Comparison of secreted and extracted forms of rat pituitary prolactin

Prolactin secreted by rat anterior pituitary explants into organ culture medium was purified by salt fractionation and gel filtration. A yield of 22 mg/g was obtained, which clearly represented de novo synthesis and secretion of the hormone. Comparative characterization studies were performed on the secreted prolactin and pituitary extracted rat prolactin obtained from the National Institute of Arthritis, Metabolism and Digestive Diseases. The biological and immunological activity estimates of both forms were comparable, although the specific activities of the secreted prolactin were somewhat lower than those of the pituitary prolactin. The secreted and extracted forms of prolactin appeared to be identical in primary structure as evidenced by similar amino acid compositions and identical NH₂-terminal sequences. Circular dichroism spectra suggested that there may be differences in tertiary structure, since the positive tryptophan band at 292 nm, which was observed with extracted hormone, was absent in the secreted prolactin.     

Homologous radioimmunoassay for secreted mouse prolactin

A highly specific, homologous radioimmunoassay has been developed for the secreted form of mouse prolactin using hormone isolated and purified from the media from long-term pituitary cultures. Mouse pituitary homogenates and mouse serum give parallel dilution response curves in the assay, and no cross-reaction is seen with either mouse growth hormone or mouse placental lactogen. The assay is sensitive to 40 pg per tube, and the reproducibility and precision of the assay are within acceptable limits. Antiserum generated to secreted mouse prolactin cross-reacts with both the secreted and stored forms of the hormone; however, the secreted form shows greater immunopotency. Secreted prolactin also shows greater immunopotency when compared with stored prolactin using an antiserum generated to the stored form of the hormone.     

The Dwarf Phenotype in GH240B Mice,Haploinsufficient for the Autism Candidate Gene Neurobeachin,Is Caused by Ectopic Expression of Recombinant Human Growth Hormone

Two knockout mouse models for the autism candidate gene Neurobeachin (Nbea) have been generated independently. Although both models have similar phenotypes, one striking difference is the dwarf phenotype observed in the heterozygous configuration of the GH240B model that is generated by the serendipitous insertion of a promoterless human growth hormone (hGH) genomic fragment in the Nbea gene. In order to elucidate this discrepancy, the dwarfism present in this Nbea mouse model was investigated in detail. The growth deficiency in Nbea ^+/− mice coincided with an increased percentage of fat mass and a decrease in bone mineral density. Low but detectable levels of hGH were detected in the pituitary and hypothalamus of Nbea ^+/− mice but not in liver, hippocampus nor in serum. As a consequence, several members of the mouse growth hormone (mGH) signaling cascade showed altered mRNA levels, including a reduction in growth hormone-releasing hormone mRNA in the hypothalamus. Moreover, somatotrope cells were less numerous in the pituitary of Nbea ^+/− mice and both contained and secreted significantly less mGH resulting in reduced levels of circulating insulin-like growth factor 1. These findings demonstrate that the random integration of the hGH transgene in this mouse model has not only inactivated Nbea but has also resulted in the tissue-specific expression of hGH causing a negative feedback loop, mGH hyposecretion and dwarfism.     

Growth hormone and prolactin receptors in adipogenesis: STAT-5 activation, suppressors of cytokine signaling, and regulation of insulin-like growth factor I

