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

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

Species variation in the binding of hGH to hepatic membranes

The binding of 125I-labeled human growth hormone (hGH) to liver membranes from several different species was studied to determine the lactogenic or somatotropic hormone nature of the receptors. Liver membranes from several species of the class of Mammalia bound significant quantities of 125I-hGH. Goat, sheep, rat, mouse, and rabbit liver membranes exhibited the highest binding with cow, pig, human, and hamster liver membranes exhibiting severalfold less binding. The binding of the dog and cat liver membranes exhibited relatively high nonspecific binding. Fish and chicken liver membranes did not bind appreciable quantities of 125I-hGH. In all species except for dog and cat in which 125I-hGH bound to the membranes, hGH was the most effective competitor for binding. The mean ID50 for hGH and all membranes was 2.4 X 10(-9) M. Human liver membranes exhibited the smallest ID50, 4.9 X 10(-10) M. In sheep liver membranes, bovine growth hormone (bGH) was equipotent to hGH in competing for 125I-hGH binding. bGH also demonstrated significant competition for 125I-hGH binding in pig and cow membranes. Ovine prolactin (oPrl) exhibited significant competition for 125I-hGH only in rodent membranes. The ID50 for oPrl was 3- to 10-fold greater than for hGH in the rat, hamster, and mouse liver membranes. The ID50 for oPrl in the sheep liver membranes was 13-fold greater than that of hGH. We conclude the following: (1) There appears to be a species specificity of hGH binding that may be phylogenetically significant and may result from variations in the structure of the hormone or the receptor. (2) The competitive binding properties of hGH are fairly consistent within phylogenetic orders. (3) The simple designation of lactogenic or somatotropic for hormones and receptors is insufficient to characterize the binding properties of this group of hormones.     

Chemical modification of human growth hormone with N-acetylimidazole. Effect on binding capacity to lactogenic and somatogenic receptors.

The effect of acetylation of tyrosine residues on the binding capacity of human growth hormone (hGH) to rat liver lactogenic and somatogenic receptors was studied. When 3.7 tyrosine and 4.8 lysine residues were acetylated with N-acetylimidazole, both the in vivo and the in vitro capacities of hGH to compete with 125I-labeled bovine growth hormone for somatogenic binding sites greatly decreased. Acetylation also affected the in vitro binding capacity to lactogenic sites. Most of the somatogenic binding activity was recovered by hydroxylamine treatment, which removes O-acetyl groups from tyrosine residues but not N-acetyl groups from lysine residues. The same treatment partially restored lactogenic binding capacity. The reactivity of hGH tyrosine residues to N-acetylimidazole, together with previous evidence, suggests that: (a) Tyrosine residues 160 and 164, when acetylated, are likely to be responsible for the low binding activity of acetylated hGH. (b) Tyrosine 160 may play a significant role in hGH interaction with lactogenic receptors.     

Modulation of human growth hormone binding to somatogenic and lactogenic receptors by monoclonal antibodies to human growth hormone.

The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interaction was investigated by studying the effects of specific monoclonal antibodies (MAbs) to hGH on the binding of [125I]hGH to rabbit liver and mouse liver microsomes. Receptor binding assays were carried out using a constant dose (1 ng) of [125I]hGH and varying concentrations of MAbs. The assay was carried out in the presence of either excess ovine prolactin for the measurement of somatogenic (SOM) binding sites, or excess bovine growth hormone for the determination of lactogenic (LAC) binding sites. Anti-hGH MAbs were found to have a whole spectrum of effects on hGH binding, including inhibitory, non-effect and enhancing activities. Enhancement of the binding of [125I]hGH to both SOM and LAC receptors was observed in liver membranes of rabbit or mouse. The observed amplified signal of [125I]hGH binding to various receptors in the presence of MAb no. 8 may be due to conformational changes which occur following MAb binding to hGH. On the other hand, most of the other MAbs caused inhibition of [125I]hGH binding. A negative correlation exists between the cross-reaction of various MAbs with the N-terminus truncated forms of hGH (Met14-hGH or Met8Leu-hGH) and their respective KD/IC50 values enabled the evaluation of the crucial role of the N-terminus region in hGH binding to both LAC and SOM receptors. MAb nos 1 and 19, which are directed towards acid residues 95-134 and the C-terminus, inhibited SOM binding more potently than LAC binding. Thus, it seems that these mid-molecule and C-terminus regions are also important in hGH binding, and that they play a role in the partial overlap of SOM and LAC binding.     

