Hormone Receptors and Enzymes Regulating Gonadal and Mammary Function

调节性腺和乳腺功能的激素受体和酶

• 批准号: 7594114
• 负责人: MARIA DUFAU
• 金额: $ 106.58万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594114
• 关键词: 1-Phosphatidylinositol 3-Kinase; Accounting; Affinity; Amino Acids; Binding; Bioluminescence; Boxing; COS-1 Cells; Caseins; Cell Nucleus; Cell physiology; Cells; Chromatin Structure; Complex; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; DNA Methylation; Development; Dominant-Negative Mutation; Dose; Down-Regulation; Endocrine; Energy Transfer; Enzymes; Epigenetic Process; Equilibrium; Event; Exons; Failure; Gametogenesis; Gene Activation; Gene Expression; Gene Targeting; Generations; Genes; Genetic Screening; Genetic Transcription; Germ Cells; Gonadotropins; HMGB2 Protein; Heterodimerization; High Mobility Group Proteins; Histone Acetylation; Histone Deacetylase; Histone Deacetylation; Histones; Hormone Receptor; Human; Infertility; JAK2 gene; Japanese Population; Knockout Mice; LH Receptors; Left; Leucine; Ligands; Localized; Luteinizing Hormone; MCF7 cell; Mammary gland; Mediating; Mediator of activation protein; Messenger RNA; Missense Mutation; Modification; Molecular; Molecular Conformation; Mus; Mutation; N-terminal; Nuclear; Nuclear Export; Nuclear Localization Signal; Nuclear Orphan Receptor; Pathway interactions; Patients; Peptidyl-Dipeptidase A; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Polyribosomes; Process; Prolactin; Prolactin Receptor; Promoter Regions; Protein Overexpression; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; RNA; RNA Helicase; RNA-Binding Proteins; Receptor Activation; Receptor Gene; Recruitment Activity; Regulation; Repression; Role; SP1 gene; Second Messenger Systems; Signal Transduction; Site; Sperm Maturation; Spermatids; Spermatocytes; Spermatogenesis; Staging; Sterility; Structure; TEP1 gene; Threonine; Transcript; Transcriptional Activation; Translation Initiation; Translations; Trichostatin A; Variant; base; cell type; demethylation; derepression; dimer; disulfide bond; gonad function; helicase; inhibitor/antagonist; leydig interstitial cell; malignant breast neoplasm; men; messenger ribonucleoprotein; mutant; novel; particle; peptide hormone; prevent; promoter; protein kinase C zeta; receptor; second messenger; sperm cell; transition protein 1

The Luteinizing Hormone receptor (LHR): The LHR gene is subject to repression by histone deacetylation at its promoter region. An Sp1 site was shown to be an essential mediator of trichostatin A (TSA)-induced LHR gene activation. TSA-induced histone acetylation and gene activation prevailed in the absence of changes in Sp1/Sp3 expression, their binding activity, disassociation of the HDAC/mSin3A complex from the Sp1 site or demethylation of the promoter. This indicated the participation of an additional mechanism in TSA-induced derepression. Phosphatidylinositol 3-kinase/protein kinase C zeta (PI3K/PKCzeta)-mediated Sp1 phosphorylation at ser 641 accounts for Sp1 site-dependent LHR activation by TSA. Sp1 phosphorylation was required for the release of inhibitor p107 