Gonadal Receptors/mechanisms Of Action--Peptide Hormones

1)LH受体：促黄体激素受体（LHR）是一种G蛋白偶联受体，在性腺发育和分化中起重要作用。我们以前的研究表明，孤儿受体EAR 2和EAR 3/COUP-TF 1（抑制性）和TR 4（刺激性）对人LH受体的Sp1/Sp3驱动的TATA无启动子的调节。这些孤儿受体竞争性结合，并与雌激素反应元件半位点和第二个简并半位点（DR）组成的不完美的直接重复基序具有高亲和力。目前的研究调查了孤儿受体的大鼠和人类LHR启动子的差异结合，和他们的调制LHR转录在大鼠颗粒细胞分化培养激素治疗。这一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到黄体化分化过程中的作用。这些研究表明，EAR 2和EAR 3/COUP-TFI通过与DR结构域结合（尽管亲和力比人低2倍）抑制大鼠LHR基因转录。由于DR的第二个半位点处的单核苷酸差异，没有发现TR 4的结合或激活。大鼠中EAR 2和EAR 3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，并且归因于相邻3-prime序列中的物种差异。hCG处理显著降低了颗粒细胞中大鼠LHR的抑制，并且还降低了EAR 2和EAR 3蛋白水平。通过去阻遏消除hCG治疗后孤儿受体介导的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：高剂量促性腺激素治疗可引起类固醇生成酶的LHR非依赖性负调节（类固醇生成脱敏）和新型促性腺激素调节RNA解旋酶（GRTH）的上调。一个以前未鉴定的蛋白质，是组成性存在于Leydig细胞和下调促性腺激素最近被克隆和特征为促性腺激素调节的长链酰基辅酶A合成酶（GR-LACS）。79-kDa的胞质蛋白在大鼠睾丸的青春期和成年间质细胞中表达，并与拉克和荧光素酶家族的两个保守区域共享序列同一性，但显示出较低的总体氨基酸相似性（23-28%）。表达的蛋白质存在于转染细胞的细胞质中，显示出对长链脂肪酸底物的酰基CoA合成酶活性。除了其对能量产生和睾丸类固醇生成的潜在贡献外，GR-LACS还可以提供对酶活性、膜功能和基因表达具有调节作用的长链酰基辅酶A酯。3)催乳素受体：该实验室之前的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。这些研究表明，该基因具有复杂的结构，并适合选择性剪接，并报告了10个外显子的存在（多个非编码外显子1和共同的非编码外显子2和外显子3-10编码长型受体）。此外，最近发现人类催乳素受体的新外显子11与其啮齿动物对应物不同，并鉴定了人类催乳素受体的两种新形式（S1 a和S1 b，来自外显子10和11的选择性剪接）。这些新形式的人催乳素受体类似于常规受体，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。在几种正常组织和乳腺癌细胞系中发现的这些短形式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，短形式都不介导长形式受体所表现出的β-酪蛋白基因启动子的催乳素诱导的激活。相比之下，这些形式作为显性负阻遏物的功能的长和中间受体亚型介导的细胞生长反应在人乳腺癌细胞。由于两种形式的细胞水平的显着差异，这是由于S1 a的周转更快，S1 b形式是一个更有效的抑制剂。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，还可以表现出不同的信号传导途径。这些新的受体可能在催乳素在人体组织中的多种作用中发挥重要作用，并对控制乳腺癌细胞生长和免疫调节具有潜在的治疗意义。其他的研究已经解决了生长激素受体基因对睾丸功能的破坏的后果。这项工作提供了一个在GH受体基因敲除小鼠体内证明，LH对睾酮分泌的作用显着受损，由于睾丸LH受体的数量减少。这伴随着睾丸类固醇生成的反应性降低和雄烯二酮转化为睾酮的能力降低。这些变化可能是由于GH缺乏小鼠循环IGF-I的缺乏。这些研究表明，IGF-I在调节睾丸内分泌功能方面具有重要作用。

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.