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.

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.

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.

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.

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.

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.

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.

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.

The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation: insight from structures of the prolactin receptor.

The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane proximal domain of the human PRLR and find that the tryptophans of the motif adopt a T-stack conformation in the unbound state. By contrast, in the hormone bound state, a Trp/Arg-ladder is formed. The conformational change is hormone-dependent and influences the receptor-receptor dimerization site 3. In the constitutively active, breast cancer-related receptor mutant PRLR(I146L), we observed a stabilization of the dimeric state and a change in the dynamics of the motif. Here we demonstrate a structural link between the WSXWS motif, hormone binding, and receptor dimerization and propose it as a general mechanism for class 1 receptor activation.

Generation of mice expressing only the long form of the prolactin receptor reveals that both isoforms of the receptor are required for normal ovarian function.

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LH-R), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.

HMGN2 inducibly binds a novel transactivation domain in nuclear PRLr to coordinate Stat5a-mediated transcription.

The direct actions of transmembrane receptors within the nucleus remain enigmatic. In this report, we demonstrate that the prolactin receptor (PRLr) localizes to the nucleus where it functions as a coactivator through its interactions with the latent transcription factor signal transducer and activator of transcription 5a (Stat5a) and the high-mobility group N2 protein (HMGN2). We identify a novel transactivation domain within the PRLr that is activated by ligand-induced phosphorylation, an event coupled to HMGN2 binding. The association of the PRLr with HMGN2 enables Stat5a-responsive promoter binding, thus facilitating transcriptional activation and promoting anchorage-independent growth. We propose that HMGN2 serves as a critical regulatory factor in Stat5a-driven gene expression by facilitating the assembly of PRLr/Stat5a onto chromatin and that these events may serve to promote biological events that contribute to a tumorigenic phenotype. Our data imply that phosphorylation may be the molecular switch that activates a cell surface receptor transactivation domain, enabling it to tether chromatin-modifying factors, such as HMGN2, to target promoter regions in a sequence-specific manner.

Development of prolactin receptor antagonists with reduced pH-dependence of receptor binding.

The cytokine hormone prolactin has a vast number of diverse functions. Unfortunately, it also exhibits tumor growth promoting properties, which makes the development of prolactin receptor antagonists a priority. Prolactin binds to its cognate receptor with much lower affinity at low pH than at physiological pH and since the extracellular environment around solid tumors often is acidic, it is desirable to develop antagonists that have improved binding affinity at low pH. The pK(a) value of a histidine side chain is ∼6.8 making histidine residues obvious candidates for examination. From evaluation of known molecular structures of human prolactin, of the prolactin receptor and of different complexes of the two, three histidine residues in the hormone-receptor binding site 1 were selected for mutational studies. We analyzed 10 variants by circular dichroism spectroscopy, affinity and thermodynamic characterization of receptor binding by isothermal titration calorimetry combined with in vitro bioactivity in living cells. Histidine residue 27 was recognized as a central hot spot for pH sensitivity and conservative substitutions at this site resulted in strong receptor binding at low pH. Pure antagonists were developed earlier and the histidine mutations were introduced within such background. The antagonistic properties were maintained and the high affinity at low pH conserved. The implications of these findings may open new areas of research in the field of prolactin cancer biology.

Two independent histidines, one in human prolactin and one in its receptor, are critical for pH-dependent receptor recognition and activation.

Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL·receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL·receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.

Unexploited therapies in breast and prostate cancer: blockade of the prolactin receptor.

Breast and prostate cancers are hormone-sensitive malignancies that afflict millions of women and men. Although prolactin (PRL) is known as a survival factor that supports tumor growth and confers chemoresistance in both cancers, its precise role in these tumors has not been studied extensively. Growth hormone and placental lactogen also bind PRL receptor (PRLR) and mimic some of the actions of PRL. Blockade of the PRLR represents a novel treatment for patients with advanced breast or prostate cancer with limited therapeutic options. This review discusses different approaches for generating PRLR antagonists. Emphasis is placed on technological advances which enable high-throughput screening for small molecule inhibitors of PRLR signaling that could serve as oral medications.

Structural characterization of the stem-stem dimerization interface between prolactin receptor chains complexed with the natural hormone.

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.

New concepts in prolactin biology.

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.

Crystal structure of an affinity-matured prolactin complexed to its dimerized receptor reveals the topology of hormone binding site 2.

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.