Prolactin Affinity Research Articles

Prolactin-immunoglobulin G complexes from human serum act as costimulatory ligands causing proliferation of malignant B lymphocytes.

Several lines of evidence indicate that immunoglobulin-bound prolactin found in human serum is not a conventional complex between an anti-prolactin antibody and prolactin but a different type of association of prolactin with the Fab portion of IgG heavy chains. The complex of prolactin with IgG was purified from serum by anti-human prolactin affinity chromatography and was shown to contain close to 1 mole of N epsilon-(gamma-glutamyl)lysine crosslinks per mole of complex, a characteristic feature in structures crosslinked by transglutaminase. Interestingly, the complex caused a proliferation of cells from a subset of patients with chronic lymphocytic leukemia, while it was inactive in a cell proliferation prolactin bioassay. By contrast, human prolactin stimulated the proliferation of cells in the bioassay but had no effect on the complex-responsive cells from the patients. Competition studies with prolactin and free Fc fragment of IgG demonstrated a necessity for engaging both the prolactin and the immunoglobulin receptors for proliferation. More importantly, competition for the growth response by free prolactin and IgG suggests both possible reasons for the slow growth of this neoplasm as well as avenues for control of the disease.

Expression of the extracellular domain of the rat liver prolactin receptor and its interaction with ovine prolactin.

A clone of the extracellular domain of the rat liver prolactin receptor was generated by the RNA-based polymerase chain reaction, and the NH2-terminal 210 amino acids were expressed in HeLa cells using a vaccinia virus/T7 hybrid expression system. The protein was isolated from serum-free culture medium directly by chromatography on an ovine prolactin affinity column and yielded approximately 1.5 mg of protein/liter of suspension culture. The extracellular domain of the rat prolactin receptor inhibited the ovine prolactin-dependent mitogenesis of rat lymphoma Nb2 cells with an IC50 of 7.1 pM and bound 125I-labeled ovine prolactin with a Kd of 1.21 +/- 0.19 nM. In contrast, the binding of the 125I-labeled extracellular domain to ovine prolactin exhibited positive cooperativity with a Hill coefficient of 1.73. High pressure gel filtration chromatography was used to demonstrate the formation of a complex consisting of one molecule of ovine prolactin and two molecules of the extracellular domain of the rat prolactin receptor. Complex formation occurred with human growth hormone, but not with ovine growth hormone, a non-lactogen.

Expression of prolactin (PRL) receptor gene and PRL-binding sites in rabbit intestinal epithelial cells.

Specific binding sites for PRL were identified on isolated intestinal epithelial cells from both male and female rabbits. These receptors exhibited a very high affinity for PRL (Kd = 5 x 10(-11) M) and were immunologically very similar to the well characterized mammary PRL receptor. About 3500 PRL receptors/cell were expressed on isolated epithelial cells from rabbit jejunum. These receptors were distributed in precise portions of the gut, being highly expressed in the proximal small intestine (duodenum and jejunum), moderately expressed in the ileum, and barely detectable in the colon. The levels of intestinal epithelial PRL receptors were low in 15-day-old rabbits, moderate at 1 month, and reached adult levels at 2 months, indicating enhanced PRL receptor expression in the intestine as development proceeded. The PRL receptor gene was specifically and highly expressed in the rabbit intestine, and four PRL receptor transcripts were detected that were identical to the transcripts characterized in the mammary gland. In individual intestinal segments, the expression of PRL-binding sites was always highly correlated with the level of PRL receptor mRNA. These results show that isolated intestinal epithelial cells express the PRL receptor gene as well as specific binding sites with a high affinity for PRL. They suggest that PRL regulates intestinal functions by exerting direct actions on the intestinal epithelial cells.

Mutational analysis of the ligand-binding domain of the prolactin receptor.

