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.     

Partial purification and characterization of bovine mammary gland prolactin receptor

Prolactin (PRL) receptors from the mammary gland of the lactating cow were solubilized with 3-[(3-cholamidopropyl)dimethylamonio]-1-propane sulfonate (CHAPS). Affinite chromatography on human growth hormone (hGH) coupled to Affi-Gel 10 resulted in over 500-fold purification, as compared to microsomal fractions. Scatchard analysis of the binding of hGH indicated an increase in the affinity constant of 2.5-fold after solubilization and of further 2-fold after the affinity purification. The specific binding activity of the affinity-purified fraction was 9000 fmol hGH/mg protein. Complexes of Triton X-100-solubilized receptors with [125I]hGH were analyzed by gel filtration on Sephadex G-150, in the presence of Triton X-100. A minor fraction of the complexes eluted as high molecular weight (Mr) aggregates, whereas a major fraction eluted as a 150 kDa peak. Assuming a contribution of approximately 30% to the Mr by the bound detergent and a hormone: receptor ratio of 1:1 in the complex, a Mr of 80-85 kDa can be calculated for the receptor molecule. Affinity labelling of the receptor with [125I]hGH revealed a Mr of 37 +/- 0.5 kDa (n = 7) for the binding subunit. Specific high Mr aggregates were also observed after crosslinking; however, the size of the labelled species was not affected by reducing agents. Homologous and heterologous competitive binding studies with ovine PRL (oPRL) or hGH revealed a considerably higher affinity for hGH as compared to oPRL. The competitive displacement patterns obtained with oPRL or hGH as tracers were similar, indicating that both hormones bound to the same receptor sites with different affinities. A similar difference in affinity was retained by the affinity-purified receptors.     

Distinct placental lactogen and prolactin (lactogen) receptors in bovine endometrium.

Specific binding sites for bovine placental lactogen (bPL) and the lactogenic hormone, prolactin, have been detected in endometrial membranes isolated from uteri of mid-pregnant heifers. The specific binding of human growth hormone (hGH) (used to monitor the presence of lactogenic binding sites) and of bPL was increased approximately 4-fold following treatment of the membranes with 4 M MgCl2. Binding was found to be ligand specific, membrane protein concentration-, time- and temperature-dependent and reversible. Scatchard analysis of bPL and hGH competition binding data revealed curvilinear plots with dissociation constants for the high affinity sites of 4.1 x 10(-11) M and 6.4 x 10(-11) M, respectively. The maximum capacity of binding of bPL at the high affinity site was 21 fmol/mg). membrane protein while approximately twice the level of binding was measured for hGH (39 fmol/mg). Both hGH and bGH, but not ovine prolactin, competed with [125I]bPL for binding. The concentrations of hGH and bGH needed to effectively compete were however 100-fold higher than those required for unlabeled bPL. No specific binding of radiolabeled bGH was detected in endometrial tissue suggesting the absence of bGH receptors. Preferential competition of [125I]hGH binding was observed by prolactin and bPL. From these data it may be inferred that hGH binding is indicative of the presence of both lactogenic (prolactin) and bPL binding sites in endometrial tissue. The presence of distinct bPL receptors in the endometrium from mid-pregnant cows suggests a possible role for bPL in the maintenance of pregnancy.     

Significance of the glycan moiety of the rat ovarian luteinizing hormone/chorionic gonadotropin (CG) receptor and human CG for receptor-hormone interaction

