Gonadal Receptors/mechanisms Of Action--Peptide Hormones

性腺受体/作用机制——肽激素

• 批准号: 6534877
• 负责人: MARIA DUFAU
• 金额: --
• 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/6534877
• 关键词: animal genetic material tag; cell growth regulation; chorionic gonadotropin; gene targeting; genetic mapping; genetic promoter element; genetic transcription; genetically modified animals; gonadotropins; gonads; hormone receptor; hormone regulation /control mechanism; human genetic material tag; human tissue; laboratory mouse; laboratory rat; luteinizing hormone; peptide hormone; posttranslational modifications; prolactin; protein structure function; receptor binding; receptor expression; steroid biosynthesis; tissue /cell culture

1) The LH receptor: The luteinizing hormone receptor (LHR) is a G protein-coupled receptor that plays an essential role in gonadal development and differentiation. Our previous studies demonstrated regulation of the Sp1/Sp3-driven TATAless promoter of the human LH receptor by the orphan receptors EAR2 and EAR3/COUP-TF1 (inhibitory) and TR4 (stimulatory). These orphans receptors bound competitively and with high affinity to an imperfect direct-repeat motif composed of a estrogen response element half-site and a second degenerate half-site (DR). Current studies investigated the differential binding of orphan receptors to rat and human LHR promoters, and their modulation of LHR transcription in rat granulosa cells differentiated in culture by hormone treatment. This process resembles the induction of the LHR gene in granulosa cells of the human ovary and permits analysis of the role of orphan receptors during gonadal cell differentiation from early stages to luteinization. These studies demonstrated repression of rat LHR gene transcription by EAR2 and EAR3/COUP-TFI through their binding to a DR domain (albeit with 2-fold lower affinity than the human). No binding or activation was found for TR4 due to a single nucleotide difference at the second half-site of the DR. The lower binding affinity for EAR2 and EAR3 in the rat was associated with a smaller inhibitory effect than that observed in the human and was attributable to species differences in the adjacent 3-prime sequences. hCG treatment markedly reduced the inhibition of the rat LHR in granulosa cells and also decreased EAR2 and EAR3 protein levels. Abolition of the orphan receptor-mediated inhibition of the rLHR upon hCG treatment via derepression may contribute to the elevated LHR expression required for progression of granulosa cell maturation. 2) Gonadotropin regulation: Treatment with high doses of gonadotropins causes LHR-independent negative regulation of steroidogenic enzymes (steroidogenic desensitization) and up-regulation of a novel gonadotropin regulated RNA-helicase (GRTH). A previously unidentified protein that is constitutively present in Leydig cells and down-regulated by gonadotropin was recently cloned and characterized as a gonadotropin-regulated long chain acyl CoA synthetase (GR-LACS). The 79-kDa cytoplasmic protein is expressed in the pubertal and adult Leydig cells of the rat testis and shares sequence identity with two conserved regions of the LACS and luciferase families, but displays low overall amino acid similarities (23-28%). The expressed protein present in the cytoplasm of transfected cells displayed acyl CoA synthetase activity for long chain fatty acid substrates. In addition to its potential contributions to energy production and testicular steroidogenesis, GR-LACS could provide long chain acyl-CoA esters with regulatory effects on enzyme activity, membrane function, and gene expression. 3) Prolactin Receptors: Previous studies in this laboratory have mapped and resolved the genomic structure of the human prolactin gene of over 100 Kb . The studies demonstrated that the gene has a complex structure and is amenable to alternative splicing, and reported the presence of 10 exons (multiple non-coding Exons 1 and the common non-coding exon-2 and exon 3-10 coding for the long form of the receptor). In addition, a novel exon-11 of the human prolactin receptor was recently found to be distinct from its rodent counterpart, and two novel forms of the human prolactin receptor (S1a and S1b, which are derived from alternative splicing of exons 10 and 11) were identified. These new forms of the human prolactin receptor resemble the conventional receptor in having similar extracellular and transmembrane domain, but differ in having unique truncated intracellular domains. These short forms, which were found in several normal tissues and in breast cancer cell lines, are expressed as cell surface receptors and possess binding affinities comparable to the long form. However, unlike the long form, neither of the short forms mediates the prolactin induced-activation of the beta-casein gene promoter that is exhibited by the long form of the receptor. In contrast, these forms act as dominant negative repressors of the function of the long and intermediate receptor isoforms that mediated cell growth responses in human breast cancer cells. Due to the marked difference in the cellular levels of the two forms, which results from the more rapid turnover of S1a, the S1b form was a more effective inhibitor. These short forms with unique C-termini may exhibit distinct signaling pathways, in addition to modulating signaling from the long form of the receptor. These new receptors may have important roles in the diversified actions of prolactin in human tissues, and are of potential therapeutic relevance to the control of mammary cancer cell growth and immunoregulation. Other studies have addressed the consequences of disruption of the growth hormone receptor gene on testicular function. This work has provided an in vivo demonstration in GH receptor knockout mice that LH action on testosterone secretion is significantly impaired, due to a decrease in the number of testicular LH receptors. This is accompanied by diminished responsiveness of testicular steroidogenesis and decreased ability to convert androstenedione to testosterone. These changes are probably attributable to the absence of circulating IGF-I in the GH-deficient mice. These studies have indicated that IGF-I has a major role in the regulation of testicular endocrine function.