Allosteric effects of monoclonal antibodies on human growth hormone

We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors.The effect of MAbAE5, AC8 and F11 on hGH binding was measured by determining the formation of¹²⁵I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding¹²⁵I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes.Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case preformed¹²⁵I-MAb:hGH complexes were added to microsomes. Data showed that¹²⁵I-MAb AE5:hGH complexes bound better to the various receptors than¹²⁵I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to¹²⁵I-MAb AC8 or¹²⁵I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32–46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding.Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site. MAb would induce either positive or negative allosteric changes in the hormone region involved in its binding to lactogenic, somatogenic and hGH-specific receptors.     

Ovine prolactin and human growth hormone derivatives. Specific modification of their alpha-amino groups

The alpha-amino group of ovine prolactin (oPRL) and human growth hormone (hGH) was selectively modified by transamination with glyoxylic acid. No difference was found in the binding capacity of transaminated oPRL to rat liver lactogenic receptors with respect to its control, although both samples showed a decrease in its binding capacity with reference to the native hormone. This decrease was due to conformational changes caused by the reaction conditions and not by the transamination itself, as shown by the circular dichroism spectra. Transaminated hGH retained the full binding capacity of the hormone. These results suggest that the alpha-amino group is not relevant for the binding to lactogenic liver receptors in both lactogenic hormones.     

Induction of lactogenic receptors. I. In the liver of hypophysectomized female rats.

To identify the hormones which affect lactogenic receptors in the liver of chronically hypophysectomized female rats, hormones were injected s.c. for 7 days. Specific binding (%, SB) of labelled ovine prolactin (PRL) in liver membrane preparations (1000,000 X g pellet) of controls was 1%. Estradiol (E2), cortisone (Con), ACTH or bovine growth hormone (bGH) treatment did not induce hepatic binding sites for PRL. Human GH and a single dose of 2mg PRL (but not lower doses) increased SB of PRL. Treatment with oPRL plus ACTH was less effective than hGH plus ACTH (13 vs 28%); combinations of oPRL plus Con as well as administration of oPRL plus ACTH to hypophysectomized and adrenalectomized female rats did not induce SB for PRL. Therapy with oPRL plus hGH (26%) was more potent than oPRL plus bGH (2%). These studies suggest that PRL, GH, and ACTH induce and in concert with sex steroids, modulate the lactogenic receptors in the female rat liver. The effect of ACTH is not due to increased adrenal corticoid secretion.     

A comparison of lactogenic receptors from rat liver and Nb2 rat lymphoma cells by using cross-linking techniques.

Lactogenic receptors were analysed with the use of the cross-linking agent disuccinimidyl suberate to attach covalently 125I-labelled ovine prolactin or human growth hormone to binding sites from (1) liver from pregnant rats and (2) the rat-derived Nb2 lymphoma cell line. Analysis by SDS/polyacrylamide-gel electrophoresis of the proteins cross-linked to labelled hormone in rat liver indicated a major specifically-labelled complex with an Mr of 68,000-72,000, when run under reducing or non-reducing conditions. With Nb2 cells a major specifically-labelled complex with an Mr of 97,000-110,000 was identified, but only when electrophoresis was run using reducing conditions. Assuming one hormone molecule (Mr 22,000-24,000) per hormone-receptor complex, then the receptor proteins have an Mr of 44,000-50,000 for rat liver and 73,000-88,000 for the Nb2 cells. For both cell types the receptors were of lactogenic specificity; lactogenic hormones competed for binding whereas somatogenic hormones did not. These studies suggest that the lactogenic receptors in rat liver membranes and Nb2 cells differ in two respects. Firstly, the Mr of the labelled receptor protein in Nb2 cells is greater than that of the corresponding receptor protein in rat liver membranes; secondly, the Nb2 cell receptor appears to exist as a disulphide-linked oligomer whereas the receptor in rat liver membranes does not.     

Solubilization and characterization of a lactogenic receptor from human placental chorion membranes

Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.     

