Melatonin receptors: molecular pharmacology and signalling in the context of system bias

Abstract

Melatonin, N‐acetyl‐5‐methoxytryptamine, an evolutionally old molecule, is produced by the pineal gland in vertebrates, and it binds with high affinity to melatonin receptors, which are members of the GPCR family. Among the multiple effects attributed to melatonin, we will focus here on those that are dependent on the activation of the two mammalian MT₁ and MT₂ melatonin receptors. We briefly summarize the latest developments on synthetic melatonin receptor ligands, including multi‐target‐directed ligands, and the characterization of signalling‐biased ligands. We discuss signalling pathways activated by melatonin receptors that appear to be highly cell‐ and tissue‐dependent, emphasizing the impact of system bias on the functional outcome. Different proteins have been demonstrated to interact with melatonin receptors, and thus, we postulate that part of this system bias has its molecular basis in differences of the expression of receptor‐associated proteins including heterodimerization partners. Finally, bias at the level of the receptor, by the expression of genetic receptor variants, will be discussed to show how a modified receptor function can have an effect on the risk for common diseases like type 2 diabetes in humans.

Linked Articles

This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc

Abbreviations

4P‐PDOT

4‐phenyl‐2‐propionamidotetralin

AD

Alzheimer's disease

ASD

autism spectrum disorder

miRNA

microRNA

MUPP1

multi‐PDZ domain protein 1

PGC‐1α

PPAR‐γ coactivator

RGS

regulator of G‐protein signalling

SCN

suprachiasmatic nucleus

SIRT

sirtuin histone deacetylase

T2D

type 2 diabetes

Introduction

The http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=39 family is one of the GPCR subfamilies (Jockers et al., 2016). In higher vertebrates, the melatonin receptor family is composed of two members, http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=287&familyId=39&familyType=GPCR and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=288&familyId=39&familyType=GPCR (Reppert et al., 1994; 1995a), which have high affinity for the natural ligand http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=224. Human MT₁ and MT₂ receptors are composed of 350 and 362 amino acids and show an overall sequence homology of 55% and 70% in the transmembrane domain (Oishi and Jockers, 2016). Based on the high‐sequence homology (50%) with MT₁ and MT₂, the melatonin‐related receptor, also called http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=107, was classified as another member of the melatonin receptor family (Reppert et al., 1996a). However, GPR50 does not bind to melatonin or any other known ligand and, thus, is still considered an orphan receptor (Reppert et al., 1996a; Ngo et al., 2016). Interestingly, GPR50 has been proposed to be the mammalian orthologue of Mel1c, a high‐affinity melatonin receptor found in non‐mammalian vertebrates (Dufourny et al., 2008). The main focus of this review will be on the function of MT₁ and MT₂ receptors, whereas GPR50 will be considered as a regulatory protein that associates with melatonin receptors.

The natural ligand of MT₁ and MT₂ receptors is melatonin. Melatonin is an evolutionally very old molecule, which is synthesized in many organisms such as bacteria, protists, fungi, macroalgae, plants and animals. In vertebrates, melatonin is the main hormone produced by the pineal gland and follows a circadian pattern, synchronized to the dark phase of the environmental light/dark cycle. In humans, MT₁ and MT₂ receptors are expressed in brain and several peripheral organs (Table 1). Because both receptors are mainly coupled to Gα_i/o proteins, decreased intracellular levels of the second messenger cAMP is the most commonly reported signalling pathway triggered by melatonin. Melatonin receptors regulate circadian rhythms, sleep, seasonal reproduction, immune functions, retinal physiology and glucose homeostasis (Dubocovich et al., 2010; Jockers et al., 2016). The establishment of MT₁ (Liu et al., 1997) and MT₂ knockout mice (Jin et al., 2003) has contributed to reveal these various physiological functions and to attribute them to one or the other receptor type. Recent reviews have focused on the description of these mouse models, the relevance to human disease and physiology, and therapeutic potential (Tosini et al., 2014; Zlotos et al., 2014; Jockers et al., 2016; Liu et al., 2016). In this review, we will focus on novel pharmacological aspects of melatonin receptors, discuss their signalling diversity in the context of system bias and start to unravel the molecular basis for system bias by defining melatonin receptor‐associated protein partners. The last section will put the discovery of multiple rare variants of melatonin receptors and associated modification of receptor function in the context of disease risk.

Table 1.

Distribution of MT₁ and MT₂ melatonin receptors expressed in human tissues

		Tissue	Technique	Reference
hMT1	Brain	Cerebellum	RT‐PCR	Mazzucchelli et al. (1996)
			In situ hybridization	Al‐Ghoul et al. (1998)
		Occipital cortex	RT‐PCR	Mazzucchelli et al., (1996)
		Parietal cortex	RT‐PCR	Mazzucchelli et al. (1996)
		Temporal cortex	RT‐PCR	Mazzucchelli et al. (1996)
		Thalamus	RT‐PCR	Mazzucchelli et al. (1996)
		Frontal cortex	RT‐PCR	Mazzucchelli et al. (1996)
		Hippocampus	RT‐PCR	Mazzucchelli et al. (1996)
			Immunohistochemistry	Savaskan et al. (2001)
		SCN	In situ hybridization	Weaver and Reppert (1996)
	Peripheral tissues	Retina	Immunocytochemistry	Savaskan et al. (2001)
				Savaskan et al. (2002)
				Scher et al. (2002)
				Scher et al. (2003)
		Brown and white adipose tissue	RT‐PCR	Brydon et al. (2001)
		Fetal kidney	RT‐PCR	Drew et al. (1998)
		Coronary artery	RT‐PCR	Ekmekcioglu et al. (2001a,b)
		Granulosa cells	RT‐PCR	Soares et al. (2003)
				Niles et al. (1999)
		Myometrium	RT‐PCR, in‐situ hybridization	Schlabritz‐Loutsevitch et al. (2003)
		Pancreatic alpha and beta cells	RNA sequencing	Blodgett et al. (2015)
		Testis	RT‐PCR	Rossi et al. (2014)
hMT2	Brain	Cerebellum	In situ hybridization	Al‐Ghoul et al. (1998)
		Hippocampus	Immunocytochemistry	Savaskan et al. (2005)
		SCN	Immunocytochemistry	Wu et al. (2013)
	Peripheral tissues	Retina	RT‐PCR	Reppert et al. (1995a)
			Immunohistochemistry	Savaskan et al. (2007)
		Brown and white adipose tissue	RT‐PCR	Brydon et al. (2001)
		Fetal kidney	RT‐PCR	Drew et al. (1998)
		Granulosa cells	RT‐PCR	Soares et al. (2003)
				Niles et al. (1999)
		Placental tissues	RT‐PCR and Western blot	Lanoix et al. (2006)
		Myometrium	In‐situ hybridization	Schlabritz‐Loutsevitch et al. (2003)
			RT‐PCR	Sharkey et al. (2009)
		Pancreatic alpha and beta cells	RNA sequencing	Blodgett et al. (2015)
		Testis	RT‐PCR	Rossi et al. (2014)

