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Prolactin (PRL) has long been known to be a hormone responsible for mammary gland development and lactation in females, whereas its role in males is still unclear. Thus, we investigated male mouse (m) PRL protein and mRNA expression in spermatozoa at various differentiation stages in the testes. Quantitative RT-PCR and in situ hybridization detected the expression of PRL not only in Leydig cells but also in germ cells, in particular in spermatogonia. The nucleotide sequence of testis PRL mRNA was the same as that in the pituitary. The mPRL was detected in Leydig cells and in round and elongated spermatids of the testes by immunohistochemistry. Immunoblotting detected 2 forms of mPRL in the testes, one form was 23-kDa PRL, and the other form was smaller than full-length PRL. Based on these results, we focused on N-terminal cleaved PRL to determine its involvement in spermatogenesis. Immunohistochemistry using two sets of antibodies, one that recognized full-length PRL and N-terminal cleaved PRL and another that recognized full-length PRL and C-terminal cleaved PRL, suggested that intact PRL was localized in the nucleus of round spermatids, while N-terminal cleaved PRL variants were localized in the Golgi apparatus of the sperrmatid nuclei of round spermatids, cytoplasms of elongated spermatids and in the spermatozoa tails. These findings suggest that PRL is ectopically expressed in the spermiogenesis and spermatogenesis and that cleaved PRL variants were localized in the Golgi apparatus of spermatids and in spermatozoa tails.

The most promising approach to targeting the tumor-growth-promoting actions of prolactin (PRL) mediated by its autocrine/paracrine pathway has been the development of specific PRL receptor (PRLR) antagonists. However, the optimization of such antagonists requires a thorough understanding of the activation mechanism of PRLR. We have thus conducted a systematic X-ray crystallographic study in order to visualize the successive steps of PRLR activation by PRL. We report here the structure at 3.35 Å resolution of the 1:2 complex between natural PRL and two PRLR chains (PRLR1 and PRLR2), corresponding to the final activated state of PRLR. Further than our previously published structure involving an affinity-matured PRL variant, this structure allowed to visualize for the first time the loop L5 spanning PRLR2 residues Thr133-Phe140, revealing its central implication for the three intermolecular interfaces of the complex. We equally succeeded in obtaining a comprehensive picture of the PRLR-PRLR dimerization interface, also called stem-stem interface. Site-directed mutagenesis was conducted to probe the energetic importance of stem-stem contacts highlighted by the structure. Surprisingly, in spite of significant structural differences between the PRL/PRLR(2) complex and the 1:2 growth hormone/growth hormone receptor complex, our mutational data suggest that hot-spot residues that stabilize the receptor dimerization interface are equivalent in the two complexes. This study provides a new overall picture of the structural features of PRLR involved in stabilizing its complex with PRL., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Human prolactin (PRL) is currently viewed as a hormone of pituitary origin, whose production (i.e. serum levels) is controlled by dopamine, whose biological actions relate exclusively to lactation and reproductive functions, for which any genetic disorder is yet to be identified, and whose unique associated pathology is hyperprolactinemia. Both experimental studies and human sample/cohort-based investigations performed during the past decade have considerably widened our perception of PRL biology: i) there are now strong epidemiological arguments supporting the fact that circulating PRL is a risk factor for breast cancer, ii) in addition to the endocrine hormone, locally produced PRL has been documented in several human tissues; there is increasing evidence supporting the tumor growth potency of local PRL, acting via autocrine/paracrine mechanisms, in both rodent models, and human breast and prostate tumors, iii) the first functional germinal polymorphisms of the PRL receptor were recently identified in patients presenting with breast tumors, which involve single amino acid substitution variants exhibiting constitutive activity, iv) human PRL analogs have been engineered, which were shown in experimental models to down-regulate the effects triggered by local PRL (competitive antagonism) or by the constitutively active receptor variants (inverse agonism). The aim of this review is to discuss these novel concepts in PRL biology, including their potential pathophysiological outcomes.

Hyperprolactinaemia is a common adverse event reported in association with treatments used in schizophrenia and bipolar disorder. Recent data are suggestive that hyperprolactinaemia may have a range of significant short-and long-term clinical consequences. The objective of this review is to examine the causes, frequency and clinical consequences of hyperprolactinaemia in the severely mentally ill (SMI) with a focus on patients taking antipsychotic medications. A Medline search was carried out to identify relevant publications. Reference lists from previous review articles were also examined to search for additional data. Hyperprolactinaemia may be one of the most common adverse events associated with some antipsychotic medications. Precise rates with individual drugs had however until recently been poorly categorized. The relationship between hyperprolactinaemia and adverse outcomes in the SMI population appears similar to that in the general population. Adverse outcomes (such as sexual dysfunction) can occur acutely and in the longer term (bone fractures and possibly breast cancer), but the precise link between degree and length of hyperprolactinaemia and adverse outcome remains to be established. In conclusion, hyperprolactinaemia is a common treatment-emergent adverse event of some antipsychotic medications and may have clinical consequences. Physicians must balance the benefits and risks of treatment when determining appropriate therapy for individual patients.

Hyperprolactinaemia is one of the most common endocrinological disorders affecting the hypothalamic-pituitary axis. The increase of prolactin level in the blood interrupts the pulsatile GnRH secretion and in turn causes hypoestrogenism. Hyperprolactinaemia can occur in physiological and pathological conditions. Prolactin has 3 different isoforms: little prolactin, big prolactin and big-big prolactin. Macroprolactin is a complex of little prolactin and an immunoglobulin G antibody and the weight of the complex is more than 150 kDa. In physiological condition macroprolactin comprises up to 1% of all circulating prolactin in the serum blood. In pathological condition percentage of macroprolactin can increase in the serum blood. The prevalence of macroprolactinaemia is found in around 10-26% of all patients with hyperprolactinaemia. Women with high serum prolactin concentration in should be screened for macroprolactinaemia. Confirmation the presence of macroprolactinaemia doesn't require any pharmacological treatment or other medical procedures.

