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.     

Studies on the irreversible nature of prolactin binding to receptors

Studies on hormone-receptor interactions generally assume that the formation of a hormone-receptor complex is a reversible process. This assumption has been examined directly in three experiments using liver membrane receptor preparations from pregnant rats and ovine PRL (oPRL). In Exp 1, Receptors were preincubated with a range of concentrations of oPRL at 23 C for periods up to 60 min, washed thereafter to remove free oPRL, and subsequently incubated with [125I]iodo-oPRL (23 C) to determine specific binding. Preincubation of receptors (0.25 mg membrane protein) with oPRL (5 ng) for periods as brief as 10 min reduced subsequent binding of [125I]iodo-oPRL to receptor, suggesting incomplete dissociation of oPRL even after 30 h. In Exp 2 after preincubation for 30 min with oPRL and subsequent incubation with [125I]iodo-oPRL for 19 h, membranes were washed, and the dissociation (23 or 37 C) of [125I]iodo-oPRL from the hormone-receptor complex in the presence or absence of 1000 ng oPRL was studied. After 48 h, only 35-50% of the [125I]iodo-oPRL dissociated from the hormone-receptor complex even in the presence or excess oPRL, indicating a heterogeneity of binding sites (i.e. 50-65% irreversible; 35-50% reversible). When pregnant rat serum was used in place of oPRL or when rabbit mammary glands were used instead of rat livers to prepare receptor preparations, results were similar to those described above, except for the nearly complete dissociation (90%) obtained at 37 C using rabbit mammary gland receptors. In Exp 3 after incubation (10 min, 2 h, or 15 h) of rat liver receptors with [125I]iodo-oPRL plus various amounts of oPRL, the hormone-receptor complex could be completely dissociated with 5 M MgCl2, restoring binding affinity and capacity of receptor to their original values. Labeled oPRL dissociated by MgCl2 treatment from such a complex is capable of binding to fresh receptor. These data strongly suggest that the PRL-receptor interaction, particularly the rat liver receptor interaction with PRL under usual in vitro conditions, is not reversible to a significant degree. This is not due to hormone or receptor damage but to a significant number of binding sites (50-65%) in the receptor preparation which are not reversible except under extreme conditions.     

Specific binding of iodinated growth hormone to rat liver in vivo.

The specific uptake by rat liver of human (hGH) and bovine (bGH) GHs labeled with 125I was studied by an in vivo procedure. A significant reduction of the uptake was observed when labeled hormones were injected together with different amounts of the corresponding native GH. This reduction was dose dependent, and the concentration of native hormone that prevents 50% of the liver uptake of the labeled hormone was close to 12 microgram/100 g BW. In normal rats, only native hGH or bGH significantly decreased the liver uptake of [125I]iodo-bGH, while bovine PRL (oPRL) or heat-denatured bGH were inactive. The highest inhibition of the uptake of [125I]iodo-hGH by rat liver was obtained when this labeled hormone was injected either together with hGH or with bGH plus oPRL while partial displacement was observed with bGH or oPRL. These data suggest that hGH binds to both somatotropic and lactogenic sites in the liver of normal rats. In hypophysectomized animals, only the somatogenic binding sites could be detected.     

Antiidiotypic antibodies raised against anti-prolactin (PRL) antibodies recognize the PRL receptor

