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.     

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)     

Covalent binding of growth hormone to surface receptors on rat adipocytes

GH specifically binds to receptors on the surface of adipocytes and produces a variety of biological effects in these cells. To gain insight into the nature of the GH receptors, [125I] human GH ([125I]hGH) was cross-linked to surface binding sites on intact rat adipocytes using the bifunctional reagent disuccinimidyl suberate. Plasma membranes were isolated, and after solubilization with sodium dodecyl sulfate (SDS), the proteins were subjected to electrophoresis on 5% or 7.5% polyacrylamide gel. Autoradiography of the 7.5% gels revealed three iodinated bands corresponding to apparent molecular weights of 56, 130, and more than 240 kilodaltons. The more than 240-kilodalton band contained approximately as much 125I as the 130-kilodalton species and about twice as much as the 56-kilodalton species. When run on the more porous 5% gel, the more than 240-kilodalton band resolved into two bands, corresponding to apparent molecular weights of 240 and 310 kilodaltons. Excess unlabeled human or bovine GH, but not ovine PRL, competed with [125I]hGH for binding and prevented the formation of all of the labeled bands. Treatment of the membranes and extracted proteins with dithiothreitol resulted in the generation of additional 130-kilodalton material at the expense of both the 310- and 240-kilodalton species, but failed to alter the amount of 125I that migrated with the 56-kilodalton species. The same pattern of labeling was seen regardless of whether protease inhibitors were present during isolation of membrane proteins or when membrane proteins were isolated under conditions that favored proteolysis, suggesting that the 56-kilodalton species is not a degradative product of the higher molecular weight species. When [125I]hGH was cross-linked to adipocytes in which total binding was decreased by hypophysectomy or starvation of the donor rats or by treatment of the cells with cycloheximide, there was a proportionate diminution in labeling of all species. It thus appears that the GH receptor contains a 130-kilodalton subunit, a portion of which is in disulfide linkage with higher molecular weight complexes and, in addition, contains a 56-kilodalton species. It cannot be determined from these studies if the various labeled protein complexes are components of a single or multiple classes of GH receptors in the adipocyte membrane.     

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.     

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.     

Processing of growth hormone by rat adipocytes in primary culture: differentiation between release of intact hormone and degradative processing

Rat adipocytes in primary culture have been used to study the intracellular processing of GH. These classic target cells for GH have been shown to process GH through two pathways: a nondegradative pathway which resulted in the rapid release of intact GH, and a slower, degradative pathway which involved the degradation of GH and release of degraded ligand. Differentiation between the two pathways was on the basis of differences in their kinetics and temperature dependence. The present study has investigated the relative characteristics of the two pathways further. Incubation of [125I]human GH ([125I]hGH)-preloaded adipocytes with extracellular unlabeled hGH (400 ng/ml) resulted in an increase in the absolute amount of [125I]hGH released. The increased amount of [125I]hGH released was all intact. Extracellular, unlabeled hGH had no effect on the rate or amount of degraded [125I]hGH released. This suggests that the nondegradative pathway is sensitive to the number of internalized hormone-receptor complexes and that GH which is not immediately degraded or stored in the degradative pathway, is redirected and processed via the faster non-degradative pathway. Ammonium chloride (known to inhibit the lysosomal degradation of many polypeptide hormones) markedly inhibited the absolute amount of [125I]hGH released from preloaded adipocytes. This inhibition was due to an effect on the release of degraded [125I]hGH. NH4Cl had no effect on the rate or amount of intact [125I]hGH released. Finally, it was found that dinitrophenol and sodium fluoride (agents known to deplete cellular energy) inhibited the release of degraded GH but not intact GH suggesting that the degradative pathway involves an energy-dependent step, most likely the fusion of hormone-containing vesicles with the lysosomal membrane. The mechanism of release of intact hormone by energy-independent means is not yet known. These data indicate that the processing of GH by cultured rat adipocytes is complex and involves at least two independently regulated pathways, a predominant degradative route and a nondegradative route. Further studies are required to assess the possible roles of these pathways in the metabolic actions of GH in adipocytes.     