The role of the glycan moiety of the rat ovarian LH/CG receptor and human CG (hCG) in high-affinity receptor-hormone interaction was investigated by cross-linking and quantitative binding experiments. hCG and its derivatives, desialylated hCG and deglycosylated hCG were labeled either to the alpha-subunit (125I) or the beta-subunit (3H). The ligands were attached to ovarian membrane particles, which were treated with neuraminidase or peptide-N-glycosidase F to remove terminal sialic acids or N-linked oligosaccharides of the receptor, respectively, and the complexes formed were solubilized, cross-linked with glutaraldehyde, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All of the ligands produced similar autoradiographic patterns with the native or glycosidase-treated receptor, and only the receptor-(alpha)hCG and receptor-(alpha, beta)hCG complexes were detected. Moreover, quantitative binding studies indicated that all of the hormone derivatives had similar affinities for the native or glycosidase-treated receptor. In addition, the orientation of the carbohydrate side chains on the receptor-hormone complex was studied by digesting the complex with the glycosidases. The molecular weight of the receptor, evidenced by ligand blotting, was reduced to the same extent, whether the membrane-bound free receptor or receptor-hormone complex was treated with the glycosidases, suggesting that the oligosaccharide side chains of the receptor are apart from the hormone binding region. As peptide-N-glycosidase F treatment reduced the size of the Mr 90,000 receptor first to about Mr 67,000 and finally to about Mr 62,000, there may possibly be 2 N-linked carbohydrate chains per receptor polypeptide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the glycosidase-treated receptor-[125I]hCG complex also revealed that neuraminidase was able to remove the sialic acids from both subunits of the receptor-bound hormone. In conclusion, the results suggest that hCG interacts with the polypeptide backbone of its ovarian receptor mainly through the peptide core of its alpha-subunit. Moreover, the carbohydrate side chains of both subunits of hCG are positioned on the outward face of the receptor-hormone complex.     

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.     

Growth hormone activates phospholipase C in proximal tubular basolateral membranes from canine kidney.

To delineate pathways for "signal" transduction by growth hormone (GH) in proximal tubule, we incubated basolateral membranes isolated from canine kidney with human growth hormone (hGH) or human prolactin (hPrl) and measured levels of inositol trisphosphate (InsP3) in suspensions and of diacylglycerol extractable from the membranes. Incubation with hGH, but not hPrl, increased levels of InsP3 and diacylglycerol in a concentration-dependent manner. Half-maximal effects occurred between 0.1 and 1 nM hGH. Increased levels of InsP3 were measured after as little as 5 sec of incubation with 1 nM hGH, and increase was maximal after 15 sec. Increases were no longer detectable after 60 sec because of dephosphorylation of InsP3 in membrane suspensions. hGH did not affect rates of dephosphorylation. hGH-stimulated increases in InsP3 were detectable in membranes suspended in 0, 0.1, and 0.2 mM calcium but not in 0.3 or 1.0 microMs calcium. 125I-labeled hGH-receptor complexes with Mr values of 66,000 and 140,000 were identified in isolated basolateral membranes. Our findings establish that GH activates phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) in isolated canine renal proximal tubular basolateral membranes, potentially after binding to a specific receptor. This process could mediate "signal" transmission by GH across the plasma membrane of the proximal tubular cell and elsewhere.     

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.     

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)     

Insulin-like growth factor receptors and binding protein in rat neuroblastoma cells

B104, an established rat neuroblastoma cell line exhibiting specific neuronal qualities, was chosen as a model to study insulin-like growth factor (IGF) binding and action in the central nervous system. Specific binding of [125I]IGF-II to B104 membranes averaged 12.2 +/- 4.0% (mean +/- SD)/100 micrograms/ml protein compared with [125I]IGF-I binding of 10.1 +/- 2.9%. In competitive binding studies employing [125I]IGF-II as the radioligand, high affinity for IGF-II was demonstrated (50% displacement at 2.7 ng/ml), with none for IGF-I or insulin. Upon cross-linking [125I]IGF-I to membranes under reducing conditions, two prominent bands were observed, migrating with apparent mol wt (Mr) of 135,000 and 280,000. Both bands were inhibited by IGFs and insulin, but not by R-II-PABI, a polyclonal antibody to the type 2 receptor. These bands presumably represent the alpha-subunit and an incompletely reduced alpha-alpha-dimer of the type 1 IGF receptor. When cross-linking [125I]IGF-II to membranes under reducing conditions, the primary labeled bands migrated with apparent Mr of 260,000 and 280,000. These bands were inhibited by IGF-II and R-II-PABI, but not by insulin, and probably represent the monomeric type 2 receptor. In addition, we observed a minor band at apparent Mr 35,000, which was inhibited by IGF but not by insulin. By a modified cross-linking technique, we confirmed the existence of a small IGF-binding protein in the serum-free conditioned medium of B104 cultures, migrating as two bands with apparent Mr of 33,000-39,000. These proteins demonstrated high affinity for IGF-I and IGF-II, but none for insulin. In summary, this study demonstrates the presence in B104 rat neuroblastoma cells of 1) abundant classical type 1 and type 2 IGF receptors, and 2) a secreted and membrane-associated small IGF-binding protein.     

