Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates.

Human growth hormone (hGH) is a pituitary-derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently, there is a single FDA-approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost 3 decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions-dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific PEG conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and prolactin receptor. Moreover, these novel hGHR antagonists display distinct antagonism of GH-induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anticancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH-associated pathologies.

The short isoform of PRLR suppresses the pentose phosphate pathway and nucleotide synthesis through the NEK9-Hippo axis in pancreatic cancer.

Prolactin binding to the prolactin receptor exerts pleiotropic biological effects in vertebrates. The prolactin receptor (PRLR) has multiple isoforms due to alternative splicing. The biological roles and related signaling of the long isoform (PRLR-LF) have been fully elucidated. However, little is known about the short isoform (PRLR-SF), particularly in cancer development and metabolic reprogramming, a core hallmark of cancer. Here, we reveal the role and underlying mechanism of PRLR-SF in pancreatic ductal adenocarcinoma (PDAC). Methods: A human PDAC tissue array was used to investigate the clinical relevance of PRLR in PDAC. The in vivo implications of PRLR-SF in PDAC were examined in a subcutaneous xenograft model and an orthotopic xenograft model. Immunohistochemistry was performed on tumor tissue obtained from genetically engineered KPC (KrasG12D/+; Trp53R172H/+; Pdx1-Cre) mice with spontaneous tumors. 13C-labeled metabolite measures, LC-MS, EdU incorporation assays and seahorse analyses were used to identify the effects of PRLR-SF on the pentose phosphate pathway and glycolysis. We identified the molecular mechanisms by immunofluorescence, coimmunoprecipitation, proximity ligation assays, chromatin immunoprecipitation and promoter luciferase activity. Public databases (TCGA, GEO and GTEx) were used to analyze the expression and survival correlations of the related genes. Results: We demonstrated that PRLR-SF is predominantly expressed in spontaneously forming pancreatic tumors of genetically engineered KPC mice and human PDAC cell lines. PRLR-SF inhibits the proliferation of PDAC cells (AsPC-1 and BxPC-3) in vitro and tumor growth in vivo. We showed that PRLR-SF reduces the expression of genes in the pentose phosphate pathway (PPP) and nucleotide biosynthesis by activating Hippo signaling. TEAD1, a downstream transcription factor of Hippo signaling, directly regulates the expression of G6PD and TKT, which are PPP rate-limiting enzymes. Moreover, NEK9 directly interacts with PRLR-SF and is the intermediator between PRLR and the Hippo pathway. The PRLR expression level is negatively correlated with overall survival and TNM stage in PDAC patients. Additionally, pregnancy and lactation increase the ratio of PRLR-SF:PRLR-LF in the pancreas of wild-type mice and subcutaneous PDAC xenograft tumors. Conclusion: Our characterization of the relationship between PRLR-SF signaling, the NEK9-Hippo pathway, PPP and nucleotide synthesis explains a mechanism for the correlation between PRLR-SF and metabolic reprogramming in PDAC progression. Strategies to alter this pathway might be developed for the treatment or prevention of pancreatic cancer.

Structural Investigation of Human Prolactin Receptor Transmembrane Domain Homodimerization in a Membrane Environment through Multiscale Simulations.

It is well established that prolactin (PRL) and its receptor (PRLR) are associated with hundreds of biological functions. They have been postulated to be linked to breast and prostate cancers, and PRLR signaling has attracted considerable medical and pharmaceutical interest in the development of compounds targeting PRLR. Dimerization of the receptor through its transmembrane (TM) domain is a key step for understanding its signaling and related issues. Our multiscale simulation results revealed that its TM domain can form dimers in a membrane environment with distinct states stabilized by different residue motifs. On the basis of the simulated data, an activation mechanism of PRL with the importance of two symmetrical tryptophan residues was proposed in detail to determine the conformational change of its receptor, which is essential for signal transduction. The better knowledge of PRLR structure and its protein-protein interaction can considerably contribute to a further understanding of PRLR signaling action and thereby help to develop some new PRLR signaling-based strategies for PRL-related diseases.