Antiidiotypic antibodies capable of recognizing the PRL receptor have been raised against antibodies to ovine PRL (oPRL) and rat PRL (rPRL). Anti-oPRL or anti-rPRL antibodies were induced in rats or rabbits, respectively, and the immunoglobulin G (IgG) fractions were isolated by affinity chromatography on a Protein A-Sepharose 4B column. Specific anti-oPRL antibodies were purified on an oPRL-Sepharose 4B affinity column. The specific antibodies (0.5 mg/rabbit) in Freund's complete adjuvant were injected into rabbits at 2- to 3-week intervals for 3 months. Antiidiotypic antibodies against rat anti-oPRL specifically inhibited [125I]iodo-oPRL binding to the immunizing anti-oPRL antibody. Membrane binding of the antiidiotypic antibodies was determined by [125I]iodo-Protein A precipitation. It was found to be significantly higher toward membrane preparations rich in PRL receptors, such as liver membranes from pregnant mice or from estradiol-treated male rats or prostate membranes from adult rats. This membrane binding by the antiidiotypic antibody was competitively inhibited by the immunizing anti-oPRL antibody, suggesting that the idiotypic antibody may share common determinants with the PRL receptor. Recognition of the PRL receptor by the antiidiotypic antibodies was also assayed by indirect binding studies. After preincubation of the antiidiotypic antibodies with the membranes, the resulting complex was precipitated and the amount of free antiidiotypic antibody remaining in the supernatant estimated according to its ability to inhibit [125I]iodo-oPRL binding to anti-oPRL IgG. The lowest degree of inhibition of [125I]iodo-oPRL binding was achieved after incubation of the anti-idiotypic IgG with liver membranes from pregnant mice, while the inhibitory capacity was about 5-fold higher subsequent to parallel incubations with the same membranes, which had previously been heated for 30 min at 65 C, or with membranes of male rat liver, demonstrating a direct correlation between the binding of the antiidiotypic antibody to the membranes and the PRL receptor content of the membranes. Furthermore, significant and dose-dependent inhibition of [125I]iodo-oPRL binding to its receptors in various PRL receptor-rich membrane preparations from rats and rabbits was demonstrated with antiserum of rabbits immunized with rabbit anti-rPRL IgG. These results suggest that effective and specific anti-PRL receptor antibodies can be produced using the antiidiotypic antibody procedure, thus avoiding the necessity of isolating and purifying the PRL receptor itself.     

Induction of prolactin (PRL) receptors by PRL in the rat lung and liver. Demonstration and characterization of a soluble receptor

A soluble PRL receptor has been identified in the 100,000 X g supernatant from homogenates of lungs and livers of male and female rats treated with either estradiol (E2; 2 mg kg-1 day-1 for 7 days, sc) or ovine PRL (oPRL; 0.1 mg kg-1 day-1 for 14 days, sc). Fifty percent of the total PRL-binding activity in the liver homogenate of E2-treated male rats was found in the supernatant fraction, and only 12% in intact female rats. The soluble PRL receptor has the same specificity, protein dependence, and binding affinity (Ka = 2.8 X 10(9) M-1) characteristics as the membrane-bound receptor. A sheep anti-PRL-receptor antiserum specifically inhibited the binding of [125I]iodo-oPRL to the soluble PRL receptor. Column chromatography on Sepharose 6B revealed a single peak of [125I]iodo-oPRL-receptor complex from liver of E2-treated rats, having a mol wt of 340,000, whereas the 100,000 X g supernatant from lungs and livers of oPRL-treated rats revealed two specific peaks of [125I]iodo-oPRL complex with mol wts of 340,000 (A) and 165,000 (B), respectively. Peak A represented 25% and 27% and peak B, 35% and 49% of the total column radioactivity for liver and lung 100,000 X g supernatant fraction, respectively. Peak B coeluted with a rabbit anti-oPRL antiserum, suggesting that it is a PRL antibody. Anti-PRL-receptor antibody reduced the radioactivity associated with peak A but not peak B. Heat inactivation at 60 C (30 min) resulted in a complete loss of binding in peak A without affecting peak B. The results indicate that the soluble PRL-binding sites, increased in rat lung and liver after treatment with oPRL or E2, may represent an intermediate step in new receptor synthesis before incorporation into the membrane.     

Inhibition of binding of ovine placental lactogen to growth hormone and prolactin receptors by monoclonal antibodies