from Sp1. These findings have revealed a novel mechanism of TSA-regulated gene expression through derecruitment of a repressor from the LHR gene promoter in a PI3K/PKC-induced Sp1 phosphorylation-dependent manner. They also, supported the participation of phosphatase(s) in the control of LHR gene transcription, where a coordinate balance between PI3K/PKC (constitutive and/or induced activity) and phosphatase(s) could be critical for up- or down-regulation of LHR gene expression by its effect on the Sp1 phosphorylation status. Our studies revealed an important contribution of phosphatases PP2A and PP1 to TSA-induced activation of LHR gene transcription in a cell type-specific manner. TSA caused dose-dependent release of PP2A and PP1 from the LHR promoter in JAR and MCF-7 cells, respectively. Blockade of phosphatase activities enhanced the TSA effect, while overexpression largely prevented both the promoter induction and Sp1 phosphorylation elicited by TSA. PP2A was shown to associate with Sp1 at both its N- and C-termini of Sp1, where the phosphorylation site (at 641), that is critical for the PI3K/PKC mediated Sp1 phosphorylation induced by TSA, is located. Our findings have demonstrated that TSA-induced changes in chromatin structure cause a cell-specific release of a phosphatase that is associated with Sp1 directly (PP2A)or through HDAC (PP1). This favors the phosphorylation of Sp1 mediated by the PI3K/ PKC pathway (constitutively active) which in turn causes the release of the p107 inhibitor from SP1 and the marked transcriptional activation of the hLHR. Gonadotropin-Regulated Genes: We have previously identified a novel Gonadotropin-Regulated Testicular Helicase (GRTH/Ddx25). This enzyme, which is present in the nucleus and cytoplasm of pachytene spermatocytes and round spermatids, binds mRNA species as an integral component of messenger RNP particles, with storage in chromatoid bodies located in the cytoplasm of spermatids. GRTH-targeted null-mice are sterile due to spermatid arrest at step 8 of spermatogenesis, and failure to elongate. The transcription of messages in spermatids steps 1-8 of these mice was not altered, but their translation was abrogated in a selective manner. Our current studies are defining the function of this helicase as an RNA binding protein, and its storage and translational functions during sperm progression. We have demonstrated differential localization of two GRTH protein species in subcellular compartments of germ cells. These include the 56 kDa species and the phosphorylated 61 kDa form primarily localized in the nucleus and cytoplasm respectively of germ cells. The post-transcriptional modification appears to be related to cytoplasmic-related events induced by protein kinase A at threonine residue(s). We have recently provided evidence for dual functional roles of GRTH as a component of mRNP in RNA export from nucleus to cytoplasm and in the translation of specific RNA transcripts at specific stages in germ cell development. The 56 kDa nuclear species interacted with CRM1 and participated in mRNA transport. The phosphorylated