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 interaction 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) binding 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 between the cross-reaction of various MAbs with the N-terminus truncated forms of hGH (Met14-hGH or Met8Leu-hGH) and their respective KD/IC50 values enabled the evaluation of the crucial role of the N-terminus region in hGH binding to both LAC and SOM 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.     

The growth hormone-like factor produced by the tapeworm Spirometra mansonoides specifically binds receptors on cultured human lymphocytes

Plerocercoid larvae of the tapeworm Spirometra mansonoides produce a factor with activities similar to those of growth hormone (GH). Highly selective receptors for GH have been described on cultured human lymphocytes (IM-9 cells) and these cells have been used as a model of binding essentially restricted to human GH (hGH). We compared the displacement of [125I]hGH by hGH and partially purified plerocercoid growth factor (PGF) in assays using rabbit hepatic membranes and IM-9 cells. PGF displaced [125I]hGH from both rabbit hepatic membranes and IM-9 cells in a dose-dependent manner (r greater than 0.98). These results show that PGF specifically binds to hGH receptors on human IM-9 cells and suggest the possibility that PGF will have somatotropic activity in humans.     

Reevaluation of lipolytic activity of growth hormone in rabbit adipocytes

The lipolytic activities of porcine pituitary fractions and purified growth hormone (GH) from human (h), porcine (p), ovine (o) and rabbit (Rb) origin as well as ovine placental lactogen (oPL), were compared to that of ACTH on rabbit adipocytes. All the GH preparations and oPL were equivalent in inhibiting the binding of labelled oGH to liver plasma membranes from pregnant rabbits. ACTH, and to a lesser extent porcine pituitary fractions and hGH, stimulated free fatty acid production by isolated adipocytes. The sensitivity of the adipocytes to these factors was increased when adenosine deaminase was added to the incubation medium. But, RbGH, pGH, oGH and oPL had no effect. We conclude that GH is not directly involved in the control of lipolysis in rabbit adipocytes and that the effect of hGH is rather due to a contamination of this preparation by other pituitary factors.     

Growth hormone receptor structure in adipocytes from normal and growth hormone-deficient rats

Using cross-linking techniques, we compared the properties of the growth hormone (GH) receptor in freshly isolated adipocytes from normal rats, from GH deficient rats, and in preincubated adipocytes from normal rats. Bound [125I]iodo-hGH was cross-linked to adipocytes with disuccinimidyl suberate, and membrane proteins labelled with [125I]iodo-hGH were visualized using sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. All of the adipocytes tested exhibited a prominent Mr = 134,000 band and additional less intense bands in the presence of reductant. No significant differences in the overall banding pattern of membrane proteins were evident in reducing or nonreducing gels, using adipocytes from rats made GH deficient by hypophysectomy or by treatment with antibodies against rat GH, or in fresh and preincubated cells from normal rats. Taken together with binding studies, these findings suggest that differences in the ability of GH to stimulate glucose oxidation in rat adipose tissue probably involve differences distal to the GH receptor.     

Growth-hormone-binding protein in patients with acromegaly.

The present study was undertaken to investigate the possible regulatory effect of chronic exposure to human growth hormone (hGH), in patients with acromegaly, on growth-hormone-binding protein (GH-BP). Nineteen patients with active acromegaly, before, during or after treatment, comprised the subjects of this study. Serum GH was measured by radioimmunoassay and GH-BP by a binding assay with dextran-coated charcoal separation. The specific binding of [125I]hGH (1 ng) obtained with 50 microliters serum was expressed as a percentage of total cpm. To evaluate the impact of the lower GH-BP on GH activity, we studied the effect of acromegalic serum on hGH displacement of [125I]hGH binding to GH receptors in rabbit liver membranes. Compared to normal controls (11.43 +/- 0.37%), the acromegalic patients had low serum levels of GH-BP (5.45 +/- 0.40%; p < 0.001), which correlated negatively with serum GH levels (p < 0.01). In 7 patients, GH-BP normalized within 2-3 months of successful therapy. The lower GH-BP was due to a reduction in binding capacity, whereas binding affinity remained unchanged. Acromegalic serum, with its low GH-BP, resulted in a shift to the left of the GH displacement curve when compared with normal human sera: IC50 values were 7.47 +/- 0.29 and 11.19 +/- 0.84 ng (p < 0.02) for acromegalic and normal human sera, respectively. We conclude that acromegaly is characterized by low levels of GH-BP due to a decrease in serum-binding capacity. The decrease in GH-BP may render the acromegalic serum GH relatively more active in the GH receptor assay.     