In silico prediction of prolactin molecules as a tool for equine genomics reproduction.

The prolactin hormone is involved in several biological functions, although its main role resides on reproduction. As it interferes on fertility changes, studies focused on human health have established a linkage of this hormone to fertility losses. Regarding animal research, there is still a lack of information about the structure of prolactin. In case of horse breeding, prolactin has a particular influence; once there is an individualization of these animals and equines are known for presenting several reproductive disorders. As there is no molecular structure available for the prolactin hormone and receptor, we performed several bioinformatics analyses through prediction and refinement softwares, as well as manual modifications. Aiming to elucidate the first computational structure of both molecules and analyse structural and functional aspects related to these proteins, here we provide the first known equine model for prolactin and prolactin receptor, which obtained high global quality scores in diverse software's for quality assessment. QMEAN overall score obtained for ePrl was (- 4.09) and QMEANbrane for ePrlr was (- 8.45), which proves the structures' reliability. This study will implement another tool in equine genomics in order to give light to interactions of these molecules, structural and functional alterations and therefore help diagnosing fertility problems, contributing in the selection of a high genetic herd.

Association of prolactin receptor (PRLR) variants with prolactinomas.

Prolactinomas are the most frequent type of pituitary tumors, which represent 10-20% of all intracranial neoplasms in humans. Prolactinomas develop in mice lacking the prolactin receptor (PRLR), which is a member of the cytokine receptor superfamily that signals via Janus kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) or phosphoinositide 3-kinase-Akt (PI3K-Akt) pathways to mediate changes in transcription, differentiation and proliferation. To elucidate the role of the PRLR gene in human prolactinomas, we determined the PRLR sequence in 50 DNA samples (35 leucocytes, 15 tumors) from 46 prolactinoma patients (59% males, 41% females). This identified six germline PRLR variants, which comprised four rare variants (Gly57Ser, Glu376Gln, Arg453Trp and Asn492Ile) and two low-frequency variants (Ile76Val, Ile146Leu), but no somatic variants. The rare variants, Glu376Gln and Asn492Ile, which were in complete linkage disequilibrium, and are located in the PRLR intracellular domain, occurred with significantly higher frequencies (P < 0.0001) in prolactinoma patients than in 60 706 individuals of the Exome Aggregation Consortium cohort and 7045 individuals of the Oxford Biobank. In vitro analysis of the PRLR variants demonstrated that the Asn492Ile variant, but not Glu376Gln, when compared to wild-type (WT) PRLR, increased prolactin-induced pAkt signaling (>1.3-fold, P < 0.02) and proliferation (1.4-fold, P < 0.02), but did not affect pSTAT5 signaling. Treatment of cells with an Akt1/2 inhibitor or everolimus, which acts on the Akt pathway, reduced Asn492Ile signaling and proliferation to WT levels. Thus, our results identify an association between a gain-of-function PRLR variant and prolactinomas and reveal a new etiology and potential therapeutic approach for these neoplasms.

Novel reagents for human prolactin research: large-scale preparation and characterization of prolactin receptor extracellular domain, non-pegylated and pegylated prolactin and prolactin receptor antagonist.

