Receptor-mediated endocytosis and degradative processing of growth hormone by rat adipocytes in primary culture

At 37 degrees C, cultured rat adipocytes bound [125I]human GH ([125I]hGH) rapidly, with binding being detectable within 1 min of incubation. The bound [125I]hGH was then internalized (within 10 min) and accumulated in the cell interior until a steady state was reached (by 60 min). At this time, where the rates of GH internalization, processing, and release are equivalent, 55% of total cell-associated [125I]hGH was intracellular. Internalization of [125I]hGH by acutely isolated (noncultured) adipocytes was preceded by a 20-min lag phase indicative of a temporary postbinding defect. The lag phase was not seen with cultured adipocytes. After preloading of [125I]hGH into the cell interior, cultured cells rapidly released [125I]hGH (t1/2 = 20-30 min) into the extracellular medium as both intact (25%) and degraded (75%) GH. The release of intact vs. degraded GH was distinguishable on the basis of kinetics and temperature dependence. In order to determine when internalized [125I]hGH entered a catabolic compartment, cultured adipocytes were incubated with [125I]hGH and the composition of intracellular GH was determined as a function of time. All [125I]hGH internalized during the first 20 min was intact. Between 20 and 30 min some of the internalized [125I]hGH entered a catabolic compartment and degradation products began accumulating within the adipocytes. Release of degraded [125I]hGH from cultured adipocytes began at 60 min. The processing of GH through the complete degradative pathway (binding, internalization, degradation, release) required a period of 1 h at 37 degrees C.     

Relationship between binding and biological effects of human growth hormone in rat adipocytes

Insulin-like responses to human GH (hGH) were produced in adipocytes isolated from the epididymal fat of normal rats 3 h after excision of the tissues. Insulin-like responses consisted of increased oxidation of glucose and incorporation of its carbons into total lipid, increased oxidation of L-[1-14C]leucine, and antagonism of the lipolytic actions of epinephrine. Refractoriness to these effects of hGH in the fourth hour of incubation was produced by the addition of as little as 3 ng/ml hGH as soon as possible after excision of the tissues. These cells also responded to the delayed lipolytic effect of hGH in the presence of theophylline. The cells were found to have high affinity, low capacity, specific binding sites for 125I-labeled hGH. Monoiodination of hGH did not interfere with its capacity to produce biological responses. Specific binding equilibrated rapidly and appeared to saturate at about 100 ng/ml. In cells that were capable of exhibiting an insulin-like response to hGH, rat and ovine GH successfully competed with [125I]hGH for binding sites, but porcine insulin, at a concentration of 100 mU/ml, failed to reduce the binding of [125I]hGH, indicating that GH does not produce its insulin-like effects by interacting with the insulin receptor. Binding of [125I]hGH in cells that are refractory to the insulin-like effects of GH is indistinguishable from binding in responsive cells. Scatchard analysis of the data for both responsive and refractory cells gave linear plots consistent with a single class of about 20,000 receptors/cell, which become half-saturated at a concentration of approximately 20 ng/ml. This corresponds well with 30-50 ng/ml needed for half-maximal insulin-like responses and the 3-10 ng/ml ED50 for induction of refractoriness or lipolysis. It thus appears unlikely that there are appreciable spare receptors for insulin-like responses. These findings make it likely that refractoriness to the insulin-like effects of GH occurs at a postreceptor site.     

