hGH Binding Research Articles

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

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

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.

Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells.

GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP-2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH receptor is, except for IGFBP-3, at least in part on the mRNA level. Lastly, IGFBP-2 and IGFBP-3 are mitogenic for UMR-106.01 rat osteosarcoma cells, inducing an increase in cell number to 125% and 142% of control cell counts after 48 h of incubation with 1000 ng/ml IGFBP-2 and -3, whereas IGF-I, IGF-II and Long R3 IGF-I did not stimulate proliferation. IGFBP-2 and -3 potentiate hGH induced mitogenesis at low hGH concentrations of both factors, whereas at higher concentrations no such effect is observed.(ABSTRACT TRUNCATED AT 400 WORDS)

A single arginine residue determines species specificity of the human growth hormone receptor.

Although growth hormone (GH) receptors (GHRs) in many species bind human (h) GH as well as their own GH, the hGHR only binds primate GH. Arg43 in hGHR interacts with Asp171 of hGH. Nonprimates have a His in the position equivalent to residue 171 of primate GH and a Leu in position 43 of primate GHR. To determine whether Arg43 accounts for the species specificity of the hGHR, point mutations that changed Leu43 to Arg were introduced into the cDNAs encoding the bovine (b) GHR or the rat GH binding protein (GHBP) and these mutants or their wild-type (WT) counterparts were expressed in mouse L cells. Binding of hGH or bGH to transfected cells or to GHBP secreted into the incubation medium was assessed by displacement of 125I-labeled hGH. WT and mutant bGHR bound hGH with similar affinity, but the affinity of the mutant receptors for bGH was reduced 200-fold. Likewise, WT and mutant GHBP bound hGH with equal affinity, but only WT GHBP bound bGH. Cross-linking of 125I-labeled hGH to WT or mutant GHR produced a 141-kDa labeled complex whose appearance was blocked by unlabeled hGH, but bGH blocked cross-linking only to WT receptors. Both hGH and bGH stimulated tyrosine phosphorylation of a 95-kDa protein in cells transfected with WT GHR, but bGH was less effective in cells expressing mutant GHR. We conclude that incompatibility of Arg43 in the hGHR with His171 in nonprimate GH is the major determinant of species specificity.

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 β-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 K a of 6.2 × 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.

Evidence for Involvement of the Carboxy Terminus of Helix 1 of Growth Hormone in Receptor Binding: Use of Charge Reversal Mutagenesis to Account for Calcium Dependence of Binding and for Design of Higher Affinity Analogs

In this study we have demonstrated that the C-terminus of helix 1 of porcine GH (pGH) is a receptor-interactive region, thus extending the current binding site model of GH. This was achieved by introducing charge reversal mutations into this region of pGH, which influenced receptor affinity and Ca2+ dependence of binding. The first mutant (R34E pGH, conversion of Arg 34 to Glu) introduced a putative Ca2+ binding site which is present in human GH (hGH) [Barnard et al. (1989) J. Theor. Biol. 140, 355-367] and sits opposite E220 of receptor subunit 1. This mutant exhibited increased Ca2+ dependence of receptor binding but even at optimal Ca2+ did not display higher than wild-type affinity. Introduction of a second Ca2+ binding site adjacent to the first by a second charge reversal (K30E R34E pGH) further increased Ca2+ dependence of binding and also increased affinity for the rabbit GH receptor (2.4 +/- 0.4)-fold relative to wild-type pGH at optimal Ca2+. Equilibrium dialysis and Scatchard analysis of binding of 45Ca2+ to pGH and K30E R34E pGH revealed two Ca2+ binding sites on wild-type pGH and an additional two Ca2+ binding sites on the K30E R34E pGH mutant (Kd 0.5-0.8 mM), as predicted. A third partial charge reversal mutant in the fourth helix (H170D) also led to enhanced Ca2+ dependence of binding, supporting our proposal that E34 and D170 are responsible for the Ca2+ dependence of hGH binding to the rabbit GH receptor. Examination of the crystal structure shows that E34 and D170 are in close proximity and would interact repulsively with a cluster of acidic residues on the receptor consisting of E126, E127, and E220 unless neutralized by Ca2+ or an introduced basic residue. Accordingly, charge reversal at the adjacent pGH residue E33 (E33K pGH) led to a Ca2+ independent (3.0 +/- 0.4)-fold increase in affinity of binding. As well as extending the binding site model of GH, these studies provide a mechanistic explanation for the unique Ca2+ dependence of hGH binding to the rabbit GH receptor. They also indicate that charge reversal can be used to design higher affinity GH analogues and could assist in the mapping of interactive regions in ligand-receptor complexes generally.

