Purchase this article with an account.
Aida Sanchez-Bretano, Ting-Chung Suen, Kenkichi Baba, Jason DeBruyne, Gianluca Tosini; Melatonin Receptor Heterodimers formation and signaling in 661 W cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5305. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Our laboratory has recently demonstrated that melatonin (MEL) signaling via melatonin receptor heterodimers (MT1/MT2) signaling may protect cones from cell death during aging. Further studies have also shown that the protective action of MT1/MT2 signaling involves the activation of the AKT-FoxO1 cell survival pathway. The aim of the present study is to determine whether the photoreceptor-like cell line, 661W – which endogenously express both melatonin receptors - represent a useful model to study the biology of the melatonin receptors heterodimer and its signaling.
MT1/MT2 heterodimerization in 661W cells was assessed by proximity ligand assay (MT1 antibody from Alomone (AMR-031); MT2 antibody from Santa Cruz (sc-13177), PLA system (DuoLink, DUO92105) from Sigma). Removal of melatonin receptors type 2 (MT2) from 661W cells was accomplished using the MT2-CRISPR/Cas9 system (Santa Cruz, sc-434093). Melatonin receptors signaling was investigated by measuring cAMP levels and activation of the AKT-FoxO1 pathway. Gene expression was investigated by quantitative-PCR (qPCR).
MT1/MT2 heterodimers were detected in 661W. Consistent with the absence of MT2 mRNA or immunoreactivity, no PLA signaling was observed in the 661W-MT2 KO. The increase in cAMP production induced by forskolin was inhibited in a dose-dependent manner by MEL only in 661W, whereas no effect was observed in 661W-MT2 KO cells. MEL (100 nM) induced phosphorylation of ERK1/2, AKT and FoxO1 in 661W, but not in 661W-MT2KO. MEL treatment also increased the mRNA levels of antioxidant peptides (nrf1, nrf2 and SOD2) only in 661W.
Our results demonstrate that heterodimerization of MT1 and MT2 receptors may occur in a system in these receptors are endogenously expressed. Our data also indicate that in 661W cells MT1 and MT2 receptors are only functional when they are associated in heteromeric complex, as occur in the mouse photoreceptors. The pathways activated by MT1/MT2 heterodimer in 661-W cells are similar to those previously reported in the mouse photoreceptors. Finally, lack of the MT1/MT2 heterodimer affects the gene expression of peptides responsible to decrease ROS levels. Our data indicate that 661W cells represent a useful model to study the mechanism underlying MT1/MT2 heterodimerization and the signaling associated with this heterodimer.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
This PDF is available to Subscribers Only