Class 1 cytokines bind two receptors to create an active heterotrimeric complex. It has been argued that ligand binding to their receptors is an ordered process, but a structural mechanism describing this process has not been determined. We have previously described an obligate ordered binding mechanism for the human prolactin/prolactin receptor heterotrimeric complex. In this work we expand this conceptual understanding of ordered binding to include three human lactogenic hormones: prolactin, growth hormone, and placental lactogen. We independently blocked either of the two receptor binding sites of each hormone and used surface plasmon resonance to measure human prolactin receptor binding kinetics and stoichiometries to the remaining binding surface. When site 1 of any of the three hormones was blocked, site 2 could not bind the receptor. But blocking site 2 did not affect receptor binding at site 1, indicating a requirement for receptor binding to site 1 before site 2 binding. In addition we noted variable responses to the presence of zinc in hormone-receptor interaction. Finally, we performed Förster resonance energy transfer (FRET) analyses where receptor binding at subsaturating stoichiometries induced changes in FRET signaling, indicative of binding-induced changes in hormone conformation, whereas at receptor:hormone ratios in excess of 2:1 no additional changes in FRET signaling were observed. These results strongly support a conformationally mediated obligate-ordered receptor binding for each of the three lactogenic hormones.

We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL.PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural components of PRL site 2 ("three-pin plug"): the conserved glycine 129 of helix alpha3, the hydrogen bond network involving surrounding residues (glycine cavity), and the N terminus. The model provides a molecular basis for the properties of the different PRL analogs designed to date, including PRLR antagonists. Finally, comparison of our 1:2 PRL.PRLR(2) structure with those of free PRL and its 1:1 complex indicates that the structure of PRL undergoes significant changes when binding the first, but not the second receptor. This suggests that the second PRLR moiety adapts to the 1:1 complex rather than the opposite. In conclusion, this structure will be a useful guiding tool for further investigations of the molecular mechanisms involved in PRLR dimerization and activation, as well as for the optimization of PRLR antagonists, an emerging class of compounds with high therapeutic potential against breast and prostate cancer.

Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10-30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be approximately 4-fold lower than that of hPRL, its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL approximately 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by approximately 7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11-15 IU mg(-1)) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Macroprolactin is a large, heterogeneous form of prolactin with limited bioavailability. Detection of macroprolactin by different immunoassays varies widely. The objectives of this study were to determine the immunoreactivity of macroprolactin by the Ortho Clinical Diagnostics Vitros((R)) ECi prolactin immunoassay, establish the most effective method for interpreting the prolactin concentration after PEG-precipitation, and correlate the clinical features of hyperprolactinemia with the presence of macroprolactin., Methods: PEG-precipitation was performed on 120 hyperprolactinemic specimens. Of these, 31 specimens with a recovery<80% were fractionated by GFC. Four different approaches for identifying true hyperprolactinemia were investigated. Clinical symptoms of hyperprolactinemia were determined by chart review., Results: Macroprolactin was detected by the Vitros ECi prolactin immunoassay. Use of a PEG modified prolactin reference interval was effective for identifying hyperprolactinemia in the presence of macroprolactin. There was no difference in the prevalence of abnormal menses, galactorrhea, or abnormal MRI between those with and without macroprolactin (p>0.05). Accounting for macroprolactin in patients with hyperprolactinemia reduced the number of idiopathic cases., Conclusions: The Vitros ECi prolactin immunoassay detects macroprolactin. PEG-precipitation is an acceptable surrogate to detect hyperprolactinemia in the presence of macroprolactin when using a prolactin reference interval derived from PEG precipitated reference sera. Although testing for macroprolactin should not substitute for standard evaluation of hyperprolactinemia, identification of macroprolactin may clarify a diagnosis and direct appropriate therapy., (Copyright 2009 Elsevier B.V. All rights reserved.)

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22 mg/L from 100 mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.

Vasoinhibins are a family of N-terminal prolactin (PRL) fragments that inhibit blood vessel growth, dilation, permeability, and survival. The aspartyl endoprotease cathepsin D is active at acidic pH and can cleave rat PRL to generate vasoinhibins. We investigated whether and where vasoinhibins could be generated by cathepsin D in the adenohypophysis of rats and mice and whether their production could be gender dependent. Vasoinhibins were detected in primary cultures of rat adenohypophyseal cells by Western blot with antibodies directed against the N terminus of PRL but not the C terminus. Ovariectomized, estrogen-treated females show greater levels of adenohypophyseal vasoinhibins than males. Peptide sequencing analysis revealed that the cleaved form of PRL in rat adenohypophyseal extracts contains the PRL N terminus and a second N terminus starting at Ser(149), the reported cleavage site of cathepsin D in rat PRL. In addition, cathepsin D inhibition by pepstatin A reduced vasoinhibin levels in rat adenohypophyseal cell cultures. Confocal and electron microscopy showed the colocalization of cathepsin D and PRL within rat adenohypophyseal cells and secretory granules, and a subcellular fraction of rat adenohypophysis enriched in secretory granules contained cathepsin D activity able to generate vasoinhibins from PRL. Of note, vasoinhibins were absent in the adenohypophysis of mice lacking the cathepsin D gene but not in wild-type mice. These findings show that cathepsin D is the main protease responsible for the generation of adenohypophyseal vasoinhibins and that its action can take place within the secretory granules of lactotrophs.

Introduction: Macroprolactin (MPRL) is an important source of interference that may lead to misdiagnosis and mismanagement of hyperprolactinaemic patients. Prolactin assays exhibit considerable variation in the detection of MPRL. In this study, we examine the requirement for polyethylene glycol (PEG) precipitation in the analysis of hyperprolactinaemia detected by the Advia Centaur as it has a relatively low reactivity with MPRL., Methods: Four hundreds and sixty-four hyperprolactinaemic samples were collected from the laboratory information system for Saint James University Hospital over a three-year period. These samples were screened for MPRL using PEG precipitation protocol. Monomeric prolactin concentration post-PEG precipitation was compared with a reference range determined by PEG precipitation in normal subjects., Results: MPRL was the cause of hyperprolactinaemia in 4% of patients (16/409) over the three-year period studied. Nine subjects with MPRL also had elevated monomeric prolactin., Conclusions: PEG screening is still needed for assays with low MPRL reactivity such as the Advia Centaur and this should be performed with a locally derived reference range for monomeric prolactin.

Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3'-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3'-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia.