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.     

Cloning and expression of ovine placental lactogen

Ovine placental lactogen (oPL) is active in a wide range of GH and PRL assays, a property that it shares with human GH (hGH). In addition, oPL is one of a small number of hormones that bind the human GH receptor with high affinity. In order to compare the sequence of oPL to the sequences of other members of the GH family, full-length cDNA clones have been isolated. These clones predict that the full sequence of oPL contains 198 amino acids preceded by a 38 amino acid signal sequence. The mature oPL sequence includes six cysteine and two tryptophan residues and shows substantially more identity to bovine PL (67%) and oPL (49%) than to mouse (31%) or human (25%) PL or to oGH (28%) or (26%) hGH. Like the natural hormone, oPL expressed in mammalian tissue cells binds with high affinity to a soluble form of the recombinant hGH receptor. Thus, oPL binds to the human receptor in spite of having a sequence that is considerably divergent from hGH. Interestingly, the sequence of oPL differs from hGH at most of the amino acids recently found by mutagenesis studies to be important residues in the binding of hGH to the human receptor.     

Suppression of Receptors for Prolactin and Estrogen in Rat Liver Due to Treatment with the Growth Hormone Analogue Produced by the Tapeworm Spirometra Mansonoides

Abstract

Somatogenic hormones play an important role in regulation of receptors for prolactin (PRL) and estrogen. Plerocercoids of the tapeworm, S. mansonoides produce a factor which mimics some, but not all of the actions reported for GH. Intact female rats were subjected to a constant infusion of plerocercoid growth factor (PGF) via a subcutaneous infection for two weeks to determine if PGF influences receptors for PRL, GH or estradiol. The rate of weight gain in the PGF-treated rats was accelerated in spite of a marked reduction in serum GH. Partially-purified PGF specifically displaced [¹²⁵I]hGH from rat liver receptors but microsomes prepared from rats treated with PGF specifically bound significantly less [¹²⁵I]hGH than microsomes from control rats. The reduction in [¹²⁵I]hGH binding was not due to occupancy or to a change in affinity but to a suppression in receptor concentration. Scatchard analysis of [³H]estradiol binding in rat liver cytosols shows a 50% reduction in receptor concentration in the PGF-treated group. Specific binding of [³H]estradiol in anterior pituitary was also suppressed by PGF treatment.     

Heterogeneity of growth-hormone receptors detected with monoclonal antibodies to human growth hormone.

The specificity of hormone-receptor interactions has been examined with the aid of monoclonal antibodies (MABs) (EB1, EB2, QA68 and NA71) defining four non-overlapping antigenic determinants on human growth hormone (hGH). The results indicate that growth-hormone receptors in liver obtained from different sources differ with regard to their affinities and relative numbers; they may also differ with respect to the region of the growth-hormone molecule to which they bind. Antibody NA71 effectively inhibited hormone binding to all receptor preparations tested, although with various degrees of potency. Monoclonal antibody EB1 demonstrated a graded inhibition with respect to its ability to block 125I-hGH binding to receptors from various sources, the maximum inhibition being seen in receptor preparations from mouse and ovine liver and the minimum in rat liver. MABs EB2 and QA68 also showed various abilities to inhibit hormone-receptor interaction, depending on the origin of the receptor preparation. Furthermore, the receptor-binding characteristics of hormone-antibody complexes were dependent on whether the binding-site preparation was derived from pregnant, lactating or 'normal' animals. A particularly striking difference between the ability of hormone-MAB complexes to bind to receptors from different sources was seen for microsomes (microsomal fractions) derived from livers of animals of the 'Little' mouse strain. These animals become progressively obese, and it was shown that MABs were considerably more effective in inhibiting 125I-labelled hGH binding to microsomes from phenotypically obese mice than to those derived from their non-obese littermates. The results can be explained by the presence of multiple receptor types for GH, the relative proportions of which vary according to the physiological state of the animal, and possibly between species.     