1）LH受体：黄体生成素受体（LHR）是一种G蛋白偶联受体，在性腺发育和分化中发挥重要作用。我们之前的研究证明了孤儿受体 EAR2 和 EAR3/COUP-TF1（抑制性）和 TR4（刺激性）对人类 LH 受体 Sp1/Sp3 驱动的 TATAless 启动子的调节。这些孤儿受体以高亲和力竞争性地结合到由雌激素反应元件半位点和第二个简并半位点（DR）组成的不完美直接重复基序。目前的研究调查了孤儿受体与大鼠和人类 LHR 启动子的差异结合，以及它们对通过激素处理在培养物中分化的大鼠颗粒细胞中 LHR 转录的调节。该过程类似于人类卵巢颗粒细胞中 LHR 基因的诱导，并允许分析孤儿受体在性腺细胞从早期分化到黄体化过程中的作用。这些研究证明，EAR2 和 EAR3/COUP-TFI 通过与 DR 结构域的结合（尽管亲和力比人类低 2 倍）来抑制大鼠 LHR 基因转录。由于 DR 后半位点的单核苷酸差异，未发现 TR4 的结合或激活。大鼠中对 EAR2 和 EAR3 的较低结合亲和力与比在人类中观察到的更小的抑制作用相关，并且可归因于相邻 3 引物序列的物种差异。 hCG 治疗显着降低了颗粒细胞中大鼠 LHR 的抑制，并降低了 EAR2 和 EAR3 蛋白水平。通过去阻遏 hCG 治疗后，消除孤儿受体介导的 rLHR 抑制可能有助于颗粒细胞成熟进展所需的 LHR 表达升高。 2）促性腺激素调节：用高剂量促性腺激素治疗会导致类固醇生成酶的不依赖于LHR的负调节（类固醇脱敏）和新型促性腺激素调节的RNA解旋酶（GRTH）的上调。最近克隆了一种以前未鉴定的蛋白质，该蛋白质组成型存在于 Leydig 细胞中并被促性腺激素下调，并被定性为促性腺激素调节的长链酰基辅酶 A 合成酶 (GR-LACS)。 79-kDa 胞质蛋白在大鼠睾丸的青春期和成年 Leydig 细胞中表达，与 LACS 和荧光素酶家族的两个保守区域具有序列同一性，但总体氨基酸相似性较低 (23-28%)。转染细胞细胞质中表达的蛋白质对长链脂肪酸底物表现出酰基辅酶A合成酶活性。除了对能量产生和睾丸类固醇生成的潜在贡献外，GR-LACS 还可以提供长链酰基辅酶 A 酯，对酶活性、膜功能和基因表达具有调节作用。 3）催乳素受体：本实验室前期研究已经绘制并解析了超过100 Kb的人类催乳素基因的基因组结构。研究表明，该基因具有复杂的结构，易于选择性剪接，并报道了 10 个外显子的存在（多个非编码外显子 1 和常见的非编码外显子 2 和外显子 3-10 编码长形式）受体）。此外，最近发现人类催乳素受体的一个新的外显子11与其啮齿类动物的对应物不同，并且人类催乳素受体的两种新形式（S1a和S1b，源自外显子10和11的选择性剪接）被识别出来。这些新形式的人催乳素受体与传统受体相似，具有相似的细胞外和跨膜结构域，但不同之处在于具有独特的截短的细胞内结构域。这些短形式存在于几种正常组织和乳腺癌细胞系中，它们表达为细胞表面受体，并具有与长形式相当的结合亲和力。然而，与长形式不同，短形式都不介导由长形式受体表现出的催乳素诱导的β-酪蛋白基因启动子激活。相反，这些形式充当长受体亚型和中间受体亚型功能的显性负阻遏物，介导人乳腺癌细胞中的细胞生长反应。由于两种形式的细胞水平存在显着差异（这是由于 S1a 更新速度更快），因此 S1b 形式是更有效的抑制剂。这些具有独特 C 末端的短形式除了调节来自长形式受体的信号传导之外，还可能表现出不同的信号传导途径。这些新受体可能在催乳素在人体组织中的多样化作用中发挥重要作用，并且与控制乳腺癌细胞生长和免疫调节具有潜在的治疗相关性。其他研究已经解决了生长激素受体基因破坏对睾丸功能的影响。这项工作在 GH 受体敲除小鼠体内证明，由于睾丸 LH 受体数量减少，LH 对睾酮分泌的作用显着受损。这伴随着睾丸类固醇生成的反应性减弱和将雄烯二酮转化为睾酮的能力下降。这些变化可能归因于 GH 缺陷小鼠体内循环 IGF-I 的缺失。这些研究表明IGF-I在睾丸内分泌功能的调节中具有重要作用。