Phys & Genetic Effects Of Disease-Causing Mutations of t

促黄体生成素/绒毛膜促性腺激素受体(LHR)在人类男性性发育中起核心作用。LHR的突变会导致睾酮的异常产生和性发育疾病。虽然LHR在黄体生成素/绒毛膜促性腺激素信号传导中的作用已被证实，但其作用机制仍不完全清楚。我们对大量LHR激活和失活突变患者的LHR进行了分子遗传学研究。这些研究有助于我们了解疾病的不同表现形式以及受体信号转导的分子机制。 携带激活突变的LHR个体会发展为家族性男性受限性早熟(FMPP)，并经常有行为问题。FMPP患者的这种行为问题可能与受体突变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期发生在妊娠早期，此时hCG水平较高，LHR在脑细胞中表达，因此hCG-LHR途径长期以来一直被怀疑参与了早期大脑的发育。我们试图使用大鼠神经细胞系PC12来评估LHR在脑细胞中的活性，PC12是双能的，在暴露于神经促进剂或分化诱导剂时，可以分化为神经细胞或嗜铬细胞。表达hLHR激活突变Asp578His的PC12细胞有10.8±1.8%的细胞突起生长。有突起生长的细胞比例明显高于载体和野生型hLHR。在稳定表达野生型LHR的细胞的培养液中加入hCG，与不加hCG处理的对照或载体表达对照相比，突起细胞的数量显著增加。人绒毛膜促性腺激素的作用呈剂量和时间依赖性。分化细胞最早出现在hCG作用后24小时，72小时后出现明显的形态变化。分化的细胞表达早期神经元标志物神经元特异性Tublin III和神经丝68，表明LHR通过受体本身的基因突变或与其配体结合而激活，诱导PC12细胞向神经细胞型分化。进一步的研究表明，p44/42和p38通路是hCG诱导野生型hLHR诱导PC12细胞分化所必需的，而SAPK/JNK MAPK和Akt通路可能不参与。CAMP在受体激活向相关信号通路传递信号的过程中起着重要作用。这些结果证实了hCG/LHR通路的神经功能。它还显示了hCG的神经促动活性及其作为神经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具有生殖系和体细胞激活突变的LHR的存在，提出了突变的LHR的致瘤潜力的问题。Asp578His是一种体细胞突变，因为携带该突变的hLHR仅在睾丸肿瘤组织中发现，而在任何FMPP患者中均未发现。另一方面，Asp578Gly是在FMPP患者中检测到的最常见的突变，可以通过生殖系传播。到目前为止，动物研究还未能建立携带Asp578His突变的LHR的雄性或雌性转基因创始小鼠品系。我们推测，尽管Asp578Gly和Asp578His涉及相同氨基酸的突变，但这两个突变的hLHR具有不同的生物学效应，并触发不同组基因的表达。为了验证我们的假设，我们比较了携带胚系激活突变(Asp578Gly)的突变LHR(Asp578Gly)和表达体细胞激活突变(Asp578His)的突变LHR的MA10细胞的表达谱。结果发现，Asp578Gly突变和Asp578His突变导致LHR的表达模式不同。我们正在描绘受这些突变的LHR影响的生物途径。

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.