Growth hormone binding and stimulation of amino acid uptake in epithelial cells of rat ventral prostate

The binding of [125I]-human growth hormone (hGH) was studied in epithelial cells isolated from rat ventral prostate. Binding and degradation were dependent on time and temperature. The effect of a lysosomotropic agent suggested internalization and lysosomal degradation of the hormone. Dissociation and stoichiometric studies indicated the existence of a single class of GH receptors with a Kd of 0.7 nM and a binding capacity of 46 fmol hGH bound mg-1 cell protein. The receptor appeared to possess a somatotrophic nature since lactogenic hormones such as human placental lactogen and rat prolactin exhibited a very low degree of competition (whereas a variety of unrelated hormones and neuropeptides showed no effect). GH-stimulated leucine uptake by the cells in a time- and dose-dependent manner, half maximal effect being observed at 0.32 nM GH thus suggesting a direct relationship with the binding step.     

Allosteric effects of monoclonal antibodies on human growth hormone

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 hGH-specific receptors.The effect of MAbAE5, AC8 and F11 on hGH binding was measured by determining the formation of¹²⁵I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding¹²⁵I-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 preformed¹²⁵I-MAb:hGH complexes were added to microsomes. Data showed that¹²⁵I-MAb AE5:hGH complexes bound better to the various receptors than¹²⁵I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to¹²⁵I-MAb AC8 or¹²⁵I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb 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.     

Chimeric bovine-human growth hormone prepared by recombinant DNA technology: binding properties and biological activity

A chimeric bovine GH (amino acids Met-Asp-Gln-greater than 1-23) and human GH (hGH) (amino acids 24-191) plasmid was constructed and expressed in Escherichia coli. The purified protein (chimeric GH) exhibited a 2-3 order of magnitude lower affinity toward lactogenic receptors in Nb2 lymphoma cells, microsomal fractions from bovine mammary gland and male rat liver. The affinity towards somatogenic receptors in IM-9 human lymphocytes and male rat liver was decreased to a much lesser degree. This diminished affinity towards lactogenic receptors was accompanied by a parallel decrease in the ability of the chimeric GH to stimulate proliferation of Nb2-11C lymphoma cells and the lipogenesis in bovine mammary gland. This implies that occupation of the respective receptors by either chimeric GH or hGH leads to identical postreceptoral effects. The chimeric GH was also capable of down-regulating the lactogenic receptors in Nb2 lymphoma cells and was recognized by three anti-hGH monoclonal antibodies. These and previously published results indicate that the N-terminal part of hGH is essential for the high affinity binding to lactogenic receptors and subsequent biological effect. Removal or replacement by a corresponding part of bovine GH converts the hormone, respectively to weak antagonist or agonists. Analysis of our data, based on hydropathy index leads us to suggest that the high affinity binding site of the hGH towards lactogenic receptors is mainly confined to amino acids nos. 8-18.     

Specific binding sites for growth hormone in cultured mouse thymic epithelial cells

Growth Hormones bound specifically to murine Thymic epithelial cells, which represent the major component of thymic micro-environment and can be modulated by pituitary hormones. The Kds found with human growth hormone and bovine growth hormone were 0.14 and 0.27 nM with a Bmax 0.56 and 0.35 fmol/10(6) cells respectively. Competition experiment analysis showed ED50 of 0.24 nM for hGH, 0.46 nM for rGH, 0.71 nM for bGH, 11.8 nM for hPRL and 11.2 nM for oPRL. No specific binding of [125I]-oPRL was observed under the same conditions. Both hPRL and bGH showed a negative regulatory effect on the number of the hGH binding sites when incubated with the culture for three days. The presence of GH receptors on Thymic epithelial cells provides biochemical evidence for the effect of GH on thymic function.     

Antigenic, receptor-binding and mitogenic activity of proteolytic fragments of human growth hormone.