The recent isolation and sequencing of the rat liver prolactin (PRL) receptor cDNA (clone F3) revealed that the receptor is a small molecular weight protein (nonglycosylated form, Mr 33,000; glycosylated form, Mr 42,000) comprised of 291 amino acids. A second form of the PRL receptor exists (591 amino acids) that contains a much longer cytoplasmic domain. In the present study, site-directed point mutations of the 5 conserved cysteine (Cys) residues and of the three potential N-linked glycosylation sites in the extracellular domain of the rat PRL receptor were constructed to assess their involvement in hormone binding. In addition, a truncation mutant (T delta 237) lacking 55 of 57 intracellular amino acids was constructed to determine the influence of the cytoplasmic domain on ligand-receptor interactions. Binding studies of transiently transfected COS-7 cells demonstrated that serine substitution of the first 4 Cys residues (Cys12, Cys22, Cys51, and Cys62) completely eliminated binding of 125I-ovine PRL and 125I-U5 and -U6, two monoclonal antibodies that bind the receptor molecule outside the PRL-binding domain. RNA blot analysis of the transfected cells showed that both the wild-type and mutant clones had similar levels of expression of receptor mRNA. Immunoblot analysis demonstrated that lack of PRL binding in these mutants was not due to incomplete processing of the protein, since the fully glycosylated Mr 42,000 form of the receptor was seen. Mutation of Cys184 had no effect on affinity or dimerization capacity of the receptor, suggesting the 5th cysteine is not directly involved in the binding domain. Carbohydrate groups of some receptors have been shown to be involved in ligand-receptor interactions as well as intracellular trafficking. This does not appear to be the case for the PRL receptor, since there was no corresponding decrease in affinity for PRL or cell surface receptor expression, following mutation of each of the 3 asparagine residues to aspartate. Interestingly, T delta 237 showed a 4-5-fold increase in affinity for PRL as well as a marked increase in the number of receptor sites. Whole cell binding assays also demonstrated that loss of the cytoplasmic domain lead to inefficient recycling of the receptor. These studies suggest that the first 4 conserved Cys residues are crucial for ligand binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphorylated variant of bovine prolactin

Bovine pituitary explants and cell cultures were incubated with [ 32P]orthophosphate. Extracts were prepared from the explants and analyzed by sodium dodecyl sulfate-containing acrylamide gel electrophoresis and autoradiography revealing a phosphoprotein that co-migrated with authentic bovine prolactin. Clonal antibodies to bovine prolactin were produced, purified and used to prepare affinity columns. Extracts of [ 32P]orthophosphate-labeled explants and cells or media were applied to prolactin affinity columns and a radiolabeled protein was eluted with a pH 2.8 wash. The eluted protein was identified as prolactin by co-migration with standard on gel electrophoresis and by amino acid analysis. Treatment of immunoaffinity-purified pituitary prolactin with alkaline phosphatase reduced the phosphate associated with prolactin in a time-dependent manner, indicating a covalent phosphate linkage. Autoradiography of gels revealed prolactin from explants, cells and their associated media to be a phosphoprotein. A phosphorylated variant of bovine prolactin is synthesized and secreted in both explant and cell cultures.

Increased prolactin binding and morphological changes in the wound epithelium of regenerating limbs of Notophthalmus viridescens.

Distribution of prolactin has been examined in regenerating forelimbs from the newt Notophthalmus viridescens. Specific prolactin binding was demonstrated in homogenates of unamputated tissue, and of regenerating limbs at from 3 to 21 days postamputation. Labeled prolactin that was injected intraperitoneally into animals with one regenerating limb accumulated in the most distal portion of the regenerate at 7 and 14 days postamputation. Light-microscopic autoradiography demonstrated that labeled prolactin was localized most heavily in the apical, outer layer of the wound epithelium. Scanning electron microscopy demonstrated that, in addition to changes in prolactin affinity following amputation, morphological changes occurred in the apical wound epithelium as well. Cell surfaces of the stump epidermis were characterized by periodic dispersion of papillae among a network of interconnecting structures 1-2 microns across. By contrast, the surfaces of cells from the area in which labeled prolactin was found to localize most intensely were characterized by lack of papillae and, depending on the stage of regeneration, a pattern of microvilli and microplicae. These morphological alterations appear to reflect functional and biochemical differences between stump epidermis and wound epithelium.