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.     

Human liver growth hormone receptor and plasma binding protein: characterization and partial purification

The human liver GH receptor has been further characterized using several biochemical approaches. Crosslinking of [125 I]human GH (hGH) to microsomal receptors and to particulate and solubilized plasma membrane receptors, followed by gel electrophoresis and autoradiography, revealed two predominant receptor-hormone complexes with a mol wt of 124,000 and 75,000, respectively. As previously shown, the 70-80 k band appears to be generated from the 124 k band in the presence of beta-mercaptoethanol, suggesting intersubunit disulfide linkages. A minor complex of mol wt 150,000 was sometimes found. By immunoblot, using a polyclonal antibody raised against a synthetic peptide (residues 391-405 of the mature human GH receptor), a single band of 100 k was detected. Human liver GH receptor and plasma GH-binding protein (BP) were purified 1,000- and 4,000-fold, respectively. The partially purified membrane receptor, analyzed by ligand-blot, showed two major bands of 55 and 32 k and minor bands of 68 and 47 k. Crosslinking of the purified GH-BP or purified receptor with [125I]hGH revealed a 75 k receptor-hormone complex. Polyclonal antibodies raised against the hepatic GH receptor inhibited the binding of hGH to the human receptor and were able to immunoprecipitate the GH receptor and also the GH-BP complex. Our findings demonstrate the existence of multiple forms of the GH receptor in human liver (major components of 100 and 50-55 k, minor component of 130 k); they lend more support to the possible subunit structure of the GH receptor; and finally, they also suggest a close relationship, with common antigenic properties, of the membrane receptor and the plasma GH-BP.     

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.     

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.     

Cellular processing of growth hormone in IM-9 cells: evidence for exocytosis of internalized hormone.

IM-9 cells extensively internalize [125I]human (h) GH at physiological temperatures, yet little is known regarding the final destination of internalized hormone and its receptor. We studied this by first binding [125I]hGH to the cell surface at 4 C, and then following its fate during a subsequent incubation at 30 C in isotope-free medium. Cell-associated radioactivity decreased with time at 30 C, with a biphasic pattern suggestive of a rapid (but minor) and a slow component. The kinetics of the latter were critically influenced by NH4Cl and were abolished at 20 C. Intracellular (acid-resistant) [125I]hGH first increased with time at 30 C until it reached a maximum after 1 h, then declined continuously upon prolonged incubation. The radioactivity released by the cells was recovered in the medium as both trichloroacetic acid-precipitable material and trichloroacetic acid-soluble fragments. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, one major band migrating with an estimated mol wt of 22,000 was identified, presumably corresponding to intact [125I]hGH. These data suggest exocytosis of intact hormone via recycling endosomes and degradation in the lysosomes, respectively. Computer modelling was consistent with two intracellular compartments acting partially in series and probably corresponding to these two organelles. When analyzed by computer curve fitting, this model accurately described the kinetics of [125I]hGH internalization. So, receptor-mediated endocytosis and subsequent exocytosis are part of the GH pathway in IM-9 cells. In as much as they reflect pathways of GH receptors, these processes contribute to receptor down-regulation and could provide an explanation for release into the medium of the high affinity GH-binding protein.     

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.     

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.     

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.     

Certain large forms of circulating immunoreactive human growth hormone are in fact immunoglobulins

