Phys & Genetic Effects Of Disease-Causing Mutations of t

促黄体激素/绒毛膜促性腺激素受体（LHR）在人类男性性发育中起着重要作用。LHR的突变导致睾酮的异常产生和性发育疾病。虽然LHR在促黄体生成素/绒毛膜促性腺激素信号转导中的作用已得到充分证实，但其作用机制仍不完全清楚。我们研究了大量LHR激活和失活突变患者的LHR分子遗传学。这些研究有助于我们了解疾病的不同表现以及受体信号转导的分子机制。 携带激活突变的LHR个体发展为家族性男性限制性早熟（FMPP），并且经常有行为问题。FMPP患者的这种行为问题可能与突变的受体引起的脑细胞功能障碍有关。由于哺乳动物脑的主要生长期发生在妊娠的前三个月，此时hCG高，LHR在脑细胞中表达，因此长期以来一直怀疑hCG-LHR途径参与早期脑的发育。我们试图使用大鼠神经元细胞系PC 12来评估LHR在脑细胞中的活性，PC 12是双能的，并且在暴露于神经营养素或分化诱导剂后可以分化成神经元或嗜铬细胞。表达携带激活突变Asp 578 His的hLHR的PC 12细胞在10.8？1.8%的细胞。具有神经突生长的细胞的百分比显著高于用载体或用野生型hLHR转染的细胞。向表达野生型LHR的稳定转染细胞的培养基中加入hCG，与无hCG处理对照或表达载体的对照相比，引起了神经突裸露细胞数量的显著增加。hCG的作用具有剂量和时间依赖性。hCG给药后24小时出现分化细胞，并在72小时内表现出深刻的形态学变化。分化的细胞表达早期神经元标记物，神经元特异性微管蛋白III和神经丝68，表明LHR激活通过受体本身的基因突变或与其配体结合诱导PC 12细胞向神经元细胞类型分化。进一步的研究表明，p44/42和p38通路参与了转染野生型hLHR并经hCG诱导的PC 12细胞向神经元分化，而SAPK/JNK、MAPK和Akt通路可能不参与。cAMP在将信号从受体激活传递到相关信号通路中起重要作用。这些结果证明了hCG/LHR通路的神经功能。它还显示了hCG的亲神经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存在具有生殖系和体细胞激活突变的LHR，提出了突变的LHR的致瘤潜力的问题。Asp 578 His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，而在任何患有FMPP的患者中均未发现。另一方面，Asp 578 Gly是在FMPP患者中检测到的最常见的突变，并且可以通过生殖系传播。迄今为止，动物研究未能建立携带Asp 578 His突变LHR的雄性或雌性转基因创始小鼠的品系。我们推测，尽管Asp 578 Gly和Asp 578 His涉及相同氨基酸的突变，但这两种突变型hLHR具有不同的生物学效应，并触发不同基因组的表达。为了证明我们的假设，我们比较了携带种系激活突变（Asp 578 Gly）的突变LHR转染的MA 10细胞与表达体细胞激活突变（Asp 578 His）的MA 10细胞的表达谱。结果显示，不同的表达模式相应的表达的LHR与Asp 578 Gly或Asp 578 His突变。我们正在描绘受这些突变LHRs影响的生物学途径。

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.