Journal of Animal Reproduction and Biotechnology (한국동물생명공학회지)

The Korean Society of Animal Reproduction and Biotechnology (한국동물생명공학회)
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Signal transduction of C-terminal phosphorylation sites for equine follicle stimulating hormone receptor (eFSHR)

  • Seong, Hoon-Ki (Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University) ;
  • Choi, Seung-Hee (Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University) ;
  • Byambaragchaa, Munkhzaya (Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University) ;
  • Min, Kwan-Sik (Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University)
  • Received : 2020.05.19
  • Accepted : 2020.05.29
  • Published : 2020.06.30
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Abstract

Equine follicle stimulating hormone receptor (eFSHR) has a large extracellular domain and an intracellular domain containing approximately 10 phosphorylation sites within the G protein-coupled receptor. This study was conducted to analyze the function of phosphorylation sties at the eFSHR C-terminal region. We constructed a mutant of eFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 641 (eFSHR-t641). This removed 10 potential phosphorylation sites from the C-terminal region of the intracellular loop. The eFSHR-wild type (eFSHR-wt) and eFSHR-t641 cDNAs were subcloned into the pCMV-ARMS1-PK2 expression vector. These plasmids were transfected into PathHunter CHO-K1 Parental cells expressing β-arrestin 2 enzyme acceptor fusion protein and analyzed for agonist-induced cAMP response. The cAMP response in cells expressing eFSHR-t641 was lower than the response in cells expressing eFSHR-wt. EC50 values of eFSHR-wt and eFSHR-t641 were 1079 ng/mL and 1834 ng/mL, respectively. eFSHR-t641 was approximately 0.58-fold compared with that of eFSHR-wt. The maximal response in eFSHR-wt and eFSHR-t641 was 24.7 nM and 16.7 nM, respectively. The Rmax value of phosphorylation sites in eFSHR-t641 was also decreased to approximately 68.4% of that in eFSHR-wt. The collective data implicate that the phosphorylation sites in the eFSHR C-terminal region have a pivotal role in signal transduction in PathHunter CHO-K1 cells, and indicate that β-arrestin is involved in coupling the activated receptors to the internalization system.

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References

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