Thermodynamic analysis of the interaction of prolactin with its receptor in the rabbit mammary-gland microsomes.
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ABSTRACT:
SUBMITTER: Sakai S
PROVIDER: S-EPMC1131636 | biostudies-other | 1990 Aug
REPOSITORIES: biostudies-other
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Project description:Receptors for human, simian, ovine, bovine and murine prolactin, human growth hormone and human placental lactogen have been identified in plasma-membrane-containing subcellular particles isolated from rabbit mammary glands. The association and dissociation of (125)I-labelled prolactin are time- and temperature-dependent processes, both being maximal at 37 degrees C. (125)I-labelled prolactin prepared by the enzymic iodination procedure with lactoperoxidase binds better to receptors than does the preparation obtained by using chloramine-t as the oxidizing agent. The binding of (125)I-labelled prolactin to receptors is strongly influenced by pH and ionic composition but not by many low-molecular-weight compounds tested, e.g. steroids, nucleotides and several drugs. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipid moieties are essential for the binding of (125)I-labelled prolactin. The binding of (125)I-labelled prolactin to receptors is a saturable and reversible process. Scatchard and Lineweaver-Burk analyses suggest that (125)I-labelled prolactin has a high affinity for its receptor. Binding of (125)I-labelled prolactin to receptors does not result in the destruction of the hormone. Considerable prolactin-binding activity is also observed in subcellular fractions isolated from the adrenal gland, liver, ovary and kidney of the pregnant rabbit, a finding that is consistent with other reported actions of prolactin in these organs.
| S-EPMC1168001 | biostudies-other
Project description:Using microsomes prepared from rabbit mammary gland, the dissociation of prolactin (PRL) from its receptor was determined in the presence of peptide hormones or various concentrations of PRL. Among the hormones tested, PRL (ovine, mouse and bullfrog), human growth hormone and human placental lactogen each accelerated the dissociation of PRL in a manner proportional to their receptor-binding activities. Hormone-dependent dissociation was observed at higher concentrations than those at which the binding of PRL was completely inhibited by lactogenic hormones. In the concentration range 0.1 ng/ml-10 micrograms/ml, PRL increased the rate of dissociation in a logarithmic concentration-dependent manner. It was concluded that the dissociation of PRL from its receptor caused by lactogenic hormones is dependent on the hormone concentration. Arrhenius plot analysis revealed that PRL changed the frequency factor for the dissociation reaction. PRL in the medium inhibited the re-association of dissociated PRL. The data also suggested that PRL regulates the rate of dissociation by interacting with the PRL-receptor complex.
| S-EPMC1151627 | biostudies-other
Project description:The structure of prolactin (PRL) receptor in the rabbit mammary gland was examined using a receptor-specific monoclonal antibody (MAb). The PRL receptor preparation used was purified by making use of a PRL-affinity column. MAb inhibited the binding of PRL to the receptor, in a dose-dependent manner and completely at a high concentration. Using the receptor directly labelled by 125I, the preparation was incubated with MAbs and the immune complex was collected by Pansorbin and examined by SDS/polyacrylamide-gel electrophoresis. The autoradiography showed that three species with apparent Mr values of 77,000, 41,000 and 25,000 specifically reacted with MAbs. The pattern changed little in the presence or absence of dithiothreitol. Western blot analysis showed that two species (Mr 77,000 and 41,000) reacted with MAb. Affinity labelling of the receptor with labelled PRL revealed three bands with Mr values of 96,000, 60,000 and 43,000 on SDS gels. The high-Mr complex (Mr greater than 200,000) was always present at the top of the gel. These results show that the mammary gland contains at least three PRL-binding subunits. The differences in Mr before and after PRL binding were close to the Mr of PRL. This would suggest that each PRL binding subunit reacts with one PRL molecule.
| S-EPMC1135503 | biostudies-other
Project description:Two lambda gt11 clones containing fragments of cDNA encoding the prolactin receptor from rabbit mammary gland were isolated using a rat liver prolactin receptor cDNA probe. An 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids that contains three domains: (i) the extracellular, amino-terminal, prolactin-binding region of 210 residues; (ii) the transmembrane region of 24 residues; and (iii) the intracellular, carboxyl-terminal domain of 358 residues. This latter domain is much longer than the cytoplasmic domain (57 residues) previously described for the rat liver prolactin receptor. In addition, the sequence identity of this form of prolactin receptor with the growth hormone receptor is extended in the cytoplasmic domain.
