Proiactin Binding in Rat Langerhans Islets

Abstract

Membrane preparations of collagenase-dispersed Langerhans islets of female Wistar rats exhibit specific binding sites for ¹²⁵I labelled ovine prolactin (¹²⁵I-oPrl). Almost negligible binding was detected in islets of male animals. The binding is a saturable and time-temperature dependent process, equilibrium being reached after 16 h incubation at 0°C. The bound oPrl is not displaceable by hFSH, hLH, bGH or hGH. In contrast with other cell fractions, the 12,000 g pellet accounts for more than 80% of the specific binding of ¹²⁵I-oPrl. Scatchard plots of data obtained in saturation studies indicate a single class of binding sites with K_a = 0.21 × 10¹⁰ M^-1. Protein and phospholipid moieties are essential for the receptor activity, since after trypsin or phospholipase C digestions marked loss of binding was verified. In islets of streptozotocin diabetic rats a marked reduction in the number of binding sites was observed. These findings may suggest that some of the actions of prolactin on endocrine pancreas could be explained by its specific interaction with islet cell membranes.     

Endogenous Prolactin Maintains its own Binding Sites in the Pigeon Crop Sac Mucosa

Abstract

The specific binding of lactoperoxidase-labelled ¹²⁵I-labelled ovine prolactin was determined in a membrane particulate of the pigeon crop-sac mucosal epithelium. Binding was found to be dependent upon the particulate preparation used, its protein concentration and the length of the incubation at 5°C. Scatchard analysis of the binding to crop-sacs from saline or prolactin-injected (1.9 μg per pigeon) revealed that prolactin stimulated 7-fold its own receptors by increasing the number of binding sites per mg protein: saline - 392±75 fmol/mg protein and prolactin 2736±602 fmol/mg protein (p<0.01). This increase did not affect the affinity constant (Ka): saline - 5.28±0.75x10⁸ l/mol and prolactin-3.28±0.40x10⁸ l/mol (N.S.), in keeping with the stimulatory effect of prolactin in the rat liver and mammary gland. This study further demonstrates the physiological role of endogenous prolactin in maintaining its own binding-sites in the pigeon crop-sac, since the administration of 0.8 ml anti-serum to prolactin resulted in a 63% reduction in the specific binding of the labelled hormone in vitro. These results confirm the prolactin binding to the pigeon crop-sac mucosa, quantify the stimulation of this binding by prolactin itself, and demonstrate the role of the endogenous hormone in the maintenance of these receptors.     

Prolactin stimulation of prolactin receptors in rat liver.

Hypophysectomy of the female rat results in a loss of prolactin receptors from the liver. Seventy percent of specific prolactin binding is lost within 24 hours; by 48 hours receptor levels are less than 5% of those found in livers from intact rats. A single dose of ovine prolactin to hypophysectomized rats causes a partial restoration of prolactin receptors between 12 and 18 hours after injection. As little as 0.5 mg prolactin significantly increases receptors, while doses above the 2 mg optimum are apparently less effective. These restored receptor sites are unaltered in their affinity for prolactin. Estradiol (E₂), progesterone, hydrocortisone, triiodothyronine (T₃) or E₂ plus T₃ could not mimic the prolactin effect. Neither combinations of prolactin with E₂ or T₃ nor repeated daily injections of prolactin alone increase receptors more than does a single prolactin injection. It appears that prolactin modulates the level of its own receptor in rat liver.     

Partial Proteolytic Digestion of the Mammary Prolactin Receptor: Identification of Smaller Prolactin Binding Fragments

Abstract

Partial proteolytic digestion of the mammary prolactin (PRL) receptor was used to generate receptor fragments and analyze their immunoreactivity and PRL binding properties. Tryptic digestion of the PRL receptor produced two immunoreactive fragments (Mr ≈ 30,000 and ≈ 15,000) that reacted with a monoclonal anti-PRL receptor antibody and still specifically bound PRL, while the complete immunoreactive PRL binding unit (Mr ≈ 42,000) disappeared. Neither chymotrypsin nor V8 protease were able to generate any immunoreactive receptor fragments. These receptor fragments may represent smaller PRL binding receptor form(s) of biological significance.     