Analysis of the subtilisin-digested, two-chain form of human growth hormone (hGH) and its constituent polypeptide fragments has been aided by the use of monoclonal antibodies which bind specifically to four distinct epitopes on the native hormone. Using the SDS-polyacrylamide immunoblotting technique, it was shown that one epitope (shared with human chorionic somatomammotropin) detected by EB1 (or EB3) antibody was expressed to a similar extent by both the N-terminal (15 K) and C-terminal (7 K) polypeptides. This epitope is unique in that it represents a repeating determinant within the single chain structure of the hormone. Another three epitopes detected by monoclonal antibodies QA68/NA27, NA71 or NA39/EB2 were absent from the 7-K fragment but were expressed on the 15-K fragment to a similar extent to that on unmodified growth hormone. Binding of NA71 antibody was demonstrated only by radioimmunoassay since this, presumably conformational epitope could not be detected by immunoblotting. The functional importance of the 15-K peptide was demonstrated by its ability to bind specifically to hormone receptors on IM9 human lymphoblastoid cells and by its retention of mitogenicity for the NB2 rat lymphoma cell line. However, all tested monoclonal antibodies inhibited the binding of [125I]15-K to IM9 cell receptors by either steric hindrance or by an allosteric mechanism and therefore could not be further related topographically to the receptor-binding moiety of hGH.     

Characterization of the slowly dissociable human growth hormone binding component of isolated rat hepatocytes

Human growth hormone (hGH) bound to specific sites on rat hepatocytes. The time course of hGH dissociation was comprised of more than one component. Dissociation was resolved into rapid (t1/2 = 10.5 min) and slow (t 1/2 = 6.4 h) fractions. The amount of slowly dissociable hormone increased for the first 75 min during which time cells and [125I]hGH associated. Subsequently, the amount of slowly dissociable hGH was constant. The time courses of hGH receptor binding and subsequent retention of slowly dissociable label were similar. The capacity of hepatocytes to accumulate slowly dissociable label was saturated by hGH over the same concentration range as the high-affinity binding site (KD approximately 2 nM). This suggested that a receptor-mediated process was responsible for the accumulation of slowly dissociable hGH. Rapidly dissociable label was intact [125I]hGH and fragments resulting from growth hormone degradation. Slowly dissociable hGH recovered from hepatocytes by acid extraction was intact and immunocompetent. There was a large increase in the extent of [125I]hGH degradation between 23 and 37 degrees C. Over this temperature range, the proportion of hGH not in rapid equilibrium with the medium decreased. High concentrations of hGH decreased the amount of slowly dissociable [125I]hGH retained by hepatocytes by competing for high-affinity sites. The interaction of [125I]hGH with low-affinity degradative systems was favored by the presence of hGH. The temperature and concentration dependencies of hGH retention and degradation distinguished these proceses.     

Effect of cell density on induction of growth hormone receptors by dexamethasone in primary cultured rat hepatocytes

The effect of cell density on the regulation of growth hormone (GH) receptors was studied by measuring specific binding of [125I]hGH to primary cultured hepatocytes with or without dexamethasone, which induces GH receptors. In cell cultures without dexamethasone, the cell density did not affect the level of binding of labeled GH appreciably. On the other hand, in the presence of dexamethasone, which induced an increase in the level of GH receptors on the cells, GH-binding by cultured cells at low cell density (3.3 x 10(4) cells/cm2) was about one-third of that of cells at high cell density (10(5) cells/cm2). Scatchard plot analysis showed that the cell-density dependent change in induction of GH binding, by dexamethasone was due to change in the number of binding sites without significant change in their affinity. The binding capacity of glucocorticoid receptors, measured as specific binding of [3H]dexamethasone to the hepatocytes, was not significantly affected by cell density. These results suggest that cell density modulates GH receptor induction by dexamethasone via events after glucocorticoid receptor binding.     