Engineering an interfacial zinc site to increase hormone-receptor affinity

Background: Human growth hormone (hGH) binds to both the hGH and human prolactin (hPRL) receptors. Binding to the hPRL receptor, however, is ∼ 50-fold tighter and requires a single Zn 2+ cation, unlike binding of hGH to the hGH receptor. Previous mutational studies have identified putative ligands from hGH and the hPRL receptor responsible for coordinating the interfacial Zn 2+. Results: One of these ligands was introduced at a structurally analogous site in the extracellular domain of the hGH receptor by mutating Asn218 to His, and the resulting mutant protein showed a 20-fold increase in hGH binding in the presence of ZnCl 2. Alanine-scanning mutagenesis showed that the binding site on hGH for the Asn218→His hGH receptor in the presence of Zn 2+ resembled that for the hPRL receptor. Conclusions: It is possible to introduce the metal-binding site from the hPRL receptor into the homologous hGH receptor. More generally, these studies indicate that affinity between two proteins may be enhanced by design of an interfacial metal-binding site.

Dexamethasone increases and serum decreases growth hormone receptor binding to UMR-106.01 rat osteosarcoma cells.

Dexamethasone (DEX) is known to exert major effects on functions of osteoblast-like cells. We investigated its action on the regulation of GH receptors in the osteoblast-like osteosarcoma cells UMR-106.01. DEX stimulated [125I]human GH (hGH) binding to UMR-106.01 cells. This effect was dose dependent and significant in a concentration range of 10(-8)-10(-6) M. The maximum effect was an increase of 42 +/- 1.4% (n = 3; mean +/- SE) above control, P < 0.01, at 10(-7) M DEX. Time dependence of this stimulation was observed, with a peak between the 12th and the 16th h of incubation, an effect being still detectable at 48 h. Cycloheximide decreased [125I]hGH binding and completely abolished the stimulating effect of DEX, suggesting that modulation of [125I]hGH binding by DEX is fully dependent on protein synthesis. Addition of fetal calf serum (FCS) resulted in a dose-dependent decrease of [125I]hGH binding to 24 +/- 2% of control (n = 3; mean +/- SE), P < 0.001, without interfering with the stimulatory effect of DEX, the ratio of DEX vs. control being higher with increasing FCS doses. Taken together, these results suggest the existence of different pathways for the regulation of GH receptor binding to UMR-106.01 cells, including a stimulatory one at the pretranslational level for DEX and an inhibitory one for (growth) factors present in FCS.

Identification of two specific prolactin binding sites on Nb2 rat lymphoma cells.

Several laboratories have earlier identified and characterized a specific prolactin binding site (receptor) on Nb2 cells which has a Kd of about 2 x 10(-10) M and a binding capacity of about 12,000 receptor sites per cell. Using [I125]-labeled human growth hormone (hGH), we have confirmed the results of these earlier reports. In addition, a second, "high-affinity" receptor site for prolactin has been identified. This site has a Kd of about 2 x 10(-12) M and there are an average of 67 sites per cell. Binding of [125]-hGH to the high-affinity receptor site is saturated by 1 min after incubation at 37 degrees C, whereas saturation of the lower affinity site requires a 1-3 hr incubation at 37 degrees C. Specificity of prolactin binding to both the high- and low-affinity "receptors" was established by the fact that several nonlactogenic hormones, including bovine growth hormone and insulin, do not displace [I125]-hGH from the receptor sites. When Nb2 cells were incubated for 1 hr at 0 degrees C with 100 pg/ml [I125]-hGH, no binding to the high-affinity receptor was detected, thus demonstrating the temperature dependence of binding to this receptor. The kinetics of binding of hGH to the high-affinity binding site correlates well with the dose-response effects of lactogenic hormones on mitogenesis in the Nb2 cells. In addition, the rapid binding of hGH to the high-affinity receptor may be related to specific effects of lactogenic-hormones which occur within seconds or minutes after adding these hormones to Nb2 cells.

