RESUMO
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.
Assuntos
Prolactina/química , Prolactina/genética , Receptores da Prolactina/química , Receptores da Prolactina/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia , Dimerização , Desenho de Fármacos , Glicina/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Lactogênio Placentário/química , Lactogênio Placentário/genética , Prolactina/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Receptores da Prolactina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ovinos , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Difração de Raios XRESUMO
There is currently no known genetic disease linked to prolactin (Prl) or its receptor (PrlR) in humans. Given the essential role of this hormonal system in breast physiology, we reasoned that genetic anomalies of Prl/PrlR genes may be related to the occurrence of breast diseases with high proliferative potential. Multiple fibroadenomas (MFA) are benign breast tumors which appear most frequently in young women, including at puberty, when Prl has well-recognized proliferative actions on the breast. In a prospective study involving 74 MFA patients and 170 control subjects, we identified four patients harboring a heterozygous single nucleotide polymorphism in exon 6 of the PrlR gene, encoding Ile(146)-->Leu substitution in its extracellular domain. This sole substitution was sufficient to confer constitutive activity to the receptor variant (PrlR(I146L)), as assessed in three reconstituted cell models (Ba/F3, HEK293 and MCF-7 cells) by Prl-independent (i) PrlR tyrosine phosphorylation, (ii) activation of signal transducer and activator of transcription 5 (STAT5) signaling, (iii) transcriptional activity toward a Prl-responsive reporter gene, and (iv) cell proliferation and protection from cell death. Constitutive activity of PrlR(I146L) in the breast sample from a patient was supported by increased STAT5 signaling. This is a unique description of a functional mutation of the PrlR associated with a human disease. Hallmarks of constitutive activity were all reversed by a specific PrlR antagonist, which opens potential therapeutic approaches for MFA, or any other disease that could be associated with this mutation in future.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroadenoma/genética , Fibroadenoma/metabolismo , Mutação de Sentido Incorreto , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Adulto , Estudos de Casos e Controles , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Éxons/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Imuno-Histoquímica , Estudos Prospectivos , Receptores da Prolactina/agonistas , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologiaRESUMO
There is increasing evidence that prolactin (PRL) and growth hormone (GH) act as growth-promoters of breast tumors. Recent arguments have accumulated to suggest that when they are locally-produced within the mammary tissue, these hormones, acting by an autocrine-paracrine mechanism may have enhanced, or even specific functions compared to endocrine PRL and GH. Classical drugs blocking pituitary hormone production (dopamine and somatostatin analogs) are ineffective on extrapituitary expression of PRL/GH genes, therefore the undesirable effects of these locally-produced hormones remain a target of interest for alternative strategies. This has encouraged the development of competitive PRL and/or GH receptor antagonists, which involve engineered variants of natural receptor ligands (PRL or GH) aimed at blocking receptor activation rather than hormone production in peripheral tissues. This article overviews the rational design of this new class of molecules, their specific molecular features (receptor specificity, biological properties, etc.) and whenever available, the data that have been obtained in cell or animal models of breast cancer.
Assuntos
Desenho de Fármacos , Prolactina/metabolismo , Receptores da Prolactina/antagonistas & inibidores , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/antagonistas & inibidores , Receptores da Somatotropina/metabolismo , Animais , Sítios de Ligação , Humanos , Mutação/genética , Receptores da Prolactina/química , Receptores da Prolactina/genéticaRESUMO
The molecular mechanisms that promote progression of localized prostate cancer to hormone-refractory and disseminated disease are poorly understood. Prolactin (Prl) is a local growth factor produced in high-grade prostate cancer, and exogenously added Prl in tissue or explant cultures of normal and malignant prostate is a strong mitogen and survival factor for prostate epithelium. The key signaling proteins that mediate the biological effects of Prl in prostate cancer are Signal Transducer and Activator of Transcription (Stat)-5a/5b via activation of Janus kinase-2. Importantly, inhibition of Stat5a/b in prostate cancer cells induces apoptotic death. Using a specific Prl receptor antagonist (Delta1-9G129R-hPRL), we demonstrate here for the first time that autocrine Prl in androgen-independent human prostate cancer cells promotes cell viability via Stat5 signaling pathway. Furthermore, we examined a unique clinical material of human hormone refractory prostate cancers and metastases and show that autocrine Prl is expressed in 54% of hormone-refractory clinical human prostate cancers and 62% prostate cancer metastases. Finally, we demonstrate that autocrine Prl is expressed from both the proximal and distal promoters of the Prl gene in clinical human prostate cancers and in vivo and in vitro human prostate cancer models, independently of pituitary transcription factor-1 (Pit-1). Collectively, the data provide novel evidence for the concept that autocrine Prl signaling pathway is involved in growth of hormone-refractory and metastatic prostate cancer. The study also provides support for the use of Prl receptor antagonists or other therapeutic strategies to block the Prl-Janus kinase-2-Stat5 signaling pathway in advanced prostate cancer.