Growth hormone receptors in isolated rat adipocytes

Specific GH binding sites in isolated rat adipocytes have been partially characterized. Binding of [125I]iodohuman(h)GH was rapid, reversible, and was time and temperature dependent. Maximum specific binding occurred at 37 C in approximately 40 min at pH 7.4. Bound labeled hGH was rapidly dissociable, with the addition of excess unlabeled hormone. Specific binding is inhibited by as little as 1.0-1.5 ng/ml hGH, and 50% inhibition was obtained with 15-20 ng/ml. No inhibition was observed with insulin, glucagon, hPRL, or hTSH at concentrations up to 1 micrograms/ml. This receptor does not discriminate between monkey GH, rat GH, bovine GH, and porcine GH. Specific binding varied linearly with cell concentration. Scatchard analysis revealed linear plots with a Ka of approximately 10(9) M-1 and 15,000 sites per cell. There was less than 15% degradation of [125I]iodo-hGH over 90 min at 37 C. There was a striking increase in [125I]iodo-hGH binding to adipocytes at pH 4.85. Scatchard analysis of binding at pH 4.85 revealed a curvilinear plot with an apparent increase of sites per cell from 15,000 to 60,000, and a modest increase in the apparent affinity constant of the high affinity, low capacity sites using the two-compartment model for curvilinear plots. The GH receptors in rat fat cells displayed no ability to bind labeled hPRL or human placental lactogen, consistent with minimal recognition of lactogenic peptides by these receptors. Thus, the rat adipocyte contains specific binding sites for GH that fulfill the major criteria for receptor binding. The presence of such receptors in these cells may facilitate the study of GH receptors in relation to the biological effects of the hormone on adipose tissue in various metabolic settings.     

Characterization and modulation of growth hormone and prolactin binding in mouse liver.

The specific binding of 125I-labeled insulin, human hormone ([125I]hGH), bovine growth hormone ([125I]bGH), and ovine prolactin ([125I]oPRL) was studied in mouse liver membranes. [125I]hGH and [125I]oPRL bound to adult liver membranes. Pregnancy increased the specific binding of [125I]hGH but not that of [125I]oPRL. [125I]hGH was displaced from membranes of pregnant mice by hGH, oPRL, and bGH, but only by hGH and oPRL from liver membranes of nonpregnant mice. Significant specific binding of [125I]bGH was seen only in pregnancy. The binding of [125I]bGH to pregnant mouse liver membranes increased with increasing concentration of either membrane protein or [125I]bGH. Both the specific binding and dissociation of [125I]bGH were greatly influenced by the time and temperature of incubation. Binding of [125I]bGH was inhibited by growth hormones, including hGH and rat GH, and not by lactogenic hormones (various prolactins and human placental lactogen), ACTH, glucagon, or insulin. The inhibition of [125I]hGH binding by hGH and bGH, in the presence of excess (2 mug/ml) of PRL, was very similar to that seen with [125I]bGH. Scatchard plots of displacement dose-response curves obtained under steady state conditions of 4C were nonlinear and very similar with either [125I]bGH or [125I]hGH. This contrasted with the linear Scatchard plots obtained from displacement dose-response curves of either [125I]oPRL or [125I]hGH in the presence of excess (2 mug/ml) bGH. Termination of pregnancy, either naturally or by hysterectomy, reduced [125I]bGH specific binding to nonpregnant levels by 24 to 36 h. Estrogen administration did not increase [125I]bGH binding in hepatic membranes. Nonpregnant mice possess hepatic lactogen binding sites which are uninfluenced by pregnancy. GH specific binding sites are markedly augmented during pregnancy. The close correlation between the level of these sites and pregnancy suggests that they are regulated by a product of the fetoplacental unit.     

Specific growth hormone receptors on human peripheral mononuclear cells: reexpression, identification, and characterization

Although specific GH receptors have been demonstrated in various tissues of a number of species, the presence of GH receptors on human peripheral mononuclear cells (PMC) is controversial. Binding of human GH (hGH) to its receptor as the hypothesized initial step of hormone action was consequently studied using mononuclear cells from peripheral venous blood of normal subjects. Specific binding of [125I]hGH was rapid, reversible, and time and temperature dependent. Specific GH binding to PMC was maximal after 8-24 h of preincubation. Binding of hormone was maximal at 37 C after incubation of cells for 2 h. Dissociation of GH was maximal at 37 C after the addition of 6 M NaCl. A linear relationship between specific GH binding and cell number was found. Saturation of GH binding to 10(6) PMC was obtained with 25 ng iodinated hormones. Half-maximal inhibition of GH binding occurred at 12-25 ng unlabeled hGH/tube. Hypothalamic and pituitary hormones as well as insulin did not interfere with specific hGH binding to PMC. Scatchard analysis of [125I]hGH binding to PMC revealed a receptor with a mean affinity constant of 1.5 +/- 0.2 (+/- SD) X 10(9)/M-1 (n = 72) and a maximal binding capacity of 7.1 +/- 2.0 X 10(-11) M/10(6) cells. The concentrations of calcium, sodium, and magnesium ions in the incubation medium strongly influenced GH binding, whereas pH or potassium concentration did not. As interassay variation of the binding assay was low (14% for total binding; 6% for specific hGH binding), this direct approach to study tissue receptors for hGH in a human in vitro test was reproducible and should encourage the investigation of receptor regulation as well as the study of binding in human disease.     