The growth hormone (GH)-binding protein cloned from human IM-9 lymphocytes modulates the down-regulation of GH receptors by 22- and 20-kilodalton human GH in IM-9 lymphocytes and the biological effects of the hormone in Nb2 lymphoma cells.

The cDNA coding for the 246-amino acid long N-terminal extracellular portion of the human (h) GH receptor, corresponding to the circulating GH-binding protein (hGHBP), was cloned by polymerase chain reaction from human IM-9 lymphocytes. The cDNA sequence was identical to that reported for human liver and placenta and demonstrated alternative splicing of exon 3. The protein with the exon 3-encoded domain was expressed and secreted in glycosylated form from baby hamster kidney (BHK) cells, purified to homogeneity, and sequenced; the amino acid sequence was identical to that predicted from liver cDNA. The cloned hGHBP competed in a dose-dependent fashion for binding of 125I-labeled 22-kilodalton (kDa) hGH, and at higher concentrations for binding of 125I-labeled 20-kDa hGH, to IM-9 lymphocytes. hGHBP decreased the association rate of [125I]hGH to the cells without decreasing the dissociation rate. hGHBP blocked the down-regulation of GH receptor in IM-9 cells by both 22- and 20-kDa hGH. hGHBP also blocked the binding of [125I]hGH to PRL receptors on Nb2 lymphoma cells and the effect of the hormone on thymidine incorporation. Binding of both 22- and 20-kDa hGH to the binding protein was demonstrated directly by immunoprecipitation with monoclonal antibody 263. The present work thus establishes the identity of the IM-9 human GHBP from those of liver and placenta, and demonstrates its ability to bind both 22- and 20-kDa hGH with good affinity and to block their biological actions mediated though both somatogenic and lactogenic receptors. The modulation of receptor down-regulation by the BP may be a relevant facet of its physiological role.

Site-directed mutagenesis of hGH at the 54–74 loop selectively modifies its lactogenic receptor-mediated biological activity

Four analogues of human growth hormone (hGH) mutated by site-directed mutagenesis at the 54–74 loop-MethGH(P59A), Met-hGH(P6lA), Met-hGH(P59A,P61A) and Met-hGH(Des 62–67) were analyzed for: (1) their biological activity mediated through lactogenic receptors using rat lymphoma Nb 2-llC cell proliferation and mouse mammary gland HC-11 cell β-casein synthesis bioassays and (2) their ability to interact with recombinant hGH binding protein (hGHBP). The analogues Met-hGH(P59A), Met-hGH(P61A) and Met-hGH(P59A,P61A) partially lost their activity relative to native hGH in the HC-11, but not in the Nb 2-llC cell bioassay. These analogues were nevertheless capable of forming a 1:2 complex with a recombinant hGH binding protein (hGHBP), despite the fact that the affinity of Met-hGH(P61A) and Met-hGH(P59A,P61A) analogues had decreased 8- and 14-fold, respectively. Met-hGH(Des 62–67) failed to form 1:1 or 1:2 complexes with hGHBP and did not compete with [ 125I]hGH for binding to hGHBP. It lost all biological activity in HC-11 cells, but retained 0.4% of its activity, in the Nb 2-11C cell proliferation bioassay. These results confirm the involvement of Pro-61 in the hGH binding and activity mediated through somatogenic receptors, while the activity mediated through two different types of lactogenic receptors was selectively modified. These findings emphasize the fact that lactogen receptors in different species or organs are not identical.