Melatonin receptor pharmacology

State of the art on melatonin receptor ligands

The pharmacological characterization of melatonin receptors started in the late 80s, when melatonin binding sites were detected by using the radiolabelled ligand http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1344 (Dubocovich, 1988). The cloning and expression of recombinant melatonin receptors in the 90s allowed further advances in the pharmacological and functional characterization of MT₁ and MT₂ receptors (Reppert et al., 1994, 1995a,b,1996b). In some studies, tritiated melatonin (http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1357) is also used as a radioligand. However, its low specific activity hampered its broader use despite the fact that it is structurally closer to melatonin than 2‐[¹²⁵I]‐iodomelatonin. Of note, a recent study comparing the pharmacology of melatonin receptors in 2‐[¹²⁵I]‐iodomelatonin and [³H]‐melatonin binding experiments revealed that these radioligands do not behave identically. Whereas [³H]‐melatonin detects two melatonin binding sites in both MT₁ and MT₂ receptors, 2‐[¹²⁵I]‐iodomelatonin detects only one binding site (Legros et al., 2014). Using G‐protein uncoupling agents, the authors concluded that the two binding sites detected by [³H]‐melatonin represent two different receptor populations, that is, in their activated and inactivated state, while 2‐[¹²⁵I]‐iodomelatonin binds only to activated receptors. Novel iodinated radioligands like http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7779 and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7771, which are the first MT₂‐selective radioligands, and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7770, an analogue of 2‐[¹²⁵I]‐iodomelatonin, were recently added to the tool box (Legros et al., 2013; 2016). Although these are promising ligands, it must be noted that they did not detect melatonin binding sites in some brain areas known to express melatonin receptors, like the SCN, pointing to the need for further improvements of these tools.

Considerable efforts are currently being made to develop fluorescently labelled melatonin receptor ligands. These are promising compounds for investigating the pharmacology and localization of melatonin receptors; they can replace radioligand binding assays and be used to visualize receptors in imaging assays respectively. The ligands described so far include the 7‐azamelatonin analogue (Wu et al., 2007), coumarin‐based compounds (de la Fuente Revenga et al., 2015) and bodipy‐fused analogues (Thireau et al., 2014; Viault et al., 2016; Gbahou et al., 2017). Some of these compounds bind with good affinity (nM range) and non‐selectively to MT₁ and MT₂ receptors, but data on their full pharmacological and functional properties are only fragmentary.

Competition binding experiments, mainly with 2‐[¹²⁵I]‐iodomelatonin, contributed largely to the identification of melatonin receptor‐selective ligands as recently reviewed (Zlotos et al., 2014; Jockers et al., 2016). The competitive non‐selective antagonist http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1363 is still the most used pharmacological tool for characterizing membrane receptor‐mediated effects, while the MT₂‐selective antagonist 4‐phenyl‐2‐propionamidotetralin (http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1358) is the main pharmacological tool used to discriminate between MT₁‐ and MT₂‐mediated effects (IUPHAR database). Reliable MT₁‐selective ligands are still unavailable. Recently, pharmacoinformatic approaches have revealed an intriguing class of compounds that are able to bind melatonin receptors with μM affinity: carbamate‐derived insecticides (carbaryl and carbofuran) (Popovska‐Gorevski et al., 2017). In fact, previous observations indicated that these compounds affect the function of the pineal gland, resulting in altered levels of nocturnal plasma melatonin (Attia et al., 1991).

Therapeutic applications of melatonin receptor ligands and multi‐target‐directed ligands

The following melatonin receptor ligands are currently available as marketed drugs for the treatment of conditions linked to circadian dysfunction: http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1356 [previously named TAK‐375, commercialized as Rozerem® to treat insomnia (Uchikawa et al., 2002; Erman et al., 2006)], http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=198 [previously named S20098, commercialized as Valdoxan® to treat depression (de Bodinat et al., 2010; Guardiola‐Lemaitre et al., 2014)] and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7393 (VEC‐162) [previously named BMS‐214778, commercialized as Hetlioz® to treat sleep and circadian disturbances (Vachharajani et al., 2003; Rajaratnam et al., 2009)]. A recent review summarized the clinical and preclinical effects of these currently marketed drugs targeting melatonin receptors (Liu et al., 2016).

Several recent studies have proposed treatments based on so‐called multi‐target‐directed ligands (Talevi, 2015). Molecules designed to display at least two complementary functions are attractive as (i) they take into account the fact that diseases are often complex multifactorial conditions and, as such, better results are expected; (ii) they abolish the risk of drug interaction, in which case they can replace the co‐administration of two different drugs; and (iii) they facilitate pharmacokinetic aspects, compared with the co‐administration condition. Melatonin‐derived multi‐target‐directed ligands were developed mainly for the treatment of Alzheimer's disease (AD) in an attempt to combine the beneficial effects of melatonin as a neuroprotective, antioxidant and anti‐amyloidogenic agent with other neuroprotective molecules or with classical anti‐cholinesterase inhibitors currently used as the sole treatment for AD. Examples of these hybrid compounds are tacrine–melatonin (Spuch et al., 2010; Zawadzka et al., 2013); melatonin–N,N‐dibenzyl(N‐methyl)amine hybrid ITH91/IQM157 (Buendia et al., 2015a); (−)‐meptazinol–melatonin hybrids (Cheng et al., 2015); curcumin–melatonin (Chojnacki et al., 2014; Gerenu et al., 2015); melatonin–sulforaphane hybrid ITH12674 (Egea et al., 2015) and donepezil–melatonin hybrids (Wang et al., 2016). In vitro and in vivo characterization studies confirmed that many of these molecules show the expected combined effects, validating the multi‐target‐directed ligand approach. The melatonin agonist piromelatine (N‐(2‐[5‐methoxy‐1H‐indol‐3‐yl]ethyl)‐4‐oxo‐4H‐pyran‐2‐carboxamide, or Neu‐P11) (Tian et al., 2010), tested in phase I clinical trial to treat insomnia and currently under phase II clinical trial to treat AD (Neurim Pharmaceuticals Ltd, 2011a,b, 2017), can also be classified as multi‐target‐directed ligand, as it acts also as an agonist at the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1 and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=3 receptors (He et al., 2013; Liu et al., 2014). Similarly, the anti‐depressive melatonin analogue agomelatine (Valdoxan®) is also a multi‐target ligand, with agonistic properties at MT₁ and MT₂ receptors and antagonistic properties at the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=8 receptor (Millan et al., 2011).

