Banerjee Antara A, Dupakuntla Madhavi, Pathak Bhakti R, Mahale Smita D
Division of Structural BiologyNational Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, IndiaNational Institute for Research in Reproductive Health (Indian Council of Medical Research)ICMR Biomedical Informatics Centre, Jehangir Merwanji Street, Parel, Mumbai 400 012, India.
Division of Structural BiologyNational Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, IndiaNational Institute for Research in Reproductive Health (Indian Council of Medical Research)ICMR Biomedical Informatics Centre, Jehangir Merwanji Street, Parel, Mumbai 400 012, India Division of Structural BiologyNational Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, IndiaNational Institute for Research in Reproductive Health (Indian Council of Medical Research)ICMR Biomedical Informatics Centre, Jehangir Merwanji Street, Parel, Mumbai 400 012, India
J Mol Endocrinol. 2015 Jun;54(3):193-204. doi: 10.1530/JME-14-0275. Epub 2015 Mar 19.
The extracellular loop 2 (EL2) of FSH receptor (FSHR) plays a pivotal role in various events downstream of FSH stimulation. Because swapping the six FSHR-specific residues in EL2 (chimeric EL2M) with those from LH/choriogonadotropin receptor resulted in impaired internalization of FSH-FSHR complex and low FSH-induced cAMP production, six substitution mutants of EL2 were generated to ascertain the contribution of individual amino acids to the effects shown by chimeric EL2M. Results revealed that L(501)F mainly and I(505)V to a lesser extent contribute to the diminished receptor function in chimeric EL2M. HEK293 cells stably expressing WT and chimeric EL2M FSHR were generated to track the fate of the receptors post FSH induction. The chimeric EL2M FSHR stable clone showed weak internalization and cAMP response similar to transiently transfected cells. Furthermore, reduced FSH-induced ERK phosphorylation was also observed. The interaction of activated chimeric EL2M and L(501)F FSHR with β-arrestins was weak compared with WT FSHR, thus explaining the impaired internalization of chimeric EL2M and corroborating the indispensable role of EL2 in receptor function.
促卵泡激素受体（FSHR）的细胞外环2（EL2）在FSH刺激后的各种下游事件中起关键作用。由于将EL2中六个FSHR特异性残基（嵌合EL2M）与促黄体生成素/绒毛膜促性腺激素受体的相应残基进行交换，导致FSH-FSHR复合物的内化受损以及FSH诱导的cAMP生成减少，因此生成了EL2的六个替代突变体，以确定单个氨基酸对嵌合EL2M所表现出的效应的贡献。结果显示，L(501)F起主要作用，I(505)V起较小作用，导致嵌合EL2M中的受体功能减弱。构建了稳定表达野生型和嵌合EL2M FSHR的HEK293细胞，以追踪FSH诱导后受体的命运。嵌合EL2M FSHR稳定克隆表现出较弱的内化和cAMP反应，类似于瞬时转染细胞。此外，还观察到FSH诱导的ERK磷酸化减少。与野生型FSHR相比，活化的嵌合EL2M和L(501)F FSHR与β-抑制蛋白的相互作用较弱，从而解释了嵌合EL2M内化受损的原因，并证实了EL2在受体功能中的不可或缺的作用。