| S-EPMC286858 | biostudies-other
Project description:1. Explants of mammary tissue from pseudopregnant rabbits were cultured at 37 degrees C in air for 24-48h in Medium 199 buffered with 20mm-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]. The medium contained insulin and corticosterone, or insulin, corticosterone and sheep prolactin in the presence or absence of ouabain, an inhibitor of Na(+)/K(+)-dependent adenosine triphosphatase. The responses of explants were assessed histologically, by measuring the tissue concentration of K(+), and by rates of synthesis of RNA, protein and fatty acids. The effect of ouabain on Na(+) and K(+) concentrations in slices of lactating rabbit mammary-gland tissue incubated for 1h at 37 degrees C in Krebs bicarbonate buffer was also studied. 2. Prolactin increased the concentration of K(+) in mammary explants, an effect prevented by ouabain. In slices of lactating tissue, there was a linear relationship between the log dose of ouabain (from 0.1 to 10mum) and increased Na(+) and decreased K(+) concentrations in the tissue. 3. Ouabain at concentrations up to 1mum did not affect the rate of synthesis of RNA, protein or fatty acids by explants cultured with insulin and corticosterone. By contrast, the stimulatory effect of prolactin on protein synthesis was diminished and the induction of medium-chain fatty acid synthesis by prolactin was almost abolished. RNA synthesis was unaffected. Histological examination showed no tissue damage by 1mum-ouabain. 4. Explants cultured in the presence of 2mum-ouabain for 24h retained their ability to respond to prolactin when the ouabain was removed from the culture medium. Between 24 and 48h they showed responses to prolactin of a magnitude similar to those of explants never exposed to ouabain. 5. These results show that a fully functional Na(+)/K(+)-dependent adenosine triphosphatase system is necessary for prolactin to promote secretory activity in rabbit mammary gland.
| S-EPMC1185725 | biostudies-other
Project description:PI 3-kinase enhancer A (PIKE-A) is critical for the activation of Akt signalling, and has an essential function in promoting cancer cell survival. However, its physiological functions are poorly understood. Here, we show that PIKE-A directly associates with both signal transducer and activator of transcription 5a (STAT5a) and prolactin (PRL) receptor, which is essential for PRL-provoked STAT5a activation and the subsequent gene transcription. Depletion of PIKE-A in HC11 epithelial cells diminished PRL-induced STAT5 activation and cyclin D1 expression, resulting in profoundly impaired cell proliferation in vitro. To confirm the function of PIKE-A in PRL signalling in vivo, we generated PIKE knockout (PIKE-/-) mice. PIKE-/- mice displayed a severe lactation defect that was characterized by enhanced apoptosis and impaired proliferation of mammary epithelial cells. At parturition, STAT5 activation and cyclin D1 expression were substantially reduced in the mammary epithelium of PIKE-/- mice. The defective mammary gland development in PIKE-/- mice was rescued by overexpression of a mammary-specific cyclin D1 transgene. These data establish a critical function for PIKE-A in mediating PRL functions.
| S-EPMC2837170 | biostudies-literature
Project description:Rabbit mammary-gland prolactin (Prl) receptors in the microsomal fraction were solubilized in 7.5 mM-Chaps) or 1% Triton X-100 and analysed by ion-exchange chromatography using DEAE-Bio-Gel A. Prl receptors in the presence of 7.5 mM-Chaps were separated into two different fractions (Fr. A and B), both of which showed identical specificity of binding to peptide hormones as those in the Chaps or Triton extract. oPrl and human growth hormone (hGH) bound to the same site, but other non-lactogenic hormones (follicle-stimulating hormone, oGH, luteinizing hormone and insulin) failed to bind to the Prl receptors. The dissociation constant (Kd) for Prl binding to the receptors in Fr. A was about 50% of those in Fr. B, suggesting that the rabbit mammary gland contains two types of Prl receptors, one with a high, and one with a low, Kd for Prl binding. A decrease in the concentration of Chaps in the column buffer to 4 mM caused aggregation of the receptors in Fr. A. H.p.l.c.-gel filtration, using Shim pack 150 and 300 columns connected in series, separated the receptor as a protein with an Mr of 74,000 +/- 4,900 (mean +/- S.D.) in the presence of 5 mM-Chaps, or of 36,800 +/- 2,100 in the presence of 7.5 mM-Chaps. Sucrose-gradient-centrifugation analysis showed that the Prl-receptor complexes in the presence of 5 mM-Chaps were sedimented between gamma-globulin and bovine serum albumin (5.56 +/- 0.22 S). As the Chaps concentration was increased to 7.5 mM, a further peak of the Prl-receptor complexes (4.01 +/- 0.23 S) appeared below ovalbumin. The present data suggest that the binding subunit causes the monomeric subunit to aggregate with itself or with another specific associated protein of similar Mr.