Biased melatonin receptor ligands

Both melatonin receptors are mainly coupled to the Gα_i/o proteins and, thus, melatonin classically signals through dampening of the cAMP/PKA pathway. Additional intracellular cascades that are commonly measured include the melatonin‐induced activation of MEK/ERK kinases and the recruitment of β‐arrestins (Jockers et al., 2016). Studies on biased ligands for melatonin receptors, that is, ligands preferentially modulating one pathway over another as compared with melatonin, are still in their infancy. The first melatonin receptor ligand characterized as a biased ligand was recently described and named ICOA‐9 (Gbahou et al., 2017). ICOA‐9 shows preferential signalling for the G_i/o/cAMP pathway over the β‐arrestin2 and ERK pathways following human MT₁ receptor activation. Furthermore, the clinically active antidepressant agomelatine shows functional properties on MT₂/5‐HT_2C heteromers that are biased towards the G_i/cAMP pathway and, thus, distinct from those of melatonin and 5‐HT_2C‐specific antagonists (see next section for more details). Molecular modelling together with molecular docking studies could provide clues about the structural requirements for biased melatonin receptor ligands, similar to the modelling‐assisted study that helped to design ligands with MT₁ versus MT₂ receptor selectivity and MT₂‐specific antagonistic properties (Pala et al., 2013; Spadoni et al., 2015).

System bias of melatonin receptor pharmacology

The above‐mentioned multi‐target drugs add a new twist to the system bias of melatonin receptor pharmacology, since it implies a context‐dependent efficacy of the ligand depending on the presence or absence of heteromeric receptor complexes. Interestingly, agomelatine not only targets MT₁, MT₂ and 5‐HT_2C receptors separately but also the heteromeric complex comprising MT₂ and 5‐HT_2C receptors (Kamal et al., 2015), which introduces a previously underappreciated system bias. In cells expressing only MT₂ receptors, melatonin has no effect on the inositol phosphate (IP) signalling pathway, while it behaves as an agonist at this pathway in the presence of the 5‐HT_2C receptor. Similarly, agomelatine has no effect on this pathway in cells expressing only MT₂ receptors, but it antagonizes melatonin‐induced IP production in cells co‐expressing MT₂ and 5‐HT_2C receptors (Kamal et al., 2015). In addition, whereas melatonin shows agonistic properties on the cAMP and IP pathway, agomelatine is an agonist of the cAMP pathway but a neutral antagonist of the IP pathway compared with melatonin in cells expressing the MT₂/5‐HT_2C heteromer. Conversely, luzindole and 4P‐PDOT, which are competitive antagonists of MT₁ and MT₂ receptors in the absence of the 5‐HT_2C receptor, behave as agonists of the cAMP pathway and full or partial agonists, respectively, of the IP pathway in cells expressing the MT₂/5‐HT_2C heteromer.

Evidence for the existence of system bias in melatonin receptor pharmacology also comes from studies showing that ligand efficacy depends on the cell context, on the receptor expression levels in a given tissue and on their active or inactive state (Legros et al., 2014). For example, luzindole and 4P‐PDOT (at high concentrations) are usually competitive melatonin receptor antagonists, but behave as inverse agonists at MT₁ receptors in the presence of constitutively activated receptors, as shown in rat artery cells expressing endogenous MT₁ receptors (Ersahin et al., 2002). Constitutive activation was also observed for MT₁ and MT₂ receptors in transfected CHO and Neuro2A cell lines, and MT₂‐specific inverse agonists (UCM 549 and UCM 724) effectively decreased the constitutive activity of MT₂ receptors (Devavry et al., 2012). The pharmacological properties of the competitive melatonin receptor antagonist 4P‐PDOT seem to be even more complex, as not only antagonistic and inverse agonistic effects have been described, as mentioned before, but also partial agonistic activity is also reported for native and recombinant MT₂ receptors (Nonno et al., 1999; Ayoub et al., 2002; Dubocovich et al., 2003). The impact of the cell context on the pharmacological properties of melatonin receptors is further confirmed by the study of Logez et al. (2014). They observed a marked decrease in ligand binding affinities of recombinant human MT₁ receptors when expressed in the eukaryotic microorganism Pichia pastoris compared with MT₁ receptors expressed in CHO cells. Intriguingly, after purifying MT₁ receptors from P. pastoris, the pharmacological profile of the receptor resembled that observed in CHO membranes (Logez et al., 2014). The authors suspect that differences in the membrane lipid composition, most likely the cholesterol content, between these two cell types are at the origin of the differences observed in the pharmacological profiles.

Collectively, cell context‐dependent receptor expression, receptor heterodimerization and context‐ and ligand‐dependent bias are among the main factors underlying system bias and are proven to be relevant to the elucidation of the pharmacology of melatonin receptors.