Effect of diethylstilbesterol and prolactin on the induction of follicle stimulating hormone receptors in immature and cycling rats

Induction of follicle stimulating hormone receptor in the granulosa cells of intact immature rat ovary by diethylstilbesterol, an estrogen, has been studied. A single injection of 4 mg of diethylstilbesterol produced 72 h later a 3-fold increase in follicle stimulating hormone receptor concentration as monitored by [¹²⁵I]-_oFSH binding to isolated cells. The newly induced receptors were kinetically indistinguishable from the preexisting ones, as determined by Lineweaver-Burk plot of the binding data. The induced receptors were functional as evidenced by increased ability of the granulosa cells to incorporate [³H]-leucine into cellular proteins. Neutralization of endogenous follicle stimulating hormone and luteinizing hormone by administering specific antisera had no effect on the ability of diethylstilbesterol to induce follicle stimulating hormone receptors, whereas blockade of endogenous prolactin secretion by ergobromocryptin administration significantly inhibited (∼ 30 %) the response to diethylstilbesterol; this inhibition could be completely relieved by ovine prolactin treatment. However, ovine prolactin at the dose tried did not by itself enhance follicle stimulating hormone receptor level. Administration of ergobromocryptin to adult cycling rats at noon of proestrus brought about as measured on diestrusII, (a) a reduction of both follicle stimulating hormone (∼ 30 %) and luteinizing hormone (∼ 45 %) receptor concentration in granulosa cells, (b) a drastic reduction in the ovarian tissue estradiol with no change in tissue progesterone and (c) reduction in the ability of isolated granulosa cells to convert testosterone to estradiol in response to follicle stimulating hormone. Ergobromocryptin treatment affected only prolactin and not follicle stimulating hormone or luteinizing hormone surges on the proestrus evening. Treatment of rats with ergobromocryptin at proestrus noon followed by an injection of ovine prolactin (1 mg) at 1700 h of the same day completely reversed the ergobromocryptin induced reduction in ovarian tissue estradiol as well as the aromatase activity of the granulosa cells on diestrus II, thus suggesting a role for proestrus prolactin surge in the follicular maturation process     

The action of carboxyl modifying reagents on the ryanodine receptor/Ca2+ release channel of skeletal muscle sarcoplasmic reticulum

Summary

In this work we show that ryanodine binding to junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor (RyR) is inhibited in a time — and concentration-dependent fashion by prior treatment with the carboxyl reagent dicyclohexylcarbodiimide (DCCD). Exposure of the membrane-bound RyR to the water soluble carboxyl reagents 1-ethyl-3 (3-(dimethylamino) propyl carbodiimide (EDC) or N-ethyl-pheny-lisoxazolium-3 -sulfonate (WRK) only slightly affects their ryanodine binding capacity. The amphipathic reagent N-ethoxy cabonyl-2-ethoxy-1, 2-dihydroquinaline (EEDQ) inhibited ryanodine binding at relatively high concentrations. DCCD-modifica-tion of the SR decreased the binding affinities of the RyR for ryanodine and Ca²⁺ by about 3- and 18-fold, respectively.

The single channel activity of SR membranes modified with DCCD and then incorporated into planar lipid bilayers is very low (5–8%) in comparison to control membranes. Application of DCCD to either the myoplasmic (c/s) or luminal (trans) side of the reconstituted unmodified channels resulted in complete inhibition of their single channel activities. Similar results were obtained with the water soluble reagent WRK applied to the myoplasmic, but not to the luminal side. The DCCD-modified non-active channel is re-activated by addition of ryanodine in the presence of 250_üM Ca²⁺ and is stabilized in a sub-conductance state. With caffeine, ryanodine re-activated the channel in the presence of 100_üM of Ca^2+. The results suggest that a carboxyl residue(s) in the RyR is involved either in the binding of Ca²⁺, or in conformational changes that are produced by Ca²⁺ binding, and are required for the binding of ryanodine and the opening of the Ca²⁺ release channel.     

Antisense Strategy Unravels a Dopamine Receptor Distinct from the D2 Subtype, Uncoupled with Adenylyl Cyclase, Inhibiting Prolactin Release from Rat Pituitary Cells

The antisense strategy was used to unravel the functional contribution of the mRNAs encoding dopamine (DA) receptors to the multiple transduction mechanisms operated by DA in rat pituitary cells. An antisense oligonucleotide was designed to recognize seven nucleotides upstream and 11 nucleotides downstream from the initiation translation codon of the mRNA that encodes the DA D2 receptor. Addition of the antisense oligonucleotide for 7 days to primary culture of rat pituitary cells resulted in a decreased expression of DA D2 receptor as shown by (a) the virtual disappearance of [³H]spiroperidol binding sites and (b) the marked reduction in the levels of both the long and the short splice variant of the D2 receptor mRNAs. After this treatment, the DA D2 receptor agonist bromocriptine lost its capability both to inhibit adenylyl cyclase activity and to reduce prolactin mRNA levels. On the contrary, the inhibition of prolactin release induced by bromocriptine was affected minimally by the antisense oligonucleotide treatment. These data indicate that (a) translation of the mRNA encoding DA D2 receptors results in receptors that are negatively coupled with adenylyl cyclase and functionally linked to inhibition of prolactin synthesis; and (b) the release of prolactin might be regulated, at least in part, by a DA receptor that is encoded by mRNA species distinct from those encoding the D2 receptor.     

