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
Munkhzaya Byambaragchaa | Ayoung Park | So-Jin Gil | Hae-Won Lee | Yun-Jeong Ko | Seung-Hee Choi | Myung-Hwa Kang | Kwan-Sik Min
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 β-subunit: single mutants, eCGβV102G/α, eCGβF103P/α, and eCGβR104K/α; double mutants, eCGβV102G;F103P/α, eCGβV102G;R104K/α, and eCGβF103P;R104K/α; triple mutant, eCGβV102G;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 eCGβV102G/α, eCGβR104K/α, and eCGβV102G;R104K/α in the culture supernatant were markedly lower than those of eCGβ/α-wt. The other mutants and rec-eCGβ/α-wt were efficiently secreted into the culture supernatant. The LH-like activities of eCGV104G/α, eCGβV102G;R104K/α, and eCGβF103P;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 eCGβR104K/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 βV102G;R104K/α and βF103P;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 β-subunit are dispensable and that the residue 104 of the eCG β-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.
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