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Keywords:

  • antioxidant;
  • melatonin;
  • melatoninergic receptor;
  • MT3;
  • quinone reductase 2

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Membrane melatonin receptors
  5. Is MT3 a melatonin membrane receptor or quinone reductase 2?
  6. Is melatonin a natural co-substrate of QR2?
  7. Conclusion
  8. References

Abstract:  The nature of the MT3 melatonin receptor/binding site has been a long pondered mystery for scientists. Even though it is a presumptive membrane receptor, neither its transduction cascade nor its biological consequences, after its stimulation, have been uncovered. Moreover, solid data support the idea that the MT3 melatonin binding site is an enzyme, quinone reductase 2 (QR2), rather than a membrane melatonin receptor. Based on the data available and our preliminary studies, we hypothesize that melatonin is a co-substrate of QR2. We surmise that melatonin binds to a co-substrate binding site (MT3 binding site) donating an electron to the enzyme co-factor, flavin adenine dinucleotide (FAD). FAD can be reduced to either FADH or FADH2 while melatonin is converted to N1-acetyl-N2-formyl-5-methoxykynuramine and/or cyclic 3-hydroxymelatonin. QR2 is considered to be a detoxifying and antioxidant enzyme and its behavior changes depending on available co-substrates. As a naturally occurring substance, melatonin’s levels fluctuate with the light/dark cycle, with aging and with health/disease state. As a result, these alterations in melatonin production under physiological or pathological conditions would probably influence the activity of QR2.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Membrane melatonin receptors
  5. Is MT3 a melatonin membrane receptor or quinone reductase 2?
  6. Is melatonin a natural co-substrate of QR2?
  7. Conclusion
  8. References

Melatonin, a highly conserved molecule, serves as an antioxidant in unicellular organisms and subsequently has evolved to be a biological signal of the light/dark cycle in vertebrates; this probably occurred about 2 billion years ago [1, 2]. Since its discovery, many physiological functions of melatonin have been reported. These include its free radical scavenging and antioxidant capacity, immune-enhancement, reproductive regulation in seasonal breeders, tumor suppression, anti-inflammation and strengthening of circadian rhythms [3, 4]. Several of these important functions are mediated by melatoninergic receptors.

Membrane melatonin receptors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Membrane melatonin receptors
  5. Is MT3 a melatonin membrane receptor or quinone reductase 2?
  6. Is melatonin a natural co-substrate of QR2?
  7. Conclusion
  8. References

To date, three major melatoninergic membrane receptors have been identified in mammals. They are denoted as MT1, MT2 and MT3 receptors. Among them, MT1 and MT2 receptors have been thoroughly studied and their physiological functions and pharmacological properties are well documented [5]. These two receptors share a common seven-transmembrane structure and are negatively coupled to adenylate cyclase via a pertussis toxin-sensitive G-protein. MT1 and MT2 are also described as high affinity melatoninergic receptors; picomolar levels of melatonin (in the range of circulating melatonin concentrations) bind to these receptors and trigger a signal transduction cascade which produces biological consequences as do other classical receptors.

Distinguished from MT1 and MT2, MT3 is designated a low affinity melatonin membrane receptor with a Kd in the nanomolar range [6]. Whether MT3 is in fact a classic melatonin membrane receptor and, if so, what is the nature of the biological consequences when this receptor is stimulated are major unanswered questions. First, serum physiological melatonin levels in mammals rarely reach the nanomolar range and, secondly, the temperature-dependency and the very rapid ligand association/dissociation kinetics suggests the behavior of MT3 is more closely akin to that of an enzyme rather than to a receptor. Actually, the signal transduction cascade of the so-called MT3 receptor has not been identified as yet. Two reports indicate that MT3 receptor stimulation results in a drop in intraocular pressure [7, 8]. This conclusion was deduced from the observation that 5-methoxycarbonylamino-N-acetyl-tryptamine (MCA-NAT), a putative MT3 agonist, produces a decremental change in the intraocular pressure of rabbits but, again, no related signal transduction pathway has been identified for this effect.

Is MT3 a melatonin membrane receptor or quinone reductase 2?