From a single cell fusion, five stable hybridomas secreting antiovine placental lactogen (oPL) antibodies were obtained. Three of these secrete immunoglobulin (Ig)G subclass, and the other two secrete IgM class antibodies. Ascites fluids were raised in mice for each clone and were used as the antibody component for the development of solid phase RIA. Three solid-phase RIAs were successfully established using individual IgG subclass monoclonal antibodies, but the IgM class antibodies were ineffective. In all three individual solid-phase RIAs, the binding of [125I]iodo-oPL to the immobilized antibody was inhibited by unlabeled oPL, but not by ovine pituitary PRL (oPRL), ovine GH (oGH), or ovine pituitary extract. Two of the IgG subclass antibodies were able to inhibit the binding of [125I] iodo-oPL to PRL receptors(s) and to GH receptor(s) in rabbit mammary gland and liver, respectively. One of these two IgG subclass antibodies was more effective at inhibiting the binding of oPL to PRL receptor(s) in rabbit mammary gland, whereas the other one is more effective in inhibiting the binding of oPL to GH receptor(s) in rabbit liver. These antibodies, however, could only weakly inhibit the binding of [125I]iodo-oPRL to rabbit mammary gland and were ineffective in inhibiting the binding of [125I]iodo-oGH to rabbit liver. The addition of monoclonal antibodies in both radioreceptor assay (RRA) for PRL (RRA-PRL) and for GH (RRA-GH) did not affect the parallelism of the displacement curve of oPL standard. Our results suggest that oPL might contain two distinct binding sequence(s): one responsible for the binding of oPL to PRL receptor(s) and the other responsible for the binding of oPL to GH receptor(s). These two binding sequences might overlap or be located adjacent to one another. The interaction of monoclonal antibodies with these binding sequences of oPL may block the binding of oPL with PRL and GH receptor(s). Alternatively, our studies suggest that the monoclonal antibodies do not bind to hormone receptor(s)-binding sequence(s) in oPL, but the interaction between oPL and monoclonal antibody might alter the conformational structure of the oPL which will consequently lead to a lower binding of oPL to PRL and GH receptor(s).     

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.     

The ontogeny of somatotropic binding sites in ovine hepatic membranes

The binding of human [125I]GH, ovine [125I]GH, bovine [125I]GH, and ovine [125I]PRL, to hepatic microsomal membranes (100,000 g) prepared from fetal, neonatal, and infant lambs and adult ewes has been examined. The specific binding of hGH increases (P less than 0.01) from 3.4 +/- 0.8% in the fetus (n = 7) to 20.0 +/- 2.1% (n = 6) in lambs at least 6 days postpartum. The binding of oGH is low in the fetus (0.7 +/- 0.2%; n = 13) and neonatal lamb (1.3 +/- 0.5%; n = 5) and increases (P less than 0.01) in older lambs (14.6 +/- 4.7%; n = 6) and adult sheep (14.9 +/- 5.3%; n = 4). Similarly, the binding of bGH is less (P less than 0.05) to fetal tissues. In contrast, the binding of oPRL is similar in fetal and postnatal preparations. Cross-reaction studies suggest that the binding of GHs is to a site with lactogenic characteristics in the fetus. In contrast, in lambs at least 4 days postpartum and in adult sheep, binding is to a site with somatogenic characteristics. The inability to detect somatogenic sites in the fetal liver is not due to saturation by endogenous GH or chorionic somatomammotropin, as the binding characteristics do not change after MgCl2 pretreatment of the membrane fractions. No change in binding is observed 25 days after fetal decapitation at 69 days (n = 3), suggesting that circulating GH does not down-regulate the fetal GH receptor. These observations suggest an immaturity of the somatogenic receptor in the ovine fetal liver and its appearance in the perinatal period. Immaturity of this receptor is likely to be the basis for the lack of a major effect of fetal GH on fetal somatic growth.     

The glycogenic effects of placental lactogen and growth hormone in ovine fetal liver are mediated through binding to specific fetal ovine placental lactogen receptors