To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for large-scale preparation of untagged human PRL (hPRL), a hPRL antagonist (del 1-9-G129R hPRL) that acts as a pure antagonist of hPRL in binding to hPRL receptor extracellular domain (hPRLR-ECD), and hPRLR-ECD are demonstrated. The interaction of del 1-9-G129R hPRL with hPRLR-ECD was demonstrated by competitive non-radioactive binding assay using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, by formation of stable 1:1 complexes with hPRLR-ECD under non-denaturing conditions using size-exclusion chromatography, and by surface plasmon resonance methodology. In all three types of experiments, the interaction of del 1-9-G129R hPRL was equal to that of unmodified hPRL. Del 1-9-G129R hPRL inhibited the hPRL-induced proliferation of Baf/LP cells stably expressing hPRLR. Overall, the biological properties of del 1-9-G129R hPRL prepared by the protocol described herein were similar to those of the antagonist prepared using the protocol reported in the original study; however, the newly described protocol improved yields by >6-fold. To provide long-lasting hPRL as a new reagent needed for in vivo experiments, we prepared its mono-pegylated analogue and found that pegylation lowers its biological activity in a homologous in vitro assay. As its future use will require the development of a PRL antagonist with highly elevated affinity, del 1-9-G129R hPRL was expressed on the surface of yeast cells. It retained its binding capacity for hPRLR-ECD, and this methodology was shown to be suitable for future development of high-affinity hPRL antagonists using a library of randomly mutated open reading frame of del 1-9-G129R hPRL and selecting high-affinity mutants by yeast surface display methodology.

Subdomain 2, Not the Transmembrane Domain, Determines the Dimerization Partner of Growth Hormone Receptor and Prolactin Receptor.

Growth hormone receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane class I cytokine receptors. In humans, GH interacts with GHR homodimers or PRLR homodimers and PRL interacts with only PRLR homodimers to promote signaling. In human breast cancer cells endogenously expressing both receptors, GHR and PRLR specifically coimmunoprecipitate. We previously devised a split luciferase complementation assay to study GHR and PRLR assemblages. In this technique, firefly luciferase is split into two fragments (N- and C-terminal fragments of the luciferase), each without enzyme activity and tethered to the tails of two receptors. The fragments restore luciferase activity when brought close to each other by the receptors. Real-time ligand-induced complementation changes reflect the arrangement of receptors and indicate that GHR/PRLR is arranged as a heteromultimer comprised of GHR-GHR homodimers and PRLR-PRLR homodimers. We now dissect determinants for GHR and PRLR homodimerization versus heteroassociation. GHR and PRLR have extracellular domains comprised of the ligand-binding N-terminal subdomain 1 and a membrane-proximal subdomain 2 (S2), which fosters receptor-receptor contact. Based on previous studies of S2 versus the transmembrane domain (TMD) in GHR dimerization, we constructed GHR(PRLRS2), GHR(PRLRS2-TMD), and GHR(PRLRTMD), replacing GHR's S2 alone, S2 plus TMD, and TMD alone with PRLR's counterpart. We tested by complementation the ability of these chimeras and GHR or PRLR to homodimerize or heteroassociate. Comparing various combinations, we found GHR(PRLRS2) and GHR(PRLRS2-TMD) behaved as PRLR, whereas GHR(PRLRTMD) behaved as GHR regarding their dimerization partners. We conclude that S2 of GHR and PRLR, rather than their TMDs, determines their dimerization partner.

Development of a new anti-prolactin receptor (PRLR) antibody, F56, which can serve as a PRLR antagonist.

In this work, we developed a new prolactin receptor (PRLR) antagonist using the hybridoma technique. A series of monoclonal antibodies against prolactin receptor (PRLR) was prepared, from which we characterized and selected one anti-PRLR antibody, F56. Epitome mapping showed that F56 and prolactin (PRL) share a common binding epitope on PRLR, and therefore, F56 could compete with prolactin (PRL) for binding to PRLR. Subsequent experiments indicated that F56 could effectively neutralize PRLR-mediated intracellular signalling molecules, such as signal transducer and activator of transcription (STAT) and extracellular signal-regulated kinase-1 and kinase 2 (ERK1/2), either by endogenously expressed PRLR or in a cell model transfected with PRLR. In addition, further experiments showed that F56 could effectively inhibit PRL-induced cell proliferation. The current study suggests that F56 has potential applications in PRLR-related studies.

Whole-exome identifies RXRG and TH germline variants in familial isolated prolactinoma.