  1. Top of page
  2. Abstract
  3. Introduction
  4. Membrane melatonin receptors
  5. Is MT3 a melatonin membrane receptor or quinone reductase 2?
  6. Is melatonin a natural co-substrate of QR2?
  7. Conclusion
  8. References

A breakthrough concerning the properties of MT3 occurred in 2000. Nosjean et al. [9] first observed that the MT3 receptor might be an enzyme, quinone reductase 2 (QR2). To further examine this novel possibility, this group transfected the recombinant human QR2 gene into CHO cells and also expressed it in this cell line. The experimental results show that the MT3 binding activity in the transfected CHO cells is two orders of magnitude higher than that in naïve CHO cells. The elevated MT3 binding sites positively associate with the QR2 activity [9, 10]. In addition, in QR2 gene knockout mice, tissues from the QR2–/– mice were depleted of MT3 binding sites [11].

Additional studies showed that MT3 binding sites essentially exist in all tissues and organs of a variety of species including hamsters, mice, dogs and monkeys. Without exception, the highest binding is in liver, kidneys and brain [12]. In contrast to previous concepts of a membrane receptor, in excess of 90% of the MT3 binding sites in transfected CHO cells or in hamster kidneys are located in cytosol rather than in membranes [10].

Interestingly, MT3 binding is temperature dependent; when the temperature reaches 37°C neither melatonin nor the so-called specific MT3 receptor agonist, MCA-NAT, binds to the MT3 site. By contrast, temperature does not influence melatonin binding to MT1 and MT2 melatoninergic receptors [12] (Fig. 1). A reasonable explanation for this unusual phenomenon related to a so-called membrane receptor is that 37°C might be the optimal temperature for the activity of the enzyme, QR2, and under these conditions, the extremely rapid substrate/enzyme association/dissociation kinetics make stable ‘binding’ impossible. It is noted that previous MT3 binding studies were performed at 4°C and thus, these results do not support the idea the MT3 is a classical receptor. Based on the data mentioned above, there is little doubt that the previously identified ‘MT3 binding site is cytosolic QR2, rather than a classic melatonin membrane receptor.

image

Figure 1.  Binding of 2-[125I]-melatonin to MT1, MT2 and MT3 melatoninergic receptors at different temperatures. The radioligand concentrations were 25, 179 and 200 pm for MT1, MT2 and MT3, respectively. As illustrated, the binding of 2-[125I]-melatonin to MT1 and MT2 is not influenced by temperature; however, the binding of 2-[125I]-melatonin to MT3 is suppressed by the increased temperature. Data from Mailliet et al. [10].

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Quinone reductase 2 was discovered in 1961 as an unknown mammalian cytosolic flavin adenine dinucleotide (FAD)-dependent flavoprotein that reduces menadione and other quinones by using N-ribosyl- and N-alkyldihydronicotinamides as the co-substrates [13]. The physiological and pathological nature of QR2 is still a mystery. Currently available data favor it being a detoxifying enzyme and related to antioxidant defense. The reductive mechanisms of QR2 have been described in terms of ping-pong kinetics, as QR2 uses a unique catalytic site for both substrate and co-substrate binding. First, the co-substrate (electron donor) occupies the site, and only after its release does the substrate (electron acceptor) enter the site. The enzymatic reactions of QR2 can involve the transfer of either 1, 2, or 4 electrons depending on the nature of the substrates. It seems that QR2 behavior differs as a function of its available co-substrate(s) [14]. However, to date, only small numbers of the natural co-substrates of QR2 have been identified.

Is melatonin a natural co-substrate of QR2?

  1. Top of page
  2. Abstract
  3. Introduction
  4. Membrane melatonin receptors
  5. Is MT3 a melatonin membrane receptor or quinone reductase 2?
  6. Is melatonin a natural co-substrate of QR2?
  7. Conclusion
  8. References

Based on the published information and our preliminary observations, we hypothesize that melatonin may be a natural co-substrate of QR2. The reported binding of melatonin to MT3 is likely an interaction between the co-substrate (melatonin) and the enzyme (QR2). The temperature-dependency and very rapid ligand association/dissociation kinetics of melatonin binding to MT3 indicate the likely reactions of a substrate and an enzyme. Unlike the picomolar levels of serum melatonin, intracellular melatonin concentrations may reach high nanomolar or even low micromolar ranges [3]. These high (relative to serum) intracellular melatonin concentrations would satisfy the kinetics of QR2 which interacts with substrates and allows QR2 to produce its optimal detoxifying effects. Indeed, melatonin and another known QR2 co-substrate, dihydrobenzylnicotinamide (BNAH), compete for the same co-substrate binding site of QR2 [10]. It seems that the affinities of melatonin and BNAH binding to the co-substrate sites of QR2 are in the similar range since the IC50 of melatonin is 130 μm when competed with BNAH (100 μm) for the binding sites of QR2.