Solubilization of a growth hormone-specific receptor from rabbit liver.

Membrane preparations from rabbit liver, known to possess GH-specific binding sites, have been solubilized with Triton X-100 and the binding characteristics of [125I]-human GH (hGH) and [125I]-bovine GH (bGH) subsequently studied. Specific binding of the hGH and bGH by the solubilized preparation was demonstrated of bound and free hormone by either polyethylene glycol precipitation or by Sephadex G-100 chromatography. Binding of hGH was both rapid and reversible and was displaced only by other growth hormones (bovine and ovine) and not by lactogenic hormones (ovine and human prolactins, human placental lactogen). As shown by Scatchard analysis specific binding of [125I]-bGH exhibited a lower binding affinity and capacity than did [125I]-hGH. Overall, the characteristics of the binding reaction for hGH were not significantly different from those reported for the particulate membrane preparation. The solubilization process did not appear to alter the binding protein(s) therefore, and permits a further study of the isolation, purification and properties of the binding protein(s) itself.     

Growth hormone maintains its own receptors in rat adipocytes

Hypophysectomy decreased the capacity of adipocytes isolated from epididymal fat to bind [125I]human GH [( 125I]hGH) specifically without changing the apparent affinity for hGH. Specific binding of hGH by adipocytes of both normal and hypophysectomized rats appeared saturated when incubated with 75-80 ng/ml or higher concentrations of GH regardless of whether binding was studied for 2 h at 37 C or for 16 h at 0 C. Maximum binding of hGH by normal adipocytes was approximately 0.45 ng/10(6) cells, and that by adipocytes of hypophysectomized rats ranged from 0.15-0.25 ng/10(6) cells. In cells of both normal and hypophysectomized rats, only 25-30% of the hormone specifically bound at 37 was removed by digestion with trypsin, and about 75% was displaced by incubation with 5 M magnesium chloride, suggesting that these adipocytes internalized a significant fraction of bound hormone and that hypophysectomy did not alter the extent of internalization. Previously bound hormone was lost from normal adipocytes with a half-time of about 32 min and from adipocytes of hypophysectomized rats with a half-time of about 45 min, suggesting that hypophysectomy slowed the rate of processing bound hormone. To determine which pituitary hormone(s) might be required to maintain GH binding, we measured the binding of [125I]hGH at 3 or 30 ng/ml by fat cells prepared from hypophysectomized rats after various treatment regimens. Administration of bovine GH ip at a dose of 10 micrograms/rat every 4 h for 24 h doubled the binding of [125I]hGH by adipocytes prepared 4 h after the last injection. Similar results were obtained in fat cells examined 4 h after only one injection of 60 micrograms bovine GH to rats hypophysectomized 2-4 weeks previously. When binding was measured 16-24 h after GH administration, there was no apparent effect on restoration of binding even after treatment with 100 micrograms GH/day for up to 6 days, suggesting that the effects of GH in maintaining receptor number are transient. In accord with the apparently short-lived ability of GH to maintain its receptors on fat cells, GH binding was significantly reduced in adipocytes obtained form both hypophysectomized and sham-operated rats as early as 4 h after surgery, and by 8 h after surgery, declined to a level as low as that in adipocytes of chronically hypophysectomized rats. Twenty-four hours after surgery, GH binding by cells of sham-operated animals returned to normal. Fasting for 24 h also reduced GH binding by adipocytes of normal rats to a level comparable to that in adipocytes of fed hypophysectomized animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of growth hormone binding protein in rat serum