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.     

Phosphorylation and glycosylation of the luteinizing hormone receptor.

Purified testicular and ovarian luteinizing hormone/human chorionic gonadotropin (hCG) receptors are phosphorylated at serine and threonine residues by the catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). Occupancy of the receptors by hCG significantly increased the rate but not the extent of phosphorylation. However, prolonged preincubation of receptors with hCG reduced the subsequent rate of receptor phosphorylation. Identical phosphopeptide maps were obtained for the phosphorylated ovarian and testicular receptors. The phosphorylated receptor, like the native receptor, bound to wheat germ lectin and hCG-Sepharose and migrated as a single band of Mr 90,000 (testis) and Mr 85,000 (ovary) on NaDodSO4/PAGE. Neuraminidase treatment of receptors caused reductions of molecular weight to 82,000 (testis) and 77,000 (ovary), and further treatment with O-Glycanase had minimal effect on molecular size. However, deglycosylation with N-Glycosidase and endoglycosidase F produced a single labeled polypeptide of Mr 59,000 for both gonadal receptors. Treatment of native receptors with neuraminidase caused no apparent change in binding of gonadotropin to blotted receptors, whereas deglycosylated receptors showed a major reduction in hormone binding. These results indicate that luteinizing hormone/hCG receptors are sialoglycoproteins with predominantly N-linked glycosyl residues that account for the size difference between testicular and ovarian receptors and that may participate in the interaction with gonadotropin. Receptor occupancy by agonist leads to a conformational change that facilitates its phosphorylation during initial binding and reduces the rate of phosphorylation after more prolonged exposure to hCG.     

Identification of circulating growth hormone-binding proteins in domestic poultry: an initial characterization

Multiple growth hormone (GH)-binding proteins (GHBPs) were identified in serum and plasma samples from domestic chickens and turkeys. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 10% acrylamide, 2.7% bis discontinuous gels under reducing conditions and electrotransferred to nitrocellulose paper. Western blots were incubated with 125I-labelled recombinant chicken GH (cGH) or bovine GH and GHBPs visualized by means of autoradiography. In fresh samples (less than 2 h from collection to gel electrophoresis), multiple minor high Mr bands were evident between approximately 72,000 and 175,000. Two major bands were observed at approximately 69,500 and 27,500. The latter is consistent with previous reports for the rat and mouse serum GHBPs based on nucleotide sequence analysis. The minor bands were essentially undetectable after storage at -25 degrees C for several months, and an additional major band at Mr approximately 52,500 appeared. The Mr-69,500 major protein contained N-linked carbohydrate, as determined by a reduction in molecular size by treatment with peptide N-glycosidase F. Binding of 125I-labelled GH was partially inhibited by co-incubation with 50 micrograms unlabelled pituitary-derived cGH/ml and excess unlabelled porcine GH as well as ovine prolactin, but not by bovine insulin. Non-specific binding of 125I-labelled GH by serum albumin was also observed. A comparison was made between these GHBPs and the hepatic GH receptor (e.g. molecular weight estimates, affinity for homologous versus heterologous GHs, cross-reactivity with prolactin, presence of N-linked carbohydrate). The origin and relationship among the various molecular weight species of GHBPs identified, and their potential role in regulation of the biological activity of GH in birds, remain to be determined.     

