Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor

To identify the specific region of eCG involved in FSH-like activity, the following mutant expression vectors were constructed targeting the amino acid residues 102-104 of the eCG beta-subunit: single mutants, eCG beta V102G/alpha, eCG beta F103P/alpha, and eCG beta R104K/alpha; double mutants, eCG beta V102G;F103P/alpha, eCG beta V102G;R104K/alpha, and eCG beta F103P;R104K/alpha; triple mutant, eCG beta V102G;F103P;R104K/alpha. The LH-like and FSH-like activities of eCG mutants were examined in CHO-K1 cells expressing rat LH/CG receptor and rat FSH receptor. The levels of eCG beta V102G/alpha, eCG beta R104K/alpha, and eCG beta V102G;R104K/alpha in the culture supernatant were markedly lower than those of eCG beta/alpha-wt. The other mutants and rec-eCG beta/alpha-wt were efficiently secreted into the culture supernatant. The LH-like activities of eCGV104G/alpha, eCG beta V102G;R104K/alpha, and eCG beta F103P;R104K/alpha were approximately 61%, 52%, and 54%, respectively, of those of eCG-wt. The Rmax values of the mutants were 58.9%-78.8% those of eCG-wt with eCG beta R104K/alpha exhibiting the lowest value. The FSH-like activities of single mutants were only 16%-20% of those of eCG-wt. Additionally, the FSH-like activity of double mutants was less than 10% of that of eCG-wt. In particular, the FSH-like activities of beta V102G;R104K/alpha and beta F103P;R104K/alpha were 2.5-2.9% of that of eCG-wt. These results suggest that the amino acid residues 102-104 of the eCG beta-subunit are dispensable and that the residue 104 of the eCG beta-subunit plays a pivotal role in signal transduction through the rat FSH receptor. Thus, these mutants may aid future studies on eCG interactions with mammalian FSH receptors in vitro and in vivo.