Melatonin receptor signalling

Common aspects of melatonin receptor signalling

As yet, the intrinsic affinity of melatonin receptors for different G proteins has not been systematically determined. The G‐protein coupling profile also depends on the relative expression levels of the different G proteins in a given cellular context, which accounts for the system bias of melatonin receptor pharmacology. In most experimental systems, both MT₁ and MT₂ receptors are mainly coupled to G_i proteins, thus leading to inhibition of AC activity (Figure 1). A more detailed analysis in HEK293 cells shows that MT₁ receptors co‐immunoprecipitate preferentially with Gα_i2 and Gα_i3 proteins and to a lesser extent to G_q/11, while no coupling to Gα_i1, Gα_z, Gα_o, Gα₁₂ or Gα_s was detected (Brydon et al., 1999b). An illustrative example of system bias is the Gα₁₆ protein, which is exclusively expressed in haematopoietic cells (Amatruda et al., 1991). Co‐transfection of MT₁ or MT₂ receptors together with Gα₁₆ in COS‐7 cells leads to the potentiation of melatonin signalling through the JNK pathway, indicating coupling of both melatonin receptors to Gα₁₆ (Chan et al., 2002). In addition to the frequently observed modulation of cAMP levels by melatonin receptors, modulation of diacylglycerol, inositol trisphosphate and Ca²⁺ levels have been observed in a cell context‐dependent manner (Brydon et al., 1999a,b). Tissues and cells in which G_q/11 coupling to endogenously expressed melatonin receptors have been observed include the myometrium (Steffens et al., 2003), prostate epithelial cells (Shiu et al., 2010), pancreatic cells (Bähr et al., 2012) and human mesenchymal stem cells (Lee et al., 2014), in addition to non‐mammalian cells (Hotta et al., 2000) and cells expressing recombinant receptors (MacKenzie et al., 2002).

Figure 1.

Melatonin receptor signalling pathways. Melatonin activation of MT₁ receptors triggers Gα_i activation, decreasing the levels of the secondary messanger cAMP, and Gβγ‐dependent activation of PI3K/Akt, PKC and ERK pathways. MT₁ coupling to Gq leads to PLC activation and increase in intracellular Ca²⁺. Melatonin‐induced modulation of neuronal action potential is mediated by MT₁‐dependent activation of the potassium and calcium ion channels (K_ir3 and Ca_v2.2). The physical interaction of MT₁ receptors with Ca_v2.2 channels tonically inhibits Ca_v2.2‐mediated calcium entry through Gβγ subunits. Melatonin activation of MT₂ receptors triggers Gα_i‐dependent cAMP and ERK signalling pathways and inhibits cGMP levels. Melatonin induced β‐arrestin recruitment to both MT₁ and MT₂ receptors, but β‐arrestin‐dependent down‐streaming signalling is not yet reported. See text for details. β‐ARR, β‐arrestin; Ca_v2.2, voltage‐gated calcium channel; ccgs, clock‐controlled genes; CREB, cAMP‐responsive element binding; K_ir3, G protein‐coupled inwardly rectifying potassium channel; sGC, soluble GC.

Melatonin can also regulate ion channels, and multiple pathways seem to be involved. Melatonin modulates muscle contractile responses in arteries (Geary et al., 1998; Masana et al., 2002) and in the myometrium (Steffens et al., 2003) by regulating the activity of large‐conductance Ca²⁺‐activated K⁺ (http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=380, also known as K_Ca1.1) channels. In the myometrium, the modulation of BK_Ca channels by melatonin was shown to be dependent on the activation of both G_q/PLC/Ca²⁺ and G_i/cAMP/PKA pathways. Interestingly, the final outcome of the effect of melatonin on BK_Ca activity depends on the physiological context, as opposite effects are observed in myocytes obtained from pregnant and non‐pregnant rats (Steffens et al., 2003). At the transcriptional level, melatonin signalling typically inhibits the transcriptional factor cAMP‐responsive element binding (CREB) and activates the transcription of genes under the control of the ERK pathway. Up to now, the major difference between MT₁ and MT₂ receptors at the signalling level concerns their ability to inhibit cGMP production, which is only observed in MT₂‐transfected cells (Petit et al., 1999). This effect was confirmed in human non‐pigmented ciliary epithelial cells expressing endogenous MT₂ receptors (Dortch‐Carnes and Tosini, 2013). The general signalling pathways triggered by melatonin receptors are depicted in Figure 1, but the impact of system bias should be kept in mind. We next present further melatonin functions for which at least some of the signalling intermediates have been described, thus expanding the molecular components of melatonin receptor signalling.

Diversity of melatonin's effects and signalling cascades – further evidence of system bias

The regulation of circadian rhythms by melatonin has been extensively studied (Dubocovich et al., 2010). Melatonin has been shown to affect the firing rate of hypothalamic suprachiasmatic nucleus (SCN) neurons, which constitutes the master clock in mammals. This effect is mediated by both receptors in a G_i‐dependent, but cAMP‐independent manner. For MT₁ receptors it involves the activation of http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=74, like K_ir3 (Nelson et al., 1996; van den Top et al., 2001; Hablitz et al., 2015), while the MT₂‐induced phase advance in neuronal activity involves the PKC signalling pathway (McArthur et al., 1997; Hunt et al., 2001). In the presence of pituitary AC activating peptide (PACAP)‐induced cAMP production, both receptors can modulate neuronal activity by the G_i/cAMP pathway (Jin et al., 2003). Melatonin regulation of clock gene expression has been reported in the SCN and both MT₁ and MT₂ receptors are involved in this effect (Pfeffer et al., 2012; Nagy et al., 2015; Kandalepas et al., 2016). In the striatum, melatonin has been reported to modulate clock gene expression through MT₁ receptors in a G_i‐dependent manner (Imbesi et al., 2009). In cerebellar Purkinje cells, the neuronal firing rate is modulated by MT₁ receptors through inhibition of http://www.guidetopharmacology.org/GRAC/FamilyIntroductionForward?familyId=80 (Ca_v2.1) in a G_i/Gβγ/PI3K/PKCδ signalling‐dependent manner (Zhang et al., 2015). Together with the SCN, the hypophyseal pars tuberalis is the main target of melatonin involved in its synchronizing effects. Melatonin transduces photic information through MT₁ receptors by regulating the expression of mPer1, mCry1, Clock and Bmal1 genes through a heterologous repressive/sensitization of the cAMP pathway that requires not only MT₁ receptors but also the adenosine A_2B receptor (von Ball et al., 2002; Dardente et al., 2003; von Gall et al., 2005; Wood and Loudon, 2014). The regulator of G‐protein signalling (RGS)4 (Dupre et al., 2011) and the basic helix‐loop‐helix Per‐Arnt‐Sim domain transcription factor NPAS4 are also suggested to participate in this signalling cascade (West et al., 2013). Although the circadian machinery is present in every cell, the effect of melatonin on rhythmic clock gene expression in other tissues is not clear and seems to be cell type‐dependent (Muhlbauer et al., 2009; Owino et al., 2016). The precise mechanism underlying the effect of melatonin on clock genes is not well defined and might involve transcriptional and post‐translational modulation of clock proteins (reviewed by Vriend and Reiter, 2015).