We disagree with the conclusion of Mailliet et al. [10] that melatonin is an inhibitor of QR2 based on the competitive inhibition of melatonin versus BNAH binding to the co-substrate binding sites of QR2. If melatonin is a co-substrate of this enzyme, it would replace another co-substrate by means of competitive inhibition; this does not necessarily mean that melatonin inhibits QR2 activity in which the end-point is to biotransform the substrates. If melatonin is a better electron donor to FAD, a co-factor of QR2, than that of BNAH, QR2 activity would be enhanced. In addition, a different co-substrate renders QR2 behavior different toward a variety of substrates. We have noticed that the chemistry does not favor melatonin donating electron(s) to FAD because of the relatively higher redox potential of melatonin compared with that of FAD. However, this interaction occurs in a hydrophobic pocket of QR2 which is surrounded by several amino acid residues [14]. A suitable microevironment may allow melatonin to donate electron(s) to FAD with ease. The potential interaction of melatonin as a co-substrate with the QR2 may occur at the catalytic site of the enzyme. A melatonin molecule would donate an electron and generate a melatonin cation radical while the co-factor of QR2, FAD, as an electron acceptor, is reduced to FADH (semi-reduced form). If this process repeats itself, FADH will be fully reduced to FADH2. The melatonin cation radical then either interacts with a superoxide anion radical to form N1-acetyl-N2-formyl-5-methoxykynuramine or is converted to the melatonin neutral radical at physiological pH. The melatonin neutral radical scavenges a hydroxyl radical to form cyclic 3-hydroxymelatonin. This presumed pathway is summarized in scheme 1.

image

Figure Scheme 1..  The speculated reactions of melatonin as a co-substrate of QR2. QR2-FAD, quinine reductase with the co-factor FAD; QR2-FADH, semi-reduced QR2-FAD; QR2-FADH2, fully reduced QR2-FAD; MEL, melatonin; MEL.+, melatonin cation radical; MEL.; melatonin neutral radical; AFMK, N1-acetyl-N2-formyl-5-methoxykynuramine; C-3OHM, cyclic 3-hydroxymelatonin; O2.– superoxide anion; .HO..

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Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Membrane melatonin receptors
  5. Is MT3 a melatonin membrane receptor or quinone reductase 2?
  6. Is melatonin a natural co-substrate of QR2?
  7. Conclusion
  8. References

The significance of melatonin being a co-substrate of QR2 is obvious. It would solve the long pondered mystery of the physiological or pathological function and the biological consequences of melatonin’s association with MT3 binding sites. It also provides a plausible explanation regarding the potent endogenous antioxidative potential of melatonin [15, 16]. Melatonin may not only scavenge existing radicals but also is involved in radical avoidance by targeting QR2 [17]. As a co-substrate of QR2 may modify the behavior of the enzyme, Vella et al. [14] have already questioned whether the naturally occurring co-substrates are similarly available in various cell types and/or in various physio-pathological conditions, e.g. during aging, elevated oxidative stress, etc. It is well known that melatonin levels change according to the light/dark cycle and its production wanes with aging [18]. In addition, in some pathological conditions including diabetes, hypertension, coronary heart disease and Alzheimer’s disease, melatonin levels are reportedly substantially reduced [19–23]. Whether the altered melatonin levels are associated with the changes in the activity of QR2 and, as a result, potentially altered antioxidative defense processes deserve further investigation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Membrane melatonin receptors
  5. Is MT3 a melatonin membrane receptor or quinone reductase 2?
  6. Is melatonin a natural co-substrate of QR2?
  7. Conclusion
  8. References
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