Familial isolated pituitary adenoma (FIPA) is a rare genetic disorder. In a subset of FIPA families AIP germline mutations have been reported, but in most FIPA cases the exact genetic defect remains unknown. The present study aimed to determine the genetic basis of FIPA in a Brazilian family. Three siblings presented with isolated prolactin genes. Further mutation screening was performed using whole-exome sequencing and all likely causative mutations were validated by Sanger sequencing. In silico analysis and secreting pituitary adenoma diagnosed through clinical, biochemical and imaging testing. Sanger sequencing was used to genotype candidate prolactinoma-mutated additional predictive algorithms were applied to prioritize likely pathogenic variants. No mutations in the coding and flanking intronic regions in the MEN1, AIP and PRLR genes were detected. Whole-exome sequencing of three affected siblings revealed novel, predicted damaging, heterozygous variants in three different genes: RXRG, REXO4 and TH. In conclusion, the RXRG and TH possibly pathogenic variants may be associated with isolated prolactinoma in the studied family. The possible contribution of these genes to additional FIPA families should be explored.

A combined computational and structural model of the full-length human prolactin receptor.

The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg.

Bio-nanocapsule-based scaffold improves the sensitivity and ligand-binding capacity of mammalian receptors on the sensor chip.

Mammalian receptors are recognized as target molecules for drug discovery, and chemical libraries have been screened for both potential antagonists and agonists mainly by ligand-binding assays using immobilized receptors. A bio-nanocapsule (BNC) of approximately 30 nm that displays a tandem form of the protein A-derived immunoglobulin G (IgG) Fc-binding Z domains (denoted as ZZ-BNC) has been developed for both clustering and oriented immobilization of IgGs on the solid phase of immunosensors. In this study, human IgG1 Fc-fused vascular endothelial growth factor (VEGF) receptor was immobilized through ZZ-BNC on the sensor chip of quartz crystal microbalance (ZZ-BNC-coating). When compared with direct adsorption and protein A-coating, the sensor chip showed higher sensitivity (∽46- and ∽165-fold, respectively) and larger ligand-binding capacity (∽4- and ∽18-fold, respectively). Furthermore, the number of VEGF molecules bound to its receptor increased from 0.20 (direct adsorption) to 2.06 by ZZ-BNC-coating, strongly suggesting that ZZ-BNC reduced the steric hindrance near ligand recognition sites through oriented immobilization. Similarly, the sensitivity and ligand-binding capacity of leptin and prolactin receptors were both enhanced at a level comparable to that observed for the VEGF receptor. Thus, the combination of ZZ-BNC and Fc-fused receptors could significantly improve the function of ligand-binding assays.

Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes.

Class 1 cytokine receptors regulate essential biological processes through complex intracellular signalling networks. However, the structural platform for understanding their functions is currently incomplete as structure-function studies of the intracellular domains (ICDs) are critically lacking. The present study provides the first comprehensive structural characterization of any cytokine receptor ICD and demonstrates that the human prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) ICDs are intrinsically disordered throughout their entire lengths. We show that they interact specifically with hallmark lipids of the inner plasma membrane leaflet through conserved motifs resembling immuno receptor tyrosine-based activation motifs (ITAMs). However, contrary to the observations made for ITAMs, lipid association of the PRLR and GHR ICDs was shown to be unaccompanied by changes in transient secondary structure and independent of tyrosine phosphorylation. The results of the present study provide a new structural platform for studying class 1 cytokine receptors and may implicate the membrane as an active component regulating intracellular signalling.

Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling.

BACKGROUND: Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response. FINDINGS: Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway. CONCLUSIONS: The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.

Residue 146 regulates prolactin receptor folding, basal activity and ligand-responsiveness: potential implications in breast tumorigenesis.

PRLR(I146L) is the first identified gain-of-function variant of the prolactin receptor (PRLR) that was proposed to be associated with benign breast tumorigenesis. Structural investigations suggested this hydrophobic core position in the extracellular D2 domain to be linked to receptor dimerization. Here, we used a mutational approach to address how the conservative I-to-L substitution induced constitutive activity. Using cell-based assays of different I146-PRLR variants in combination with spectroscopic/nuclear magnetic resonance analyses we found that chemical manipulation of position 146 profoundly altered folding, PRL-responsiveness, and ligand-independent activity of the receptor in a mutation-specific manner. Together, these data further add to the critical role of position 146, showing it to also be crucial to structural integrity thereby imposing on the biological PRLR properties. When stably introduced in MCF-7 (luminal) and MDA-MB231 (mesenchymal) breast cancer cells, the most potent of the PRL-insensitive mutants (PRLR(I146D)) had minimal impact on cell proliferation and cell differentiation status.