cytoplasmic 61kDa was associated with polyribosomes and selectively regulates the translation of specific genes. Phosphorylation of GRTH, in addition to facilitating the initiation of translation of its target genes, might induce conformational changes to recruit protein(s) to gain access of mRNAs to chromatoid bodies for storage and/or degradation. The N-terminal leucine-rich region was identified as the nuclear export signal that participates in the CRM1-dependent nuclear export pathway. A 14 amino acid GRTH sequence at 100-114 residues was identified as the nuclear localization signal. GRTH selectively regulated the translation of specific genes including histone 4 H4 and high mobility group protein HMG2 in germ cells. In addition, GRTH participated in the nuclear export of RNA messages including phosphoglyrate kinase 2 PGK2, testicular angiotensin converting enzyme (tACE), transition protein 1 & 2 TP1 & TP2. The cytoplamic levels of these proteins are markedly reduced in GRTH null mice. Our studies have demonstrated that GRTH/Ddx25 is a multifunctional RNA helicase that is an essential regulator of sperm maturation. Two polymorphic forms of GRTH were identified by genetic screening of GRTH/DDX25 in fertile and infertile Japanese men with non-obstructive azoospermia. We found heterozygous mutations in exons 8 (missense) and exon 11 (sense). The missense mutation Arg242 His in exon 8 was identified in 5.8% of infertile patients and 1% of normal subjects, and the silent was observed in 2% of infertile patients. Although the mutant protein was efficiently expressed in COS-1 cells, only the 56 kDa nuclear/cytoplasmic non-phosphorylated species was present and the 61 kDa species was absent. This finding highlights the relevance of the R242 residue for the post-transcriptional modification leading to generation of this relevant species. The absence of the phospho-GRTH species could be of relevance to some of the functional aspects of the protein that impact on germ cell development and/or function. Prolactin Receptors Prolactin:Short forms of the prolactin receptor (PRLR) with abbreviated cytoplasmic sequences (S1 a and S1b) exert dominant-negative effects on prolactin (PRL)-induced activation of transcription by the long form (LF). The LF homodimer is the only PRLR form able to activate the JAK2/STAT5 pathway, which is essential for PRL-induced gene transcription. The inhibitory action of SFs results from their heterodimerization with the LF. The presence of preexisting homodimers of the LF receptor in the absence of prolactin indicated that PRL is a conformational modifier rather that a dimer-inducer. Bioluminescence resonance energy transfer analysis (BRET) demonstrated PRL-independent homo- and heterodimer association of hPRLR variants. However, PRL binding could alter dimerized PRLR conformation to facilitate signal transduction. Mutation of four conserved extracelular cys residues, which form intramolecular disulfide bonds (36-46, 75-86 aa) that are necessary for PRL binding to the PRLR, completely eliminated the PRL-stimulated JAK phosphorylation that is normally observed