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

在一例由LH/CG-R失活引起的间质细胞发育不全（LCH）患者中，通过对LH/CG-R的核苷酸测序，发现了一种新的杂合突变A340T，该突变导致Phe取代保守的氨基酸Ile-114。该突变位于LH/CG-R外域的第三个富亮氨酸重复序列（LRR）上。体外表达研究表明，这种突变导致配体结合和受体信号转导减少。用不同氨基酸取代保守的Ile-114的LH/CG-R结构的研究表明，受体活性对侧链的大小、形状和电荷的变化很敏感。建立了野生型LH/CG-R外结构域的同源性模型，说明了保守的疏水侧链在蛋白质核心的包装。用苯丙氨酸取代il -114可能会破坏激素和受体之间的分子间接触。这种突变也可能影响LH/ cg - r -二聚体的相互作用。因此，I114F突变减少了LH/CG-R的配体结合和信号转导，是患者LCH的部分原因。

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.