To examine the relative roles of placental lactogen (PL) and GH in fetal metabolism, we have examined the effects of ovine PL (oPL), ovine GH (oGH), and ovine PRL (oPRL) on glycogen metabolism in cultured ovine fetal hepatocytes and have examined the binding of these hormones to hepatic membranes from fetal and neonatal lambs. In ovine fetal hepatocytes, oPL (150 ng/ml-20 micrograms/ml) stimulated dose-dependent increases in [14C]glucose incorporation into glycogen (18-167%) and total cellular glycogen content (10-69%). oGH and oPRL also stimulated glycogen synthesis in fetal hepatocytes, but the potencies of these hormones were only 12% and 4% that of oPL. The dose-response curves of the three hormones were parallel, and their maximal effects were identical, suggesting a common mechanism of action. In hepatic membranes from fetal lambs, the maximal specific binding of [125I]oPL was 26.3% while the maximal specific binding of [125I]oGH was only 0.9-1.5%. The binding of [125I]oPL was saturable and reversible and varied with incubation time and temperature. Unlabeled oPL (1 ng/ml-5 micrograms/ml) caused a dose-dependent inhibition of the binding of [125I]oPL to fetal hepatic membranes, with half-maximal displacement of [125I]oPL by 5-7 ng unlabeled oPL/ml. oGH and oPRL caused parallel displacement of [125I]oPL, but with potencies only 2% and 0.1% that of oPL. Scatchard analysis of oPL dose-response curves indicated that the hormone bound to a single class of receptors with a dissociation constant of 1.1 X 10(-10) M. The maximal specific binding of [125I]oGH to hepatic membranes of neonatal lambs (20.1%) greatly exceeded the binding of oGH to fetal hepatic membranes. In addition, the potency of oGH in competing for [125I]oPL binding sites in neonatal liver greatly exceeded the potency of oGH in competing for [125I]oPL binding sites in fetal liver. Although the biological effects of both oPL and oGH in postnatal subprimate tissues may be mediated through binding to nonprimate GH receptors, the results of these studies suggest that the glycogenic effects of oPL in ovine fetal liver are mediated through binding to specific fetal oPL receptors. The relatively weak biological effects of oGH and oPRL in ovine fetal liver appear to be mediated through the binding of the hormones to fetal oPL receptors. The presence of specific, high affinity PL receptors in ovine fetal tissues provides a mechanism whereby oPL may function as a GH in the ovine fetus.     

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.     

Evolution of prolactin and placental lactogen receptors in ewes during pregnancy and lactation

The present paper describes a method of membrane preparation from ewe mammary gland using a sucrose cushion (1.3 M) to select smooth membranes; this results in a membrane preparation richer in PRL receptors than the microsomal preparation classically used. This method was used for the characterization and measurement of PRL and ovine placental lactogen (oPL) receptors in three organs of the ewe (mammary gland, liver, and adipose tissue). PRL receptors were measured by competition of iodinated human GH ([125I]hGH) with ovine PRL (oPRL). This hormone, which has both growth and lactogenic activities, appears to interact with PRL receptors with a higher affinity than oPRL itself and is a good probe for the determination of PRL receptors in the ewe. oPL receptors were measured by the specific binding of [125I]oPL. This hormone appears to bind exclusively to a somatogenic site in the ewe, since various GHs compete efficiently for binding, whereas oPRL is without effect. The evolution of PRL and oPL receptors was determined during pregnancy and lactation and at different periods after an estradiol and progesterone treatment, which provokes growth of the mammary gland and milk secretion. During pregnancy, PRL receptors increased in the mammary gland up to day 100. During the last trimester, receptor content remained stable, and a second increase occurred during early lactation. No additional significant changes were observed either for PRL receptors in liver or adipose tissue or for oPL receptors in any of the organs studied (mammary gland, liver, adipose tissue). Injections of large doses of estradiol and progesterone to nonpregnant ewes were able to reproduce effectively the pattern of PRL receptors observed during pregnancy, but had no effect on oPL receptor levels. These studies demonstrate the independence of PRL and PL receptor sites in the ewe and suggest a different hormonal regulation for each type of receptor.     

Lactogenic and somatotropic binding sites in liver membranes of rats with renal insufficiency

Binding sites for human GH (hGH) were studied in liver membranes of rats with chronic renal insufficiency (CRI) associated with marked growth retardation. A subtotal nephrectomy was performed in young female rats. One month after the nephrectomy, the animals with a plasma creatinine level 3 times or more that of controls were studied; their mean statural gain was 56% that of controls. The specific binding of [125I]hGH to microsomal membranes of rats with CRI was low (40% that of controls). The number of binding sites rather than the affinity of the binding was affected; both the lactogenic and somatotropic sites were decreased, as judged from the binding of ovine [125I]PRL and bovine [125I]GH. The binding sites of the plasma membranes as well as those of the Golgi fractions, were reduced. In plasma membranes of rats with CRI, the specific binding of glucagon was low, and the specific binding of insulin was elevated; these modifications were associated with a high plasma glucagon level and a decreased insulinemia in rats with CRI, but no modification of plasma GH and PRL levels was found. Thus, the hormone level does not appear to regulate the GH-binding sites in this system. The link between the growth defect and the decreased number of GH-binding sites in the liver membranes of rats with CRI remains to be established.     