Delta 41-52 hPRL (human prolactin with residues 41-52 removed) is a lead compound for a new class of hPRL antagonists. The deleted sequence contains residues that functionally couple sites 1 and 2, the two hormone surfaces that each bind receptors. Delta 41-52 hPRL retains 0.03% agonist activity in FDC-1 cell bioassays, a 3054-fold reduction in activity, and displays approximately 100-fold less agonist activity than G129R hPRL, an antagonist that reduces the binding of hPRL receptor at site 2 during the formation of the heterotrimeric hormone/receptor complex. Replacement of various numbers and types of residues into the gap created by the deletion of residues 41 through 52 created hPRLs with varying agonist activities, suggested that manipulation of the sequence connecting the C-terminal of helix 1 with the disulfide bond (cysteines 58 with 174) linking helices 1 and 4 modulates articulation of these helices and influences agonist activity. We have compared the antagonist activities of G129R and Delta 41-52 hPRLs to induce apoptosis in Jurkat cells, a human lymphoid cell line displaying an autocrine/paracrine hPRL/receptor system. Delta 41-52 hPRL induces apoptosis in a time and dose-dependent fashion. Under these same conditions G129R hPRL fails to induce apoptosis. We conclude Delta 41-52 hPRL is a lead compound of a new class of hPRL antagonists capable at low concentrations of inducing apoptosis in human cells expressing an autocrine/paracrine hPRL/receptor system.

S179D prolactin (S179D PRL) is a pseudophosphorylated form of human PRL which has potent antitumor and anti-angiogenic activities in vivo. This molecule binds to the same forms of the PRL receptor (PRLR) as unmodified PRL, yet this binding results in different intracellular signaling and biological end points. Since it is now clear that PRLRs are predimerized and therefore that ligand binding must initiate signaling by inducing a conformational change in the receptor dimer, we hypothesized that S179D PRL had an altered conformation compared to unmodified PRL. The conformation of the ligand-receptor ternary complex would therefore also have an altered conformation, and thus, different signaling molecules would be activated. Here we present evidence in support of this hypothesis by demonstrating, in contrast to unmodified PRL, that S179D PRL has reduced nickel and zinc binding capacity and a higher affinity for heparin and DEAE. Conformational changes have occurred since these features are counterintuitive on the basis of the simple substitution of a serine with a negatively charged aspartate residue. To demonstrate that these particular properties of S179D PRL were not due to misfolding of the molecule during production, S179D PRL was expressed in two different mammalian cell lines. Also investigated was the potential for production of S179D PRL as a soluble cytoplasmic, or secreted periplasmic, protein in Escherichia coli.

Background: The three pituitary hormones, viz. prolactin (PRL), growth hormone (GH) and somatolactin (SL), together with the mammalian placental lactogen (PL), constitute a gene family of hormones with similar gene structure and encoded protein sequences. These hormones are believed to have evolved from a common ancestral gene through several rounds of gene duplication and subsequent divergence., Principal Findings: In this study, we have identified a new PRL-like gene in non-mammalian vertebrates through bioinformatics and molecular cloning means. Phylogenetic analyses showed that this novel protein is homologous to the previously identified PRL. A receptor transactivation assay further showed that this novel protein could bind to PRL receptor to trigger the downstream post-receptor event, indicating that it is biologically active. In view of its close phylogenetic relationship with PRL and also its ability to activate PRL receptor, we name it as PRL2 and the previously identified PRL as PRL1. All the newly discovered PRL2 sequences possess three conserved disulfide linkages with the exception of the shark PRL2 which has only two. In sharp contrast to the classical PRL1 which is predominantly expressed in the pituitary, PRL2 was found to be mainly expressed in the eye and brain of the zebrafish but not in the pituitary. A largely reduced inner nuclear layer of the retina was observed after morpholino knockdown of zebrafish PRL2, indicating its role on retina development in teleost., Significance: The discovery of this novel PRL has revitalized our understanding on the evolution of the GH/PRL/SL/PL gene family. Its unique expression and functions in the zebrafish eye also provide a new avenue of research on the neuroendocrine control of retina development in vertebrates.

A member of the family of hematopoietic cytokines human prolactin (hPRL) is a 23k kDa polypeptide hormone, which displays pH dependence in its structural and functional properties. The binding affinity of hPRL for the extracellular domain of its receptor decreases 500-fold over the relatively narrow, physiologic pH range from 8 to 6; whereas, the affinity of human growth hormone (hGH), its closest evolutionary cousin, does not. Similarly, the structural stability of hPRL decreases from 7.6 to 5.6 kcal/mol from pH 8 to 6, respectively, whereas the stability of hGH is slightly increased over this same pH range. hPRL contains nine histidines, compared with hGH's three, and they are likely responsible for hPRL's pH-dependent behavior. We have systematically mutated each of hPRL's histidines to alanine and measured the effect on pH-dependent global stability. Surprisingly, a vast majority of these mutations stabilize the native protein, by as much as 2-3 kcal/mol. Changes in the overall pH dependence to hPRL global stability can be rationalized according to the predominant structural interactions of individual histidines in the hPRL tertiary structure. Using double mutant cycles, we detect large interaction free energies within a cluster of nearby histidines, which are both stabilizing and destabilizing to the native state. Finally, by comparing the structural locations of hPRL's nine histidines with their homologous residues in hGH, we speculate on the evolutionary role of replacing structurally stabilizing residues with histidine to introduce pH dependence to cytokine function.

The mature segment gene of prolactin (PRL) in White Goose was amplified from pituitary by RT-PCR and then cloned into the pMD18-T vector. Sequencing analysis showed that the cDNA has a length of 690 bp including the termination codon and encodes a protein composed of 230 amino acids, which differs from the published PRL cDNA sequence. There is a homology of 99.57% in base and 99.56% in amino acids with that of Wanxi White Goose, respectively. A prokaryotic expression vector, pET-32a(+), was used to construct the recombinant plasmid pET-32a(+)-PRL to produce protein. Having been induced by IPTGthe host cell carrying the recombinant plasmid expressed the recombinant PRL. The optimal condition for expression is 1 mmol/L IPTG at 37. Based on this condition, the expression rose to the highest level by 4 h of induction, accounting for 28.96% of the total bacterial protein.

