Differential in vivo activities of bovine growth hormone analogues (original) (raw)
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Journal of Endocrinology, 1998
We have examined the regulation of hepatic growth hormone receptors (GH-R) and serum GH binding proteins (GHBP) in transgenic mice expressing an antagonist of bovine growth hormone (bGH), G119K-bGH, and consequently exhibiting a growth suppressed dwarf phenotype. Specific GHBP could be measured in transgenic dwarf mouse serum only by immunological methods (RIA), because these mice have a very high concentration of mutated bGH in circulation (>1 µg/ml) and, therefore, almost all GHBP is bound to G119K-bGH and cannot be quantitated in binding assays. The concentrations of GHBP were 0·6 0·4 nM and 1·7 0·4 nM for normal and dwarf mice respectively. The concentrations of free GHBP in normal mice and in transgenic mice expressing wild-type GH can be calculated using chromatographic techniques as the dissociation constant (K d ) and the ratio of bound 125 I-GH to free 125 I-GH in the serum ([GHBP] free =B/F.K d ). In agreement with the assumption that GHBP reflects GH-R status, liver uptake of injected labeled bGH was greatly reduced in transgenic dwarfs in comparison with normal mice or with transgenic mice expressing wild-type bGH (liver/blood ratio of 0·48 0·21, 2·7 0·2, and 1·3 0·3 respectively) indicating that the high concentration of the mutated bGH (G119K-bGH) prevents labeled bGH uptake, as was expected from the dwarf phenotype. 125 I-bGH taken up by the liver of transgenic dwarf mice was found in a smaller molecular species than in normal mice, compatible with the presence of 1:1 [(GH-R):GH] complexes instead of the 2:1 [(GH-R) 2 :GH] or 2:2 [(GHBP) 2 :(GH) 2 ] complexes found in normal mice.
Sex differences in binding of human growth hormone to isolated rat hepatocytes
1976
Since liver is a target for growth hormone action, binding of 125I-labeled human growth hormone to enzymatically isolated rat hepatocytes was studied. Specific binding was shown with hepatocytes from both male and female animals. There was a single class of receptors for human growth hormone on cells from males (affinity constant, Ka = 1.16 X 109 liters/mole; sites per cell, q = 6200). In males, bovine growth hormone was almost as potent as human growth hormone in displacing bound 123I-labeled human growth hormone, while ovine prolactin was about 1000 times less potent. Cells from female rats bound more 25I-labeled human growth hormone than cells from males. The cells from females contained at least two classes of receptors for human growth hormone. The receptor of highest affinity had the same affinity for human growth hormone as the single receptor found in males (Ka = 0.96 X 109 liters mole). However, there were three to four times as many of these receptors per cell in females (q = 21,000). In females, bovine growth hormone and ovine prolactin were both about 20 times less potent than human growth hormone. Treatment of male rats with estrone produced cells that show the same binding characteristics as females. These results indicate that human growth hormone binds to a somatogenic receptor in hepatocytes from male rats. In females and estrogen-treated males, the receptors that bind human growth hormone recognize lactogenic as well as somatogenic properties. This suggests that the lactogenic and growth-promoting effects of human growth hormone in the rat are mediated by different receptors.
Allosteric effects of monoclonal antibodies on human growth hormone
Molecular and Cellular Biochemistry, 1994
We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGHspecific receptors. The effect of MAbAE5,AC8 and F11 on hGH binding was measured by determining the formation ofl25I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding 125I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes. Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case preformed lZSI-MAb:hGH complexes were added to microsomes. Data showed that 125I-MAb AE5:hGH complexes bound better to the various receptors than 125I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to ~2SI-MAb AC8 or ~25I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding ofMAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32-46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding. Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site. MAb would induce either positive or negative allosteric changes in the hormone region involved in its binding to lactogenic, somatogenic and hGH-specific receptors. (Mol Cell Biochem 136: 35-42, 1994)
The Journal of biological chemistry, 1990
We have recently established that the human growth hormone-variant (hGH-V) gene is functional in vivo by documenting its expression in the placenta. We have subsequently generated transformed murine cell lines stably expressing the genes for normal pituitary growth hormone (hGH-N), hGH-V, and each of two chimeric genes generated by exon 3 exchanges, hGH-NV3 and hGH-VN3. In the present study, we utilize these cell lines as sources of hormone to characterize and compare the receptor binding profiles of hGH-N with hGH-V. hGH-V was found to displace 125I-ovine prolactin bound to rat liver microsomes (lactogen binding) and to displace 125I-hGH bound to rabbit liver microsomes (somatogen binding). Therefore, hGH-V would be predicted to display both somatogenic and lactogenic bioactivity, a dual specificity previously thought to be unique to hGH-N. The concentrations of hormone necessary to displace 50% (IC50) of the 125I-hGH from somatogen receptors and 125I-ovine prolactin from lactogen ...