Growth hormone GH stimulates lipolysis in mature adipocytes and primary preadipocytes but promotes adipogenesis in preadipocyte cell lines. The lactogenic hormones (prolactin PRL and placental lactogen) also stimulate adipogenesis in preadipocyte cell lines but have variable lipolytic and lipogenic effects in mature adipose tissue. We hypothesized that differences in expression of GH receptors GHR and PRL receptors PRLR during adipocyte development might explain some of the differential effects of the somatogens and lactogens on fat metabolism. To that end, we compared: (a) the expression of GHR and PRLR mRNAs in 3T3-L1 preadipocytes during the course of adipocyte differentiation; (b) the induction of STAT-5 activity by GH and PRL during adipogenesis; and (c) the acute effects of GH and PRL on the suppressors of cytokine signaling (SOCS-1-3 and cytokine-inducible SH2-domain-containing protein CIS) and IGF-I. In confluent, undifferentiated 3T3-L1 cells, the levels of GHR mRNA were approximately 250-fold higher than the levels of PRLR mRNA. Following induction of adipocyte differentiation the levels of PRLR mRNA rose 90-fold but GHR mRNA increased only 0.8-fold. Expression of both full-length (long) and truncated (short) isoforms of the PRLR increased during differentiation but the long isoform predominated at all time points. Mouse GH mGH stimulated increases in STAT-5a and 5b activity in undifferentiated as well as differentiating 3T3-L1 cells; mouse PRL mPRL had little or no effect on STAT-5 activity in undifferentiated cells but stimulated increases in STAT-5a and 5b activity in differentiating cells. mGH stimulated increases in SOCS-2 and SOCS-3 mRNAs in undifferentiated cells and SOCS-1-3 and CIS mRNAs in differentiating cells; mPRL induced CIS in differentiating cells but had no effect on SOCS-1-3. mPRL and mGH stimulated increases in IGF-I mRNA in differentiating cells but not in undifferentiated cells; the potency of mGH (3-6-fold increase, p < 0.01) exceeded that of mPRL (40-90% increase, p < 0.05). Our findings reveal disparities in the expression of PRLR and GHR during adipocyte development and differential effects of the hormones on STAT-5, the SOCS proteins, CIS, and IGF-I. These observations suggest that somatogens and lactogens regulate adipocyte development and fat metabolism through distinct but overlapping cellular mechanisms.     

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.     

Nucleotide sequence of mouse prolactin and growth hormone mRNAs and expression of these mRNAs during pregnancy

The mRNAs for mouse prolactin and growth hormone have been isolated from anterior pituitary glands and cloned as cDNAs. The nucleotide sequences of these mRNAs have been determined, and these sequences, along with the predicted amino acid sequences, are compared to those of other mammalian prolactin and growth hormone mRNAs. Levels of prolactin and growth hormone mRNAs during pregnancy have been monitored by hybridization to the cloned cDNA probes. We find the levels of these mRNAs to remain nearly constant during mid-to-late gestation.     

Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice

Prolactin receptors were monitored by measuring ¹²⁵I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (K_d) of 0.99 · 10^?9 M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (K_d) of the receptors.     

Purification, cloning, and expression of the prolactin receptor

The rat liver prolactin receptor has been purified to homogeneity, and partial amino acid sequences have been obtained. The structure of the receptor has been deduced from a single complementary DNA clone. The mature protein of 291 amino acids has a relatively long extracellular region, a single transmembrane segment, and a short (57 amino acids) cytoplasmic domain. With the rat cDNA used as a probe, the prolactin receptor in rabbit mammary gland and human hepatoma cells has also been isolated. These tissues contain a second, longer form of the receptor (592 and 598 amino acids, respectively). Both the short and long forms of the prolactin receptor show regions of strong sequence identity with the human and rabbit growth hormone receptors, suggesting that the prolactin and growth hormone receptors originate from a common ancestor.     

Cloning of the cDNAs coding for cat growth hormone and prolactin

Characterization of the prolactin (PRL) amino acid (aa) or cDNA sequences has not been reported for any member of the Felidae family. We cloned cat growth hormone (cGH) and cat PRL (cPRL) cDNA sequences from a feline pituitary cDNA library. High homology between species allowed bovine PRL (bPRL) and bGH cDNA clones to be used to identify clones encoding the 229-aa cPRL and 216-aa cGH sequences. The cGH protein is most homologous to pig and dog GH. Similarly, cPRL shares the most aa identity to pig PRL (pPRL). Northern blot analysis revealed the mRNA size for cGH and cPRL to be approx. 1 and 1.1 kb, respectively. These results reveal that GH and PRL from the Felidae family are highly conserved to other families of GH and PRL.     