The structural and functional properties of human prolactin (hPRL), a 23 kDa protein hormone and cytokine, are pH-dependent. The dissociation rate constant for binding to the extracellular domain of the hPRL receptor increases nearly 500-fold over the relatively narrow and physiologic range from pH 8 to 6. As the apparent midpoint for this transition occurs around pH 6.5, we have looked toward histidine residues as a potential biophysical origin of the behavior. hPRL has a surprising number of nine histidines, nearly all of which are present on the protein surface. Using NMR spectroscopy, we have monitored site-specific proton binding to eight of these nine residues and derived equilibrium dissociation constants. During this analysis, a thermodynamic interaction between a localized triplet of three histidines (H27, H30, and H180) became apparent, which was subsequently confirmed by site-directed mutagenesis. After consideration of multiple potential models, we present statistical support for the existence of two negative cooperativity constants, one linking protonation of residues H30 and H180 with a magnitude of approximately 0.1 and the other weaker interaction between residues H27 and H30. Additionally, mutation of any of these three histidines to alanine stabilizes the folded protein relative to the chemically denatured state. A detailed understanding of these complex protonation reactions will aid in elucidating the biophysical mechanism of pH-dependent regulation of hPRL's structural and functional properties.

The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5A resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Comparison of the structure of the receptor-bound PRL variant with the structure reported for the unbound form of a similar analogue ( Jomain, J. B., Tallet, E., Broutin, I., Hoos, S., van Agthoven, J., Ducruix, A., Kelly, P. A., Kragelund, B. B., England, P., and Goffin, V. (2007) J. Biol. Chem. 282, 33118-33131 ) demonstrates that receptor-induced changes in the backbone of the four-helix bundle are subtle, whereas large scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. Hydrogen exchange mass spectrometry data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at binding site 1.

Dopamine (DA) holds a predominant role in the regulation of prolactin (PRL) secretion. Through a direct effect on anterior pituitary lactotrophs, DA inhibits the basally high-secretory tone of the cell. It accomplishes this by binding to D2 receptors expressed on the cell membrane of the lactotroph, activation of which results in a reduction of PRL exocytosis and gene expression by a variety of intracellular signalling mechanisms. The hypothalamic dopaminergic neurons, which provide DA to the anterior pituitary gland, are themselves regulated by feedback from PRL through a 'short-loop feedback mechanism'. A variety of other modulators of prolactin secretion act at the hypothalamic level by either disinhibition of the dopaminergic tone (e.g. serotonin, GABA, oestrogens and opioids) or by reinforcing it (e.g. substance P). All typical antipsychotic medications are associated with sustained hyperprolactinaemia due to their high affinity for the D2 receptor and their slow dissociation from the receptor once bound, but atypicals differ quite dramatically in their propensity to cause prolonged high prolactin levels. Of those atypicals that are associated with prolactin elevation, the main causative factor appears to be a higher peripheral-to-central dopamine receptor potency of either the parent drug or its active metabolite (e.g. risperidone, 9-hydroxy-risperidone and amisulpride). Antipsychotics that easily cross the blood-brain barrier and exhibit fast dissociation from the dopamine receptor once bound do not result in sustained hyperprolactinaemia.

The formation of new blood vessels has become a major focus of mammary gland research stimulated by the therapeutic opportunities of controlling angiogenesis in breast cancer. Normal growth and involution of the mammary gland are profoundly affected by the expansion and regression of blood vessels, whereas dysregulation of angiogenesis is characteristic of breast cancer growth and metastasis. Prolactin stimulates the growth and differentiation of the mammary gland under normal conditions, but its role in breast cancer is controversial. Its action is complicated by the fact that prolactin itself is angiogenic, but proteases cleave prolactin to generate vasoinhibins, a family of peptides that act on endothelial cells to suppress angiogenesis and vasodilation and to promote apoptosis-mediated vascular regression. This review summarizes our current knowledge about the vascular effects of prolactin and the generation and action of vasoinhibins, and discusses their possible contribution to the regulation of blood vessels in the normal and malignant mammary gland.

Prolactin (PRL) is a 23-kDa protein hormone that binds to a single-span membrane receptor, a member of the cytokine receptor superfamily, and exerts its action via several interacting signaling pathways. PRL is a multifunctional hormone that affects multiple reproductive and metabolic functions and is also involved in tumorigenicity. In addition to being a classical pituitary hormone, PRL in humans is produced by many tissues throughout the body where it acts as a cytokine. The objective of this review is to compare and contrast multiple aspects of PRL, from structure to regulation, and from physiology to pathology in rats, mice, and humans. At each juncture, questions are raised whether, or to what extent, data from rodents are relevant to PRL homeostasis in humans. Most current knowledge on PRL has been obtained from studies with rats and, more recently, from the use of transgenic mice. Although this information is indispensable for understanding PRL in human health and disease, there is sufficient disparity in the control of the production, distribution, and physiological functions of PRL among these species to warrant careful and judicial extrapolation to humans.

The bovine placenta produces an array of proteins structurally similar to pituitary prolactin (PRL). At least ten genes of the bovine placental PRL family, including bovine placental lactogen (bPL) and ten bovine PRL-related protein-I to -X (bPRP-I to -X), encode hormones/cytokines predicted to be involved in the establishment and maintenance of pregnancy. Targets and biological roles for most members of the bovine PRL family have yet to be specified. This study focused on three members of bovine PRL family, bPL, bPRP-I, and bPRP-VI. An alkaline phosphatase (AP) tagging strategy was used to monitor interactions of the ligands with their targets. AP-bPRP-I and AP-bPRP-VI specifically bound to tissue sections of the bovine placentome. AP-bPRP-I and AP-bPRP-VI binding within the placentome mimicked the distribution of the extracellular matrix (ECM). Consequently, AP fusion protein binding to individual ECM components (heparin, laminin, fibronectin, type I collagen, and type IV collagen) was evaluated. AP-bPRP-I specifically bound to type IV collagen, but not to the other ECM components. AP-bPRP-VI exhibited weak interactions with ECM components, while AP-bPL and AP did not show significant binding to any of the ECM components. Binding of AP-bPRP-I to type IV collagen was concentration-dependent, influenced by salt concentrations, and specific to the N-terminal cross-linking domain (7S) of type IV collagen but not its triple-helical domain. The interaction of bPRP-I with type IV collagen suggests that bPRP-I accumulates in the ECM where it likely acts on cells traversing the bovine placentome.