Investigation of the influence of external factors on the conformational dynamics of rhodopsin-like receptors by means of molecular dynamics simulation

Abstract

The study reports about the influence of binding of orthosteric ligands on the conformational dynamics of β-2-adrenoreceptor. Using molecular dynamics (MD) simulation, we found that there was a little fraction of active states of the receptor in its apo (ligand-free) ensemble. Analysis of MD trajectories indicated that such spontaneous activation of the receptor is accompanied by the motion in intracellular part of its alpha-helices. Thus, receptor’s constitutive activity directly results from its conformational dynamics. On the other hand, the binding of a full agonist resulted in a significant shift of the initial equilibrium towards its active state. Finally, the binding of the inverse agonist stabilized the receptor in its inactive state. It is likely that the binding of inverse agonists might be a universal way of constitutive activity inhibition in vivo. Our results indicate that ligand binding redistribute pre-existing conformational degrees of freedom (in accordance to the Monod–Wyman–Changeux Model) of the receptor rather than cause induced fit in it. Therefore, the ensemble of biologically relevant receptor conformations is encoded in its spatial structure, and individual conformations from that ensemble might be used by the cell in conformity with the physiological behavior.     

Development of prolactin receptor antagonists with reduced pH‐dependence of receptor binding

The cytokine hormone prolactin has a vast number of diverse functions. Unfortunately, it also exhibits tumor growth promoting properties, which makes the development of prolactin receptor antagonists a priority. Prolactin binds to its cognate receptor with much lower affinity at low pH than at physiological pH and since the extracellular environment around solid tumors often is acidic, it is desirable to develop antagonists that have improved binding affinity at low pH. The pK_a value of a histidine side chain is ～6.8 making histidine residues obvious candidates for examination. From evaluation of known molecular structures of human prolactin, of the prolactin receptor and of different complexes of the two, three histidine residues in the hormone–receptor binding site 1 were selected for mutational studies. We analyzed 10 variants by circular dichroism spectroscopy, affinity and thermodynamic characterization of receptor binding by isothermal titration calorimetry combined with in vitro bioactivity in living cells. Histidine residue 27 was recognized as a central hot spot for pH sensitivity and conservative substitutions at this site resulted in strong receptor binding at low pH. Pure antagonists were developed earlier and the histidine mutations were introduced within such background. The antagonistic properties were maintained and the high affinity at low pH conserved. The implications of these findings may open new areas of research in the field of prolactin cancer biology. Copyright © 2010 John Wiley & Sons, Ltd.     

Thyroid hormone regulation of prolactin binding to mouse mammary glands.

Membranes from mammary glands of mildly hypothyroid mice show a 70–85% reduction in prolactin binding while those from hyperthyroid mice bound 66% more prolactin compared to similar preparations from euthyroid animals. The prolactin binding data for mammary glands correlate well with the ability of the tissue from animals in various thyroid states to respond to prolactin $\text{in}\text{vitro}$ with increased lactose synthetase activity. Binding of prolactin to mammary membranes is enhanced when explants from mid-pregnant mice are cultured overnight in the presence of insulin, hydrocortisone and 10^?9 M L-T₃. This enhancement is not blocked by puromycin. These data suggest that thyroid hormones control the level of prolactin binding in mouse mammary tissue. This may be accomplished, at least in part, by activation of preexisting receptor molecules.     

Reaction of the histidines of prolactin with ethoxyformic anhydride. A binding site modification.

The seven histidines of bovine prolactin were modified with ethoxyformic anhydride and two classes of reactivity were apparent: 5 histidines were in the more reactive class (k = 0.097 min-1) and 2 histidines were less reactive (k = 0.011 min-1). The activity of the modified prolactins was determined by measuring their ability to bind to prolactin receptors from rabbit mammary glands. This assay showed that prolactin was fully active when 0 to 5 histidines were modified. If all 7 residues were modified, the hormone was unable to bind to its receptor. Circular dichroism studies indicated no significant differences in conformation for prolactins which had 2 to 7 histidines modified. Modification of human growth hormone and human placental lactogen with ethoxyformic anhydride resulted in a loss of the ability of these lactogenic hormones to bind to the prolactin receptor. For all three hormones, essentially full activity was recovered when the modifying group was removed by treatment with hydroxylamine. Sequence comparisons indicate that only 2 of the 3 growth hormone histidines and 2 of 7 placental lactogen histidines were homologous with histidines in bovine prolactin and that, in each case, they correspond to His-27 and His-30 in bovine prolactin. It is postulated that these residues serve to identify a portion of the binding domain of bovine prolactin.     

