Park Jong-Ju, Seong Hun-Ki, Kim Jeong-Soo, Munkhzaya Byambaragchaa, Kang Myung-Hwa, Min Kwan-Sik
Animal Biotechnology, Graduate School of Future Convergence Technology, Department of Animal Life Science, Institute of Genetic Engineering, Hankyong National University, Ansung 17579, Korea.
Department of Food Science and Nutrition, Hoseo University, Asan 31499, Korea.
Dev Reprod. 2017 Jun;21(2):111-120. doi: 10.12717/DR.2017.21.2.111. Epub 2017 Jun 30.
Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.
马绒毛膜促性腺激素（eCG）是一种独特的分子，在其他物种中能引发促卵泡激素（FSH）和促黄体生成素（LH）的反应特征。本实验室先前的研究表明，来自中国仓鼠卵巢（CHO-K1）细胞的重组eCG（rec-eCG）在大鼠颗粒细胞和睾丸间质细胞中表现出FSH样和LH样活性。在本研究中，我们分析了通过rec-eCG、野生型eCG（eCGβ/α）和α亚基Asn处带有N-连接寡糖的突变型eCG（eCGβ/αΔ56）引起的受体内化。两种rec-eCG均从CHO-K1细胞获得。通过测量rec-eCG的刺激时间和浓度来分析受体激动剂的激活情况。在用10 ng的rec-eCGβ/α刺激后50分钟时，稳定选择的大鼠促卵泡激素受体（rFSHR）和大鼠促黄体生成素/绒毛膜促性腺激素受体（rLH/CGR）中的内化值最高。当使用10 ng的rec-eCGβ/α时，剂量依赖性反应最高。去糖基化的eCGβ/αΔ56突变体不会增强激动剂刺激的内化。我们得出结论，rFSHR和rLH/CGR的激活状态可通过激动剂刺激的内化来调节。我们的结果表明，rec-eCG的激动剂刺激可使eLH/CGRs在60分钟内大部分内化。我们还提出，去糖基化的eCGβ/αΔ56缺乏反应性可能是因为糖基化位点在表达rFSHR和rLH/CGR的细胞中激动剂刺激的内化过程中起关键作用。