Many studies have demonstrated that melatonin plays an important role in regulating different aspects of retinal physiology. The clock machinery of the retina is responsive to melatonin, and both receptors are involved, but the precise signalling pathway has not, as yet, been elucidated. In MT₁ receptor knockout mice, the rhythmic expression of Per1 was not abolished, but the phase was significantly affected (Dinet et al., 2007). Significant changes in the pattern of expression of other clock genes, as well as clock‐controlled genes (ccgs, like cfos) were also observed in melatonin receptor knockout mice (Hiragaki et al., 2014; Kunst et al., 2015). Melatonin controls the retinal light sensitivity at night (Baba et al., 2013), an effect that was shown to depend on MT₁/MT₂ heteromers (further discussed in the Melatonin receptor oligomers section), which preferentially activate the G_q/PLC/Ca²⁺ pathway (Baba et al., 2013), while regulation of photoreceptor viability is believed to depend on the survival‐related Akt/FOXO1 signalling pathway (Gianesini et al., 2016).

In addition to retinal cells, melatonin also modulates the viability of neurons under physiological and pathological conditions. The neuroprotective and anti‐apoptotic properties of endogenous and exogenous melatonin have been extensively investigated, and different signalling pathways underlie these effects. In neural stem cells, the effect of melatonin on cell survival, maturation and differentiation is melatonin receptor‐dependent, as it is prevented by the competitive melatonin receptor antagonist luzindole (Ramirez‐Rodriguez et al., 2009; Tocharus et al., 2014; Chu et al., 2016; Ortiz‐Lopez et al., 2016). In pluripotent stem cells melatonin‐induced neural differentiation involves the PI3K/Akt pathway and is also blocked by luzindole (Shu et al., 2016). However, prolonged exposure of embryonic stem cells to melatonin favours the pluripotency state of the cells in a MT₁‐dependent manner that involves a synergism between the effect of the PI3K/Akt and ERK pathways that results in the up‐regulation of the glucose transporter GLUT1 (Wu et al., 2017). In an in vivo rat model of neuro‐inflammation, endogenous melatonin protects cerebellar neurons from LPS toxicity, while neuronal death is observed in the presence of the competitive melatonin receptor antagonist luzindole (Pinato et al., 2015). Similarly, Wang et al. (2011) observed that neurons were more vulnerable to cell death in the presence of luzindole and in MT₁‐silenced cells. In ischaemia/reperfusion models, the protective effect of melatonin relies on its anti‐apoptotic and antioxidant effects, as it is known to up‐regulate several antioxidant enzymes, including SOD1 and glutathione peroxidase (Parada et al., 2014; O'Neal‐Moffitt et al., 2015; Ramos et al., 2017). In cerebral ischaemia, the protective effect of melatonin and agomelatine was linked to activation of the nuclear factor erythroid‐related factor 2 (Nrf2), which regulates the expression of antioxidant enzymes (Ding et al., 2014; Chumboatong et al., 2017). In in vitro and in vivo ischaemic models, the multi‐target‐directed 5‐HT and melatonin receptor Neu‐P11 ligand promoted neuronal survival through the activation of PI3K/Akt, ERK and JAK2 pathways (Buendia et al., 2015b) (Figure 2).

Figure 2.

Extended, context‐specific melatonin receptor signalling pathways. Depending on the cell type or the presence of cell stressors, melatonin can activate additional melatonin receptor‐dependent signalling cascades. These pathways have been reported mainly for MT₁ receptors, but the participation of MT₂ receptors cannot be excluded. Melatonin modulation of mitochondrial function is reported under oxidative stress condition and in neurodegenerative diseases. Proposed signalling pathways involve the regulation of the activity and/or translocation of Bcl2/Bax and SIRT proteins. Activation of JAK2, ERK and the Akt/FOXO1 complex are suggested to mediate melatonin‐induced cell survival and to modulate pluripotency/differentiation of stem cells, while melatonin‐induced inhibition of these pathways is reported in cancer cells. MT₁‐dependent activation of SIRT1 might underlie melatonin anti‐inflammatory and anti‐oxidative effects through regulation of transcription factors like Nrf2, PGC1α and NF‐κB. MT₁‐coupling to G₁₆ protein occurs in haematopoietic cells and triggers the JNK pathway. In several cell types, including cancer cells, melatonin is reported to modulate the expression of different miRNAs. See text for details. SIRT, sirtuin.

A number of observations suggest that the antioxidant and anti‐apoptotic effects of melatonin depend largely on mitochondrial function and dynamics (Tan et al., 2016). Melatonin has been reported to modulate the expression levels and localization of Bax and Bcl‐2 proteins and to inhibit the release of cytochrome c and the activation of caspase‐3 (Radogna et al., 2008; Wang et al., 2009; Luchetti et al., 2010). The JAK2/STAT3 pathway is suspected to mediate melatonin‐induced Bax/Bcl‐2 translocation in cardiomyocytes (Yang et al., 2013), while ERK activation and p38 MAPK inhibition were proposed to mediate the anti‐apoptotic effect of melatonin in monocytes (Luchetti et al., 2009). Additional mitochondria‐associated signalling cascades activated by melatonin include activation of http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=848 (SIRTs) (reviewed by Mayo et al., 2017) through AMPK/SIRT3/SOD2 and SIRT1/PPAR‐γ coactivator (PGC‐1α), as shown in hepatocytes (Guo et al., 2014; Chen et al., 2015; Pi et al., 2015). Of note, the transcription factor PGC‐1α is also regulated by MT₁ receptors in the retina (Kunst et al., 2015). Melatonin signalling through SIRT might also underlie the well‐known anti‐inflammatory action of melatonin through inhibition of the inflammatory transcription factor NF‐κB (Tajes et al., 2009; Zhao et al., 2017). In mouse models of neurodegenerative diseases, such as AD, amyotrophic lateral sclerosis and Huntington's disease, the neuroprotective effect of melatonin was linked to MT₁‐dependent modulation of mitochondrial function (Dragicevic et al., 2011; Wang et al., 2011; Zhang et al., 2013). Interestingly, the inhibitory effect of melatonin on cytochrome c release could be reproduced in purified brain mitochondria, presumably through mitochondrial MT₁ receptors, as suggested by the authors (Wang et al., 2011). Recently, further evidence for the intriguing mitochondrial localization of MT₁ receptors was obtained by using the first cell‐impermeable melatonin receptor agonist, which allowed us to discriminate between MT₁‐triggered G_i/cAMP signalling at the cell surface and in mitochondria (Gbahou et al., 2017). Whether the neuroprotective effect of melatonin linked to mitochondrial function is due to mitochondrial MT₁ signalling is currently under investigation.