Biology of Reproduction, 1997
The effect of continuous human GH (hGH) secretion on female reproduction was studied in adult female transgenic rats expressing the hGH gene with a mouse whey acidic protein (mWAP) promoter. Two lines of transgenic female rats carrying the mWAP/hGH gene were established and used in the study. One was characterized by relatively high levels of serum hGH (high line), and the other had relatively low levels (low line). High-line female rats had recurring, pseudopregnancy-like estrous cycles accompanied by increased serum progesterone levels for 2 wk after ovulation, and they were fertile. In these rats, luteinization occurred spontaneously without cervical stimulation, probably due to high levels of serum hGH, which has prolactin (PRL)-like activity in the rat. Although low-line female rats had recurring, regular 4-day estrous cycles, they were sterile. In these rats, pseudopregnancy could not be induced by mating or by mechanical cervical stimulation. PRL surges following cervical stimulation were not detected, and PRL secretion was not induced by a dopamine antagonist, metoclopramide. The ovarian tissue in this line had a much higher number of corpora lutea and grew much heavier than in normal littermates, suggesting impairment of PRL-induced structural luteolysis. Suppression of PRL secretion in low-line rats was, at least in part, due to a marked decrease in the number of lactotrophs in the pituitary. The present study shows that the serum hGH level plays a crucial role in regulating luteal function in female transgenic rats expressing the hGH gene.
Growth Hormone & IGF Research, 2006
Expression plasmids containing DNA sequences optimized for expression in Escherichia coli were prepared encoding human pituitary (hGH-N 20K) and placental (hGH-V 20 and 22K) growth hormones. The proteins were expressed in bacteria, refolded and purified to homogeneity by anion-exchange chromatography on Q-Sepharose according to a unique protocol developed for each protein. The yields from 5 l of fermentation culture varied between 400 and 700 mg of electrophoretically pure, over 95% monomeric protein. Circular dichroism (CD) analysis revealed similarity of the purified hGHs' secondary structure to that of the pituitary hGH-N 22K, except for hGH-V 20K, in which the a-helix content was lower. The purified proteins were stable as a 0.1% sterile solution held at pH 10-11 at 4°C for at least one month. All three purified hGH molecules formed a 1:2 complex with hGH receptor extracellular domain (hGHR-ECD), similar to hGH-N 22K. Binding experiments using hGHR-ECD revealed that the differences between the two 22K variants or between the two 20K variants were not significant, except that hGH-V 20K exhibited slightly lower affinity. Somatogenic activity was tested in vitro using FDC-P1 cell lines. Whereas the bioactivity of 22K hGHs and hGH-N 20K in FDC-P1-9D11 cells stably transfected with hGHR was almost equal and two to threefold higher than that of hGH-V 20K, in FDC-P1 3B9 cells stably transfected with rabbit (rb) GHR, the bioactivity of both 20K analogues was significantly (five to ninefold) lower than that of the 22K hormones. The lactogenic activity measured in heterologous assays (Nb2-11C cells and Baf/3 cells stably transfected with the long form of rabbit prolactin receptor) revealed that the activity of hGH-N 20K was close to that of hGH-N 22K in the Baf/3 cells, but 4.5-fold lower in the Nb2 cells. The activity of hGH-V 22K was ninefold less in Nb2 cells and 55-fold less in Baf/3 cells, whereas hGH-V 20K had no lactogenic activity in either bioassay. In contrast, in a homologous lactogenic assay using Baf/3 LP cells stably transfected with hPRLR, the activity of both placental hGHs was nil and the activity of hGH-N 20K was 4.3-fold lower than that of hGH-N 22K. The latter finding raises the question of whether the lack of intrinsic lactogenic activity in the placental hGHs that dominate during pregnancy has any physiological relevance.