To explain frequent discordances between serum GH levels and clinical manifestation of acromegaly, we investigated the possibility that certain immunoglobulins G (IgGs) might be responsible for the displacement of [125I]human (h) GH in the hGH RIA. We incubated dilute sera from seven active acromegalics (basal immunoreactive hGH, 22-313 micrograms/L) with rat adipocyte plasma membranes adsorbed on polystyrene plates. IgGs that bound to GH receptor sites in the absence and presence of 250 nM hGH (for nonspecific binding) were detected using anti-hIgG (Fc-specific) antibody conjugated with alkaline phosphatase. In this system two of the seven sera studied tested positive for IgGs against GH-binding sites (serum 4 in 1:400 dilution, and serum 7 in 1:10 dilution). We studied further the serum with the highest titer. On Sephadex G-100, most of the GH-like immunoreactivity (assayed by RIA) present in serum 4 coeluted with IgGs (assayed by immunodiffusion) as a high mol wt (greater than or equal to 150 kDa) component. To confirm its IgG nature, this material was then adsorbed on protein-A-Sepharose and eluted with 0.1 M sodium citrate, pH 3.0. The protein-A-purified IgGs from serum 4 bound specifically to GH receptor sites in adipocyte membranes and displaced [125I]hGH in the hGH RIA. In contrast, IgGs purified from another acromegalic patient (313 micrograms/L hGH) repeatedly tested negative in the membrane binding assay and hGH RIA.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Release of growth hormone binding protein from IM-9 lymphocytes by endopeptidase is dependent on sulfhydryl group inactivation

The hydrophilic GH-binding protein of serum is a derivative of the GH receptor. Little is known how this GH binding protein is released from the receptor which is firmly anchored in the plasma membrane. The IM-9 lymphocytes provide a useful laboratory model for studying this process because they are richly endowed with GH receptors and, under special conditions, are able to shed these receptors during incubation. Incubation of IM-9 cells for 90 min at 30 C did not result in the appearance of significant [125I]hGH binding in conditioned medium as determined with an ultrogel AcA 44 minicolumn. When iodoacetamide, 20 mM, or N-ethylmaleimide, 5 mM, was added during incubation, the conditioned medium bound 20-35% of [125I]human(h)GH. p-Chloromercuriphenyl sulfonic acid was less effective in promoting shedding of GH-binding protein. In contrast, aprotinin, phenylmethylsulfonylfluoride (PMSF), bacitracin, leupeptin, pepstatin, phosphoramidon, or chloroquine did not promote release of GH binding protein and did not affect iodoacetamide-induced release. Release was not inhibited by the addition of serum lacking GH binding protein. GH binding protein release was markedly temperature sensitive and practically ceased at 4 C. GH binding protein incubated with [125I] hGH was cross-linked with disuccinimidyl suberate. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol the complex migrated with an estimated molecular weight of 100,000 whereas [125I]hGH cross-linked to the membrane-bound GH receptor of the IM-9 cells migrated with an estimated molecular weight of 135,000. The smaller size of the binding protein is consistent with its derivation from the extracellular domain of the GH receptor. Because the release of this GH binding is greatly augmented by iodoacetamide and N-ethylmaleimide, two known sulfhydryl reactive reagents, we suggest that a free sulfhydryl group, either on the GH receptor or on a neighboring protein normally maintains the integrity of the receptor. The loss of this sulfhydryl group destabilizes the receptor and permits a membrane endopeptidase to release the GH binding protein. Cleavage is not dependent on lysosomal action and is not inhibited by protease inhibitors.     

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.     

Pituitary and recombinant deoxyribonucleic acid-derived human growth hormones alter glucose metabolism in 3T3 adipocytes

3T3-F442A adipocytes were used to compare the effects on glucose metabolism of pituitary human (h) GH and methionyl-hGH produced by recombinant DNA techniques (met-hGH 22K). Pituitary hGH and met-hGH 22K were similar in their ability to inhibit glucose oxidation and lipid accumulation after 48 h. After 4 h of incubation, both forms of hGH stimulated glucose oxidation transiently in 3T3 adipocytes. A bacterially produced form of the 20,000-dalton variant of hGH (met-hGH 20K) also stimulated glucose oxidation at 4 h and inhibited glucose oxidation and lipid synthesis after 48 h. All three forms of hGH had a similar ability to inhibit [125I]iodo-met-hGH 22K binding to 3T3-adipocytes. Thus, met-hGH 22K and 20K directly produce in 3T3 adipocytes the transient stimulation and delayed inhibition of glucose metabolism attributed to pituitary hGH, indicating that these metabolic effects are intrinsic to the hGH molecule.