth hormone promoter which can be negatively hormone responsive (nTRE). The receptors have approximately 7-fold lower affinity in vitro for the nTRE than for the thyroid hormone-responsive enhancer of the rat growth hormone gene (TRE). Proteins extracted with high salt concentration from rat pituitary cell nuclei enhance binding of the receptors to both the TRE and nTRE. A modification of the avidin-biotin complex DNA binding assay which enhances the sensitivity of the assay approximately 100-fold was used in these studies. The immediate proximity of a receptor binding site to the rat growth hormone TATA box suggests that direct interaction between receptor and TFIID (the TATA binding protein) mediates nTRE activity.     

Ovine prolactin and human growth hormone derivatives. Specific modification of their alpha-amino groups

The alpha-amino group of ovine prolactin (oPRL) and human growth hormone (hGH) was selectively modified by transamination with glyoxylic acid. No difference was found in the binding capacity of transaminated oPRL to rat liver lactogenic receptors with respect to its control, although both samples showed a decrease in its binding capacity with reference to the native hormone. This decrease was due to conformational changes caused by the reaction conditions and not by the transamination itself, as shown by the circular dichroism spectra. Transaminated hGH retained the full binding capacity of the hormone. These results suggest that the alpha-amino group is not relevant for the binding to lactogenic liver receptors in both lactogenic hormones.     

Comparative study of biosynthetic human growth hormone immunogenicity in growth hormone deficient children

The immunogenicities of six recombinant human growth hormone (rhGH) preparations, from KABI (A rhGH191 and B rhGH192), Eli Lilly (C), Nordisk (D), Sanofi (E) and Serono (F), used to treat 260 GH-deficient children, have been compared using a common specific and sensitive procedure for antibody determination. For this purpose we developed two immunoassays: a competitive liquid radioimmunoassay using 125I-rhGH, and an immunometric solid enzymoimmunoassay in which the rhGHs were immobilized. Blood samples were collected from the GH-deficient children before treatment and after 3, 6, 9, 12, 18 and 24 months of therapy. Human GH antibodies were detected in children treated with 3 of the 6 rhGH preparations. Seven percent of the patients treated with hormone A, 14% with hormone B and 22% with hormone C formed antibodies against the respective rhGH. Differences in capacity and affinity of the hGH antibodies were observed between these anti-GH-positive groups. They could be divided into 2 groups according to their immunopotency. One group (7, 14 and 6% of the patients treated with hormones A, B and C, respectively) developed anti-hGH antibodies with very low binding capacities (30-100 fmol/ml). The other group (16% of the patients treated with hormone C) developed IgG-type antibodies to hGH with higher binding capacities (200-1,200 fmol/ml) and a measurable binding affinity (Ka = 10(8) M-1). These hGH antibodies partially inhibited the binding of labeled GH to its specific liver membrane receptor. However, because of their low titer, they did not inhibit growth in the treated children.(ABSTRACT TRUNCATED AT 250 WORDS)