Binding inhibition by monoclonal antibodies of ovine placental lactogen to growth hormone receptors in human liver

In the radioreceptor assay for growth hormone (RRA-GH) using [125I]iodo-hGH, hGH and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, ovine placental lactogen (oPL) is capable of inhibiting the binding of [125I]iodo-hGH in a parallel manner with hGH and in equipotency. Similarly, in the RRA-GH by employing [125I]iodo-oPL, oPL and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, hGH is also equipotent as oPL in inhibiting the binding of [125I]iodo-oPL in a parallel fashion. The addition of monoclonal antibodies against oPL in the assay was effective in inhibiting the binding of [125I] iodo-oPL to human liver, but could not, however, inhibit the binding of [125I]iodo-hGH to human liver. Furthermore, the addition of the monoclonal antibodies in the RRA-GH did not affect the parallelism of the oPL standard but lowered the total binding of oPL. Our studies indicate that the structure of the binding sequence in oPL which binds to the GH receptor of human liver is not identical to the equivalent sequence of hGH and that the monoclonal antibodies compete with GH receptors in human liver for the binding of oPL.     

The receptor binding properties of the 20K variant of human growth hormone explain its discrepant insulin-like and growth promoting activities

The 20K variant of native (22K) hGH is a full agonist for the growth promoting and lactogenic properties of the hormone in vivo but has been reported to have weak or absent insulin-like properties. To explore if these differences may be explained at the receptor level, we compared the ability of 22K and 20K hGH to inhibit the binding of 125I-22K hGH to receptors in isolated rat adipocytes, a target for the insulin-like effects of the hormone and in IM-9 cultured human lymphocytes, more specific for growth effects. Our data show that while 20K hGH is a potent agonist of native 22K hGH in the IM-9 lymphocyte assay, its potency in the rat adipocyte binding assay is only 3%, even when both cells are incubated together in identical conditions. Thus, the receptors for hGH appear to be different on various target cells, explaining why the 20K variant has different relative biological potencies at different sites of action.     

Mitogenic and binding properties of monoclonal antibodies to the prolactin receptor in Nb2 rat lymphoma cells. Selective enhancement by anti-mouse IgG

Three monoclonal antibodies (mAbs) (T6, U5, and U6) against prolactin (PRL) receptors in rat liver were studied in the rat lymphoma lactogen-dependent (Nb2-11C) and autonomous (Nb2-SP) cell lines. The mAbs had strong affinity for lactogen receptors (Ka = 12-14 nM-1), similar to that of human growth hormone (hGH) which is a lactogenic hormone. T6 and hGH competed for the same binding site, while U5 and U6 interacted with another epitope. The 125I-hGH-receptor complex could be immunoprecipitated by either U5 or U6, but not by T6. Affinity labeling and immunoblotting revealed that hGH and U6 bind to a protein of 63-65 kDa. T6, U5, and U6 were mitogenic in Nb2-11C cells but their respective potencies were 185-, 70-, and 4700-fold lower than that of hGH. Anti-mouse IgG enhanced the mitogenic effect of all three mAbs and almost completely abolished the differences between them, although their mitogenic activity was still 60-120-fold lower than hGH. Des-13-hGH, a competitive antagonist of hGH which hardly effected the binding of 125I-U5, inhibited the U5-stimulated proliferation of Nb2-11C cells in a noncompetitive manner, indicating that simultaneous binding of both ligands fixed the receptor in a nonactive conformation. A Fab fragment of T6 was not mitogenic, and inhibited the hGH-induced mitogenesis in a competitive manner, but its mitogenicity could be restored by anti-mouse IgG. We suggest that the dimerization or oligomerization of the lactogen receptor in Nb2-11C cells is an obligatory step in the transduction of the mitogenic signal. It may be induced by binding of the mAb to a site, which can be either identical or may even be distinct from that which binds the lactogenic hormone.     

Identification of residues outside the two binding sites that are critical for activation of the lactogenic activity of human growth hormone

Human growth hormone (hGH) binds lactogenic or somatotrophic receptors, creating active heterotrimeric complexes. Comparison of hGH structures, either free or bound to a single lactogenic or somatotrophic receptor, shows binding is associated with structural changes. Changes in hGH structure are unique when binding either lactogenic or somatotrophic receptors and they influence the spatial arrangement of residues constituting the second receptor-binding site. Using site-directed mutagenesis, we identified a contiguous set of largely hydrophobic residues that forms a motif communicating between the two receptor-binding sites of hGH. The residues are external to the receptor-binding epitopes and were identified when their mutation reduced site 2 function without changing site 1 function. The motif includes Phe44, Leu93, Tyr160, Leu163, and Tyr164, located in two hydrophobic clusters between the receptor-binding sites. Their mutation to Glu disrupts hydrophobic interactions and reduces lactogenic activity between 4.7- and 85-fold with little effect on somatotrophic activity or spectroscopic properties. These differential effects indicate that loss of lactogenic activity is not a result of global mis-folding. We propose the loss of lactogenic activity results from disruption of specific hydrophobic clusters that disables the site 1 binding-induced structuring of the second receptor-binding site.     

