Effects of mutation of the LH/CG- receptor on its signaling activity

在1例LH/CG-R失活导致的Leydig细胞发育不全（LCH）患者中，通过LH/CG-R核苷酸序列测定，发现了一个新的杂合突变A340 T，导致Phe取代了保守的Ile-114氨基酸。 该突变位于LH/CG-R胞外域的第三个富含亮氨酸重复序列（LRR）。 体外表达研究表明，这种突变导致受体的配体结合和信号转导减少。 LH/CG-R结构的研究，其中各种氨基酸取代保守的Ile-114表明，受体活性是敏感的大小，形状和侧链的电荷的变化。 构建了野生型LH/CG-R胞外域的同源模型，说明了蛋白质核心中保守的疏水侧链的堆积。 Ile-114被Phe取代可能会破坏激素与受体之间的分子间联系。 该突变也可能影响LH/CG-R-二聚体相互作用。 因此，I114 F突变减少了LH/CG-R的配体结合和信号转导，并且它是患者中LCH的部分原因。 LH/CG-R的激活突变导致家族性男性限制性早熟（Familial Male-limited Precocious Puberty，FMPP）的发生。 在睾丸肿瘤患者中发现LH/CG-R存在生殖系和体细胞激活突变，提出了突变的LH/CG-R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LH/CG-R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FMPP患者中检测到的最常见突变，可以通过种系传播。 迄今为止，动物研究未能建立携带Asp 578 His突变LH/CG-R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LH/CG-Rs具有不同的生物学效应，并触发不同基因/途径的表达。 为了验证我们的假设，我们用cDNA微阵列和系统生物学方法比较了携带生殖系激活突变（Asp 578 Gly）的人突变LH/CG-R转染的MA 10细胞和表达体细胞激活突变（Asp 578 His）的MA 10细胞的表达谱。 结果表明，表达野生型人LH/CG-R和两种突变体的细胞的基因表达可以通过系统聚类和多维尺度分析来区分。 通过比较两种突变体与野生型，观察到132个基因的差异表达。 也有特定的突变基因。Asp 578 Gly突变特异性基因54个，Asp 578 His突变特异性基因49个。 每个突变独特的新的调控途径被确定，9个网络的Asp 578 Gly和12个Asp 578 His。进一步的分析表明，c-Myc和c-Sr分别是Asp 578 Gly和Asp 578 His突变细胞中的关键调节因子。这两个因素的参与，证实了分子和功能测定。这些结果为hLH/hCG-R突变在睾丸肿瘤发生中的作用提供了新的解释。

In a patient with Leydig Cell Hypoplasia (LCH) caused by inactivation of the LH/CG-R, a novel heterozygous mutation A340T leading to the substitution of Phe for the conserved amino acid Ile-114 was identified by nucleotide sequencing of the LH/CG-R. This mutation is located in the third leucine-rich repeat (LRR) in the ectodomain of the LH/CG-R. In vitro expression studies demonstrated that this mutation results in reduced ligand binding and signal transduction of the receptor. Studies of LH/CG-R constructs where various amino acids were substituted for the conserved Ile-114 showed that receptor activity is sensitive to changes in size, shape, and charge of the side chain. A homology model of the wild-type LH/CG-R ectodomain was made, illustrating the packing of conserved hydrophobic side chains in the protein core. Substitution of Ile-114 by Phe might disrupt intermolecular contacts between hormone and receptor. This mutation might also affect an LH/CG-R-dimer interaction. Thus, the I114F mutation reduces ligand binding and signal transduction by the LH/CG-R, and it is partially responsible for LCH in the patient. Activating mutations of the LH/CG-R lead to the development of Familial Male-limited Precocious Puberty (FMPP). Discovery of the presence of LH/CG-R with germline and somatic activating mutations in patients with testicular tumor raised the question of the tumorigenic potential of mutated LH/CG-R. Asp578His is a somatic mutation since LH/CG-R 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 LH/CG-R 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 LH/CG-Rs have distinct biological effects and trigger expression of different sets of genes/pathways. To prove our hypothesis we compared the expression profile of MA10 cells transfected with human mutated LH/CG-R carrying the germline activating mutation (Asp578Gly) with those expressing the somatic activating mutation (Asp578His) using cDNA microarray and systems biology approach. Results showed that gene expression of cells expressing the wild type human LH/CG-R and the two mutants could be distinguished by hierarchical clustering and multi-dimensional scaling analysis. By comparing both mutants to the wild type, 132 genes were observed to be differentially expressed. There were also mutation-specific genes. Fifty four genes were found to be specific to Asp578Gly mutation while 49 genes were specific to Asp578His. Novel regulatory pathways unique to each mutation were identified, with 9 networks for Asp578Gly and 12 for Asp578His. Further analyses revealed that c-Myc and c-Sr are the key regulators in Asp578Gly and Asp578His mutant cells, respectively. The involvement of these two factors was confirmed by molecular and functional assays. These results open a new dimension and provide novel explanation for the role of hLH/hCG-R mutation in testicular tumorigenesis.