Growth Hormone Receptors in Avian Epiphyseal Growth-Plate Chondrocytes

Growth hormone receptor (GH-R) gene expression was evaluated in avian growth-plate cartilage by Northern blot and hybridization using the avian GH-R probe. A single transcript of ∼5.2 kb was demonstrated in cultured growth-plate chondrocytes as well as in growth-plate extracts. GH receptor gene expression was inhibited by chicken GH (cGH) in a dose-and time-dependent manner. Chicken GH was more potent in down-regulating the GH-R gene expression than hGH, but on the other hand cGH exhibited a lower affinity to avian chondrocytes receptor than did the human hormone. Addition of ascorbic acid to the culture media caused cell differentiation: induction of alkaline phosphatase activity and attenuation of collagen type II gene expression. No differences in the GH-R gene expression were observed in the nondifferentiated cells compared with the differentiated cells. Chicken GH did not form any complex with the purified hGH binding protein (hGHBP), did not bind to human lymphocytes GH receptor, and did not affect Nb2 cell proliferation. These systems represent somatogenic and lactogenic types of GH receptors, respectively. In summary, avian growth-plate chondrocytes in situ and in culture exhibit GH-R and these receptors are capable of binding GH. Thus, the failure of GH to affect avian chondrocytes' proliferation was not due to either the absence of receptors on the cell membrane or to a lack in its binding activity, but rather may be due to events farther downstream.

Pregnancy lactogens in the rat conceptus and fetus: circulating levels, distribution of binding, and expression of receptor messenger ribonucleic acid.

To clarify the roles of the rat placental lactogens in embryogenesis and fetal development, we measured the concentrations of rat placental lactogen-II (rPL-II) in fetal rat serum and examined the distribution and expression of rPL-I- and rPL-II-binding sites in rat uteroplacental and fetal tissues. The concentration of rPL-II in fetal rat serum on day 20 of gestation was 28.3 +/- 0.8 ng/ml (mean +/- SEM; n = 6), approximately 1/14th its concentration in maternal serum (398.3 +/- 45.3 ng/ml; n = 6). In the midgestational uterus and placenta, rat PL-I bound specifically to mesometrial decidua and to a capsular layer of stroma overlying the antimesometrial decidua. The binding of radiolabeled rPL-I to these tissues was inhibited by unlabeled rat PRL and human (h) GH, but not by rat GH, suggesting that the rPL-I-binding sites are lactogenic in nature. In the late gestational fetus, rat PL-II bound specifically to fetal adrenal, kidney, small intestine, liver, and pancreas; its binding, like that of rPL-I, was inhibited by rPRL, but not by rGH. rPL-II-binding sites in fetal adrenal were detected as early as day 16, whereas rPL-II-binding sites in fetal kidney and small intestine were not demonstrable until day 18. Lactogenic binding sites in fetal liver and pancreas did not appear until days 19-20. The relative amounts of specific binding of rPL-II to fetal tissues correlated positively with tissue levels of expression of the 4.2- and 1.8-kilobase PRL receptor mRNA transcripts. Radiolabeled hGH, which interacts with somatogenic receptors as well as lactogenic receptors, bound specifically to mesometrial decidua, fetal adrenal, kidney, small intestine, liver, and pancreas. In addition, radiolabeled hGH bound specifically, but with low intensity, to fetal brain. In mesometrial decidua and fetal adrenal, kidney, and small intestine, the binding of hGH was blocked by rPL-II and rPRL, but not by rGH or ovine GH, suggesting the predominance of lactogenic receptors. In contrast, in fetal brain, the binding of hGH was inhibited by rGH, but not by rPL-II, suggesting that the fetal brain contains somatogenic receptors. The presence of rPL-I-binding sites in maternal decidua suggests a paracrine role for the hormone in decidual function at midgestation. The presence of rPL-II in fetal serum and the widespread distribution of rPL-II-binding sites in fetal tissues indicate a role for rPL-II in fetal development.