Binding and cross-linking of iodinated rat prolactin to rat hepatic prolactin receptor

Basal parameters for binding and cross-linking of 125I-rat prolactin (rPRL) to lactogenic (PRL) binding species present in crude membrane fraction (CMF) or detergent-solubilized preparations of rat liver have been investigated. (1) The highest specific binding to CMF was obtained with an incubation time of 50 h at 20 degrees C and with a 50 mM potassium phosphate buffer adjusted to pH 8.5. (2) Cross-linking of 125I-rPRL to binding sites in CMF with disuccinimidyl suberate (DSS) showed the autoradiographic appearance of an Mr 40,000 binding species. (3) No specific binding or cross-linking of rPRL was seen in Triton X-100-solubilized CMF. This is probably due to Triton X-100-induced changes in the physical properties of rPRL. (4) Specific binding of 125I-rPRL was detected in CHAPS-solubilized CMF. Following cross-linking the autoradiographic appearance of a binding species with an Mr value of 40,000 was shown. 125I-hGH was cross-linked to three PRL binding species with Mr 82,000, 40,000 and 35,000 in CHAPS-solubilized preparations. (5) In Golgi-enriched low-density membrane preparation 125I-rPRL was cross-linked to Mr 82,000, 40,000 and 35,000 species. It is proposed that the inability of rPRL to be cross-linked to Mr 82,000 and 35,000 species present in CHAPS-solubilized preparation is the result of CHAPS-induced changes of rPRL binding properties and low solubilizing capacity of CHAPS. (6) In conclusion, this study shows that also the iodinated endogenous hormone, rat prolactin, and not only hGH identifies high and low molecular forms of the rat liver prolactin receptor.     

Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: evidence for processing of cell-bound IGFBP-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coli-derived recombinant human IGFBP-3 (hIGFBP-3E. coli) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3CHO) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [125I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3E. coli or hIGFBP-3CHO produced a dose-dependent inhibition of IGF-I but not insulin-stimulated [3H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGAFBP-3E. coli or hIGFBP-3CHO potentiated subsequent IGF-I-stimulated [3H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3E. coli for 24 h, [125I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25%. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [3H]AIB uptake. The increase in [125I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3E. coli with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [125I]IGF-I to cell-associated 29,000 Mr hIGFBP-3E. coli seen after 24 h of incubation was reduced approximately 70% after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3E. coli forms of 12,000-27,000 Mr. Cell-associated IGFBP-3E. coli (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3E. coli in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF-I action.     

Binding sites of human growth hormone and ovine and bovine prolactins in the mammary gland and the liver of lactating dairy cow

Membrane preparations and Triton X-100 solubilized fractions from the mammary gland and liver of the lactating dairy cow were capable of specific binding of [125I]hGH and [125I]oPRL. The specific binding of the latter was significantly lower and could not be increased by higher receptor levels. Displacement studies of [125I]hGh by hGH, bPRL and oPRL revealed that the two latter hormones have a 20-40-fold lower affinity for the receptor than hGH, although strong indications exist that they all bind or the same sites. This feature is unique for cows and does not exist or is much less pronounced in rodents.     

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.     

Monoclonal antibodies as probes to study the human growth hormone-binding domain to lactogenic rat liver receptors.

A set of monoclonal antibodies (MAb) to human GH (hGH) was used to study the hormone binding orientation to its receptors (R) from female rat liver. The hGH antigenic region left exposed after its binding to liver microsomes was detected by measuring the ability of various [125I]MAb to bind to the preformed hGH-R complexes. Results indicated that a cluster of epitopes defined by the MAb, termed AE5, AC8, and AE12, remains accessible in the hGH-R complex whereas overlapping epitopes 3C11 and HG3 would define a hGH region involved in the binding site. Supporting these findings, solubilization and HPLC gel filtration of [125I]MAb-hGH-R complexes showed a radioactive peak of about 450,000 mol wt for MAb AE5 or AC8, but not for MAb 3C11 or HG3. [125I]MAb AE12 behaved differently, suggesting that epitope AE12 may be masked or altered in hGH-R-solubilized complexes. MAb directed to the putative hGH-binding site (MAb 3C11, HG3, and the closely related MAb 10C1 and NA71) failed to inhibit binding of the preformed [125I]MAb AE5-hGH complex to the receptors, suggesting a hormone modification after MAb AE5 binding. Accordingly competition experiments indicated an increase in the affinity of hGH for its receptors induced by this MAb. A higher hGH concentration was required to obtain 50% [125I]hGH binding to liver microsomes in the presence of MAb AE5 than in its absence. As the MAb used define epitopes that were previously correlated with the hGH structure, we concluded that a high flexible region (sequences 134-150) is exposed in the hGH-R complex. Furthermore, some MAb directed to this region enhance the hormone affinity for its rat liver receptors, probably through an induced conformational change.     

