Functional MT1 and MT2 melatonin receptors in mammals

Melatonin, dubbed the hormone of darkness, is known to regulate a wide variety of physiological processes in mammals. This review describes well-defined functional responses mediated through activation of high-affinity MT₁ and MT₂ proteinteoupled receptors viewed as potential targets for drug discovery. MT₁ melatonin receptors modulate neuronal firing, arterial vasoconstriction, cell proliferation in cancer cells, and reproductive and metabolic functions. Ativation of MT₂ melatonin receptors phase shift circadian rhythms of neuronal firing in the suprachiasmatic nucleus, inhibit dopamine release in retina, induce vasodilation and inhibition of leukocyte rolling in arterial beds, and enhance immune responses. The melatonin-mediated responses elicited by activation of MT₁ and MT₂ native melatonin receptors are dependent on circadian time, duration and mode of exposure to endogenous or exogenous melatonin, and functional receptor sensitivity. Together, these studies underscore the importance of carefully linking each melatonin receptor type to specific functional responses in target tissues to facilitate the design and development of novel therapeutic agent.     

C-terminal domains within human MT1 and MT2 melatonin receptors are involved in internalization processes

Abstract:  Melatonin, a molecule implicated in a variety of diseases, including cancer, often exerts its effects through G-protein-coupled melatonin receptors, MT₁ and MT₂. In this study, we sought to understand further the domains involved in the function and desensitization patterns of these receptors through site-directed mutagenesis. Two mutations were constructed in the cytoplasmic C-terminal tail of each receptor subtype: (i) a cysteine residue in the C-terminal tail was mutated to alanine, thus removing a putative palmitoylation site, and a site possibly required for normal receptor function (MT₁C7.72A and MT₂C7.77A) and (ii) the C-terminal tail in the MT₁ and MT₂ receptors was truncated, removing the putative phosphorylation and β-arrestin binding sites (MT₁Y7.64 and MT₂Y7.64). These mutations did not alter the affinity of 2-[¹²⁵I]-iodomelatonin binding to the MT₁ or MT₂ receptors. Using confocal microscopy, it was determined that the putative palmitoylation site (cysteine residue) did not play a role in receptor internalization; however, this residue was essential for receptor function, as determined by 3',5'-cyclic adenosine monophosphate (cAMP) accumulation assays. Truncation of the C-terminal tail of both receptors (MT₁Y7.64 and MT₂Y7.64) inhibited internalization as well as the cAMP response, suggesting the importance of the C-terminal tail in these receptor functions.     

Melatonin receptor subtype MT2 (Mel 1b) and not mt1 (Mel 1a) is associated with melatonin-induced enhancement of cell-mediated and humoral immunity

Individuals of many vertebrate species undergo seasonal changes in immune function in addition to marked seasonal changes in reproductive, metabolic, and other physiological processes. Despite growing evidence that photoperiod mediates seasonal changes in immunity, little is known regarding the neuroendocrine mechanisms underlying these changes. Enhanced immune function in short days is correlated with increased duration of nightly melatonin secretion, and recent studies indicate that melatonin can act directly on immune cells to enhance immune function. It remains unknown, however, which melatonin receptor subtype mediates immune enhancement by melatonin. The present study examined the contribution of specific melatonin receptor subtypes, mt1 (Mel 1a) and MT2 (Mel 1b), in mediating melatonin-induced enhancement of cell-mediated and humoral immune function in mice. Melatonin enhanced both splenocyte proliferation and anti-keyhole limpet hemocyanin (KLH) IgG concentrations in both wild-type (WT) and mice lacking a functional gene for melatonin receptor mt1 (mt1 -/-), suggesting that the mt1 receptor does not mediate these responses. In addition, luzindole, an MT2 receptor antagonist, attenuated melatonin-induced enhancement of splenocyte proliferation in both WT and mt1 -/- mice. Taken together, these results suggest that receptor subtype mt1 is not necessary for mediating melatonin-induced enhancement of immune function and provide the first evidence for a specific melatonin receptor subtype, MT2, that may be involved in melatonin-induced immune enhancement.     