Alteration by concanavalin A of the slow dissociable component in the human growth hormone-receptor interaction

In mice liver plasma membranes (PM), the binding affinity of receptors for [125I] human growth hormone (hGH) was dependent on the association time: after 18 hours, a high affinity receptor form with KA = 6.8 X 10(9) M-1 accumulated and, as compared to after 1 hour, an increase up to 88%, in a slow dissociating component was observed. Preincubation of PM with concanavalin A (Con A) or other lectins from Lens culinaris (LCA), Ricinus communis (RCA I), Wheat germ agglutinin (WGA) specifically inhibited the binding of hGH to receptors by 54, 28, 50 and 25%, respectively. Furthermore, PM pretreatment with Con A concomitantly increased the rate of dissociation of the hormone-receptor (H-R) complex to 92 or 65% after association for 1 or 18 hours. These Con A-induced alterations resulted from a reduced fraction of the slow dissociable component together with an increased rate constant. The treatment of PM with Con A subsequent to incubation with the hormone did not decrease hormone binding but caused the conversion of the class of hGH receptors exhibiting fast dissociation kinetics towards a form exhibiting slow ones. These data strongly suggest a role for glycoproteins of the N-acetyllactosaminic type in the affinity state of liver membrane hGH receptors.     

Human growth hormone binds to lactogenic receptors in bovine, ovine and rat adrenals

The distribution of 125I radioactivity in the liver, kidneys, adrenals and serum of male rats was measured 10 minutes after an intravenous bolus of 125I-labelled human growth hormone (hGH) was administered in the presence or absence of a large excess of ovine growth hormone or ovine prolactin. The hGH binding sites in the adrenals had displacement properties characteristic of lactogenic receptors, whereas those in the liver had displacement properties characteristic of somatogenic receptors. Bovine and ovine adrenal microsomal membrane fractions contained high affinity (Ka = 1.4-3.3 nM-1) binding sites for hGH which showed ligand specificity typical of lactogenic receptors. It is concluded that the hGH binding site in the adrenal gland is a classical lactogenic receptor and that this tissue is a convenient and rich (42.6 +/- 6.4 fmol hGH specifically bound/mg protein) source of receptor suitable for further characterization.     

The specificity of binding of growth hormone and prolactin to purified plasma membranes from pregnant-rabbit liver.

The binding of 125I-labelled human growth hormone (hGH) to a purified plasma membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction with Triton X-100, was dependent on time, temperature, the cations used and the receptor concentration. Solubilization did not affect the binding properties of the receptors at low concentrations of Triton X-100. Some somatogenic hormones, such as bovine GH, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled hGH from purified plasma membranes and solubilized receptor preparations, but GHs and prolactins from various other species were rather ineffective. The results indicate that although there are binding sites for hGH in these pregnant rabbit liver membranes, few of these are specifically somatogenic or lactogenic. The binding properties of the purified plasma membranes are similar to those of a microsomal preparation studied previously, suggesting that the complex nature of the binding of hGH is not due to the heterogeneity of cellular membranes used to study binding, but is a property of the receptors associated with plasma membranes.     

In vitro insulin-like actions of the growth factor from the tapeworm, Spirometra mansonoides

In vitro actions of purified plerocercoid growth factor (PGF) were compared with those of insulin and human growth hormone (hGH) in adipose tissue from normal male rats. Insulin-like effects were measured by the ability of PGF, insulin, or hGH to stimulate oxidation of [U-14C]glucose to 14CO2, to stimulate lipogenesis, and to inhibit epinephrine-induced lipolysis. PGF and insulin stimulated significant increases in glucose oxidation and lipogenesis in adipose tissue that had not been preincubated as well as in tissue that had been preincubated. hGH stimulated insulin-like effects only in tissue that had been preincubated for 3 hr. Insulin, hGH, and PGF inhibited epinephrine-induced lipolysis of preincubated (3 hr) adipose tissue. hGH produced a dramatic lipolytic response in tissue freshly removed from normal rats but no dose of PGF was lipolytic. PGF did not displace 125I-insulin from its receptors on adipocytes but did competitively inhibit 125I-hGH binding to adipocytes. These results suggest that PGF has direct insulin-like actions which are initiated by binding a GH receptor, but PGF had no anti-insulin action and the insulin-like activity of PGF was unaffected by refractoriness of adipose tissue to GH.