Assuntos
Janus Quinase 2/metabolismo , Prolactina/metabolismo , Neoplasias da Próstata/patologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Oligodesoxirribonucleotídeos Antissenso/genética , Fosforilação/efeitos dos fármacos , Prolactina/antagonistas & inibidores , Prolactina/genética , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/genética , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacos , Transplante Heterólogo , Proteínas Supressoras de Tumor , Tirfostinas/farmacologiaRESUMO
The N-terminus is the most divergent region within the prolactin (PRL)/placental lactogen (PL)/growth hormone (GH) family. Since all of these ligands are able to activate the lactogen receptor, it has been usually assumed that the N-terminus plays no major role in biological actions of any family member. In this study, we generated several analogs of human PRL in which the N-terminus was truncated by 9 and iteratively up to the 14 first residues. Truncation did not alter protein folding, and it even decreased the formation of PRL aggregates that appear during the purification of refolded protein. Removal of the entire N-terminal loop (14 residues) decreased the affinity for the receptor by two-three-fold, and reduced the ability of the hormone to activate the human lactogen receptor. In contrast, removal of 13 or less residues improves receptor activation since these analogs are able to produce supra-maximal activities in a transcriptional bioassay, or in proliferation assays exhibit dose-response curves that are less bell-shaped, which reflects enhanced stabilization of receptor dimers. Altogether, these data suggest that the N-terminus of PRL is actually slightly detrimental to bioactivity, but may be required for other properties of the hormone.
Assuntos
Prolactina/química , Prolactina/farmacologia , Sequência de Aminoácidos , Diferenciação Celular , Humanos , Dados de Sequência Molecular , Prolactina/análogos & derivados , Prolactina/metabolismo , Deleção de SequênciaRESUMO
Human prolactin (PRL) and its receptor (PRLR) are markedly induced during human uterine decidualization, and large amounts of PRL are released by decidual cells as differentiation progresses. However, the role of PRL in decidualization is unknown. In order to determine whether PRL plays an autocrine role in decidualization, human uterine fibroblast cells that were decidualized in vitro with medroxyprogestrerone acetate (1 microM), estradiol (10 nM), and prostaglandin E(2) (1 microM) were exposed to exogenous PRL and/or the pure PRLR antagonist delta1-9-G129R-PRL. As measured by quantitative PCR, cells that were decidualized in the presence of exogenous PRL (0.25-2 microg/ml) expressed significantly lower levels of mRNA for the genes that encode insulin-like growth factor binding protein 1 (IGFBP1), left-right determination factor 2 (LEFTY2), PRL, decorin (DCN), and laminin alpha 1 (LAMA1), all of which are known to be induced during decidualization. These effects were blocked when the cells were exposed simultaneously to PRL and the PRLR antagonist, which confirms the specific inhibitory action of PRL on the expression of decidualization markers. In addition, cells exposed to the PRLR antagonist alone expressed higher levels of the marker gene mRNAs than cells that were decidualized in control media. Taken together, these results strongly suggest that PRL acts via an autocrine mechanism to regulate negatively the extent of differentiation (decidualization) of human uterine cells.
Assuntos
Comunicação Autócrina/fisiologia , Decídua/fisiologia , Prolactina/fisiologia , Útero/fisiologia , Western Blotting , Caspase 3/biossíntese , Caspase 3/genética , Decídua/efeitos dos fármacos , Decorina , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Proteína Ligante Fas/biossíntese , Proteína Ligante Fas/genética , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Laminina/biossíntese , Laminina/genética , Fatores de Determinação Direita-Esquerda , Prolactina/antagonistas & inibidores , Prolactina/farmacologia , Proteoglicanas/biossíntese , Proteoglicanas/genética , RNA/biossíntese , RNA/genética , Receptores da Prolactina/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Útero/efeitos dos fármacosRESUMO
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.