Nonlinear Regression Analysis of the time Course of Ligand Binding Experiments

Abstract

The binding and release of hormones and growth factors are often relatively slow processes under biological conditions. Consequently, a knowledge of the underlying rate constants may be of greater physiological relevance than the equilibrium constant. Here we show how, by following a single time course of binding, the rate constants for both binding and release can be determined. The ratio of these rate constants allows the binding constant to be calculated. A nonlinear regression computer program is described which facilitates these calculations and which provides estimates and standard errors of the constants determined. The method is illustrated by the binding of human growth hormone to the human growth hormone binding protein, and the binding of ovine prolactin to the rabbit prolactin receptor.     

Chemical modification studies of tryptophan,arginine and lysine residues in maize chloroplast ferredoxin:sulfite oxidoreductase

The ferredoxin-dependent sulfite reductase from maize was treated, in separate experiments, with three different covalent modifiers of specific amino acid side chains. Treatment with the tryptophan-modifying reagent, N-bromosuccinimide (NBS), resulted in a loss of enzymatic activity with both the physiological donor for the enzyme, reduced ferredoxin, and with reduced methyl viologen, a non-physiological electron donor. Formation of the 1:1 ferredoxin/sulfite reductase complex prior to treating the enzyme with NBS completely protected the enzyme against the loss of both activities. Neither the secondary structure, nor the oxidation-reduction midpoint potential (E _m) values of the siroheme and [4Fe–4S] cluster prosthetic groups of sulfite reductase, nor the binding affinity of the enzyme for ferredoxin were affected by NBS treatment. Treatment of sulfite reductase with the lysine-modifying reagent, N-acetylsuccinimide, inhibited the ferredoxin-linked activity of the enzyme without inhibiting the methyl viologen-linked activity. Complex formation with ferredoxin protects the enzyme against the inhibition of ferredoxin-linked activity produced by treatment with N-acetylsuccinimide. Treatment of sulfite reductase with N-acetylsuccinimide also decreased the binding affinity of the enzyme for ferredoxin. Treatment of sulfite reductase with the arginine-modifying reagent, phenylglyoxal, inhibited both the ferredoxin-linked and methyl viologen-linked activities of the enzyme but had a significantly greater effect on the ferredoxin-dependent activity than on the reduced methyl viologen-linked activity. The effects of these three inhibitory treatments are consistent with a possible role for a tryptophan residue the catalytic mechanism of sulfite reductase and for lysine and arginine residues at the ferredoxin-binding site of the enzyme.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Effect of nitration on prolactin activities.

The seven tyrosines of ovine prolactin were modified with tetranitromethane and the resulting products were assayed for α-lactalbumin production in a mouse mammary gland explant assay system, for binding activity in a radioreceptor assay, and by radioimmunoassay. The five tyrosines which were exposed can be nitrated without loss of activity. The two remaining tyrosines can be nitrated only under denaturing conditions, a reaction that caused a loss of binding and biological activities. The loss of activity was not a consequence of the denaturants but was due to modification of either or both of the two relatively unreactive tyrosines. It is postulated that this activity loss is the result of alterations of conformation rather than the modification of a tyrosine which is in contact with the prolactin receptor.     

Synthesis and activity of phosphono desmuramyldipeptide analogs

Summary Two phosphono desmuramyldipeptide analogs, 6 and 11, were synthesized and evaluated for immunomodulatory activity. Phthalimido-and adamantyl-substituted side chains as a replacement for the N-acetylmuraminic acid were coupled with appropriate dipeptides using DPPA as the coupling reagent. Introduction of a phosphonamidate moiety in compound 6 resulted in lower biological activity.     

The role of arginine residues in interleukin 1 receptor binding.