Blockade of estrogen-stimulated proliferation by a constitutively-active prolactin receptor having lower expression in invasive ductal carcinoma.

A comprehensive understanding of prolactin's (PRL's) role in breast cancer is complicated by disparate roles for alternatively-spliced PRL receptors (PRLR) and crosstalk between PRL and estrogen signaling. Among PRLRs, the short form 1b (SF1b) inhibits PRL-stimulated cell proliferation. In addition to ligand-dependent PRLRs, constitutively-active varieties, missing the S2 region of the extracellular domain (ΔS2), naturally occur. Expression analysis of the ΔS2 version of SF1b (ΔS2SF1b) showed higher expression in histologically-normal contiguous tissue versus invasive ductal carcinoma. To determine the function of ΔS2SF1b, a T47D breast cancer line with inducible expression was produced. Induction of ΔS2SF1b blocked estrogen-stimulated cell proliferation. Unlike intact SF1b, induction of ΔS2SF1b had no effect on PRL-mediated activation of Stat5a. However induction inhibited estrogen's stimulatory effects on serine-118 phosphorylation of estrogen receptor α, serine-473 phosphorylation of Akt, serine-9 phosphorylation of GSK3ß, and c-myc expression. In addition, induction of ΔS2SF1b increased expression of the cell cycle-inhibiting protein, p21. Thus, increased expression of ΔS2SF1b, such as we demonstrate occurs with the selective PRLR modulator, S179D PRL, would create a physiological state in which estrogen-stimulated proliferation was inhibited, but differentiative responses to PRL were maintained.

Prolactin (PRL) in adipose tissue: regulation and functions.

New information concerning the effects of prolactin (PRL) on metabolic processes warrants reevaluation of its overall metabolic actions. PRL affects metabolic homeostasis by regulating key enzymes and transporters associated with glucose and lipid metabolism in several target organs. In the lactating mammary gland, PRL increases the production of milk proteins, lactose, and lipids. In adipose tissue, PRL generally suppresses lipid storage and adipokine release and affect adipogenesis. A specific case is made for PRL in the human breast and adipose tissues, where it acts as a circulating hormone and an autocrine/paracrine factor. Although its overall effects on body composition are both modest and species-specific, PRL may be involved in the manifestation of insulin resistance.

Impact of subdomain D1 of the short form S1b of the human prolactin receptor on its inhibitory action on the function of the long form of the receptor induced by prolactin.

BACKGROUND: Long-form (LF) homodimers of the human prolactin receptor (PRLR) mediate prolactin's diverse actions. Short form S1b inhibits the LF function through heterodimerization. Reduced S1b/LF-ratio in breast cancer could contribute to tumor development/progression. Current work defines the structural and functional relevance of the D1 domain of S1b on its inhibitory function on prolactin-induced LF function. METHODS: Studies were conducted using mutagenesis, promoter/signaling analyses, bioluminescence resonance energy transfer (BRET) and molecular modeling approaches. RESULTS: Mutation of E69 in D1 S1b or adjacent residues at the receptor surface near to the binding pocket (S) causes loss of its inhibitory effect while mutations away from this region (A) or in the D2 domain display inhibitory action as the wild-type. All S1b mutants preserved prolactin-induced Jak2 activation. BRET reveals an increased affinity in D1 mutated S1b (S) homodimers in transfected cells stably expressing LF. In contrast, affinity in S1b homodimers with either D1 (A) or D2 mutations remained unchanged. This favors LF mediated signaling induced by prolactin. Molecular dynamics simulations show that mutations (S) elicit major conformational changes that propagate downward to the D1/D2 interface and change their relative orientation in the dimers. CONCLUSIONS: These findings demonstrate the essential role of D1 on the S1b structure and its inhibitory action on prolactin-induced LF-mediated function. GENERAL SIGNIFICANCE: Major changes in receptor conformation and dimerization affinity are triggered by single mutations in critical regions of D1. Our structure-function/simulation studies provide a basis for modeling and design of small molecules to enhance inhibition of LF activation for potential use in breast cancer treatment.