The tertiary structure and backbone dynamics of human prolactin

Human prolactin is a 199-residue (23 kDa) protein closely related to growth hormone and placental lactogen with properties and functions resembling both a hormone and a cytokine. As a traditional hormone, prolactin is produced by lactotrophic cells in the pituitary and secreted into the bloodstream where it acts distally to regulate reproduction and promote lactation. Pituitary cells store prolactin in secretory granules organized around large prolactin aggregates, which are produced within the trans layer of the Golgi complex. Extrapituitary prolactin is synthesized by a wide variety of cells but is not stored in secretory granules. Extrapituitary prolactin displays immunomodulatory activities and acts as a growth factor for cancers of the breast, prostate and tissues of the female reproductive system. We have determined the tertiary structure of human prolactin using three-dimensional (3D) and four-dimensional (4D) heteronuclear NMR spectroscopy. As expected, prolactin adopts an "up-up-down-down" four-helical bundle topology and resembles other members of the family of hematopoietic cytokines. Prolactin displays three discrete structural differences from growth hormone: (1) a structured N-terminal loop in contact with the first helix, (2) a missing mini-helix in the loop between the first and second helices, and (3) a shorter loop between the second and third helices lacking the perpendicular mini-helix observed in growth hormone. Residues necessary for functional binding to the prolactin receptor are clustered on the prolactin surface in a position similar to growth hormone. The backbone dynamics of prolactin were investigated by analysis of 15N NMR relaxation phenomena and demonstrated a rigid four-helical bundle with relatively mobile interconnecting loops. Comparison of global macromolecular tumbling at 0.1mM and 1.0mM prolactin revealed reversible oligomerization, which was correlated to dynamic light scattering experiments. The existence of a reversible oligomerization reaction in solution provides insight into previous results describing the in vitro and in vivo aggregation properties of human prolactin.     

Studies on prolactin 48: Isolation and properties of the hormone from horse pituitary glands

Isolation of prolactin from equine pituitary glands has been described. It has a potency of 42 IU/mg in the pigeon crop-sac test and consists of 199 amino acids. The hormone has only four half-cystine residues in contrast to other mammalian prolactins which have six residues. From NH₂-terminal sequence analysis and amino acid composition of cyanogen bromide fragments, the NH₂-terminal disulfide loop is missing in the equine prolactin molecule. Circular dichroism spectra indicate that the α-helical content of equine prolactin appears to be lower (50%) than that found in the ovine hormone (65%).     

Prolactin modifies the prostaglandin synthesis, prolactin binding and fluidity of mouse liver membranes

The objective of these studies was to determine if prolactin, known to induce its own receptors, alters the prostaglandin (PG) synthesis which could, in turn, modify the fluidity of the membrane and thus alter the functionality of prolactin receptors. Adult male C₃H mice were injected subcutaneously with 100 μg of oPRL every 4 h for 0, 24 or 48 h and sacrificed 8 h after receiving the last injection. Liver 100,000 × $\text{g}$ membrane pellets were used in the measurement of these parameters. The amount of binding of prolactin to these membranes increased with the duration of injections, the values being 179 and 244% of control values after 24 and 48 h of injections, respectively. The amounts of PGF_2α and PGE synthesized also increased after these injections, the values being 127 and 270% of control for PGF_2α and 634 and 695% of control values for PGE after 24 and 48 h of injections, respectively. Fluorescence polarization, an index of microviscosity, was decreased by 14 and 20% after 24 and 48 h of PRL administration, respectively. Previous studies have demonstrated simultaneous in vitro effects of prostaglandin on both prolactin receptors and membrane fluidity. The current data are in agreement with those observations and suggest that prolactin may modulate its own receptor by increasing the fluidity of the membrane in which it exists by alterations within the PG cascade. Such biochemical changes may then modify existing restraints and allow the hormone receptor to assume a more functional configuration.     

Effects of colchicine and 2-Br-alpha-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone by functional pituitary tumor cells in culture