In addition to the above‐mentioned examples, the impact of system bias on melatonin receptor signalling can also be nicely exemplified in the cancer field, where the repertoire of signalling pathways modulated by melatonin is highly context‐specific. In general, melatonin is reported to display anti‐tumoural properties, inhibiting proliferation and inducing apoptosis. In breast cancer models, the anti‐tumoural effect of melatonin appears to involve mainly MT₁ receptors through inhibition of the phosphorylation of signalling molecules such as AKT, ERK and PKC (Hill et al., 2011; 2015). In these cells, melatonin has also been reported to activate the p53 DNA‐protective pathway in a receptor‐dependent manner (Santoro et al., 2013), while in ovarian cancer, melatonin inhibits Akt, p38 MAPK and mTOR signalling (Ferreira et al., 2014).

New players contributing to system bias of melatonin receptor function

MicroRNAs

Additional complexity to the study of melatonin receptors signalling emerges when considering the growing number of reports pointing to the role of microRNAs (miRNA) in mediating the effects of melatonin. In prostate cancer cells, the anti‐angiogenic effect of melatonin was linked to an up‐regulation of miRNA3195 and miRNA374b (Sohn et al., 2015). MiR‐24 is a miRNA often up‐regulated in several types of cancer, and melatonin was able to down‐regulate it in a luzindole‐sensitive manner (Mori et al., 2016). Regulation of miRNAs by melatonin has been also demonstrated in hepatocytes (Kim et al., 2015; 2017) and neurons (Carloni et al., 2016). Interestingly, miRNAs can also regulate the expression of MT₁ receptors (Zhu et al., 2014). It has recently been reported that the expression of miRNAs can vary in a daily pattern (Marcola et al., 2016), which introduces an additional bias regarding the time when data are collected. Because miRNAs are highly context‐dependent, it is likely that they greatly contribute to the system bias on melatonin receptors signalling. Altogether, an extended melatonin receptor signalling network is emerging from these studies in the light of the system bias concept (Figure 2).

Melatonin receptor oligomers

The work from Ayoub et al. (2002) was the first to propose that, similar to other GPCRs, melatonin receptors could exist as homomers and/or heteromers. By BRET experiments using transfected HEK293 cells, we demonstrated that melatonin receptors form homomers and heteromers in living cells, with prevalence for the formation of the heteromers (Ayoub et al., 2002; 2004). Melatonin activation did not have any apparent effect on the oligomerization state of the receptors. Importantly, the heteromer showed a distinct pharmacological profile in BRET experiments compared with MT₂ homomers, with the competitive antagonist luzindole being 100 times more potent on heteromers (Ayoub et al., 2004). Both binding sites seem to be preserved in the heteromer when inspected individually and are not subject to negative cooperativity (Ayoub et al., 2004). Although many GPCRs have a natural tendency to oligomerize (Ferre et al., 2014), such oligomers appear to be less abundant in tissues at natural expression levels, and their physiological relevance has been proven only in some cases. MT₁/MT₂ heteromers were detected in retinal photoreceptor cells and were shown to be key players in improving retinal light sensitivity at night (Baba et al., 2013). The in vitro characterization of the heteromer revealed that the PKC signalling pathway is potentiated by melatonin under this condition and, indeed, the in vivo effect is also PKC‐dependent (Baba et al., 2013). Inasmuch as many tissues express both melatonin receptors, including the SCN, the involvement of MT₁/MT₂ heteromers in mediating melatonin's effects deserves further investigation. Interestingly, different melatonin signalling patterns are detected in cerebellar granular cells expressing endogenous MT₁ and MT₂ receptors. In this cellular context, melatonin at a low nM concentration inhibits rather than stimulates ERK and Akt pathways, while a stimulatory response is detected if either receptor is silenced. Conversely, melatonin decreases forskolin‐simulated cAMP production only in cells expressing both MT₁ and MT₂ receptors, while no effect is detected in cerebellar granular cells expressing only one type of melatonin receptor (Imbesi et al., 2008). It is likely that the responsiveness of these cells to melatonin relies on the MT₁/MT₂ heteromer.

The orphan receptor of the melatonin receptor family, GPR50, has also been demonstrated to engage in oligomeric complexes with MT₁ and MT₂ receptors (Levoye et al., 2006). Contrary to what was observed for MT₁/MT₂ heteromers, in this case, the dimer formation markedly altered ligand binding and signalling properties of MT₁, but not of MT₂ receptors, as melatonin binding and G_i protein and ß‐arrestin coupling of MT₁ receptors are lost in the MT₁/GPR50 heterodimer. The negative modulation of MT₁ receptors by GPR50 was confirmed in hCMEC/D3 cells expressing both receptors endogenously, as melatonin signalling was observed only after GPR50 silencing (Levoye et al., 2006). The physiological relevance of the MT₁/GPR50 heterodimer, as well as the regulatory factors of their association and dissociation in vivo, remain to be elucidated. Nevertheless, the fact that GPR50 is expressed in the pituitary gland, in several hypothalamic nuclei and in the median eminence, which are main loci of melatonin's action, as well as in other central areas (Batailler et al., 2012), implies that a GPR50‐dependent regulation of melatonin receptors signalling might be physiologically relevant.

As mentioned before, the pharmacological properties of agomelatine lead to the investigation of the existence of heteromers comprising melatonin and 5‐HT receptors. In transfected HEK293 cells, it was observed that both MT₁ and MT₂ receptors are able to associate with the 5‐HT_2c receptor in a heteromeric complex (Kamal et al., 2015). The pharmacology of MT₂ receptors seems to be altered in the heteromer, as melatonin activates not only G_i‐dependent signalling but also G_q/PLC signalling, which is not observed in cells expressing the MT₂ receptor alone (Kamal et al., 2015). We proposed a transactivation model in which the melatonin‐activated MT₂ receptor is able to allosterically transactivate 5‐HT_2c‐dependent G_q signalling. These heteromers were also targeted by agomelatine suggesting that MT₂/5‐HT_2c heteromers might participate in the antidepressant effect of this drug. The biased pharmacology of melatonin receptor dimers is shown in Figure 3.

