Glycoprotein hormones have a common α-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the α-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the NH2-terminus of the α-subunit to the COOH-terminus of the β-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form Δ96, Lys was substituted at position 95 to form Δ95, His was inserted at position 93 to form Δ93 and Tyr was substituted at position 87 to form Δ87. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and Δ96, Δ95, and Δ93 mutants were efficiently secreted into the medium but the Δ87 mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the Δ87 mutant. However, the Δ87 mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The Δ95 and Δ93 mutants were completely inactive in both the LH- and FSH-like activity assays. The Δ96 mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the Δ96 mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the α-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the α-subunit is very important for the secretion and functioning of this hormone.

ABSTRACT   INTRODUCTION   MATERIALS AND METHODS    Materials    Construction of the Tethered-eCG Mutant TransferVectors    Transient and Stable Transfection of the CHO CellLines    Hormone Quantitation and Western Blot Analysis ofTethered rec-eCGs    RT-PCR, Real-time PCR and Northern Blotting Analysis    Collection of Intracellular Proteins by Cell Lysis    In Vitro Bioassy for LH- and FSH-like Activities    Metabolic Clearance Rate of Tethered rec-eCGβαand rec-eCGβαΔ3   RESULTS    Production of Transient and Stably Tethered receCGMutants    RT-PC, Real-time PCR and Northern Blotting Analyses    Collection and Western Blotting Analysis of the IntracellularProtein Fraction obtained after Cell Lysis    Biological Activity of Tethered rec-eCG    Clearance Rates of Tethered eCGβα and rec-eCGβαMutants   DISCUSSION

• Youn-Hee Jeoung(Animal Biotechnology, Graduate School of Bio. & Information Technology, Institute of Genetic Engineering,)
• Jong-Taek Yoon(Animal Biotechnology, Graduate School of Bio. & Information Technology, Institute of Genetic Engineering,)
• Kwan-Sik Min(Animal Biotechnology, Graduate School of Bio. & Information Technology, Institute of Genetic Engineering,) Corresponding author