Isolation, Characterization, and Regulation of the Prolactin Receptor

The prolactin, or lactogenic hormone, receptor has been purified (∼ 80%) from lactating mouse liver and human term placenta by the nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl) -dimethylammonio] -1-propane sulfonate and a prolactin affinity column. The isolated “core-binding unit” has a molecular weight of 37,000±2,000 daltons. It retains the specificity for lactogenic hormones and binds prolactin with an affinity (Ka = 2 to 6×109M−1) similar to that of the receptor as it occurs in its membranous environment (Ka = 3 to 5×109M−1). Whether this “core-binding unit” exists on the cell surface in a cryptic or active form is influenced greatly by its association with other membrane proteins and the concentration of phosphatidylcholine within its local membranous environment.

Purification of rabbit mammary prolactin receptor by acidic elution from a prolactin affinity column.

Membrane-bound prolactin receptors from the mammary gland of 6-7-day postpartum lactating rabbits were solubilized using the zwitterionic detergent, Zwittergent 3-12 (3- dodecyldimethylammonio )-1-propanesulfonate). The solubilized receptor from one rabbit was bound to an ovine prolactin-agarose affinity gel and eluted at pH 4.2. The receptor appeared as a single band upon staining sodium dodecyl sulfate-polyacrylamide gels with Coomassie blue. The protein yield from one rabbit was about 8 micrograms and the overall yield of receptor was over 50%. The apparent molecular weight was 42,000 on sodium dodecyl sulfate gels but varied on molecular weight columns due to the type of detergent. Receptor inactivated by iodination had an apparent molecular weight of 21,000 on sodium dodecyl sulfate gels. The purified receptor did not bind to concanavalin A-agarose or Lens culinaris-agarose.

Influence of infused prolactin on hormone binding to tissue slices.

The influence of circulating prolactin on the binding of labeled hormone to tissue slices in vitro was investigated in rats bearing dimethylbenzanthracene-induced mammary tumors. Prolactin was infused into four animals over four 30-min periods to give serum levels ranging from 20 to 2000 ng/ml. Serial biopsies of liver and tumor were taken during infusion, and binding of labeled prolactin and insulin measured in tissue slices. Whereas insulin binding remained constant, prolactin binding was significantly depressed in both tissues when samples were obtained at serum prolactin levels greater than 300 ng/ml. This decrease was due to diminished numbers of available binding sites rather than a change in binding-site affinity for prolactin. The data indicate that under the conditions of this experiment, endogenous circulating prolactin may interfere with in vitro measurements of hormone uptake when serum levels excee 300 ng/ml.

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 (E 2), progesterone, hydrocortisone, triiodothyronine (T 3) or E 2 plus T 3 could not mimic the prolactin effect. Neither combinations of prolactin with E 2 or T 3 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.

A Receptor Site for Prolactin in Lactating Mouse Mammary Tissues

Prolactin iodinated by lactoperoxidase method showed immunologically, electrophoretically and biolo9gically similar properties to native prolactin and possessed enough specific radioactivity for receptor studies. 1251-prolactin was incubated with mouse mammary tissues at 8 days of lactation. Both binding and release of 1251-prolactin depended on incubation time and temperature and were maximal at 37 degrees C. Michaelis constant was estimated to be 1.4 X 10(-9) M from Lineweaver-Burk plot and to be 1.2 X 10(-9) M from id-value of the dose-response curve for displacement with native prolactin. Total number of binding sites for prolactin was 1.38 X 10(-15) mole per mg weight of tissue. Ovine prolactin, human growth hormone and human placental lactogen complete with 1251-prolactin and dose-response curves for these three hormones were all parallel. These results suggest the existence of a specific receptor site with high affinity for prolactin in lactating mouse mammary glands.