MT1 and MT2 melatonin receptors are expressed in nonoverlapping neuronal populations

Melatonin (MLT) exerts its physiological effects principally through two high‐affinity membrane receptors MT1 and MT2. Understanding the exact mechanism of MLT action necessitates the use of highly selective agonists/antagonists to stimulate/inhibit a given MLT receptor. The respective distribution of MT1 and MT2 within the CNS and elsewhere is controversial, and here we used a “knock‐in” strategy replacing MT1 or MT2 coding sequences with a LacZ reporter. The data show striking differences in the distribution of MT1 and MT2 receptors in the mouse brain: whereas the MT1 subtype was expressed in very few structures (notably including the suprachiasmatic nucleus and pars tuberalis), MT2 subtype receptors were identified within numerous brain regions including the olfactory bulb, forebrain, hippocampus, amygdala and superior colliculus. Co‐expression of the two subtypes was observed in very few structures, and even within these areas they were rarely present in the same individual cell. In conclusion, the expression and distribution of MT2 receptors are much more widespread than previously thought, and there is virtually no correspondence between MT1 and MT2 cellular expression. The precise phenotyping of cells/neurons containing MT1 or MT2 receptor subtypes opens new perspectives for the characterization of links between MLT brain targets, MLT actions and specific MLT receptor subtypes.     

Chimeric Galphaq subunits can distinguish the long form of the Xenopus Mel1c melatonin receptor from the mammalian mt1 and MT2 melatonin receptors

The family of melatonin receptors is composed of the mt1, MT2, and Mel1c subtypes. The Mel1c is further divided into one long and two short isoforms. A recent study has shown that, unlike mt1 and MT2, the long form of Mel1c is incapable of activating the pertussis toxin-insensitive G16. Here we used three well-characterized Galphaq chimeras to explore the coupling specificity of the melatonin receptors. The qi5, qo5, and qz5 chimeras can link numerous Gi-coupled receptors to the stimulation of phosphoinositide-specific phospholipase C. Both mt1 and MT2 receptors interacted productively with the Galphaq chimeras, while the long form of Mel1c was totally ineffective. Among the Galphaq chimeras, qo5 was less efficiently coupled to the melatonin receptors. Such differential coupling is best explained by structural differences between the melatonin receptors as well as among the Galphaq chimeras. Since the long form of Mel1c receptor possesses an exceptionally large C-terminal tail, we tested the ability of four melatonin receptor C-terminal tail chimeras (Chi 1-4) to interact with the Galphaq chimeras. The presence of the large C-terminal tail of Mel1c in Chi 1 and Chi 3 markedly hindered their coupling to the Galphaq chimeras. On the other hand, the attachment of either the mtl or MT2 C-terminal tail to a Mel1c backbone produced chimeras (Chi 2 and Chi 4) that were capable of activating the Galphaq chimeras. These findings suggest the involvement of C-terminal regions of melatonin receptors in the recognition of G proteins.     

The antidepressant-like effect of the melatonin receptor ligand luzindole in mice during forced swimming requires expression of MT2 but not MT1 melatonin receptors