Figure 3.

Signalling of melatonin receptor homomers and heteromers. Melatonin activation of MT₁/MT₁ homomers triggers intracellular signalling predominantly through the G_i pathway over the G_q pathway, while MT₂/MT₂ homomers signal exclusively through G_i proteins. In the MT₁/MT₂ heteromer, melatonin signalling is biased towards G_q activation over G_i. When dimerizing with GPR50, the MT₁ receptor loses its ability to bind melatonin, to trigger G_i signalling and to recruit β‐arrestin (β‐ARR). Melatonin activation of the MT₂/5‐HT_2c heteromer triggers 5‐HT_2C receptor dependent G_q signalling through a MT₂ receptor transactivation mechanism. See text for details. MLT, melatonin.

Melatonin receptor‐associated protein complexes

Formation of receptor heteromers is only one of the possible ways that GPCRs have to shape their cellular micro‐environment to ultimately determine the signalling outcome. Other proteins that might be constitutive or agonist‐induced components of these receptor‐associated complexes have also been identified for melatonin receptors. By combining different proteomic and genomic approaches and different biological resources expressing endogenous melatonin receptors, an interactome of MT₁ and MT₂ receptors composed of 366 individual proteins was built (Figure 4) (Daulat et al., 2007; Benleulmi‐Chaachoua et al., 2016). This represents one of the most complete and diverse GPCR interactomes currently available. Out of the 366 interactors, only 52 were identical between the two receptors. Many of these common interactors belong to the family of small G proteins (Rab and Rho GTPases), heterotrimeric G proteins, molecular chaperones (calnexin and calreticulin), cytosqueleton components (filamin, myosin, etc.) and ubiquitin ligases. This suggests common functions and highlights the previously underappreciated link between melatonin receptors and small G proteins and cytosqueleton organization that warrants further attention.

Figure 4.

Melatonin receptors interactome. MTR1A (MT₁)‐ and MTR1B (MT₂)‐interacting proteins were identified in 20 different screens and are clustered based on the different identification methods: dark blue for the MYTH, blue for Cter peptide purification, green for the Y2H and brown for the TAP methods. Thick lines correspond to confirmed protein–protein interactions and node colours refer to predicted gene ontology biological function. See text for details. CTER, carboxyl terminus peptide affinity chromatography; MYTH, membrane yeast two‐hybrid; TAP, tandem affinity purification; Y2H, yeast two‐hybrid. Modified from Benleulmi‐Chaachoua et al. (2016), with permission.

MT₁ and MT₂ receptors are known to undergo agonist‐dependent and ‐independent internalization (Gerdin et al., 2003; Guillaume et al., 2008). The melatonin receptor interactome contains trafficking proteins such as caveolin, dynamin 1, AP2 and AP4 adaptor proteins that act in concert with GPCRs kinases and ß‐arrestins to promote receptor internalization (Levoye et al., 2006; Bondi et al., 2008; Maurice et al., 2008).

The interactome also revealed that most of the interactions are unique for each receptor (168 and 143 for MT₁ and MT₂ respectively). This result was unexpected given the fact that previous studies revealed only few functional differences between MT₁ and MT₂ receptors. These novel results will be a rich source of investigation for the next few years to unravel and understand the functional differences between both receptors. Among the possible new functions are links with several ion transporters/channels like the electroneutral potassium‐chloride co‐transporter 1, the zinc transporters and the electroneutral Na/HCO₃ co‐transporter in the MT₂ interactome. Another remarkable finding was the exclusive presence of several synaptic proteins in the MT₁ interactome such as synapsin, SNAP25 and 47, the voltage‐gated calcium channel Ca_v2.2, Munc‐18, rabphilin and snapin. The absence of these proteins from the MT₂ interactome suggested differences in the subcellular localization of MT₁ and MT₂ receptors in neurons, a notion that was confirmed in primary hippocampal neurons. The different localization of melatonin receptors in neurons is likely to increase our understanding of the different roles of both receptors in the brain and, at the same, adds a not yet appreciated spatial bias to melatonin receptor function. The interactome also provides some clues as to how the different localization of both receptors is achieved as the MT₁, but not the MT₂ receptor, interacts with kinesin‐1, which has been shown to transport the Na⁺ channel to axons (Barry et al., 2014). The interaction of MT₁ receptors with the Ca_v2.2 channel was shown to be of functional importance as in vitro experiments in CHO cells showed that the presence of MT₁ receptors tonically inhibits the Ca_v2.2‐promoted calcium current through Gβγ subunits (Benleulmi‐Chaachoua et al., 2016). Indeed, voltage‐dependent inhibition involving direct binding of Gβγ subunit to the channel (Zamponi and Currie, 2013) is the most widespread mechanism by which GPCRs regulate voltage‐gated calcium channels. Localization of MT₁ receptors in the presynaptic membrane suggests its involvement in synaptic functions such as neurotransmission. This conclusion corroborates earlier reports suggesting a possible influence of melatonin on neurotransmitter release and uptake in some brain regions, like the ventral hippocampus, medulla pons, preoptic area and median and posterior hypothalamus but not in others (Cardinali et al., 1975; Zisapel and Laudon, 1982). This new input from the interactome analysis will renew the interest in the possible role of MT₁ receptors in neurotransmitter regulation.

Among the best‐characterized interactors of the MT₁ receptor is RGS20 (Maurice et al., 2010) and the multi‐PDZ domain protein, MUPP1 (Guillaume et al., 2008). Whereas RGS20 regulates the speed of G_i‐protein signalling of MT₁ receptors by accelerating the activation kinetics of K_ir3 channels, the expression of MUPP1 is obligatory for MT₁ receptors to inhibit cAMP production by a yet poorly defined mechanism. Altogether, these data demonstrate the tremendous influence of melatonin receptor‐associated proteins on their function and underline the necessity to define the interactome of melatonin receptors in a given cell type to fully understand the functional outcome of melatonin stimulation in a given tissue.

Melatonin receptor variants

Receptor variants introduce a bias at the level of the receptor that contributes to inter‐individual differences in receptor function, which are suspected to contribute to the risk of common diseases. Genetic variation may also have important consequences on drug action. This variation may occur at the level of different ethnic groups (typically for frequent variants) or at the level of individuals (typically for rare or very rare variants). Consequently, information of the existence of receptor variants within cohorts for clinical trials helps to stratify and homogenize the cohorts to decrease their genetic variability and to help improve the outcome for clinical trials.

