Design and validation of the first cell‐impermeant melatonin receptor agonist

Abstract

The paradigm that GPCRs are able to prolong or initiate cellular signalling through intracellular receptors recently emerged. Melatonin binds to G protein-coupled MT 1 and MT 2 receptors. In contrast to most other hormones targeting GPCRs, melatonin and its synthetic analogues are amphiphilic molecules easily penetrating into cells, but the existence of intracellular receptors is still unclear mainly due to a lack of appropriate tools. EXPERIMENTAL APPROACHWe therefore designed and synthesized a series of hydrophilic melatonin receptor ligands coupled to the Cy3 cyanin fluorophore to reliably monitor its inability to penetrate cells. Two compounds, one lipophilic and one hydrophilic, were then functionally characterized in terms of their affinity for human and murine melatonin receptors expressed in HEK293 cells and their signalling efficacy. KEY RESULTSAmong the different ligands, ICOA-13 showed the desired properties as it was cell-impermeant and bound to human and mouse MT 1 and MT 2 receptors. ICOA-13 showed differential activities on melatonin receptors ranging from partial to full agonistic properties for the G i /cAMP and ERK pathway and β-arrestin 2 recruitment. Notably, ICOA-13 enabled us to discriminate between G i /cAMP signalling of the MT 1 receptor initiated at the cell surface and neuronal mitochondria. CONCLUSIONS AND IMPLICATIONSWe report here the first cell-impermeant melatonin receptor agonist, ICOA-13, which allows us to discriminate between signalling events initiated at the cell surface and intracellular compartments. Detection of mitochondrial MT 1 receptors may have an important impact on the development of novel melatonin receptor ligands relevant for neurodegenerative diseases, such as Huntington disease.

GPCR signalling from within the cell

“…As with nuclear and endosomal GPCRs, there is a growing list of mitochondrial GPCRs. These include the purine, P2Y 1 and P2Y 2 receptors (Belous et al ., ), 5‐HT 4 receptor (Wang et al ., ), angiotensin (Ang II) types 1 and 2 receptors (Abadir et al ., ), melatonin MT 1 receptors (Gbahou et al ., ) and cannabinoid CB 1 receptors (Benard et al ., ; Ma et al ., : Hebert‐Chatelain et al ., ,b, ; Xu et al ., ; Melser et al ., ). To date, activation and/or inhibition of mitochondrial GPCRs appears to affect processes such as mitochondrial Ca 2+ uptake, ATP production, ROS production and even apoptosis.…”

Melatonin receptor structures shed new light on melatonin research

“…The lateral access route of melatonin to its receptor highlights also the likely impact of the lipid environment on melatonin receptor function and ligand properties. The impact might be particularly important in lipid rafts and in mitochondrial membranes, known to have a particular lipid composition and shown to express functional MT 1 receptors , …”

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

“…Immunofluorescence analysis was also performed in HEK293T cells expressing MTRs. Forty‐eight hours post‐transfection, cells were fixed in 2% PFA (15 minutes, −20°C) and permeabilized with Triton X‐100 (0.2%, 1 hour), as described . Tissue slice and fixed cells were blocked with horse serum and incubated with mABs (10 µg/mL, overnight, 4°C), followed by incubation with anti‐TAG antibodies (anti‐FLAG 1:500, Sigma‐Aldrich; anti‐MYC (1:500, Santa Cruz Biotechnology) or anti‐BIP (endoplasmic reticulum marker; 1:3000, Sigma‐Aldrich) antibody and the secondary antibodies Alexa Fluor 555 anti‐mouse and Alexa Fluor 647 anti‐rabbit (1:200, 2 hours, RT).…”

“…Forty-eight hours post-transfection, cells were fixed in 2% PFA (15 minutes, −20°C) and permeabilized with Triton X-100 (0.2%, 1 hour), as described. 34 Tissue slice and fixed cells were blocked with horse serum and incubated with mABs (10 µg/mL, overnight, 4°C), followed by incubation with anti-TAG antibodies (anti-FLAG…”