The present report describes the initial characterization of a specific, high-affinity growth hormone binding protein (GH-BP) in adult male rat serum. GH-BP activity was measured by incubation of rat serum with [125I]hGH and [125I]rGH and separation of bound from free GH by dextran-coated charcoal. [125I]hGH binding to rat serum was dependent on serum concentration and incubation time, equilibrium being reached within 10 min both at 4 and 37 degrees C. Binding was rapidly and completely reversible and specific for somatogenic (but not lactogenic) hormones. Scatchard analysis yielded a linear plot with an affinity (Ka) of 1.51 +/- 0.63 x 10(8) M-1. Preliminary data obtained in various physiological conditions showed that GH-BP activity in adult male rats was 5.95 +/- 0.20%/0.1 ml serum. Significantly higher values were obtained in sera of female (21.66 + 0.79%/0.1 ml serum) and pregnant rats (23.02 +/- 1.15%/0.1 ml serum). A closer analysis of these binding values by Scatchard analysis revealed that the binding capacity in pregnant rats (50.5 +/- 5.8 pmol/0.1 ml serum) was significantly higher than in adult female estrous rats (19.2 +/- 6.5 pmol/0.1 ml serum), both being much higher than in adult male rats (2.5 +/- 0.6 pmol/0.1 ml serum). The GH-BP activity of 10-day-old rats was only approximately 63% of the adult male rat value. The presence of high-affinity GH-specific binding protein in rat serum suggests a probable action in regulation of GH activity. The detailed physiological role of rat serum GH-BP is currently being investigated.     

Reduction of lactogenic receptors in female hamster liver due to the human growth hormone analog produced by plerocercoids of the tapeworm, Spirometra mansonoides

The inductive effect of GH on hepatic lactogenic receptors is suspected of being due to a direct somatogenic action. Plerocercoid larvae of the tapeworm, Spriometra mansonoides, produce a factor that stimulates body growth, suppresses endogenous GH, and specifically displaces [125I]human (h) GH from hepatic receptors. Plerocercoid growth factor (PGF) mimics the growth-promoting actions of GH, but it has not been shown to duplicate all of the activities reported for GH. An important function of GH is its role in the maintenance of liver receptors for lactogenic hormones. This study was undertaken to determine if treatment of female hamsters with PGF would increase, decrease, or have no effect on liver receptors that bind hGH. Since hGH binds to somatogenic as well as lactogenic receptors, it was necessary to demonstrate the specificity of PGF's effects on [125I]hGH binding. PGF-treated (15 pleocercoids sc) hamsters had accelerated body growth, suppressed serum GH, and a marked reduction in [125I]hGH and [125I]ovine PRL binding to hepatic microsomes. Specific binding of [125I] bGH was unaltered by PGF treatment. The difference in [125I] hGH binding was due to a reduction in receptor number and not to receptor occupancy or reduced affinity. Serum GH was normalized after 10 days of estradiol benzoate (25 micrograms/day) injections, but the binding capacity for [125I]hGH of the PGF-treated group was less than half that of the control group. The fact that estrogen injections normalized serum GH, but not hGH binding, indicates that down-regulation of these receptors by PGF cannot be entirely explained on the basis of reduced levels of serum GH. The lack of any effect of PGF treatment on [125I]bGH binding suggests that the hepatic somatogenic receptors were not involved and that the reduction in receptors for [125I]hGH was associated with the lactogenic component of hGH.     

Role of disulfide bonds in human growth hormone binding and dissociation in isolated rat hepatocytes and liver plasma membranes

The role of disulfide bonds and sulfhydryl groups in rat hepatocytes and rat liver plasma membranes in the binding of human GH (hGH) has been studied. Since hGH binding involves uptake and irreversible binding, the effect of disulfide reducing agents [dithiothreitol (DTT) and 2-mercaptoethanol (ME)] and an alkylating agent [N-ethylmaleimide (NEM)] on the time course of binding and displacement of [125I]hGH was determined in the hepatocytes and membranes. The time course of binding and displacement of [125I]hGH was similar in membranes and cells, indicating that the irreversible nature of hGH binding is not dependent upon an intact cellular structure. Both 1% ME and 10 mM DTT prevented further binding of [125I]hGH when added at 60 min of the 300-min binding incubation for both hepatocytes and plasma membranes. The ME caused some initial dissociation of bound [125I]hGH, with subsequent binding to levels that were present when the ME was added. Only ME caused an increase in nonspecific binding with the plasma membranes. Both ME and DTT caused an increase in displacement of [125I]hGH in the presence of excess unlabeled hGH. The amount of [125I]hGH remaining bound in the presence of DTT and unlabeled hGH approached nonspecific levels by 240 min of incubation. NEM caused an increase in the total [125I]hGH bound, but this was apparently due to increased nonspecific binding in the presence of NEM. The effect of the reducers on binding was not secondary to an effect on hGH, since the disulfides of [125I]hGH were not reduced under the conditions of binding with either ME or DTT. The effect of the ME or DTT on binding could be reversed or prevented by subsequent or simultaneous addition of an oxidizer such as NAD or oxidized glutathione. The data indicate that disulfide bonds in the membranes are intimately involved in the maintenance of a receptor structure necessary for hGH binding. The disruption of the disulfides also results in increased dissociation and displacement of the bound [125I]hGH, indicating a possible role in the irreversible nature of hGH binding. This represents a partial delineation of the hGH binding process.     