in the wild type LFs and SFs. The wild type SF (S1b), in contrast to the LF, displayed high constitutive JAK2 phosphorylation that was not observed in S1bX mutant. This indicates that intra-molecular Cys-bridge(s)are required for the correct conformation to allow JAK binding to box-1. Also, the inhibitory action of the wild type S1b on LF mediated -casein promoter activity was not observed in the S1bX. BRET analysis showed reduced heterodimeric association of LF with S1bX versus wild type and an increased affinity in homodimers of SFX. The increased association of S1bX homodimers leaves the LFs to form homodimers for ligand-induced signaling.

黄体生成激素受体（LHR）：LHR基因在其启动子区域的组蛋白脱乙酰化受到抑制作用。 SP1位点被证明是Trichostatin A（TSA）诱导的LHR基因激活的必不可少的介体。 TSA诱导的组蛋白乙酰化和基因激活在没有SP1/SP3表达变化的情况下占了上风，其结合活性，HDAC/MSIN3A复合物与SP1位点的分离或启动子的去甲基化。这表明另一种机制参与了TSA诱导的抑制。磷脂酰肌醇3-激酶/蛋白激酶C Zeta（PI3K/PKCZETA）在Ser 641处介导的SP1磷酸化介导了TSA的SP1位点依赖性LHR激活。 SP1磷酸化是从SP1释放抑制剂P107所必需的。这些发现揭示了通过以PI3K/PKC诱导的SP1磷酸化依赖性方式从LHR基因启动子中脱落阻遏物的新机理。他们还支持磷酸酶（S）参与LHR基因转录的控制，其中PI3K/PKC（组成型和/或诱导的活性）和磷酸酶之间的坐标平衡对于通过对LHR基因表达的上调节而对SP1磷酸化的效果的上调节可能至关重要。我们的研究揭示了磷酸酶PP2A和PP1对TSA诱导的LHR基因转录激活的重要贡献。 TSA分别从JAR和MCF-7细胞中的LHR启动子中引起PP2A和PP1的剂量依赖性释放。磷酸酶活性的阻断增强了TSA效应，而过表达在很大程度上阻止了TSA引起的启动子诱导和SP1磷酸化。显示PP2A在其SP1的N和C末端都与SP1相关，其中磷酸化位点（在641）对PI3K/PKC介导的TSA诱导的SP1磷酸化至关重要。我们的发现表明，TSA诱导的染色质结构的变化会导致与SP1直接相关的磷酸酶的细胞特异性释放（PP2A）或通过HDAC（PP1）。这有利于PI3K/ PKC途径介导的SP1磷酸化（组成型活性），进而导致p107抑制剂从SP1释放以及HLHR的明显转录激活。 促性腺激素调节的基因：我们先前已经鉴定出一种新型的促性腺激素调节的睾丸解旋酶（GRTH/DDX25）。这种酶存在于黄体精子细胞和圆形精子的细胞核和细胞质中，将mRNA物种结合为Messenger RNP颗粒不可或缺的成分，并在精子细胞质中的色素体中储存。靶向GRTH的零鼠，由于精子发生的第8步，未能拉长，由于精子停滞而导致无菌。这些小鼠的精子步骤1-8中消息的转录没有改变，但它们的翻译被选择性地废除。 我们目前的研究将这种解旋酶作为RNA结合蛋白的功能及其在精子进展过程中的储存和翻译功能定义。我们已经证明了两种GRTH蛋白质物种在生殖细胞亚细胞区室中的差异定位。这些包括56 kDa物种和主要定位于生殖细胞细胞核和细胞质的磷酸化的61 kDa形式。转录后的修饰似乎与苏氨酸残基（S）蛋白激酶A诱导的细胞质相关事件有关。 最近，我们提供了GRTH作为MRNP的双重功能作用的证据，在RNA从核向细胞质的RNA导出以及在生殖细胞发育中特定阶段的特定RNA转录物的翻译中。 56 kDa核种与CRM1相互作用并参与mRNA运输。磷酸化的细胞质61kDa与多核糖体相关，并有选择地调节特定基因的翻译。除了促进其靶基因翻译的启动外，GRTH的磷酸化还可能引起构象变化以募集蛋白质以使mRNA访问染色体体以储存和/或降解。富含N末端亮氨酸的区域被确定为参与CRM1依赖性核出口途径的核出口信号。在100-114个残基处的14个氨基酸GRTH序列被确定为核定位​​信号。 GRTH选择性地调节了特定基因的翻译，包括组蛋白4 H4和高迁移率组蛋白HMG2在生殖细胞中。 此外，GRTH参加了RNA消息的核输出，包括磷酸酯激酶2 PGK2，睾丸血管紧张素转化酶（TACE），过渡蛋白1和2 TP1和TP2。这些蛋白质的细胞环素水平在GRTH NULL小鼠中明显降低。我们的研究表明，GRTH/DDX25是一种多功能RNA解旋酶，是精子成熟的必不可少的调节剂。 通过在具有非刺激性植物植物的肥沃和不育的日本男性中对GRTH/DDX25的遗传筛查，通过对GRTH/DDX25进行遗传筛查来鉴定出两种多态性形式。 我们在外显子8（错过）和外显子11（有义务）中发现了杂合突变。在5.8％的不育患者和1％的正常受试者中，发现了外显子8中的错义突变Agr242，在2％的不育患者中观察到了沉默。尽管突变蛋白在COS-1细胞中有效表达，但仅存在56 kDa核/细胞质非磷酸化物质，并且不存在61 kDa物种。这一发现突出了R242残基与转录后修饰的相关性，从而导致该相关物种的产生。缺乏磷酸化物种可能与影响生殖细胞发育和/或功能的蛋白质的某些功能方面有关。 催乳素受体催乳素：催乳素受体（PRLR）的短形式，具有缩写的细胞质序列（S1 A和S1B）对长形式（LF）对催乳素（PRL）诱导的转录激活产生了显着的阴性作用。 LF同二聚体是唯一能够激活JAK2/STAT5途径的PRLR形式，这对于PRL诱导的基因转录至关重要。 SFS的抑制作用是由于其与LF的异二二聚化导致的。 在没有催乳素的情况下，LF受体的同型同二聚体的存在表明PRL是一种构象修饰剂，而不是二聚体诱导剂。生物发光谐振能量转移分析（BRET）证明了HPRLR变体的非PRL独立均和异二聚体关联。 然而，PRL结合可以改变二聚PrlR构象以促进信号转导。 在与PRL结合的PRL结合所必需的四个保守外Cys残基的突变，它们形成了分子内二硫键（36-46，75-86 AA），完全消除了PRL刺激的JAK磷酸化，通常在野生型LFS和SFS中观察到。与LF相比，野生型SF（S1B）显示出高的组成型JAK2磷酸化，在S1BX突变体中未观察到。这表明正确的构象需要进行分子内Cys桥（S）才能使Jak结合到Box-1。同样，在S1BX中未观察到野生型S1b对LF介导的-casein启动子活性的抑制作用。 BRET分析显示，LF与S1BX与野生型的异二聚体关联降低，而SFX同型二聚体的亲和力增加。 S1BX同型二聚体的关联增加使LFS形成同型二聚体，以进行配体诱导的信号传导。