Recent large‐scale sequencing studies revealed considerable inter‐individual genetic variability in GPCR coding genes. In humans, an average of 32 non‐synonymous variants has been estimated to exist per GPCR in a sample of 10 000 individuals (Nelson et al., 2012; Karamitri and Jockers, 2014). In the case of melatonin receptors, 8 and 42 non‐synonymous variants have been demonstrated to occur in the MTNR1A and MTNR1B genes coding for the MT₁ and MT₂ receptor respectively (Jockers et al., 2016). These numbers are likely to increase with the increased number of sequenced human genomes and targeted exon sequencing. Based on reports indicating that an alteration in melatonin synthesis is associated with autism spectrum disorders (ASDs), non‐synonymous variants have been identified in the MTNR1A and MTNR1B genes in 300 ASD patients and a matched control population. No significant difference in the prevalence of these variants was found indicating that they do not contribute to ASD risk (Chaste et al., 2010). A similar conclusion was reached after sequencing of MTNR1A and MTNR1B in individuals with attention deficit hyperactivity disorder (ADHD; Chaste et al., 2011). From a pharmacological point of view, it is important to note that the majority of the 16 non‐synonymous variants identified in these studies showed altered receptor function with some of them showing a biased signalling profile compared with the wild‐type receptor (Chaste et al., 2010). This was the first report to show that variability of melatonin action does exist at the receptor level.

Inspired by genome‐wide association studies revealing a robust association of the minor allele of the common rs10830963 variant located in the intron of MTNR1B with increased type 2 diabetes (T2D) risk (Bouatia‐Naji et al., 2009; Lyssenko et al., 2009), the two exons of the MTNR1B gene were sequenced in 7632 individuals including 2186 individuals with T2D (Bonnefond et al., 2012). Forty non‐synonymous MT₂ variants were identified including 38 rare and the two common variants. Those variants with a loss‐of‐function phenotype, but not the functionally neutral ones, were associated with increased T2D risk, establishing for the first time a link between melatonin receptor function and the risk for a common disease such as T2D. Subsequent studies that attempted to understand the functional basis of the association of the common rs10830963 variant with T2D risk indicated that risk allele carriers express two to four times more MTNR1B mRNA in their human pancreatic islets (van de Bunt et al., 2015). Taken together with other experimental findings, a model was proposed based on the assumption that MT₂ protein levels are increased, exaggerating the putative inhibitory effect of melatonin on pancreatic insulin production in risk allele carriers (Tuomi et al., 2016). Obviously, this hypothesis contrasts with the conclusion drawn from rare loss‐of‐function variants, namely, that defective melatonin receptor function is associated with T2D risk. This apparent controversy is discussed in several recent commentaries (Bonnefond et al., 2016; Mulder, 2017). Drawing the right conclusions will be of relevance for human health, as it will be important to know whether melatonin supplementation (as practised by millions of people in the world) is beneficial or detrimental in terms of glucose homeostasis and T2D risk. From a pharmacological point of view, functional evidence for the expression of MT₂ receptors in human pancreatic beta cells is weak. The MT₂ receptor has not been detected at the protein level, and mRNA expression is only detectable in less than 5% of the cells at very low (close to background) levels (Segerstolpe et al., 2016; Thomsen et al., 2016). Evidence for the inhibitory effect of melatonin on cAMP levels and insulin production is substantial in rodents at the cellular and animal level (Peschke et al., 2007); however, conflicting results are reported in human cells with some studies even showing a stimulation/potentiation of insulin production by melatonin (Ramracheya et al., 2008; Costes et al., 2015). It is important to note that the physiological effect of melatonin on metabolism is expected to be fundamentally different as diurnal humans are day active, whereas nocturnal rodents are night active, even though melatonin is always secreted during the night. In the light of this inconclusive evidence, further hypotheses have to be explored to understand the effect of melatonin on glucose homeostasis in humans. This includes the search for further functions of melatonin in pancreatic beta cells, such as the recently reported stimulatory role of melatonin on human beta‐cell survival (Costes et al., 2015). Whether these effects are indeed mediated through melatonin receptors or through novel pharmacological units, such as heteromeric complexes, has to be investigated. Melatonin target tissues, other than pancreatic beta cells, like the brain or the liver and adipose tissues will have to be considered. Furthermore, focusing on the inhibitory effect of melatonin receptors on the cAMP pathway might also be too restrictive and other G protein‐ and β‐arrestin‐dependent signalling events might be more relevant.

In conclusion, the currently available data show that melatonin receptor variants exist in the human population and that they are of relevance for major common diseases. However, we are only at the beginning of our understanding of the full impact of such variants on human health. An expansion of future studies towards the MTNR1A gene and other diseases like sleep and circadian rhythm disorders represents an interesting field of future research.

Conclusion and perspectives

Multiple functions have been attributed to melatonin receptors that are transmitted by the activation of a large diversity of signalling pathways. Current knowledge clearly indicates that the signalling profile of melatonin receptors is highly cell‐ and tissue‐dependent, arguing for the existence of system bias as an important determinant of the functional outcome of melatonin receptor signalling. This highly complex arrangement makes it difficult to transpose functional properties described in one cell context into another. It also implies that the exogenous expression of recombinant receptors might be only of limited predictive value for the signalling properties of endogenous melatonin receptors in a given tissue. Interesting areas of future research are the detailed investigation of the intriguing localization of melatonin receptors in intracellular compartments such as mitochondria, the widespread formation of melatonin receptor heteromers and the development of novel generations of multi‐target‐directed ligands. New radioactive and fluorescently labelled tracer molecules are likely to detect further activation states of melatonin receptors that will be highly informative in defining new melatonin receptor complexes. Finally, the generation of biased ligands for melatonin receptors is still in its infancy but warrants further attention given the huge expectation of these compounds for therapeutic application in terms of signalling specificity and reduced side effects.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015a,b,c).

Conflict of interest

The authors declare no conflicts of interest.

Cecon, E. , Oishi, A. , and Jockers, R. (2018) Melatonin receptors: molecular pharmacology and signalling in the context of system bias. British Journal of Pharmacology, 175: 3263–3280. 10.1111/bph.13950.