A standard protocol for isolation of buffalo prolactin (buPRL) was modified at the alcohol precipitation step. This modification could separate lower molecular weight prolactin from the higher molecular weight prolactin (PRL). Reloading the prolactin onto a Sephacryl S-200 gel purified the buPRL monomer. The purity of buPRL monomer was confirmed by 15% SDS PAGE. The buPRL monomer was >90% pure. It was characterized by specific anti-buPRL serum in ELISA and Western blot. A native PAGE of the PRL showed three charge isoforms. A protocol was standardized to separate prolactin monomeric least acidic isoforms using an anion exchanger.

Competitive antagonists of the human prolactin (hPRL) receptor are a novel class of molecules of potential therapeutic interest in the context of cancer. We recently developed the pure antagonist Del1-9-G129R-hPRL by deleting the nine N-terminal residues of G129R-hPRL, a first generation partial antagonist. We determined the crystallographic structure of Del1-9-G129R-hPRL, which revealed no major change compared with wild type hPRL, indicating that its pure antagonistic properties are intrinsically due to the mutations. To decipher the molecular bases of pure antagonism, we compared the biological, physicochemical, and structural properties of numerous hPRL variants harboring N-terminal or Gly(129) mutations, alone or combined. The pure versus partial antagonistic properties of the multiple hPRL variants could not be correlated to differences in their affinities toward the hPRL receptor, especially at site 2 as determined by surface plasmon resonance. On the contrary, residual agonism of the hPRL variants was found to be inversely correlated to their thermodynamic stability, which was altered by all the Gly(129) mutations but not by those involving the N terminus. We therefore propose that residual agonism can be abolished either by further disrupting hormone site 2-receptor contacts by N-terminal deletion, as in Del1-9-G129R-hPRL, or by stabilizing hPRL and constraining its intrinsic flexibility, as in G129V-hPRL.

Background: The prolactin-related proteins (PRPs) are non-classical placental-specific members of the prolactin/growth hormone family. Among ruminants, they are expressed in the cotyledonary villi of cattle and goat. We investigated placental PRP in sheep in order to gain a comprehensive understanding of the function and evolution of these molecules. We also examined the sequence properties, expression and lactogenic activation of the cloned genes., Results: We cloned two novel ovine PRPs, named oPRP1 and oPRP2. oPRP2 had a typical PRP sequence similar to bovine PRP1 (bPRP1). oPRP1 had a short sequence identical with bovine or caprine type PRP but the reading frame was shifted. Both oPRPs were expressed in trophoblast giant binucleate cells (BNC) as in cattle and goat. oPRP1 expression declined from the early to the middle stage of gestation. In contrast, oPRP2 expression remained constant throughout the gestation period. oPRP2 was translated to form a mature protein in a mammalian cell expression system. Western blotting showed a molecular mass of 35 kDa for the FLAG-tag fusion oPRP2 protein. This recombinant protein and bPRP1 were bioassayed using Nb2 lymphoma cells; it was confirmed that neither ruminant PRP had lactogenic activity because the Nb2 lymphoma cells did not proliferate., Conclusion: We have identified two novel PRPs, oPRP1 and oPRP2, in ovine placenta. Both these ovine PRPs were localized and quantitatively expressed in BNC. Absence of lactogenic activity was confirmed for the oPRP2 molecule. It is anticipated that novel and known ruminant PRPs have common functions, except for lactogenic activity.

Expression of prolactin (PRL) or prolactin-like hormone has been reported in invertebrates. We investigated the larval phase of Trichinella spiralis: (a) to express 23 kDa PRL, (b) to define its localization and (c) to test its possible biological activity. Immunostaining in isolated larvae demonstrated positive material to 23 kDa PRL by all along the stichosome, specifically in the stichocytes. Homogenized immunoblot larvae showed a 23 kDa protein band. To assess PRL release and its biological activity, larvae were incubated in culture medium and the excretory/secretory products were analyzed by the Nb2 cells bioassay. A cellular growth equivalent until 10 nM PRL and using antibody against 23 kDa PRL, the growth was blocked. In conclusion our result provides evidence that PRL-like hormone is expressed and secreted by the larvae of T. spiralis.

To investigate the consequences of freshwater (FW) transfer, we studied the prolactin (PRL) cDNA sequence and its mRNA expression, and physiological responses in the black porgy (Acanthopagrus schlegeli). We cloned and characterized cDNA encoding its PRL from the pituitary gland. Black porgy PRL cDNA consists of 1492 bp and encodes a protein of 212 amino acids including 24 signal peptides. Reverse transcription-PCR showed the PRL mRNA expression in the pituitary gland. Expression of pituitary gland PRL mRNA was significantly higher during FW acclimation. Furthermore, we studied the stress responses and osmoregulatory abilities of black porgy in changing salinities. Plasma cortisol, glucose, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) significantly increased in the fish immediately after transfer to FW. We also identified significant changes in the fish in terms of plasma ions (Na(+), Cl(-), Ca(2+)) and osmolality during the acclimation period. These results suggests that PRL plays an important role in hormonal regulation in osmoregulatory organs, thereby improving the hyperosmoregulatory ability of black porgy in freshwater.

A member of the family of hematopoietic cytokines, human prolactin (hPRL) serves a dual role both as an endocrine hormone and as an autocrine/paracrine cytokine or growth factor. During investigation of the solution structural properties of hPRL, we have noted a surprising pH dependence of its structural stability over a range from approximately pH 6.0 to pH 8.0. An analysis of backbone atom NMR chemical shift changes and backbone amide hydrogen-deuterium exchange rates due to titration of the solution pH over this same range, along with calculations of protein surface electrostatic potential, suggests the possible involvement of a localized cluster of three His residues (27, 30, and 180), which comprise a portion of the high-affinity receptor-binding epitope. Surface plasmon resonance analysis of the interaction between hPRL and the extracellular domain (ECD) of the hPRL receptor reveals a selective 500-fold change in the dissociation rate between pH 8.3 and pH 5.8. In comparison, the interaction of hGH with the same receptor ECD did not demonstrate any significant dependence on pH. We also present an initial investigation of the pH dependence of hPRL function in rat Nb2 cell proliferation assays and a STAT5 luciferase gene reporter assay in the T47D human breast cancer cell line, whose results are consistent with our biophysical studies. The potential implications of this variation in hPRL's structural stability and receptor-binding kinetics over this physiologic range of pH are discussed.

