RESUMO
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 ß-subunit: single mutants, eCGßV102G/α, eCGßF103P/α, and eCGßR104K/α; double mutants, eCGßV102G;F103P/α, eCGßV102G;R104K/α, and eCGßF103P;R104K/α; triple mutant, eCGßV102G;F103P;R104K/α. 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 eCGßV102G/α, eCGßR104K/α, and eCGßV102G;R104K/α in the culture supernatant were markedly lower than those of eCGß/α-wt. The other mutants and rec-eCGß/α-wt were efficiently secreted into the culture supernatant. The LH-like activities of eCGV104G/α, eCGßV102G;R104K/α, and eCGßF103P;R104K/α 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 eCGßR104K/α 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 ßV102G;R104K/α and ßF103P;R104K/α were 2.5-2.9% of that of eCG-wt. These results suggest that the amino acid residues 102-104 of the eCG ß-subunit are dispensable and that the residue 104 of the eCG ß-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.