Mutant prolactin receptor and familial hyperprolactinemia.

Hyperprolactinemia that is not associated with gestation or the puerperium is usually due to tumors in the anterior pituitary gland and occurs occasionally in hereditary multiple endocrine neoplasia syndromes. Here, we report data from three sisters with hyperprolactinemia, two of whom presented with oligomenorrhea and one with infertility. These symptoms were not associated with pituitary tumors or multiple endocrine neoplasia but were due to a heterozygous mutation in the prolactin receptor gene, PRLR, resulting in an amino acid change from histidine to arginine at codon 188 (His188Arg). This substitution disrupted the high-affinity ligand-binding interface of the prolactin receptor, resulting in a loss of downstream signaling by Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Thus, the familial hyperprolactinemia appears to be due to a germline, loss-of-function mutation in PRLR, resulting in prolactin insensitivity.

Prolactin stimulates cell migration and invasion by human trophoblast in vitro.

INTRODUCTION: Prolactin (PRL) is present in endometrium at the time of embryo implantation and throughout pregnancy. Extrapituitary PRL acts as a cytokine in cells expressing PRL receptor (PRLR). So far no specific function has been demonstrated for PRL in the trophoblast of early pregnancy. METHODS: PRLR in placental tissue and trophoblast cells was shown here immunochemically. The possibility that PRL could influence trophoblast cell migration and invasion was investigated in vitro using isolated cytotrophoblast of the first trimester of pregnancy placental tissue and HTR-8/SVneo cell line. Wound healing cell migration test was performed on HTR-8/SVneo cells, and both cell types were used in Matrigel invasion test. RESULTS: PRLR is expressed by extravillous cytotrophoblast of the cell column and the placental bed, as well as in isolated cytotrophoblast (CT) and HTR-8/SVneo cells. PRL (at 100 and 1000 ng/ml) stimulated HTR-8/SVneo cell migration and cell invasion in both cell types, which could be blocked by anti-PRLR. Integrins α1 and α5, and galectin-1 (gal-1) were variably increased in PRL treated CT and HTR-8/SVneo cells. DISCUSSION: To our knowledge this is the first study demonstrating that PRL stimulates trophoblast invasiveness through PRLR, which is accompanied by increased integrins and gal-1, not excluding change in other potential mediators. This finding further supports relevance of PRLR for invasive trophoblast. CONCLUSION: This report supports a possibility that PRL may have a role in trophoblast invasion in vivo.

Molecular mechanisms of prolactin and its receptor.

Prolactin and the prolactin receptors are members of a family of hormone/receptor pairs which include GH, erythropoietin, and other ligand/receptor pairs. The mechanisms of these ligand/receptor pairs have broad similarities, including general structures, ligand/receptor stoichiometries, and activation of several common signaling pathways. But significant variations in the structural and mechanistic details are present among these hormones and their type 1 receptors. The prolactin receptor is particularly interesting because it can be activated by three sequence-diverse human hormones: prolactin, GH, and placental lactogen. This system offers a unique opportunity to compare the detailed molecular mechanisms of these related hormone/receptor pairs. This review critically evaluates selected literature that informs these mechanisms, compares the mechanisms of the three lactogenic hormones, compares the mechanism with those of other class 1 ligand/receptor pairs, and identifies information that will be required to resolve mechanistic ambiguities. The literature describes distinct mechanistic differences between the three lactogenic hormones and their interaction with the prolactin receptor and describes more significant differences between the mechanisms by which other related ligands interact with and activate their receptors.