The turnover of growth hormone (GH)-binding protein and GH receptor in rabbit and rat

The present study was undertaken to further explore the comparative dynamics of growth hormone-binding protein (GH-BP) in relation to the turnover of the GH-receptor (GH-R) in vivo in rabbits and rats. The strategy used was to examine the time course of hepatic GH-R turnover over a 3 h period after cycloheximide treatment, with simultaneous measurements of serum GH-BP level. In the rabbit we sampled multiple liver biopsies and serum samples consecutively from each animal. In the rat, experiments on individual animals were conducted for each time point. In the rat, both liver GH-R and serum GH-BP declined after cycloheximide injection following first-order kinetics. The t 1 2 values for GH-R and GH-BP were 29.7–44.5 and 82.7–119.5 min (95% confidence limits), respectively. A significant positive correlation was found between rat liver GH-R and serum GH-BP ( r = 0.85; p < 0.001). In contrast, the decline in rabbit liver GH-R, following cycloheximide treatment, was accompanied by simultaneous time-dependent accumulation of serum GH-BP. The t 1 2 for rabbit serum GH-BP accumulation was 30.4–67.6 min. Scatchard analysis of [ 125I]hGH binding to rabbit GH-BP indicated that the binding capacity increased from 2818 ± 538 fmol/ml, at time zero, to 5236 ± 419 fmol/ml following 60 min cycloheximide treatment ( p < 0.05). No significant changes in affinity were observed. Pooling all rabbit data points after cycloheximide injection, a significant negative correlation was found between liver GH-R and serum GH-BP ( r = −0.58; p < 0.001). Thus, parallel disappearance of hepatic GH-R and serum GH-BP in the rat following cycloheximide treatment contrasts that in the rabbit, in which the decline in hepatic GH-R was accompanied by simultaneous time-dependent accumulation of serum GH-BP.

Growth hormone receptor regulation in growth hormone-deficient dwarf rats

In the rat, many actions of GH depend upon the sexually dimorphic pattern of exposure to GH. Hepatic human GH (hGH) receptor binding differs between the sexes and is sensitive to GH deficiency, but this has mostly been studied in acutely hypophysectomized rats, which lack all pituitary hormones. We have used a strain of GH-deficient dwarf (Dw) rats to determine whether chronic GH deficiency alters the normal developmental pattern and sexually dimorphic expression of hepatic GH receptors. Adult female Dw rats had lower levels of 125I-labelled hGH binding (reflecting predominantly lactogenic receptors) than their normal counterparts whereas there was no difference between adult Dw and normal males; binding capacity increased from 25 days of age, becoming sexually dimorphic from 40 days to adulthood in both strains (% specific binding/mg protein: normal males 1.6 +/- 0.3, normal females 13.2 +/- 1.1, Dw males 2.1 +/- 0.4, Dw females 10.0 +/- 0.6). In contrast, hepatic 125I-labelled bovine GH (bGH) binding (somatogenic receptors) was much lower, and similar in both Dw and normal animals. A sex difference in 125I-labelled bGH binding was only seen in adult animals, and was considerably less marked in Dw rats compared with normal animals (normal males 1.3 +/- 0.1, normal females 2.5 +/- 0.2, Dw males 1.9 +/- 0.2, Dw females 2.4 +/- 0.2%/mg protein). Continuous hGH infusion stimulated growth in female Dw rats, and raised somatogenic and lactogenic GH binding (3.2 +/- 0.4 and 19.6 +/- 2.5%/mg protein) compared with sham-infused controls (2.4 +/- 0.2 and 7.9 +/- 0.6%/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone-binding protein (GH-BP) levels in follicular fluid from human preovulatory follicles: correlation with serum GH-BP levels.

