Phys & Genetic Effects Of Disease-Causing Mutations of t

物理

• 批准号: 7334103
• 负责人: Owen M Rennert
• 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/7334103
• 关键词: Phys; Genetic; Effects; Disease; Causing

Luteinizing hormone/choriogonadotropin receptor (LHR) plays a central role in human male sexual development. Mutation of the LHR results in abnormal production of testosterone and diseases of sexual development. Though the role of LHR in transducing the signal of luteinizing hormone/chorionic gonadotropin is well established, the mechanism of its action is still not fully understood. We had studied the molecular genetics of the LHR of a large number of patients with activating and inactivating mutations of the LHR. These studies help us understand the varied presentation of the diseases as well as the molecular mechanism of signal transduction of the receptor. Individuals with LHR carrying activating mutations develop familial male-limited precocious puberty (FMPP) and often have behavioral problems. This behavioral problem of FMPP patients may be related to the dysfunction of brain cells caused by the mutated receptor. Since the major growth period of mammalian brain occurs in the first trimester of pregnancy when hCG is high and LHR is expressed in brain cells, the hCG-LHR pathway has long been suspected to be involved in the development of the early brain. We attempted to assess the activity of LHR in brain cells using a rat neuronal cell line, PC12, which is bipotent and can differentiate into either neuronal or chromaffin cells upon exposure to neurotropins or differentiating inducing agents. PC12 cells expressing hLHR carrying the activating mutation Asp578His demonstrated neurite outgrowth in 10.8 ? 1.8 % of cells. This percent of cells with neurite outgrowth was significantly higher than that of cells transfected with vector or with wild type hLHR. Addition of hCG to the culture medium of stably transfected cells expressing wild type LHR caused a significant increase in the number of neurite-baring cells compared to that of no hCG treatment controls or vector expressing controls. The effect of hCG was dose and time-dependent. Differentiated cells appeared as early as 24 hrs after hCG administration and demonstrated profound morphological changes in 72 hours. The differentiated cells expressed early neuronal markers, neuronal specific tubillin III and neural filament 68, indicating that LHR activation either through a genetic mutation of the receptor itself or binding with its ligand induced the differentiation of PC12 cells toward neuronal cell type. Further studies showed that both p44/42 and p38 pathways were required for the neuronal differentiation of PC12 cells transfected with wild type hLHR and induced by hCG, while the SAPK/JNK MAPK and Akt pathways might not be involved. cAMP played an important role in transmitting signals from receptor activation to the signaling pathways involved. These results demonstrated a neural function of the hCG/LHR pathway. It also shows the neurotropic activity of hCG and its potential as a therapeutic agent for neurological disorders and acute injuries of the nervous system. Discovery of the presence of LHR with germline and somatic activating mutations in patients with testicular tumor raised the question of the tumorigenic potential of mutated LHR. Asp578His is a somatic mutation since hLHR carrying this mutation has only been found in testicular tumor tissues and has not been found in any patient with FMPP. On the other hand, Asp578Gly is the most common mutation detected in FMPP patients and can be transmitted through the germ-line. Animal studies have so far failed to establish lines of male or female transgenic founder mice carrying LHR with the Asp578His mutation. We speculate that in spite of the fact that Asp578Gly and Asp578His involve mutation of the same amino acid, the two mutant hLHRs have distinct biological effects and trigger expression of different sets of genes. To prove our hypothesis we compared the expression profile of MA10 cells transfected with mutated LHR carrying the germline activating mutation (Asp578Gly) with those expressing the somatic activating mutation (Asp578His). Results revealed different expression pattern consequential to the expression of the LHR with the Asp578Gly or the Asp578His mutation. We are in the process of delineating the biological pathways affected by these mutant LHRs.

黄体生成激素/绒毛膜腺苷受体（LHR）在人类男性性发育中起着核心作用。 LHR的突变导致睾丸激素异常产生和性发育疾病。尽管LHR在转导黄体生成激素/绒毛膜促性腺激素的信号中的作用已经很好地确定，但其作用的机理仍未得到充分了解。我们研究了大量患有LHR的激活和灭活突变患者的LHR的分子遗传学。这些研究有助于我们了解疾病的各种表现以及受体信号转导的分子机制。 LHR携带激活突变的个体会出现家族性男性限制的早熟青春期（FMPP），并且通常存在行为问题。 FMPP患者的这种行为问题可能与突变受体引起的脑细胞功能障碍有关。由于哺乳动物大脑的主要生长时期发生在HCG高且LHR且在脑细胞中表达LHR的头三个月，因此长期以来一直怀疑HCG-LHR途径与早期大脑的发展有关。我们试图使用大鼠神经元细胞系PC12评估LHR在脑细胞中的活性，PC12是兼容的，并且在暴露于神经性蛋白或区分诱导药物的情况下可以分化为神经元或染色体细胞。表达携带激活突变ASP578HIS的HLHR的PC12细胞在10.8中表现出神经突的生长？ 1.8％的细胞。具有神经突生长的细胞的百分比显着高于用载体或野生型HLHR转染的细胞。与无HCG治疗对照或表达对照的载体相比，在表达野生型LHR的稳定转染细胞的培养基中添加HCG显着增加。 HCG的作用是剂量和时间依赖的。分化细胞早在HCG给药后24小时出现，并在72小时内显示出深刻的形态变化。分化的细胞表达了早期的神经元标记，神经元特异性图比林III和神经丝68，表明LHR通过受体本身的遗传突变而激活，或与其配体结合诱导PC12细胞对神经元细胞类型的分化。进一步的研究表明，p44/42和p38途径均需要用野生型HLHR转染并由HCG诱导的PC12细胞的神经元分化，而SAPK/JNK MAPK和AKT途径可能不参与。 CAMP在将信号从受体激活传输到所涉及的信号通路方面发挥了重要作用。这些结果证明了HCG/LHR途径的神经功能。它还显示了HCG的神经性活性及其作为神经系统疾病和神经系统急性损伤的治疗剂的潜力。 在睾丸肿瘤患者中发现LHR具有生殖线和躯体激活突变的存在，提出了突变LHR的致瘤潜力的问题。 ASP578HI是一种体细胞突变，因为携带该突变的HLR仅在睾丸肿瘤组织中发现，并且在任何FMPP患者中都没有发现。另一方面，ASP578GLY是FMPP患者中最常见的突变，可以通过种系传播。到目前为止，动物研究未能建立携带LHR的男性或女性转基因创始人小鼠的线条。我们推测，尽管ASP578Gly和Asp578His涉及相同氨基酸的突变，但两个突变的HLHR具有不同的生物学作用，并且触发了不同基因集的表达。为了证明我们的假设，我们将携带种系激活突变（ASP578Gly）的突变LHR转染的MA10细胞的表达谱与表达躯体激活突变（ASP578HIS）的表达谱。结果揭示了通过ASP578GLY或ASP578HIS突变与LHR表达的不同表达模式。我们正在描述受这些突变体LHR影响的生物学途径。