Continuous-elution electrophoresis (CEE) has been applied to separate putative hormones from adult Atlantic halibut pituitaries. Soluble proteins were separated by size and charge on Model 491 Prep Cell (Bio-Rad), where the homogenate runs through a cylindrical gel, and protein fractions are collected as they elute from the matrix. Protein fractions were assessed by SDS-PAGE and found to contain purified proteins of molecular size from 10 to 33 kDa. Fractions containing proteins with molecular weights of approximately 21, 24, 28 and 32 kDa, were identified as putative growth hormone (GH), prolactin, somatolactin and gonadotropins, respectively. These were analyzed further by mass spectrometry and identified with peptide mass protein fingerprinting. The CEE technique was used successfully for purification of halibut GH with a 5% yield, and appears generally well suited to purify species-specific proteins often needed for research in comparative endocrinology, including immunoassay work. Thus, the GH obtained was subsequently used as standards and iodination label in a homologous radioimmunoassay, applied to analyze GH content through larval development in normally and abnormally metamorphosing larvae. As GH is mainly found in the pituitary, GH contents were analyzed in tissue extracts from the heads only. The pituitary GH content increases proportionally to increased larval weight from first feeding to metamorphic climax. No difference in relative GH content was found between normal and abnormal larvae and it still remains to be established if GH has a direct role in metamorphosis.

Hyperprolactinaemia due to macroprolactin (MPRL) can lead to misdiagnosis and inappropriate treatment. We studied the new Roche Elecsys Prolactin assay (PRL II) which has been developed to reduce reactivity with MPRL. We investigated the performance of the PRL II assay at six laboratory sites to determine precision and establish reference intervals for total immunoreactive PRL and for monomeric PRL determined by precipitation with polyethylene glycol (PEG). We compared the reactivities with macroprolactin of the PRL II, the PRL I and seven other PRL assays. The PRL II assay reacted less strongly than the PRL I assay and similarly to the ADVIA Centaur assay with macroprolactin. PEG precipitation can be used with the PRL II assay to estimate the concentration of monomeric PRL.

Site-specific metal-catalyzed oxidation (MCO) was applied to characterize the metal-binding site (MBS) of recombinant human prolactin (hPRL), which belongs to the hematopoietic cytokine family. Copper and ascorbate of various concentrations were used to initiate the oxidation of hPRL, and the oxidation-sensitive motifs were characterized and quantitated by mass spectrometry. Based on the results obtained with 10 microM Cu(2+) and 0.3-2.0mM ascorbate, we propose that the MBS in hPRL is composed of His27, His30, and His173. This result shows the similarity of hPRL to human growth hormone (hGH), a member of the same family as hPRL, where the MBS is composed of His18, His21, and Glu174.

Vasoinhibins are a family of peptides derived from prolactin, growth hormone and placental lactogen that act on endothelial cells to suppress vasodilation and angiogenesis and to promote apoptosis-mediated vascular regression. Some of the pathways by which vasoinhibins act have now been defined, and recent developments indicate that endogenous vasoinhibins exert tonic and essential actions on blood vessel growth, dilation and regression in vivo. By studying the pathways that can generate vasoinhibins, and the nature of their receptors and key biological mediators, it should be possible to clarify the role of vasoinhibins in controlling vascular function in health and disease.

Angiogenesis is a crucial step in many pathologies, including tumor growth and metastasis. Here, we show that tilted peptides exert antiangiogenic activity. Tilted (or oblique-oriented) peptides are short peptides known to destabilize membranes and lipid cores and characterized by an asymmetric distribution of hydrophobic residues along the axis when helical. We have previously shown that 16-kDa fragments of the human prolactin/growth hormone (PRL/GH) family members are potent angiogenesis inhibitors. Here, we demonstrate that all these fragments possess a 14-aa sequence having the characteristics of a tilted peptide. The tilted peptides of human prolactin and human growth hormone induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and inhibit capillary formation both in vitro and in vivo. These antiangiogenic effects are abolished when the peptides' hydrophobicity gradient is altered by mutation. We further demonstrate that the well known tilted peptides of simian immunodeficiency virus gp32 and Alzheimer's beta-amyloid peptide are also angiogenesis inhibitors. Taken together, these results point to a potential new role for tilted peptides in regulating angiogenesis.

The synthesis, purification and characterization of G129R-hPRL and S179D-hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the first time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 microg/10(6) cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the specific denaturation, refolding and purification conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step purification process and included SDS-PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-flight mass spectral (MALDI-TOF-MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). This last technique revealed a considerable difference in hydrophobicity due to a single amino acid substitution, with S179D-hPRL less (t(RR) = 0.85 +/- 0.010) and G129R-hPRL more (t(RR) = 1.10 +/- 0.013) hydrophobic than hPRL, where t(RR) is the relative retention time. The biological characterization was based on further refinement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 x 10(-3) for S179D-hPRL and 70 x 10(-5) for G129R-hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting effects of hPRL in vivo in animal models of breast and prostate cancer.

Signal peptide peptidase (SPP) is an intramembrane aspartyl protease that cleaves remnant signal peptides after their release by signal peptidase. SPP contains active site motifs also found in presenilin, the catalytic component of the gamma-secretase complex of Alzheimer's disease. However, SPP has a membrane topology opposite that of presenilin, cleaves transmembrane substrates of opposite directionality, and does not require complexation with other proteins. Here we show that, upon isolation of membranes and solubilization with detergent, the biochemical characteristics of SPP are remarkably similar to gamma-secretase. The majority of the SPP-catalyzed cleavages occurred at a single site in a synthetic substrate based on the prolactin (Prl) signal sequence. However, as seen with cleavage of substrates by gamma-secretase, additional cuts at other minor sites are also observed. Like gamma-secretase, SPP is inhibited by helical peptidomimetics and apparently contains a substrate-binding site that is distinct from the active site. Surprisingly, certain nonsteroidal antiinflammatory drugs known to shift the site of proteolysis by gamma-secretase also alter the cleavage site of Prl by SPP. Together, these findings suggest that SPP and presenilin share certain biochemical properties, including a conserved drug-binding site for allosteric modulation of substrate proteolysis.