We previously reported an antidepressant-like effect in C3H/HeN mice during the forced swimming test (FST) following treatment with the MT1/MT2 melatonin receptor ligand, luzindole. This study investigated the role melatonin receptors (MT1 and/or MT2) may play in the effect of luzindole in the FST using C3H/HeN mice with a genetic deletion of either MT1 (MT1KO) or MT2 (MT2KO) melatonin receptors. In the light phase (ZT 9-11), luzindole (30 mg/kg, i.p.) significantly decreased immobility during swimming in both wild type (WT) (135.6 +/- 25.3 s, n = 7) and MT(1)KO (132.6 +/- 13.3 s, n = 8) as compared with vehicle-treated mice (WT: 207.1 +/- 6.0 s, n = 7; MT1KO: 209.5 +/- 6.2 s, n = 8) (P < 0.001). In the dark phase (ZT 20-22), luzindole also decreased time of immobility in both WT (89.5 +/- 13.9 s, n = 8) and MT1KO (66.5 +/- 6.4 s, n = 8) mice as compared with the vehicle treated (WT: 193.8 +/- 3.5, n = 6; MT1KO: 176.6 +/- 6.2 s, n = 8) (P < 0.001). Genetic disruption of the MT1 gene did not alter the diurnal rhythm of serum melatonin in MT1KO mice (ZT 9-11: 1.3 +/- 0.6 pg/mL, n = 7; ZT 20-22: 10.3 +/- 1.1 pg/mL, n = 8) as compared with WT (ZT 9-11: 1.4 +/- 0.7 pg/mL; ZT 20-22: 10.6 pg/mL). Swimming did not alter the serum melatonin diurnal rhythm in WT and MT1KO mice. Decreases in immobility of WT and MT1KO mice by luzindole treatment were not affected by gender or age (3 months versus 8 months). In contrast, luzindole did not decrease immobility during the FST in MT2KO mice. We conclude that the antidepressant-like effect of luzindole may be mediated through blockade of MT2 rather than MT1 melatonin receptors.     

Rhythmic expression of functional MT1 melatonin receptors in the rat adrenal gland

We previously demonstrated that melatonin is involved in the regulation of adrenal glucocorticoid production in diurnal primates through activation of MT1 membrane-bound melatonin receptors. However, whether melatonin has a similar role in nocturnal rodents remains unclear. Using an integrative approach, here we show that the adult rat adrenal gland expresses a functional MT1 melatonin receptor in a rhythmic fashion. We found that: 1) expression of the cognate mRNA encoding for the MT1 membrane-bound melatonin receptor, displaying higher levels in the day/night transition (1800-2200 h); 2) expression of the predicted 37-kDa MT1 polypeptide in immunoblots from adrenals collected at 2200 h but not 1000 h; 3) no expression of the MT2 melatonin receptor mRNA and protein; 4) specific high-affinity 2-[(125)I]iodomelatonin binding in membrane fractions and frozen sections from adrenals collected at 2200 h but not 0800 h (dissociation constant = 14.22 +/- 1.23 pm; maximal binding capacity = 0.88 +/- 0.02 fmol/mg protein); and 5) in vitro clock time-dependent inhibition of ACTH-stimulated corticosterone production by 1-100 nm melatonin, which was reversed by 1 microm luzindole (a melatonin membrane receptor antagonist). Our findings indicate not only expression but also high amplitude diurnal variation of functional MT1 melatonin receptors in the rat adrenal gland. It is conceivable that plasma melatonin may play a role to fine-tune corticosterone production in nocturnal rodents, probably contributing to the down slope of the corticosterone rhythm.     

Sleep well. Untangling the role of melatonin MT1 and MT2 receptors in sleep

The pharmacological potential of targeting selectively melatonin MT1 or MT2 receptors has not yet been exploited in medicine. Research using selective MT1/MT2 receptor ligands and MT1/MT2 receptor knockout mice has indicated that the activation of MT2 receptors selectively increases non‐rapid eye movement (NREM) sleep whereas MT1 receptors seem mostly implicated in the regulation of REM sleep. Moreover, MT1 knockout mice show an increase in NREM sleep, while MT2 knockout a decrease, suggesting an opposite role of these two receptors. A recent paper in mice by Sharma et al (J Pineal Res, 2018, e12498) found that MT1 but not MT2 receptors are expressed on orexin neurons in the perifornical lateral hypothalamus (PFH). Moreover, after injecting melatonin or luzindole into the mouse PFH, the authors suggest that melatonin promotes NREM sleep because activates PFH MT1 receptors, which in turn inhibit orexin neurons that are important in promoting arousal and maintaining wakefulness. In this commentary, we have critically commented on some of these findings on the bases of previous literature. In addition, we highlighted the fact that no conclusions could be drawn on the melatonin receptor subtype mediating the effects of melatonin on sleep because the authors used the non‐selective MT1/MT2 receptors antagonist luzindole. More solid research should further characterize the pharmacological function of these two melatonin receptors in sleep.     

Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain

The involvement of melatonin in mammalian brain pathophysiology has received growing interest, but information about the anatomical distribution of its two G‐protein‐coupled receptors, MT₁ and MT₂, remains elusive. In this study, using specific antibodies, we examined the precise distribution of both melatonin receptors immunoreactivity across the adult rat brain using light, confocal, and electron microscopy. Our results demonstrate a selective MT₁ and MT₂ localization on neuronal cell bodies and dendrites in numerous regions of the rat telencephalon, diencephalon, and mesencephalon. Confocal and ultrastructural examination confirmed the somatodendritic nature of MT₁ and MT₂ receptors, both being localized on neuronal membranes. Overall, striking differences were observed in the anatomical distribution pattern of MT₁ and MT₂ proteins, and the labeling often appeared complementary in regions displaying both receptors. Somadendrites labeled for MT₁ were observed for instance in the retrosplenial cortex, the dentate gyrus of the hippocampus, the islands of Calleja, the medial habenula, the suprachiasmatic nucleus, the superior colliculus, the substantia nigra pars compacta, the dorsal raphe nucleus, and the pars tuberalis of the pituitary gland. Somadendrites endowed with MT₂ receptors were mostly observed in the CA3 field of the hippocampus, the reticular thalamic nucleus, the supraoptic nucleus, the inferior colliculus, the substantia nigra pars reticulata, and the ventrolateral periaqueductal gray. Together, these data provide the first detailed neurocytological mapping of melatonin receptors in the adult rat brain, an essential prerequisite for a better understanding of melatonin distinct receptor function and neurophysiology.     

Hypoxia increases leptin expression in human PAZ6 adipose cells

AIMS/HYPOTHESIS: Leptin, an adipose tissue-derived cytokine involved in the control of body weight, also participates in a variety of biological functions, including angiogenesis. Because reduced oxygen availability is a major inducer of angiogenesis, we hypothesized that low cellular oxygen tension could regulate leptin expression in adipose cells. METHODS: Differentiated PAZ6 adipocytes were cultured for 48 h in the presence of chemical inducers of cellular hypoxia (cobalt chloride or desferrioxamine) or in an atmosphere containing only 6% oxygen. The effect of hypoxia on the expression of leptin and several adipose genes was assessed by semi-quantitative RT-PCR. The effect of hypoxia on leptin promoter activity was tested in PAZ6 cells transiently transfected with a luciferase reporter construct, containing 1.87 kb of the human leptin promoter. Leptin secretion in the culture medium was determined by radioimmunoassay. RESULTS: Hypoxia increased leptin mRNA expression, leptin promoter activity and leptin secretion in the culture medium by two- to threefold ( p<0.05). The expression of the glucose transporter isoform 1 (GLUT-1) mRNA, a well known hypoxia inducible gene, was also increased. In contrast, glucose transporter isoform 4 (GLUT-4), hormone sensitive lipase (HSL), fatty acid binding protein (aP2) and uncoupling protein 2 (UCP2) mRNAs were markedly reduced by hypoxia. In addition, a similar hypoxia-induced increase in leptin mRNA and secretion was observed in primary rat adipose cells. CONCLUSION/INTERPRETATION: Hypoxia markedly and specifically increased leptin gene expression through activation of the leptin gene promoter, and this resulted in an increased leptin production by human PAZ6 adipocytes.     

MT(1) melatonin receptor overexpression enhances the growth suppressive effect of melatonin in human breast cancer cells

Melatonin inhibits the proliferation of estrogen receptor alpha (ERalpha)-positive (MCF-7), but not ERalpha-negative (MDA-MB-231) breast cancer cells. Here, we assessed the effect of MT(1) melatonin receptor stable overexpression in MCF-7 and MDA-MB-231 breast cancer cells on the growth-suppressive effects of melatonin. Parental and vector-transfected MCF-7 cells demonstrated a modest, but significant, growth-suppressive response to melatonin; however, melatonin treatment of MT(1)-transfected MCF-7 cells resulted in significantly enhanced growth-suppression. This response was blocked by an MT1/MT2 melatonin receptor antagonist. Interestingly, MT(1)-overexpression did not induce a melatonin-sensitive phenotype in melatonin-insensitive MDA-MB-231 cells. Finally, Northern blot analysis demonstrated an enhanced inhibition of ERalpha mRNA expression and an enhanced induction of pancreatic spasmolytic polypeptide (pS2) by melatonin in MT(1)-transfected MCF-7 cells relative to vector-transfected MCF-7 cells. These data suggest the involvement of the MT(1) melatonin receptor in mediation of melatonin effects on growth-suppression and gene-modulation in breast cancer cells.     