Interleukin 1 (IL-1) is a family of polypeptide cytokines that plays an essential role in modulating immune and inflammatory responses. IL-1 activity is mediated by either of two distinct proteins, IL-1 alpha or IL-1 beta, both of which bind to the same receptor found on T-lymphocytes, fibroblasts and endothelial cells (Type 1 receptor). The effect of specific chemical modification of recombinant IL-1 alpha and IL-1 beta on receptor binding was examined. Modification of the proteins with phenylglyoxal, an arginine-specific reagent, resulted in the loss of Type 1 IL-1 receptor binding activity. The stoichiometry of this modification revealed that a single arginine in either IL-1 alpha or IL-1 beta is responsible for the loss of activity. Cyanogen bromide cleavage of phenylglyoxal modified IL-1 alpha and IL-1 beta, followed by sequencing of the peptides, revealed that arginine-12 in IL-1 alpha and arginine-4 in IL-1 beta, which occupy the same topology in the respective crystallographic structures, are the target of phenylglyoxal. These results suggest that an arginine residue plays an important role in ligand-receptor interaction.     

Intracellular location of the Ah receptor

The subcellular distribution of the Ah receptor from the mouse hepatoma line, Hepa-1, was investigated following cytochalasin B treatment and cell enucleation. Probing the resultant cytoplast and nucleoplast fractions with radiolabelled tetrachlorodibenzo-p-dioxin (TCDD) revealed the presence of a specifically bound peak of receptor only in the cytoplast fraction. However, the quantity of receptor recovered in these experiments was only 10–12% of the expected value. We therefore undertook an investigation to determine the fate of the Ah receptor in the presence of cytochalasin B. Incubation of Hepa-1 cells with this compound resulted in a rapid loss or inactivation of cytosolic binding activity with a concomitant decrease in the amount of receptor partitioned into the nucleus at all time periods examined. Control experiments indicated that cytochalasin B did not compete with TCDD for binding to the Ah receptor and furthermore, that its mechanism of action could not be attributed to a non-specific effect on all cytosolic proteins. The results obtained are discussed in relation to the proposed models for induction by the estrogen and glucocorticoid binding receptors.     

Unoccupied prolactin binding components of the benign and malignant human prostate in a subclinical and clinical procedure

Optimal conditions for the quantitation of free prolactin binding components of human prostatic tissue obtained by TURP were studied by applying γ receptor assay. The radioligand used was ¹²⁵I-prolactin. Significantly greater heat stability of the prostate membrane prolactin binding sites, when compared to that of androgen cytoplasmic receptors, was confirmed. The saturability and specificity of the prolactin binding components was demonstrated by the results of both Scatchard plot analysis and displacement studies. Free prolactin receptors were found in none of the poorly differentiated (G3) prostatic tumors examined, and only in 62.5% of medium differentiated (G2) prostatic malignancies. The majority of tissue specimens coming from patients with either BPH or well differentiated prostatic tumor (G1) contain measurable amounts of free prolactin membrane binding components. In the present study we report also the case in which the change in tumor differentiation toward a higher grade (G2 to G1, provoked by the successful chemohormonal treatment) is accompanied with the appearance of previously absent free prolactin binding components. In histologically proven BPH tissue specimens free prolactin receptor negative status has been found in most patients with a slight increase in serum PAP values, while receptor rich status was detected in the majority of those with elevated PSA concentrations. We believe therefore that the prolactin receptor values, when used as part of the multivariable analysis, may participate in further delineation of the role of prolactin in the development of prostate cancer, but may also play a role in a subclinical prediction related to the conversion of either an adenoma or a latent adenocarcinoma to the clinically manifest prostatic malignancy.     

Sulfhydryl Reagents Alter Epidermal Growth Factor Receptor Affinity and Association with the Cytoskeleton

Abstract

Sulfhydryl (SH) reagents are known to influence the characteristics of many ligand-receptor systems. The SH reagent N-ethylmaleimide has been demonstrated to interact with EGF receptors, and to inhibit EGF receptor kinase activity. The data presented in this paper concern the effect of SH reagents on two intriguing features of the EGF receptor system, namely the presence of low and high affinity EGF binding sites, and the interaction of EGF receptors with the cytoskeleton. SH reagents were observed to induce a disappearance of high, but not low, affinity EGF receptors from the cell surface, and an increase in receptor-cytoskeleton interaction. Comparison of the effects of membrane-permeant and membrane-impermeant SH reagents on wild type and structurally modified EGF receptors suggested that sulfhydryl groups on the cytoplasmic, rather than the extracellular, receptor domain are involved. This indicates that the cytoplasmic domain of the EGF receptor plays a role in the high affinity binding of EGF, and in the interaction of EGF receptors with the cytoskeleton. Experiments with an anti-EGF receptor antibody that specifically blocks the binding of EGF to low affinity receptors indicated that EGF induces a shift in the EGF receptor from low to high affinity. SH reagents probably affect EGF binding by inhibiting this EGF-induced receptor conversion.