Serum GH-binding protein (GH-BP), which is identical with the extracellular domain of the GH-receptor, has important implications for the distribution and physiological activity of GH and may enable evaluation of GH-receptor function. Recent studies suggest that GH plays an important role in the modulation of ovarian function and GH-receptors/BPs are found in the female reproductive system. The purpose of the present study was to investigate the presence of GH-BP in human follicular fluid (FF) and compare the levels of FF GH-BP with those detectable in serum, in 46 women undergoing in vitro fertilization. Levels of GH-BP were determined by incubation of [125I]hGH with 50 microL FF or serum, in the absence or presence of excess hGH and specific binding was expressed as a percentage of the total counts per min incubated. The mean GH-BP level in patient FF was 12.39 +/- 0.63% (mean +/- SE) and this correlated significantly with serum GH-BP levels (r = 0.82; P < 0.001). The binding of hGH to FF was dose dependent, highly specific, and of high affinity and low capacity. The affinity constants (Ka) obtained by Scatchard analysis for hGH binding to patient's FFs and sera were not significantly different. Furthermore, we have analyzed the sodium dodecyl sulfate gel electrophoretic pattern of GH-BP in FF and serum by both the ligand-blot technique and after cross-linking with [125I]hGH. Our results show that there is close similarity between serum and FF GH-BPs. Significantly lower FF GH-BP levels were measured in patients older than 36 yr compared to younger women (P < 0.05), whereas increased values were obtained both in patients with elevated E2 concentrations in serum (> 7000 pmol/L) and in FF (> 2200 nmol/L), (P < 0.02 and P < 0.05, respectively). This first demonstration of GH-BP in FF is expected to increase our understanding of the possible direct effect of GH on ovarian steroidogenesis, and suggests a possible regulatory role for GH-BP in folliculogenesis.

Growth hormone- and prolactin-binding proteins in mammalian serum.

The present study was undertaken to characterize the species specificity and diversity of lactogenic and somatogenic binding to serum among mammals and to classify GH-binding protein (GH-BP) in this group of species. Animal sera were characterized on the basis of human (h) GH and bovine (b) PRL binding levels, binding specificity toward GHs and PRLs, binding affinity constant (Ka), and the ability of a monoclonal antirabbit GH receptor antibody (MAb-7) to inhibit the binding to serum. Analyses of the results yielded a classification of the mammalian sera into five types of GH binding, which we elected to call GH-BP. The guinea pig had undetectable levels of GH-BP and was labeled type 0. Type I GH-BP was found in the mouse and the rat. hGH binding to GH-BP of type I serum was of low affinity (1.2-3.9 x 10(8) M-1) with high IC50 (approximately 100 ng/tube) and was purely somatogenic in nature. Type II GH-BP was found in the goat, sheep, and cow. Their IC50 of hGH binding was approximately 4-fold lower than that of type I GH-BP. Their binding of hGH was relatively specific and only marginally displaced by the PRLs. Type III GH-BP was found in the rabbit, horse, cat, dog, and pig. These animals' sera had high affinity binding toward hGH (4.7-9.2 x 10(9) M-1), with low IC50 (approximately 2 ng/tube) and dominant lactogenic binding. The great similarity of type III GH-BPs was further stressed by the ability of MAb-7 to inhibit [125I]hGH binding to all type III sera, but not to the other mammalian sera tested. Type IV GH-BP was found in the human and rhesus monkey sera. These were characterized by binding affinities that were intermediate between those for types II and III GH-BP and by the inability of nonprimate GHs to compete with hGH binding. To directly confirm the potent effect of the lactogenic hormones in type III GH-BP, the specific binding of bPRL to sera of type III animals was studied. [125I]bPRL-specific binding was determined under optimized assay conditions and was the highest in rabbit serum, with the following rank order being observed: rabbit >> horse = dog = pig > cat. Scatchard analysis of [125I]bPRL binding revealed linear plots with similar affinity constants (Ka) of 1.7-3.3 x 10(9) M-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of human growth hormone binding to somatogenic and lactogenic receptors by monoclonal antibodies to human growth hormone