Decreased MT1 and MT2 melatonin receptor expression in extrapineal tissues of the rat during physiological aging

Abstract:  Aging is a complex process associated with a diminished ability to respond to stress, a progressive increase in free radical generation and a decline in immune function. Melatonin, a molecule with a great functional versatility exerts anti-oxidant, oncostatic, immunomodulatory, and anti-aging properties. Melatonin levels drop during aging and it has been speculated that the loss of melatonin may accelerate aging. This study was designed to elucidate whether aging involves responsiveness to reduced melatonin. Melatonin membrane receptor (MT1 and MT2) expression and MT1 protein expression were analyzed in extrapineal tissues (thymus, spleen, liver, kidney, and heart) of 3- and 12-month-old rats using real time polymerase chain reaction and western blotting analysis. Moreover, melatonin in tissues was measured by high performance liquid chromatography. We report for the first time, an age-related reduction in mRNA MT1 and MT2 expression levels as well as MT1 protein expression in all tissues tested except the thymus, where surprisingly, both melatonin receptor levels were significantly higher in 12-month-old rats and MT1 protein expression maintained unchanged with age. Diminished melatonin concentrations were measured in spleen, liver, and heart during aging. As a conclusion, physiological aging seems to exert responsiveness to melatonin and consequently, the loss of this potent anti-oxidant may contribute to onset of aging.     

Evidence that membrane-bound G protein-coupled melatonin receptors MT1 and MT2 are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia

Melatonin is synthesized and released by the pineal gland in a circadian rhythm, and many of its peripheral actions are mediated via membrane MT1 and MT2 receptors. Apart from its metabolic functions, melatonin is a potent neuroprotective molecule owing to its antioxidative actions. The roles of MT1 and MT2 in the neuroprotective effects of melatonin and cell signaling after cerebral ischemia remain unknown. With the use of MT1 and MT2 knockout (mt1/2(-/-) ) mice treated with melatonin, we evaluated brain injury, edema formation, inducible nitric oxide synthase (iNOS) activity, and signaling pathways, including CREB, ATF-1, p21, Jun kinase (JNK)1/2, p38 phosphorylation, resulting from ischemia/reperfusion injury. We show that the infarct volume and brain edema do not differ between mt1/2(-/-) and wild-type (WT) animals, but melatonin treatment decreases infarct volume in both groups and brain edema in WT animals after middle cerebral artery occlusion. Notably, melatonin's neuroprotective effect was even more pronounced in mt1/2(-/-) animals compared to that in WT animals. We also demonstrate that melatonin treatment decreased CREB, ATF-1, and p38 phosphorylation in both mt1/2(-/-) and WT mice, while p21 and JNK1/2 were reduced only in melatonin-treated WT animals in the ischemic hemisphere. Furthermore, melatonin treatment lowered iNOS activity only in WT animals. We provide evidence that the absence of MT1 and MT2 has no unfavorable effect on ischemic brain injury. In addition, the neuroprotective effects of melatonin appear to be mediated through a mechanism independent of its membrane receptors. The underlying mechanism(s) should be further studied using selective melatonin receptor agonists and antagonists.     

