Melatonin receptor heterodimerization in a photoreceptor-like cell line endogenously expressing melatonin receptors.

Purpose: Melatonin signaling plays an important role in the modulation of retinal physiology and photoreceptor viability during aging. In this study, we investigated whether 661W cells-a photoreceptor-like cell that endogenously expresses melatonin receptor type 1 (MT1) and melatonin receptor type 2 (MT2) receptors-represent a useful model for studying the biology of heterodimerization and signaling of MT1/2 receptors. Methods: 661W cells were cultured, and MT1/MT2 heterodimerization in 661W cells was assessed with proximity ligation assay. MT2 was removed from the 661W cells using the MT2-CRISPR/Cas9 system. Melatonin receptor signaling was investigated by measuring cAMP levels and activation of the AKT-FoxO1 pathway. Results: The results demonstrated that heterodimerization of MT1 and MT2 receptors occurs in 661W cells. The pathways activated by MT1/MT2 heterodimer (MT1/2h) in 661W cells are similar to those previously reported in mouse photoreceptors. Disruption of the heterodimer formation by genetically ablating MT2 from 661W cells abolished the activation of melatonin signaling in these cells. Conclusions: The data indicated that in 661W cells, MT1 and MT2 receptors are functional only when they are associated in a heteromeric complex, as occurs in mouse photoreceptors. 661W cells represent a useful model for studying the mechanism underlying MT1/MT2 heterodimerization.