| S-EPMC1147040 | biostudies-other
Project description:Prolactin receptors present in the particulate fraction of lactating pig mammary gland were solubilized by 7.5mM-3-[(3-cholamidopropyl)dimethylammonio]-1-propane-su lph onic acid (Chaps) and purified by affinity chromatography on prolactin coupled to Affi-Gel 10. Nearly 30% of the particulate receptors were solubilized by the detergent and over a 1000-fold purification from homogenates was achieved. A water-soluble fraction rich in receptors was observed during the preparation of membranes, although this fraction has not yet been purified. Prolactin binding to the receptors was a time-dependent, reversible and saturable reaction in particulate, Chaps-solubilized and purified receptors. In all forms, receptors showed the same specificity to peptide hormones. Prolactin and human growth hormone bound to the same receptors, whereas bovine growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and insulin failed to bind. After solubilization, the dissociation constant (Kd) for prolactin was decreased 5-fold from 9.8 X 10(-11) M in the particulate receptors to 1.8 X 10(-11) M in solubilized and purified receptors, being due principally to an increase in the association rate constant from 1.0 X 10(9)M-1 X h-1 to (3.9-4.6) X 10(9)M-1 X h-1, respectively, with the dissociation rate constant remaining unchanged at (1.1-1.3) X 10(-2)h-1. Isoelectric focusing of the prolactin-receptor complex revealed two peaks, one at a pI of 5.5-5.6 and the other at 5.2-5.3. Microsomal receptors were covalently cross-linked to 125I-labelled ovine prolactin with ethylene glycol bis(succinimidyl succinate) and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Autoradiography of the gel revealed a major subunit of Mr 28 000-35 000 and a minor one of Mr 67 000-69 000. Anti-(prolactin receptor) antibodies raised against rabbit mammary gland prolactin receptors were equally effective in inhibiting prolactin binding to particulate, solubilized and affinity-purified receptors, suggesting that purified prolactin receptors have a structure indistinguishable immunologically from particulate receptors and rabbit mammary gland prolactin receptors. The present demonstration shows that particulate prolactin receptors from a domestic animal can be solubilized and purified without losing the original properties of high affinity and binding specificity for hormones.
| S-EPMC1144528 | biostudies-other
Project description:Oxytocin receptor (OXTR) is involved in social behaviors, thermoregulation, and milk ejection, yet little is known about its role in breast cancer. To investigate the role of OXTR in mammary gland development and tumorigenesis, a transgenic mouse model of OXTR overexpression (++Oxtr) was used. Overexpression of OXTR-induced progressive mammary hyperplasia, unexpected milk production, and tumorigenesis in females. OXTR-induced mammary tumors showed ERBB2 upregulation and mixed histological subtypes with predomination of papillary and medullary carcinomas. OXTR overexpression led to an activation of prolactin (PRL)/p-STAT5 pathway and created a microenvironment that promotes mammary-specific tumorigenesis. PRL inhibitor bromocriptine (Br) could mitigate OXTR-driven mammary tumor growth. The study demonstrates Oxtr is an oncogene and a potential drug target for HER2-type breast cancer.
| S-EPMC8184747 | biostudies-literature
Project description:The hormone prolactin (PRL) and its receptor (hPRLr) are significantly involved in breast cancer pathogenesis. The intermediate hPRLr (hPRLrI) is an alternatively-spliced isoform, capable of stimulating cellular viability and proliferation. An analogous truncated mouse PRLr (mPRLr) was recently found to be oncogenic when co-expressed with wild-type mPRLr. The goal of this study was to determine if a similar transforming event occurs with the hPRLr in human breast epithelial cells and to better understand the mechanism behind such transformation. hPRLrL+I co-expression in MCF10AT cells resulted in robust in vivo and in vitro transformation, while hPRLrI knock-down in MCF7 cells significantly decreased in vitro malignant potential. hPRLrL+I heterodimers displayed greater stability than hPRLrL homodimers, and while being capable of activating Jak2, Ras, and MAPK, they were unable to induce Stat5a tyrosine phosphorylation. Both immunohistochemical breast cancer tissue microarray data and RNA sequencing analyses using The Cancer Genome Atlas (TCGA) identified that higher hPRLrI expression associates with triple-negative breast cancer. These studies indicate the hPRLrI, when expressed alongside hPRLrL, participates in mammary transformation, and represents a novel oncogenic mechanism.
| S-EPMC7997966 | biostudies-literature