Expression of the melatonin receptor (MT) 1 in benign and malignant human bone tumors

The beneficial effects of melatonin on bone homeostasis have been shown in various diseases. As this indoleamine causes dose-dependent modulation of bone-forming osteoblast and bone-resorbing osteoclast activities by receptor-independent and -dependent pathways, we investigated the expression of G-protein-coupled melatonin receptors (MTs) in malignant and non-malignant human bone lesions. By TaqMan polymerase chain reaction (PCR), we analyzed 30 specimens from osteosarcoma and 11 from benign bone tumors for MT1-mRNA expression. Furthermore, we determined mRNA expression levels of the osteoclast activity-stimulating receptor activator of nuclear factor-kappa B ligand (RANKL) and its counterpart osteoprotegerin (OPG). Although mean MT1-mRNA levels were similar (P = 0.596) in malignant (4.39 +/- 4.98-fold) and benign samples (4.64 +/- 6.81-fold), the highest MT1-mRNA levels (up to 27-fold) were observed in individual osteosarcomas, particularly, in two specimens of patients with local recurrence of the tumor. Moreover, mean RANKL- and OPG-mRNA levels were similar in malignant and benign specimens (RANKL: 7.38 +/- 9.61-fold versus 3.57 +/- 3.11-fold, P = 0.207; OPG: 23.45 +/- 32.76 versus 8.07 +/- 7.23-fold, P = 0.133). Again, highest RANKL- and OPG-mRNA levels (up to 41- and 160-fold, respectively) were observed in individual osteosarcomas. Expression of MT1-mRNA was confirmed in two human osteosarcoma cell lines (HOS, MG63). High expression levels of MT1-mRNA together with low OPG-mRNA were found in both osteosarcoma cell lines, while in normal human osteoblasts and bone marrow stromal cells, high OPG-mRNA levels were associated with low MT1-mRNA levels. These data on the abundant expression of MT1-mRNA in human bone tumors and osteosarcoma cells lines suggest an important role for MT1 in bone pathology.     

Reduced hippocampal MT2 melatonin receptor expression in Alzheimer's disease

The aim of the present study was to identify the distribution of the second melatonin receptor (MT2) in the human hippocampus of elderly controls and Alzheimer's disease (AD) patients. This is the first report of immunohistochemical MT2 localization in the human hippocampus both in control and AD cases. The specificity of the MT2 antibody was ascertained by fluorescence microscopy using the anti-MT2 antibody in HEK 293 cells expressing recombinant MT2, in immunoblot experiments on membranes from MT2 expressing cells, and, finally, by immunoprecipitation experiments of the native MT2. MT2 immunoreactivity was studied in the hippocampus of 16 elderly control and 16 AD cases. In controls, MT2 was localized in pyramidal neurons of the hippocampal subfields CA1-4 and in some granular neurons of the stratum granulosum. The overall intensity of the MT2 staining was distinctly decreased in AD cases. The results indicate that MT2 may be involved in mediating the effects of melatonin in the human hippocampus, and this mechanism may be heavily impaired in AD.     

Nociceptive responses in melatonin MT2 receptor knockout mice compared to MT1 and double MT1/MT2 receptor knockout mice

Melatonin, a neurohormone that binds to two G protein-coupled receptors MT₁ and MT_2, is involved in pain regulation, but the distinct role of each receptor has yet to be defined. We characterized the nociceptive responses of mice with genetic inactivation of melatonin MT₁ (MT₁^−/−), or MT₂ (MT₂^−/−), or both MT₁/MT₂ (MT₁^−/−/MT₂^−/−) receptors in the hot plate test (HPT), and the formalin test (FT). In HPT and FT, MT₁^−/− display no differences compared to their wild-type littermates (CTL), whereas both MT₂^−/− and MT₁^−/−/MT₂^−/− mice showed a reduced thermal sensitivity and a decreased tonic nocifensive behavior during phase 2 of the FT in the light phase. The MT₂ partial agonist UCM924 induced an antinociceptive effect in MT₁^−/− but not in MT₂^−/− and MT₁^−/−/MT₂^−/− mice. Also, the competitive opioid antagonist naloxone had no effects in CTL, whereas it induced a decrease of nociceptive thresholds in MT₂^−/− mice. Our results show that the genetic inactivation of melatonin MT₂, but not MT₁ receptors, produces a distinct effect on nociceptive threshold, suggesting that the melatonin MT₂ receptor subtype is selectively involved in the regulation of pain responses.