Characterization and applications of monoclonal antibodies to the prolactin receptor

Monoclonal antibodies (mAbs) were produced in BALB/c mice immunized with partially purified PRL receptors from rat liver. Two mAbs (T1 and T6) were able to completely inhibit [125I]ovine PRL ([125I]oPRL) binding to solubilized rat liver PRL receptors, while two other mAbs (U5 and U6) showed only a small effect on PRL binding, but were able to precipitate hormone-receptor complexes. Scatchard analysis of [125I]oPRL binding to rat liver microsomes in the presence of mAbs resulted in a decrease in the number of sites without changing the affinity of PRL binding by T1 and T6, whereas U5 and U6 altered neither parameter. [125I]mAb binding to rat liver microsomes was performed in the presence of various concentrations of unlabeled mAbs or oPRL to examine the interaction between mAbs. Competition of binding to the receptor was observed, respectively, between T1 and T6, U5 and U6, and U5 and E21 (a mAb to the rat liver PRL receptor previously produced). Both [125I]T1 and [125I]T6 binding were inhibited by oPRL, although not completely (80% inhibition at the higher concentrations). When [125I]T1 binding was analyzed by Scatchard analysis, two classes of binding sites to rat liver microsomes were found, of which only the number of higher affinity sites was affected by the presence of oPRL in incubation. Similar results were observed for [125I]T6 binding. [125I]mAb binding to microsomes from other tissues and species was examined. All five mAbs were able to bind to microsomes from rat tissues (liver, ovary, adrenal, prostate, and Nb2 lymphoma cells), similar to the level of [125I]oPRL binding in these tissues. The binding characteristics of [125I]T6 or [125I]U5 were essentially identical in all rat tissues examined. Although T1, U6, and E21 showed strong species specificity, there was significant binding of T6 to rabbit liver and mammary gland and of U5 to rabbit and pig mammary gland and mouse liver Competition curves of [125I]U5 binding were parallel for rat, rabbit, and mouse tissues, while [125I]T6 binding was able to distinguish PRL receptors in rabbit mammary gland from those in rat tissues. The use of 125I-labeled mAb in immunoblot analysis of the PRL receptor resulted in a marked increase in sensitivity. All mAbs detected microsomal PRL receptors in rat liver with mol wt of 84,000, 42,000 and 40,000. As little as 4 fmol receptors can be identified using this approach. Microsomal PRL receptors from rat ovary, prostate, and Nb2 cells and purified receptors from pig and rabbit mammary gland were subjected to immunoblot analysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of solubilized prolactin receptors from Rana catesbeiana tadpole tissues

Prolactin (PRL) receptors were solubilized from Rana catesbeiana tadpole liver and tail fin and female Sprague-Dawley rat-liver membranes by treatment with 1% Triton X-100. Binding of [125I]oPRL to tadpole-liver and tail-fin solubilized extracts reached equilibrium by 12 h at 19 degrees C. The binding was linear up to 50 micrograms of tadpole liver and tail-fin protein and 30-40 micrograms of rat-liver protein. Solubilization did not affect the dissociation constant for [125I]oPRL binding but did result in a greater number of sites. The binding was specific for oPRL and hGH, which has PRL-like activity. Neuraminidase pretreatment of membranes altered the binding affinity of oPRL to tadpole-liver membranes and solubilized extracts but not to tadpole-tail fin membranes or extracts. Pretreatment of membranes with neuraminidase did not affect the binding capacity of tadpole-liver or tail-fin membranes or solubilized extracts.     