The 16 kDa N-terminal fragment of prolactin (16K-prolactin) is a potent antiangiogenic factor. Here, we demonstrate that matrix metalloproteases (MMPs) produced and secreted by chondrocytes generate biologically functional 16K-prolactin from full-length prolactin. When incubated with human prolactin at neutral pH, chondrocyte extracts and conditioned medium, as well as chondrocytes in culture, cleaved the Ser155-Leu156 peptide bond in prolactin, yielding - upon reduction of intramolecular disulfide bonds - a 16 kDa N-terminal fragment. This 16K-prolactin inhibited basic fibroblast growth factor (FGF)-induced endothelial cell proliferation in vitro. The Ser155-Leu156 site is highly conserved, and both human and rat prolactin were cleaved at this site by chondrocytes from either species. Conversion of prolactin to 16K-prolactin by chondrocyte lysates was completely abolished by the MMP inhibitors EDTA, GM6001 or 1,10-phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 cleaved human prolactin at Gln157, one residue downstream from the chondrocyte protease cleavage site, with the following relative potency: MMP-8 > MMP-13 > MMP-3 > MMP-1= MMP-2 > MMP-9. Finally, chondrocytes expressed prolactin mRNA (as revealed by RT-PCR) and they contained and released antiangiogenic N-terminal 16 kDa prolactin (detected by western blot and endothelial cell proliferation). These results suggest that several matrix metalloproteases in cartilage generate antiangiogenic 16K-prolactin from systemically derived or locally produced prolactin.

Monomeric prolactin (PRL) of molecular weight 23 kDa constitutes up to 95% of adult serum PRL. Macroprolactin is a large antigen-antibody complex of molecular weight greater than 100 kDa and constitutes less than 1% of circulating PRL. Thus, in most cases, hyperprolactinaemia is usually a result of high levels of monomeric PRL, which may be due to excess production as with a prolactinoma or due to disinhibition by compression of the pituitary stalk. Nevertheless, it must be noted that macroprolactinaemia may be a cause of hyperprolactinaemia in some individuals, which may have no association with any pathology. The presence of macroprolactin should always be suspected when a patient's clinical history and/or radiological data are incompatible with his/her PRL value. Thus, it may be useful to screen all patients with high sera PRL levels in order to prevent unnecessary investigations into the cause for hyperprolactinaemia. This has recently been facilitated with the advent of simple laboratory tests such as the polyethyleneglycol precipitation method, although gel filtration chromatography remains the gold standard. It is hoped that macroprolactinaemia is included in the differential diagnosis of hyperprolactinaemia.

Human prolactin consists of multiple forms of different sizes including three major prolactin (PRL) species, termed as little, big, and big-big PRL. If serum contains big-big PRL, it is termed macroprolactinemia; no symptoms of hyperprolactinemia develop due to low biological activity of big-big PRL. There are still few data regarding macroprolactinemia in children and adolescents. In this paper we describe six patients with macroprolactinemia, one of whom was asymptomatic and the other five either had headache, menstrual disturbance, short stature, increased hair growth or early puberty, compatible with high PRL levels. Two of the cases had microadenoma. Initial mean +/- SD PRL levels of the patients were 75.2 +/- 22.8 ng/ml (range: 42.3-105.2 ng/ml). Macroprolactin analysis revealed big-big PRL levels of the patients ranging between 21.6-98.6 ng/ml. It was noteworthy that bromocriptine (BRC) therapy started in three patients caused an abrupt decrease in PRL levels. It may be concluded that macroprolactinemia should be taken into account in the differential diagnosis of hyperprolactinemia in childhood and adolescence, whether or not they have relevant clinical symptoms.

Background: Macroprolactin is a high molecular mass complex of prolactin that may be the cause of elevated serum prolactin as determined by immunoassay. The composition of macroprolactin and its reactivity in immunoassays are variable but the complex has minimal bioactivity in vivo. Hyperprolactinaemia due to unrecognized macroprolactinaemia can lead to misdiagnosis and mistreatment., Methods: Serum from a patient with an unusual pattern of immunoreactivity was distributed to three users of each of the most popular immunoassays represented in the UK National External Quality Assessment Scheme (NEQAS) for prolactin. Clinical details were provided and participating centres were invited to investigate the prolactin content of the specimen according to their local protocol, and to comment on their results. The nature of the macroprolactin in the specimen was investigated in detail by gel filtration chromatography of the native serum and of the serum after adsorption of IgG with protein A, and by affinity chromatography with concanavalin A., Results: Gel filtration studies revealed two peaks of macroprolactin in this serum. These macroprolactins were shown to be different in their IgG content and degree of glycosylation. All eight immunoassays reacted strongly with the macroprolactin present. The majority (78%) of centres that interpreted their results either demonstrated the presence of macroprolactin in the specimen, or suggested it as a likely cause of the hyperprolactinaemia. However, two centres inappropriately excluded macroprolactinaemia as the cause of the elevated prolactin, and a further two did not consider it at all. Data from previous UK NEQAS distributions (between 1996 and 2003) of macroprolactin containing sera are presented which suggest that the frequency of recognition of macroprolactin as a possible cause of hyperprolactinaemia has increased over time., Conclusions: Very high molecular mass forms of prolactin and the presence of multiple molecular mass forms, as detected in the case presented here, are uncommon. Also, the pattern of immunoreactivity reported in this specimen was unusual as most macroprolactins studied previously react less strongly in, for example, the Bayer ADVIA Centaur assay compared to the Roche E170 assay. Both peaks of macroprolactin in this serum reacted in all assays tested. This case highlights the variable nature and immunoreactive behaviour of macroprolactin species.

Antiangiogenic molecules derived from prolactin (PRL) are not a single entity, but rather a family of peptides with different molecular masses, all containing the N-terminal region of PRL. Cleavage of PRL by cathepsin-D or by matrix metalloproteases generates N-terminal fragments that act on endothelial cells to suppress vasodilation and angiogenesis and promote vascular regression. N-terminal PRL fragments have been identified in cartilage and retina, where angiogenesis is highly restricted. In vivo experiments demonstrate that these PRL fragments exert a tonic and essential suppression of retinal blood vessel growth and dilation. Similar PRL fragments have been detected in the pituitary gland, a highly vascularized organ where the control of vascular growth may differ from that in tissues where angiogenesis is highly restricted. We have previously proposed the name vasoinhibins to describe the collection of N-terminal PRL fragments having blood vessel-blocking activity, and here we discuss their promise as factors to control vascular function in health and disease.