The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interactions was investigated by studying the effects of specific monoclonal antibodies (MAbs) to hGH on the binding of [ 125I]hGH to rabbit liver and mouse liver microsomes. Receptor binding assays were carried out using a constant dose (1 ng) of [ 125I]hGH and varying concentrations of MAbs. The assay was carried out in the presence of either excess ovine prolactin for the measurement of somatogenic (SOM) bindings sites, or excess bovine growth hormone for the determination of lactogenic (LAC) binding sites. Anti-hGH MAbs were found to have a whole spectrum of effects on hGH binding, including inhibitory, non-effect and enhancing activities. Enhancement of the binding of [ 125I]hGH to both SOM and LAC receptors was observed in liver membranes of rabbit or mouse. The observed amplified signal of [ 125I]hGH binding to various receptors in the presence of MAb no. 8 may be due to conformational changes which occur following MAb binding to hGH. On the other hand, most of the other MAbs caused inhibition of [ 125I]hGH binding. A negative correlation exists betweed the cross-reaction of various MAbs with the N-terminus truncated forms of hGH (Met 14-hGH or Met 8Leu-hGH) and their respective K D IC 50 values enable the evaluation of the crucial role of the N-terminus region in hGH binding to both LAC and SOME receptors. MAb nos 1 and 19, which are directed towards acid residues 95–134 and the C-terminus, inhibited SOM binding more potently than LAC binding. Thus, it seems that these mid-molecule and C-terminus regions are also important in hGH binding, and that they play a role in the partial overlap of SOM and LAC binding.

Immunodominant structures of human growth hormone identified by homolog-scanning mutagenesis

Homolog-scanning mutagenesis has been reported to be useful in elucidating the antigenic epitopes recognized by monoclonal antibodies and hGH binding to its receptor. However, little is known about which structures are recognized as immunodominant by murine serum antibodies. Therefore, the previously published series of hGH homologs and additional mutants of human placental lactogen (hPL), porcine growth hormone (pGH), and human prolactin (hPRL) were examined for their interaction with murine serum derived anti-hGH antibodies. As compared to wild-type hGH, nine of the nineteen segment substituted mutants tested showed a significant reduction in binding to anti-hGH sera. These disruptive substitutions mapped to 5 regions on a structural model of hGH: the length of helix 1 (residues 11–33), the loop between the first disulfide bond and helix 2 (residues 54–74), the beginning of helix 3 (residues 109–112), the carboxyl half of helix 4 (residues 167–182), and the final carboxyl terminus segment of the molecule (residues 184–191). In terms of the current structural model, three of the five immunodominant regions (the loop between residues 54–74, central portion of helix 4 to the carboxyl terminus and part of the amino terminus region of helix 1) closely overlaps the hGH receptor binding epitopes.

Effect of human growth hormone (GH)-binding protein in human serum on GH binding to rabbit liver membranes

The sequence identity of growth hormone-binding protein (GH-BP) with the extracellular domain of GH receptors raised the possibility that circulating GH-BP might affect the binding of human GH (hGH) to its receptors, and thus, its biological effects. To test this hypothesis, we tested the effects of sera with low GH-BP levels (obtained from prepubertal children, girls with anorexia nervosa [AN], and patients with hepatic cirrhosis), normal control sera, and sera with high GH-BP levels (obtained from obese patients) on hGH binding to its receptors. GH-BP activity in patients' sera was measured by incubation with [ 125I]hGH and the separation of bound hGH from free hGH with dextran-coated charcoal. The effect of GH-BP was studied by preincubation of patients' sera with increasing concentrations of hGH, followed by incubation with [ 125I]hGH and a rabbit liver membrane preparation known to be rich in GH receptors, and finally by measuring hGH bound to the receptors. In this study, we report on the ability of GH-BP to reduce the inhibitory capacity (IC 50) of hGH on [ 125I]hGH binding to GH receptors. The concentration of GH-BP in serum is positively correlated with the IC 50 of hGH incubated with different sera on [ 125I]hGH binding to its receptors (n = 21; r = .886, P < .001). In the presence of high serum GH-BP levels, such as those observed in obesity ( 20.13% ± 0.71% 0.05mL serum), the IC 50 values were significantly higher than those obtained with sera containing GH-BP levels lower than those measured in human control subjects, such as from prepubertal children, AN patients, and cirrhotic patients. Based on these findings, it is proposed that increasing levels of GH-BP may modulate GH binding to its receptor, and thus may exert a negative feedback on receptor recycling and synthesis and on GH-BP cleavage from the membrane receptor or its independent expression.