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.