Induction of prolactin receptors by prolactin in the rat lung and liver: demonstration of separate receptor and antibody entities

This study examined the role of prolactin (PRL) in inducing its own receptors in rat lung and liver beyond the parallel immunological response evoked. Ovine PRL (oPRL), mixed with polyvinylpyrrolidone (PVP) and injected s.c. daily to male rats for 7, 10 and 14 days, was shown to induce specific binding of [125I]iodo-oPRL in the lung and liver crude membrane fractions. Doses as low as 12.5 ng/kg were effective in inducing PRL-binding sites, which, however, differed qualitatively from those found in livers of 17 beta-estrogen-treated male rats. The oPRL-induced sites were highly specific for oPRL and were relatively stable to heat, suggesting the possible participation of antihormone antibodies in the binding observed. Indeed, anti-oPRL antibodies found in sera of oPRL-treated rats increasingly bound oPRL as a function of the duration of treatment. Water-washing of the membrane fraction succeeded in gradually eliminating the loosely bound antibody and in partially restoring the displacing ability of excess (1 micrograms) rat PRL on oPRL-binding sites in the lung (32.3%) and liver membranes (29.3%). Also restored were the heat lability typical of the receptor site, as well as the inhibitory effect of anti-PRL-receptor antiserum (1:100) on PRL binding (50-65% inhibition of total binding). In keeping with these results, in vitro incubation of liver membranes with rat anti-oPRL antiserum greatly reduced the ability of rat PRL to compete for oPRL binding, supporting the findings after in vivo treatment with oPRL.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A unique placental lactogen receptor: implications for fetal growth

To determine whether there are structural differences between the binding sites for placental lactogen (PL) and GH, we have compared the molecular weights of complexes formed by the covalent cross-linking of [125I]ovine (o) PL and [125I]oGH to hepatic membranes from fetal and pregnant sheep in mid- and late gestation and from postnatal nonpregnant sheep at 3 days to 7 months of age. Specific [125I]oPL binding sites in fetal liver were detected as early as midgestation, and cross-linking of [125I]oPL to fetal hepatic membranes yielded a major radiographic band with a mol wt of 60 +/- 5 K (mean +/- SD). Unlabeled oPL at low concentrations (0.9-9 nM) specifically competed with [125I]oPL for binding to the 60 K complex. In contrast, oGH and oPRL competed for binding to the 60 K complex only at much higher concentrations (greater than or equal to 90 nM). In addition, no specific cross-linking of [125I]oGH or [125I]oPRL to fetal hepatic membranes was observed. These findings suggest the presence of a distinct and unique PL binding site in ovine fetal liver. Since the mol wt of oPL is 22 K, the estimated mol wt of the oPL receptor protein is 38 +/- 5 K. During the first week after birth, there was a striking increase in the number of [125I]oGH binding sites. Cross-linking of [125I]oGH to postnatal liver yielded radiographic bands with apparent mol wts of 75 K and 140 K. The relative potencies of oPL, oGH, and oPRL in competing for binding to the 75 K and 140 K complexes were similar to the relative potencies of these hormones in competing for [125I]oGH binding sites in postnatal liver, suggesting that the 75 K and 140 K bands represent subunits of the oGH receptor bound covalently to [125I]oGH. Cross-linking of [125I]oPL to pregnant and postnatal nonpregnant liver yielded three radiographic bands with mol wts of 60 K, 75 K, and 140 K. The intensities of all three bands were reduced by low concentrations (0.9-9 nM) of oPL. Higher concentrations of oGH abolished the 75 K and 140 K bands but reduced the intensity of the 60 K band by only 20-30%. oPRL had minimal effect on band intensities. These observations suggest the presence of two functionally and structurally distinct receptors in pregnant liver: the oPL receptor, which has high affinity for oPL and low affinity for oGH and oPRL.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling

The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with [125I]ovine PRL [( 125I]oPRL). PRL receptors were solubilized from mammary microsomes with 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with 125I using the chloramine-T method. Covalent labeling of PRL receptors with [125I]oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of [125I]oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five [125I]oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit. From these observations, we conclude that this predominant 32,000 mol wt component is a major binding subunit of the PRL receptor molecule and does not aggregate with itself or with other subunits through S-S linkages.