A Comparative Study of Lactogenic Hormone Binding Sites in the Adrenal Gland,Ovary and Kidney of the Lactating Cow

Abstract

The specific binding of ¹²⁵I-oPRL to microsomal fractions from the adrenal gland, ovary and kidney of the lactating cow was significantly lower than binding of iodinated hGH. In addition, the ability of oPRL to compete with iodinated hGH as compared to hGH, was 50-fold lower in the adrenal gland 35-fold lower in the ovary and 18-fold lower in the kidney. These results are similar to those obtained in the mammary gland and liver, whereas the ability of oPRL to compete with iodinated hGH was 90-fold lower, as compared to hGH. In the kidney the difference between the binding of iodinated hGH and iodinated oPRL was smaller. Results obtained with a solubilized kidney microsomal fraction also show a slightly higher affinity for oPRL than in other tissues. Thus the phenomena of differential affinities of oPRL and hGH to lactogenic hormone binding sites, characterizes most lactogenic hormone target tissues in the lactating cow. The distribution of these sites in different parts of the tissues was also studied. In the adrenal gland, the binding capacity in the cortex was 8-fold higher than in the medulla. In the ovary most of the binding sites were found in the corpus luteum, while in the kidney the binding capacity was higher in the cortex as compared to the medulla.     

Positive cooperative effects between receptors induced by an anti-human growth hormone allosteric monoclonal antibody

Monoclonal antibodies (MAb) anti-human growth hormone (hGH) termed MAb AE5, AC8 and F11 recognize a cluster of epitopes left exposed after hormone binding to receptors. Since these MAb were able to produce either positive (MAb AE5) or negative (MAb AC8 and F11) allosteric effects on hGH binding, the purpose of this work was to further characterize MAb behavior. Results indicated a straight correlation between MAb allosteric effects and affinity constant values for binding of different hGH:MAb complexes to lactogenic receptors from rat liver. Affinity of hGH:MAb AE5 as well as hGH:Fab AE5 complexes enhanced proportionally to the fraction of occupied receptors and Hill coefficients higher than 1 were obtained, suggesting the induction of positive cooperative effects between membrane-bound receptors. On the other hand, hGH:MAb AC8 and hGH:MAb F11 complexes binding affinity to lactogenic sites could not be related to receptor occupancy degree. It is proposed that binding of hGH:MAb AE5 complexes to receptors would elicit a conformational change on adjacent receptor molecules leading to an increase of their affinity to bind subsequent hGH:MAb AE5 complexes.     

Somatotropic and lactotropic receptors in transgenic mice expressing human or bovine growth hormone genes

The somatotropic and lactotropic receptors were studied in liver microsomal preparations from transgenic mice carrying the human growth hormone (hGH) or bovine growth hormone (bGH) gene fused to mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK). Specificity studies indicated that, similarly to normal mice, liver microsomes from the transgenic animals possess a mixed population of somatotropic and lactotropic binding sites. In transgenic animals of both sexes, the binding capacity of somatotropic receptors was significantly increased without corresponding changes in affinity. Expression of the MT-hGH hybrid gene was associated with the induction of somatotropic receptors which was approximately twice as great as that measured in animals expressing the MT-bGH hybrid gene. The binding capacity of lactotropic receptors in liver microsomes (quantitated, by the use, of labelled ovine prolactin) was increased 2–3 fold in transgenic females and approximately 10-fold in transgenic males as compared to the respective normal controls. We conclude that lifelong excess of GH up-regulates hepatic GH and prolactin receptors, and that lactogenic activity of GH is not essential for induction of prolactin receptors in the liver of transgenic mice.     

Differential effects of zinc on functionally distinct human growth hormone mutations

