Melatonin Receptor Heterodimers formation and signaling in 661 W cells

Abstract

Purpose : 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.

Methods : 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).

Results : 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.

Conclusions : 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.