Growth Hormone & Igf Research, 2001
Growth hormone (GH) releasing hormone (GHRH) transgenic mice were used to examine the influence of GH on GH receptor (GHR) and membrane-associated GH binding protein (MA-GHBP) levels by means of specific radioimmunoassays and Western blot analysis, since MA-GHBP was described as the major constituent of somatogenic binding to liver membranes in mice. In transgenic animals, a 10-fold increment over normal values was found for hepatic somatogenic binding that could be accounted for by a 3-4-fold increase in GHR and a 9-fold augmentation of MA-GHBP levels. The apparent molecular weight of MA-GHBP was smaller than that of serum GHBP, a difference that was partially abolished by endoglycosidase F digestion. In vivo treatment of female mice with 17βestradiol led to an unexpected down-regulation of MA-GHBP and GHR by 60-75% only in transgenic animals. MA-GHBP and GHR levels are strongly up-regulated by GH, although MA-GHBP up-regulation is much more important than that of GHR.
Growth Hormone & IGF Research, 1998
The fate of exogenous radiolabeled growth hormone ( 125 I-hGH) was studied in Ames dwarf mice, which do not express growth hormone (GH) or prolactin (PRL) genes. Labeled GH was injected in low amounts that did not exceed the normal physiological GH concentration in mice. Binding of most of the injected 125 I-hGH by the GH-binding proteins (GHBPs) present in plasma represents the first step in the handling of this material in vivo. The decay curve followed a two-compartment model and gave the equation: Conc = 2.807e -0.0067t + 15301e -0.0647t (coefficient of determination 0.9986 ± 0.0019), while in normal mice, GH decay followed a three-compartment model as we have previously reported. The fast compartment with t 1/2 of 1-2 min was virtually absent in dwarf mice, and chromatographic studies revealed the disappearance of free GH in these mice. We also present evidence of the labeled GH-forming complexes, presumably with GHBPs under in vivo conditions. The second step of processing labeled GH in vivo is the uptake by the liver, which was slower in dwarf than in normal mice (30-45 vs 15 min). Moreover, a lower GH uptake was found in dwarf than in normal mice (L/B ratio of 1.75 ± 0.29 [30 min] vs L/B ratio of 3.68 ± 0.33 [15 min], respectively) due to lower concentration of free GH in plasma and to the reduced number of GH-receptors (GHRs). The radioactive material present in the liver was compatible with 125 I-hGH-GHR complexes with Stokes radius of 59Å. In summary, we provide evidence that plasma of dwarf mice contains proteins capable of binding GH in vivo and probably representing GHBPs not complexed with GH. The presence of these proteins modified the pharmacokinetics of 125 I-hGH in plasma and its subsequent uptake by the liver. The presence of these binding proteins in the absence of endogenous GH suggests that a fraction of total GHBPs (one class?) is independent of GH concentration.
Ovine placental lactogen and human growth hormone bind to different regions of the same receptors
Growth Hormone & Igf Research, 1999
Anti-human growth hormone (hGH) polyclonal and monoclonal antibodies (MAb) failed to recognize ovine placental lactogen (oPL), indicating that the antigenic topographies of both hormones are different. Binding assays showed that oPL completely inhibited hGH binding to lactogenic receptors from Nb2-cells and to somatogenic receptors from rabbit or sheep liver; in contrast, oPL only bound to a subpopulation of rat liver receptors. Zinc ion increased hGH and oPL binding to Nb2-cell receptors and slightly inhibited both hormones' recognition by somatogenic receptors. However, ZnCl 2 increased hGH binding to rat liver microsomes but prevented that of oPL. Furthermore, MAb R7B4, recognizing lactogenic as well as somatogenic receptors, entirely blocked hGH binding to the various receptor systems but not affected oPL binding. Therefore, results presented in this paper suggest that oPL and hGH bind to different regions of the same receptors.