Adenosine 3',5'-monophosphate-dependent loss of growth hormone binding in rat adipocytes

GH exerts a number of metabolic effects on adipose tissue. Depending on the circumstances, it may increase or decrease glucose metabolism and lipolysis. These effects appear to be mediated by a single class of receptors, which bind GH with high affinity. Incubation of isolated rat adipocytes with a variety of lipolytic agents, including catecholamines, forskolin, or (Bu)2cAMP, decreased the specific binding of [125I]human (h) GH within 10 min. In the presence of 10 microM forskolin, GH binding declined to less than 20% of the control value within 50 min. Cholera and pertussis toxins, which increase cAMP secondary to ADP ribosylation of guanine nucleotide-binding proteins associated with hormone receptors, also decreased the binding of GH. None of these agents affected the rate of loss of cell-associated 125I when added to cells that had previously equilibrated with [125I]hGH. The inhibitory effects of forskolin and (Bu)2cAMP were at least as great when binding was measured in the presence of the protease inhibitor leupeptin, suggesting that increased rates of internalization and processing of bound hormone could not account for the decline in binding. Scatchard plots of data obtained in the presence of forskolin or (Bu)2cAMP were linear and parallel to control plots, indicating that the decline in binding could be accounted for by a decrease in the number of binding sites, with no change in affinity. To determine whether phosphorylation affected binding to receptors already present in the membrane or modified the turnover of receptors, we studied adipocyte ghosts, whose cellular apparatus for receptor turnover is disrupted. Incubation of adipocyte ghosts with cAMP-dependent protein kinase decreased the binding of [125I]hGH by 25%. The data suggest that cAMP-dependent phosphorylation of the GH receptor or a closely associated membrane protein renders the receptor incapable of binding GH.     

Enhancement of rat growth hormone binding by membrane disulfide reduction

The effect of disulfide reduction on the binding of [125I]rat GH (rGH) to rat liver plasma membranes and hepatocytes was studied to determine the role of disulfide bonds in the binding of GH to its receptor. The total amount of [125I] rGH bound to the liver receptors increased severalfold in the presence of dithiothreitol and mercaptoethanol. The nonspecific binding also increased at higher concentrations of the reductant, but the amount specifically bound was still greater in the presence of disulfide reductant. In contrast, the disulfide reductant inhibited [125I] human GH (hGH) binding and enhanced its displacement from hypophysectomized female rat hepatocytes. This was similar to the effect of reductants on [125I]hGH binding to normal female rat hepatocytes. The effect of the disulfide reductants on [125I]rGH binding could be prevented or reversed by the simultaneous or subsequent addition of an oxidizing agent such as NAD or oxidized glutathione. Sulfhydryl-reactive agents such as iodoacetamide prevented additional binding of [125I]rGH when added at 30 min of the incubation. The additional [125I] rGH bound in the presence of disulfide reductant was displaceable by excess unlabeled rGH. Both rGH and hGH exhibited similar degrees of disulfide reduction in the presence of mercaptoethanol. The disulfide reductant produced effects on binding at concentrations that resulted in less than 10% reduction of the GH disulfides. We conclude that: 1) the disulfides and sulfhydryls of the hepatocyte membrane are intimately involved in the binding of GH to hepatic receptors; 2) the locus of the disulfides and sulfhydryls may be in the subunit structure of the membrane receptor, but this will require verification using soluble receptors; and 3) the effect of disulfide reducing agents reveals basic differences in the mechanism of binding of rGH and hGH to somatotropic hormone receptors on the hepatocytes.     