Human growth hormone (hGH) binds and activates lactogenic receptors by a sequential receptor dimerization mechanism. The affinity for the first lactogenic receptor is increased due to one zinc molecule linking hGH residues H18 and E174, located in helices 1 and 4, respectively, with two adjacent residues in the lactogenic receptor (D187 and H188). Two functionally unique groups of mutant hGHs have been identified. Addition of 25 microM zinc to lactogenic bioassays differentially affects mutant activities based on which group they belong to. One mutation (G120R) is located within the binding surface of hGH that interacts with the second lactogenic receptor. In the presence of endogenous zinc, G120R reduces the maximal activity of hGH without altering either the agonist or antagonist phases of the bell-shaped dose-response curve. Addition of zinc to this assay further reduces the activity of this protein. In contrast, mutations within a hydrophobic motif in hGH that functionally couples the two lactogenic receptor binding sites decrease the sensitivity (right-shift) of the agonist phase of the dose-response curve without similarly affecting the antagonist phase. The addition of zinc to these lactogenic assays increases the sensitivity (left-shifts) of the dose-response curves, largely negating the effect of these mutations. The effects of zinc differentiate between mutations within these two distinct functional motifs by limiting the pool of potential conformations that are available for binding within either of the receptor binding sites of this ligand.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Dispersed mammary epithelial cells. Receptors of lactogenic hormones in virgin, pregnant, and lactating rabbits.

The number and affinity of binding sites for lactogenic hormones have been determined in dispersed mammary cells from virgin, pregnant, and lactating rabbits. Dispersed epithelial cells, prepared from mammary glands by enzyme digestion, calcium chelation, and gentle shearing, were separated from nonepithelial cells by density centrifugation. 125I-labeled ovine prolactin (oPRL) and 125I-labeled human growth hormone (/GH) were used as tracers. Association and dissociation of 125I-oPRL or 125I-hGH were time- and temperature-dependent. The rate of association followed a second order reversible reaction with a rate constant of approximately 0.5 at 4 degrees C, approximately 2.0 at 23 degrees C, and approximately 9 x 10(7) M-1 min-1 at 37 degrees C. Maximum binding was achieved after 120 h at 4 degrees C, 48 h at 23 degrees C, and 2 to 4 h at 37 degrees C. Dissociation of 125I-oPRL or hGH from cells by unlabeled oPRL was complete at 4 degrees C after 160 h, following a first order reaction (5-1 = 9.9 x 10(-5) min) and incomplete at 23 degrees C and 37 degrees C even after prolonged time. Internalization of receptor-bound 125I-oPRL was studied by quantitative electron microscope autoradiography. Grain distribution over- and volume densities of cellular organelles was analyzed as a function of time and temperature. At 37 degrees C, there was a rapid and specific translocation of lactogenic hormones to intracellular organelles. Autoradiographic grains were found associated with vesicles, Golgi elements, lysosome-like structures, and the nucleus. One class of high affinity binding sites was estimated from Scatchard plot and direct kinetic analyses at 4 degrees C. Whereas the apparent affinity constant (approximately 10(10) M-1) did not change significantly throughout pregnancy and early lactation, the number of receptors extrapolated from Scatchard plots at 4 degrees C varied in an inverse relation to serum progesterone concentration. Thus, approximately 1900 sites were detected in virgin rabbits (progesterone, approximately 200 pg/ml), and midpregnancy (progesterone, approximately 15,000 pg/ml), and approximately 1800 during early lactation (progesterone, approximately 500 pg/ml). The binding properties of lactogenic hormones to dispersed cells was compared with those to Triton X-100 solubilized microsomal membrane preparations. Good correlation between the two systems was found indicating that cell dispersion did not alter binding properties. Our results indicate that dispersed mammary cells bind lactogenic hormones in a saturable and reversible process, that the number of exposed receptors varies throughout gestation and lactation, and finally that lactogenic hormones are internalized following interaction with their membrane receptors.     

Methionine oxidation in human growth hormone and human chorionic somatomammotropin. Effects on receptor binding and biological activities

The oxidation of the methionine residues of human growth hormone (hGH) and human chorionic somatomammotropin (hCS) to methionine sulfoxide by hydrogen peroxide has been studied. The kinetics of oxidation of individual methionine residues has been measured by reverse-phase high pressure liquid chromatography tryptic peptide mapping. Met-170 is completely resistant to oxidation in both hormones. The other 3 methionine residues in hCS (Met-64, Met-96, and Met-179) have markedly different reaction rates. Oxidation of the methionine residues does not appear to cause gross conformational changes in either hGH or hCS, as judged by CD and 1H NMR spectroscopy. Oxidation of Met-14 and Met-125 in hGH has little effect on affinity of the hormone for lactogenic receptors or on its potency in the Nb2 rat lymphoma in vitro bioassay for lactogenic hormones. The oxidation of Met-64 and/or Met-179 in hCS reduces profoundly both its affinity for lactogenic receptors and its in vitro biological potency. It is inferred by induction that residues 64 and/or 179 are critical for the binding of both hGH and hCS to lactogenic receptors and the expression of lactogenic biological activity.