Characterization of a low affinity binding protein for growth hormone in rat serum

GH forms a high Mr complex in rat serum distinct from that with GH-binding protein (GHBP). The present study investigates the nature of this complex. When subjected to AcA44 filtration chromatography, 125I-labeled human GH (hGH) in rat serum eluted in four peaks. Peak 1 eluted at the void volume, whereas peaks 2, 3, and 4 corresponded to the GHBP complex, free hGH, and iodide, respectively. Stripping of GHBP in serum by immunoaffinity chromatography depleted peak 2 but did not affect peak 1. Peak 1 accounted for 11.4 +/- 1.2% of the total radioactivity (mean +/- SEM; n = 6) in stripped serum. Addition of unlabeled hGH (0.9-9 microM) demonstrated the binding of [125I]hGH to be specific, with Scatchard analysis revealing an affinity of 0.88 +/- 0.03 x 10(5) M(-1)(n = 3)and a capacity of 2.46 +/- 0.14 microM. Sepharose CL-6B filtration chromatography showed the complex to be 260 kDa in size. The distribution of GH binding to GHBP and this high Mr serum factor was investigated by incubating [125I]hGH in sera containing a low (5 nM) and a high (35 nM) concentration of GHBP over a range of physiological GH concentrations. In sera containing a low concentration of GHBP, the proportion of GH complexed in peak 1 increased with increasing GH concentrations. In sera with a high concentration of GHBP, GH was complexed mainly in peak 2. Studies with normal rat sera revealed that more GH was complexed in peak 1 in male than in female rats (3.4 +/- 0.4% and 1.4 +/- 0.1%, respectively; P < 0.006), in contrast to that of peak 2 (1.1 +/- 0.2% and 7.6 +/- 0.4%, respectively; P < 0.002). In summary, we provide strong evidence for the existence of a factor in rat serum that binds GH with low affinity and high capacity. It has a Mr of approximately 240 kDa, assuming a 1:1 binding stoichiometry, and is immunologically distinct from GHBP. This factor may provide supplementary capacity for GH binding when binding to GHBP is saturated.     

Growth hormone binding to specific receptors stimulates growth and function of cloned insulin-producing rat insulinoma RIN-5AH cells

Binding of 125I-labeled human GH (hGH) to a cloned rat insulin-producing cell line RIN-5AH in monolayer culture was studied along with some physiological effects of the hormone on these cells. Binding was time and temperature dependent, and steady state binding was observed in 60 min at 37 C with [125I]hGH at 4.2 pM, whereas at 24 C, binding had not reached a steady state after 120 min. The binding was largely reversible, since 80% of initially bound [125I]hGH dissociated from the cells upon incubation in hGH-free buffer for 120 min. Half-maximal binding was obtained when cells were incubated in the presence of 3.0 X 10(-10) M unlabeled hGH. Rat GH as well as human placental lactogen were able to compete for binding sites, but with less affinity. Other non-GH peptides at 6.7 micrograms/ml did not affect [125I]hGH binding. Scatchard analysis revealed curvilinear plots, and approximately 2700 high affinity binding sites were calculated. Culture of RIN-5AH in the presence of 1 microgram/ml hGH for 4 days resulted in an 80% increase in insulin content as well as an 18% increase in cell number and DNA and protein content compared to those in cells cultured in the absence of hGH. The dose dependence of the insulinotropic effect showed that half-maximal and maximal stimulation were observed in cells cultured in the presence of 10 and 100 ng/ml, respectively. Insulin release to the medium during the 4-day culture period was not affected by hGH. These data suggest that GH, through binding to specific receptors in the cell membrane, directly stimulates proliferation and function of pancreatic beta-cells.