Specific binding sites for prolactin and growth hormone in the adult rabbit lung

Specific prolactin (PRL) and growth hormone (GH) binding sites were identified and characterized in lung membranes from male and female adult rabbits. The binding of iodinated human GH ([125I]iodo-hGH) and iodinated ovine PRL ([125I]iodo-oPRL) was time, temperature and protein dependent and was found to conform to the requirements defining a physiological receptor, in terms of hormonal and immunological specificities as well as kinetic properties. [125I]Iodo-hGH was displaced from lung membranes by hGH, oPRL, ovine GH and rat GH, while [125I]iodo-oPRL was effectively displaced only by oPRL and hGH. Scatchard plots of the competition curves of [125I]iodo-hGH and [125I]iodo-oPRL were both linear, suggesting, in each case, a single class of binding sites with affinity constants (Ka) of 1.74 +/- 0.64 X 10(9) M-1 and 0.78 +/- 0.28 X 10(9) M-1 and binding capacities of 6.43 +/- 0.53 and 4.16 +/- 0.69 fmol/mg protein, respectively. Anti-PRL-receptor antiserum significantly inhibited the binding of the [125I]iodo-oPRL to rabbit lung membranes, while it was less potent in preventing the binding of [125I]iodo-hGH, which has both lactogenic and somatogenic activity. Removal of endogenous ligand by treating lung membranes with 4 M MgCl2 increased specific binding of hGH about 2.5-fold, exposing additional specific binding sites without significantly changing the binding affinity. The level of binding of hGH and oPRL to rabbit lung did not show a pronounced sex differentiation. In summary, PRL and GH binding sites have been demonstrated for the first time in adult rabbit lung membranes, and they support the possibility of a physiological role for PRL and GH in the lung.     

The mouse fibroblast growth hormone receptor: ligand processing and receptor modulation and turnover

The recent observation that adipose conversion of mouse 3T3 fibroblasts is stimulated by physiological concentrations of human GH (hGH) and rat GH in vitro suggested that this cell line may be suitable for the study of GH-receptor interactions. The aim of this study was to examine the binding and subsequent processing of [125I]iodo-hGH by BALB/c 3T3 mouse fibroblasts. Binding of [125I]iodo-hGH to 3T3 fibroblasts was time and temperature dependent. Apparent steady state binding was achieved after 1 and 2 h at 37 and 30 C, respectively. At 37, 30, and 20 C specifically bound [125I]iodo-hGH became increasingly resistant to removal by acid treatment (0.15 M NaCl/0.05 M glycine, pH 2.5). In contrast at 4 C or at higher temperatures in the presence of metabolic inhibitors, a greater proportion of specifically bound hGH was removed by acid treatment. Inclusion of 0.2 mM chloroquine in the incubation medium resulted in significantly more accumulation of trichloroacetic acid (TCA)-precipitable radioactivity compared to control cells without affecting the shift of radioactivity from the acid-elutable to the acid-inaccessible compartment. After removal of [125I]iodo-hGH from the medium there was a rapid loss of radioactivity (t 1/2 = 36.5 +/- 7.2 min, SE, n = 3) from the cell monolayer with a concomitant appearance in the medium of TCA-soluble radioactive species. Chloroquine reduced the rate of efflux of radioactivity from the monolayer (t 1/2 = 4.5 +/- 0.6 h, n = 3) and the appearance of TCA-soluble material in the medium. The half-time of GH receptor loss after inhibition of protein synthesis with cycloheximide (0.1 mM) was 1.25 +/- 0.14 h, n = 3). In contrast half-time of net receptor synthesis calculated from the recovery of specific [125I]iodo-hGH binding capacity after ligand-induced down-regulation was 10.2 +/- 1.5 h, n = 3). These data reveal that after binding of [125I]iodo-hGH to specific cell surface receptors there is rapid irreversible binding of GH to its receptor with a resultant reduction in receptor concentration. Degradation of [125I]iodo-hGH occurs intracellularly and involves processes which are inhibited by lysosomotropic agents. On the basis of these studies we conclude that the binding and subsequent processing of GH by 3T3 fibroblasts is qualitatively similar to that described for other polypeptide hormones and growth factors in this and other cell lines.     