The effects of colchicine and 2-Br-α-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone have been studied in a clonal strain of rat pituitary tumor cells (GH₃) in monolayer culture. These cultures produce both prolactin and growth hormone and release both proteins spontaneously into the medium without storing them in large amounts. Immunological methods were used to measure both intracellular and extracellular concentrations of the hormones. Colchicine (5 × 10^?6 M for 3 hours) caused a 2- to 3-fold increase in intracellular concentrations of prolactin and growth hormone but, under basal conditions, had little or no measurable effect on the amounts of hormone accumulated in the medium during the course of the standard three hour treatment period. This latter finding evidently is due to a lag in the onset of drug action. Colchicine had little or no effect on accumulation of extracellular prolactin during the first two hours of treatment whereas such accumulation was depressed by over 60% during the third hour of treatment. Previous studies have shown that treatment of GH₃ cells with thyrotropin releasing hormone (TRH) and hydrocortisone (HC) increases both intra and extracellular levels of prolactin and growth hormone, respectively. In cultures treated with TRH (5 × 10^?8 M), colchicine (5 × 10^?6 M for 3 hours) increased intracellular prolactin by about 70% and decreased extracellular hormone by 10%. In cultures treated with HC (3 × 1O^?6 M), colchicine increased intracellular growth hormone by more than 100% and decreased medium concentrations of the hormone by 15%. Colchicine did not significantly alter total hormone (intracellular + extracellular) accumulation, cellular uptake of ³H-amino acids, or total cell protein synthesis. The synthetic ergot alkaloid, CB 154, (3.3 × 10^?6 M for 3 hours) caused an 80% increase in intracellular, and a nearly 50% decrease in extracellular, prolactin without affecting the accumulation of growth hormone, the uptake of ³H-labeled amino acids, or overall protein synthesis in the cultures. Elevation of medium potassium concentration from a basal value of 5.3 mM to 3–5 × 10^?2 M (by addition of KCl) decreased intracellular levels of prolactin by 85% and growth hormone by 55%. These effects of high potassium were blocked by colchicine and by CB 154. We conclude that colchicine, after a lag period of two hours, acts to inhibit the release of prolactin and growth hormone from GH₃ cells. By the end of three hours of treatment, this inhibition is over 60% complete in the case of prolactin. The qualitatively different effects of colchicine and CB 154 on prolactin and growth hormone release suggest that these two secretory blocking agents probably act on GH₃ cells by different mechanisms.     

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.     

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.     

Stereological and biochemical analysis of prolactin and growth hormone secreting rat pituitary cells in culture

Summary The secretion of prolactin is increased by treatment of prolactin producing rat pituitary cells with the hypothalamic tripeptide thyroliberin. To investigate the underlying mechanisms we used three closely related rat pituitary tumor cell strains (GH₁2C₁, GH₃ and GH₄C₁), which synthesize and spontaneously secrete prolactin and/or growth hormone. Growth hormone and prolactin released into the culture medium over a period of 24 h were measured by radioimmunoassay. Initial rates of synthesis were measured by immunoprecipitation of intracellular growth hormone and prolactin after incubation of cell cultures with ³H-leucine. The observed increase in prolactin synthesis and release was correlated with morphological effects of thyroliberin treatment. The volume density of Golgi complexes and the volume and surface densities of rough endoplasmic reticulum were compared in untreated cells and thyroliberin treated cells. As normal distribution could not be assumed, the non-parametric rank test of Wilcoxon was used whereby the densities calculated for each cell section were ranked. Alle three morphological parameters increased after thyroliberin treatment in cells secreting prolactin only (GH₄C₁), implying that the increase of prolactin secretion, at lest in part, is due to increased prolactin synthesis.     

Breakdown of corticotropin-(1-24) by mouse brain extracts.

ACTH-(1–24) was rapidly degraded to its constituent amino acids upon incubation with soluble mouse brain preparations. Breakdown had a pH optimum near neutrality, a K_m of 1 × 10^?4m, and a V_max of 63 nmol/mg protein/h. Initially a preferential liberation of the amino acids of the N-terminal region of the hormone was observed, followed shortly by a relatively uniform release of amino acids originating throughout the ACTH-(1–24) sequence. Enzymes cleaving internal bonds appear to play a major role in the degradation. The short lag period between hormone disappearance and amino acid formation indicated that few if any large peptides were accumulated during incubation. Pepstatin, chymostatin, antipain, leupeptin, bacitracin, l-1-tosylamide-2-phenylethylchloromethyl ketone, soybean trypsin inhibitor, and diisopropyl-fluorophosphate had little or no effect on amino acid release. Puromycin, bestatin, and EDTA partially inhibited amino acid formation, affecting mainly the residues in the central and carboxyl portions of ACTH-(1–24). N-Ethylmaleimide strongly inhibited the release of all amino acids, indicating extensive involvement of sulfhydryl peptidases in ACTH-(1–24) breakdown.     

Glycosylated equine prolactin and its carbohydrate moiety

Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities,G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about l/30th the mitogenic activity of bovine PRL; G-ePRL was approximately l/10th as active as ePRL. Glycosylation of G-ePRL at Asn³¹ was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29–37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man₃, GlcNAc₂, GalNAc₁, Fuc_0.6, Gal_0.2, NeuAc_0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn³¹ carbohydrate unit is a fucosylated complex mono- and/or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.