Complementary DNAs of three recombinant proteins related to the prolactin family: ovine placental lactogen (oPL), ovine prolactin (oPRL), and rabbit soluble extracellular domain of prolactin receptor (rbPRLR-ECD) were subcloned by different methods and inserted into prokaryotic expression plasmids. Escherichia coli cells transformed with those plasmids overexpressed the respective proteins either by induction or constitutively, resulting in accumulation of the recombinant proteins in insoluble inclusion bodies, which were subsequently purified, used for refolding and purifying of the proteins by one-step chromatography. The isolated oPL, oPRL, and rbPRLR-ECD were biologically active over >95% pure monomers. Ten-liter bacterial culture yielded hundreds of milligrams or more than gram quantities of recombinant proteins. The methodology described in the present chapter allows large-scale preparation of pure, monomeric, biologically active oPL, oPRL, and rbPRLR-ECD suitable for performing in vitro and in vivo experiments.

Prolactin (PRL) is a hormone that contributes to both the growth and differentiation of mammary epithelial cells, activities likely to impact breast cancer in opposite ways. Whether PRL causes growth or differentiation has been solely attributed to the coexisting steroidal environment, with PRL stimulating mammary gland growth during pregnancy, and then milk production after the postpartum drop in estrogen and progesterone. However, previous work from our laboratory has shown that the form of PRL may also be an important factor. During pregnancy, unmodified PRL (U-PRL) promotes mammary growth, while an increase in phosphorylated PRL, or administration of a molecular mimic of phosphorylated PRL (S179D PRL), inhibits growth. Unknown, however, is whether these forms of PRL have opposite effects on growth in the absence of steroids and whether effects are directly on mammary epithelial cells. To mimic the glandular epithelium in vitro, we used contact-inhibited, differentiated cells and showed that even with these minimally growing cells that treatment with U-PRL caused increased expression of cyclin D1 and cyclin-dependent kinase 4, increased activity of both cdk4 and cdk2, while having no effect on the inhibitory protein, p21. S179D PRL, by contrast, had no effect on cyclin D1 and cdk4 expression, but increased p21 expression and expression of the vitamin D receptor (VDR). We conclude that increased U-PRL or decreased phosphorylated PRL can directly affect cell cycle control proteins in relatively differentiated mammary epithelial cells, thereby implicating the balance between these two forms of PRL in the early promotion of breast cancer.

We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactin-receptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction.

We report the production of recombinant possum prolactin (posPrl), and its use in the development and validation of a highly specific homologous radioimmunoassay for the measurement of prolactin (Prl) in brushtail possums. This enabled the subsequent investigation of some basic mechanisms involved in the regulation of Prl secretion in this species. Recombinant posPrl spanning the entire coding region was expressed in Escherichia coli, resulting in a 199 amino acid protein with a molecular weight approximately 23 kDa. The potency of posPrl was 45.3 +/- 4.8% that of ovine Prl in a radioreceptor assay using possum mammary gland receptors and induced a 3.4 +/- 0.8-fold increase in progesterone secretion in primary possum granulosa cells. Antiserum (G27) was raised against recombinant posPrl and was highly specific for possum Prl (approximately 30% binding at 1:60,000 final dilution), and exhibited negligible cross-reactivity (<0.0001%) with possum growth hormone. Serial dilutions of pituitary gland extracts, and plasma samples from male and female possums gave parallel inhibition curves to recombinant posPrl standards in the assay. Biological validation of the RIA included treating possums with drugs known to alter Prl secretion in other mammals. In seasonally anoestrous female possums, administration of 20 microg thyrotropin-releasing hormone (TRH) resulted in a 15-fold increase (P < 0.01) in plasma Prl concentrations. In mid-late lactating female possums, a bolus of cabergoline (dopamine agonist; 75 microg) reduced (P < 0.05) plasma Prl levels to baseline for 24 h, while repeated administration (6 x 75 microg at 12 h intervals) suppressed (P < 0.01) plasma Prl concentrations until 24h after the last injection. Prolonged inhibition of Prl levels subsequently caused marked (P < 0.01) attenuation in rate of bodyweight increase of pouch young. The amplitude of the Prl surge in response to a bolus of TRH (15 microg) was 5-fold lower in cabergoline-treated, compared to control mid-late lactating possums. In conclusion, we report the development and validation of a robust and sensitive RIA for measuring Prl concentrations in the plasma of brushtail possums.

Two high molecular mass forms of prolactin (PRL) in serum have been identified by gel filtration chromatography (GFC): macroprolactin (big-big PRL, > 100 kDa) and big PRL (40-60 kDa). Macroprolactin has a variable composition and structure, but is most frequently a complex of PRL and IgG, with a molecular mass of 150-170 kDa. It is formed in the circulation following pituitary secretion of monomeric PRL but has a longer half-life, and the PRL in the complex remains reactive to a variable extent in immunoassays. In the majority of subjects little or no macroprolactin can be detected in serum, but in some individuals it may be the predominant immunoreactive component of circulating PRL and the cause of apparent hyperprolactinaemia. Owing to its high molecular mass, macroprolactin appears to be confined to the intravascular compartment and much evidence indicates that it has minimal bioactivity in vivo and is not of pathological significance. Nevertheless, hyperprolactinaemia due to macroprolactin can lead to diagnostic confusion and unnecessary further investigation and treatment if it is not recognized as such. Macroprolactin is a common cause of apparent hyperprolactinaemia with some assays and it is essential that laboratories introduce screening programmes to examine samples with elevated total immunoreactive PRL for the presence of macroprolactin and determine the monomeric PRL component which is known to be bioactive in vivo. A number of screening tests have been described; that based on the precipitation of macroprolactin with polyethylene glycol has been the most widely validated and applied. The reference technique of GFC should be available for confirmation and further investigation of samples, giving equivocal results in screening tests. In comparison with macroprolactin, little is known about big PRL. It is a more consistent component of total serum PRL but rarely, if ever, the cause of hyperprolactinaemia. Further research is required into the nature of macroprolactin and big PRL, the relationships between high molecular mass forms of PRL, and their clinical significance.