Effects of tunicamycin on growth hormone binding in rat adipocytes

Digestion of covalently linked [125I]human (h) GH-receptor complexes with neuraminidase or endoglycosidase F reduced the mass of the principal hormone receptor complex from about 130 kilodaltons (kDa) to 120 and 110 kDa, respectively, suggesting that about 20% of the mass of the GH receptor of rat adipocytes consists of N-linked sialocarbohydrates. Incubation of adipocytes with tunicamycin, an inhibitor of N-linked glycosylation, decreased the incorporation of [35S]methionine into membrane glycoproteins by more than 50% in 4 h and decreased specific binding of [125I]hGH by about 70% after 8 h. Decreased binding and incorporation of [35S]methionine were seen only after a lag time of about 2 h. Cross-linking of [125I] hGH to cells that had been treated with tunicamycin resulted in the appearance of a new labeled species of hormone-receptor complex with an apparent mass of about 110 kDa. This band appeared after a delay of about 3 h and reached approximately equal prominence with the 130 kDa band at 5 h. By 8 h, the 110 kDa complex was the predominant band in radioautograms, but some of the 130 kDa species remained. Scatchard analysis of binding data in tunicamycin-treated adipocytes indicated that decreased binding of [125I]hGH resulted from a 3- to 4-fold decrease in affinity accompanied by only a small (30%) decline in receptor number. Tunicamycin did not affect the rate of receptor turnover in cells that were also treated with cycloheximide to block protein synthesis, but receptor turnover decelerated with increasing time of incubation. Treatment with tunicamycin for 8 h markedly slowed the rate at which specifically bound [125I]hGH disappeared from adipocytes, suggesting that N-linked carbohydrates may play some role in internalization and processing of labeled hormone. We conclude that 1) N-linked carbohydrates contribute about 20 kDa to the apparent mass of the GH receptor of rat adipocytes; 2) N-linked glycosylation is not required for GH receptors to be inserted into the adipocyte membrane in the proper orientation and to retain their ability to recognize and bind GH; 3) N-linked sugar chains are required for maintenance of a normal high affinity of receptors for GH; 4) N-linked carbohydrates are necessary for normal rates of internalization and processing of bound hGH.     

The human liver growth hormone receptor

Human livers, obtained from donors at the time of transplant, were homogenized in 0.25 M sucrose and fractionated by differential centrifugation. The specific binding of [125I] human (h) GH to total particulate fractions from 18 livers varied from 0.4-5.1% of the total radioactivity/100 micrograms protein. Binding affinity was 2.0 +/- 0.3 X 10(9) M-1, and binding capacity ranged from 14-53 fmol/mg protein. A different proportion of receptors occupied by endogenous hGH did not explain the large variation in binding. Binding sites were specific for hGH. Dissociation of the hormone-receptor complex was extremely slow. No specific binding of [125I]hPRL was observed. Specific binding of insulin was found in fractions from all livers and varied less than hGH binding. Cross-linking of [125I]hGH to plasma membrane and microsome receptors yielded two major autoradiographic bands corresponding to an estimated mol wt of 103,000 for the receptor, with a possible subunit of 54,000. Human liver primary fractions were characterized. The binding of hGH and insulin displayed a nucleo-microsomal distribution pattern in the primary fractions; 54.2% and 27.9% of the hGH-binding activity were found in the microsomes and the nuclear fraction, respectively, whereas insulin binds equally to nuclear and microsomal elements. Our findings suggest that hGH-binding sites are present in the plasma membrane and also in one or more intracellular compartments, whereas a high proportion of insulin receptors is associated with the plasma membrane.     

