Equine chorionic gonadotropin (eCG) is a member of the glycoprotein hormone. eCG, over 40%, is a heavily glycosylated glycoprotein than other glycoprotein hormones. eCG is composed of non-covalently linked α and β subunit. The α subunit is common to all glycoprotein hormones, whereas the β subunit was known distinct sequences and specific receptor binding. Unusually, eCG shows both FSH and LH activities in other species. eCG α subunit has two N-glycosylation sites (Asn56, Asn82) and β subunit has one N-glycosylation site (Asn13) and about 13 O-glycosylation sites in the C-terminal region. We constructed 3 type mutants (βα△56: α-subunit Asn56→ Gln56; β-Da: β-subunit C-terminal deletion; β-Dα△56: β C-terminal deletion & α Asn56→Gln56) in the tethered eCGβα (wild type) and all mutants included myc-tag between first and second amino acid of β subunit. The plasmid DNAs cloned into pcDNA3 mammalian expressing vector were transiently transfected into CHO-Suspension cells. We also constructed rat LH/CG receptor and rat FSH receptor into pcDNA3 expression vector. These receptors were transiently transfected into CHO-K1 cell. Each receptor cells were used for further assays at 3 days after transfection. cAMP and IP-one were evaluated by CISBIO cAMP HiRange and IP-one kits using the rec-eCGβα mutants. According to cAMP assay results, IC50 values of 4 type ligand treatment in the rat FSH receptor cells were: eCGβα: IC50_16.8841; eCGβα56: IC50_95.6099; eCGβ-Dα: IC50_395.0087; eCGβ-Dα56: IC50_1439.8702. In the rat LH/CG receptor cells of 4 types ligand treatments, cAMP results were: eCGβα: IC50_0.9760; eCGβα56: IC50_8.3884; eCGβ-Dα: IC50_9.2550; eCG β-Dα56: IC50_45.9439. As seen in these data, β C-terminal region and α Asn56 play an important role in rat FSHR and rat LH/CGR, respectively. And rat LHCG receptor cells was remarkably stronger than rat FSH receptor cells. According to IP-one assay, IC50 values in rat FSH receptor cells, the results were: eCGβα: IC50_561.4490; eCGβα56: IC50_361.3005; eCGβ-Dα: IC50_911.8577; eCGβ-Dα56: IC50_139.1193. And in rat LH/CG receptor cells, IP-one results were: eCGβα: IC50_422.7315; eCGβα56: IC50_406.4915; eCGβ-Dα: IC50_537.8300; eCGβ-Dα56: IC50_254.2004. As shown in these data, IP-one result was a little different to cAMP result. The β eCGβ-Dα56 of IC50 value was shown generally high signal.
Now we are trying to analyse role of C-terminal region of eLH/CGR with cAMP, IP-one and ERK signal transduction assays.

• So Yun Lee(Department of Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University 327 Joongang-ro, Ansung-si, Kyonggi-do, 456-749)
• Munkhzaya Byambaragchaa(Department of Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University 327 Joongang-ro, Ansung-si, Kyonggi-do, 456-749)
• Jung Min Kim(Department of Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University 327 Joongang-ro, Ansung-si, Kyonggi-do, 456-749)
• Mi Hyang Yoo(Department of Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University 327 Joongang-ro, Ansung-si, Kyonggi-do, 456-749)
• Jeong Soo Kim(Department of Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University 327 Joongang-ro, Ansung-si, Kyonggi-do, 456-749)
• Kwan-Sik Min(Department of Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University 327 Joongang-ro, Ansung-si, Kyonggi-do, 456-749)