Relationship between increased binding and insulin-like effects of human growth hormone in adipocytes from young fa/fa rats

Binding and insulin-like effects of human GH (hGH) in inguinal adipocytes from young lean Fa/fa and obese fa/fa Zucker rats were studied. The binding of [125I]hGH per unit surface area is 2-fold higher in adipocytes prepared from 16- and 30-day-old fa/fa rats than in cells from lean littermates. A 3-h preincubation of the cells increases the hGH-binding capacity without changing the affinity of the binding, regardless of genotype. Freshly isolated adipocytes from 30-day-old lean rats fail to respond to hGH, whereas after preincubation of the cells, hGH produces a maximal 105% increase in glucose transport and a maximal 40% increase in glucose oxidation. In contrast, freshly isolated adipose cells from fa/fa rats are already responsive to hGH and the amplitude of the response is markedly elevated in preincubated cells, with a 430% stimulation of glucose transport. The concentration of hGH (1 nM) that inhibits 50% of [125I]hGH binding and that which produces half-maximal stimulation of glucose transport as well as glucose metabolism are not different, suggesting the absence of spare receptors for these insulin-like effects of hGH. Plots of GH effects on glucose transport as a function of receptor occupancy are linear, with a change in the slope after preincubation. Our results suggest a strong correlation between binding of hGH and actions on glucose transport and glucose metabolism in adipocytes of young Zucker rats.     

Binding of bovine growth hormone to bovine liver membranes

The binding of [125I]bGH and [125I]hGH to bovine liver membranes is compared to characterize the somatotrophic hormone-receptor interaction. [125I]bGH binding exhibits higher nonspecific binding than [125I]hGH while the time-course of binding and displacement with unlabeled GH are similar. Divalent and monovalent cations enhance [125I]hGH binding with well-defined peaks of binding at specific cation concentrations. Monovalent cations do not enhance [125I]bGH binding at concentrations of 100 mM while divalent cations enhance binding over a range of cation concentration (4-80 mM). The binding of [125I]bGH is dependent upon the presence of divalent cations, with minimal effect of pH upon binding in the absence of calcium. Scatchard plots of bGH and hGH binding data indicate at least two binding sites. We conclude that somatotrophic GH exhibits unique and distinguishing characteristics of binding. The characteristics of hGH binding to the bovine liver membranes suggest that its binding may differ from bGH binding to its homologous receptor.     

Monoclonal antibodies to the rabbit liver growth hormone receptor: production and characterization

Monoclonal antibodies to the GH receptor (GHR) have been produced by the application of hybridoma technology to splenic lymphocytes from BALB/C mice immunized with a human (hGH) affinity purified preparation of rabbit liver GHR. Primary screening of 384 wells yielded 4 antibodies able to immunoprecipitate [125I]iodo-hGH complexes with purified GHR and one able to inhibit binding of [125I]iodoovine GH ([125I]iodo-oGH) to rabbit liver microsomes. These cells were cloned and grown as ascitic tumors with loss of 1 of the 4 precipitators. Ascitic fluids contained monoclonal antibodies of high titer (inhibitor and 2 precipitators, 1:2.0 X 10(5); one precipitator, 1:2.0 X 10(4)) and high affinity (precipitators, 2.5-6.0 X 10(9) M-1; inhibitor, high affinity component, 6.4 X 10(10) M-1), which were isotyped as IgG1K and IgG2aK (1 precipitator). These antibodies did not cross-react with rabbit insulin or PRL receptors in the appropriate receptor assays and did not possess antihormone activity. Binding of [125I]iodo-MAb7, the inhibitory antibody, was totally blocked by the addition of excess unlabeled oGH or hGH, although these hormones had no effect on binding of the 125I-labeled precipitators. Scatchard analysis of [125I]iodo-oGH binding in the presence of MAb7 showed decreased binding by loss of sites rather than affinity. Antibody dilution curves and Scatchard plots for MAb7 binding provided evidence for two types of GHR in the rabbit liver, in accord with previously published data based on hormone binding studies. All precipitating antibodies gave an enhancement of [125I] iodo-oGH binding with purified receptor (up to 360% of polyethylene glycol-precipitated control), but only minimal enhancement with solubilized microsomal membranes. This enhancement was shown to be due to an increase in receptor number rather than affinity. After examining a number of hypotheses, we concluded that the enhancement was an artifact resulting from a nonpolyethylene glycol-precipitable species of GHR which could be totally precipitated by the monoclonal antibodies. We have produced and characterized four monoclonal antibodies to the GHR which will be of value in characterizing the structure and function of this receptor.