Human MT2 Research Articles

From methodological limitations to the function of metallothioneins - A guide to approaches for determining weak, moderate, and tight affinity zinc sites.

Mammalian metallothioneins (MTs) are small cysteine-rich proteins whose primary role is participation in zinc and copper homeostasis. Ever since their discovery, MTs have been investigated in terms of metal-binding affinity. The initial concept of seven Zn(II) ions (Zn7MT) bound with the same, undifferentiated low picomolar affinity in the α and β domains prevailed for many years and derived from spectroscopic studies. The application of fluorescent zinc probes has changed the perception of MTs, showing that they function in nano- to subnanomolar free zinc concentrations due to the presence of tight, moderate, and weak binding sites. The discovery of Zn(II)-depleted MTs in many tissues and determination of cellular free Zn(II) concentrations with differentiated zinc affinity sites revealed the critical importance of partially saturated Zn4-6MTs species in cellular zinc buffering in a wide pico- to nanomolar range of free Zn(II) concentrations. Until today, there is no clear agreement on the presence of differentiated or only tight zinc sites. Here, we present a series of spectroscopic, mass spectrometry-based, and enzymatic competition experiments which reveal how weak, moderate, or high affinity ligands interact with human MT2, with special attention to the determination of Zn(II) affinities. The results show that the simplification of the stability model is the major reason for determining significantly different stability data that obscured the actual MTs function. Therefore, we emphasize that different metal affinities are the single most reason for their presumed function, which changed over the years from tight binding and, thus, storage to one that is highly dynamic.

An ion mobility-mass spectrometry study of copper-metallothionein-2A: binding sites and stabilities of Cu-MT and mixed metal Cu-Ag and Cu-Cd complexes.

The presence of Cu, a highly redox active metal, is known to damage DNA as well as other cellular components, but the adverse effects of cellular Cu can be mitigated by metallothioneins (MT), small cysteine rich proteins that are known to bind to a broad range of metal ions. While metal ion binding has been shown to involve the cysteine thiol groups, the specific ion binding sites are controversial as are the overall structure and stability of the Cu-MT complexes. Here, we report results obtained using nano-electrospray ionization mass spectrometry and ion mobility-mass spectrometry for several Cu-MT complexes and compare our results with those previously reported for Ag-MT complexes. The data include determination of the stoichiometries of the complex (Cui-MT, i = 1-19), and Cu+ ion binding sites for complexes where i = 4, 6, and 10 using bottom-up and top-down proteomics. The results show that Cu+ ions first bind to the β-domain to form Cu4MT then Cu6MT, followed by addition of four Cu+ ions to the α-domain to form a Cu10-MT complex. Stabilities of the Cui-MT (i = 4, 6 and 10) obtained using collision-induced unfolding (CIU) are reported and compared with previously reported CIU data for Ag-MT complexes. We also compare CIU data for mixed metal complexes (CuiAgj-MT, where i + j = 4 and 6 and CuiCdj, where i + j = 4 and 7). Lastly, higher order Cui-MT complexes, where i = 11-19, were also detected at higher concentrations of Cu+ ions, and the metalated product distributions observed are compared to previously reported results for Cu-MT-1A (Scheller et al., Metallomics, 2017, 9, 447-462).

A novel built-in adjuvant metallothionein-3 aids protein antigens to induce rapid, robust, and durable immune responses.

Adjuvants are crucial components of vaccines that can enhance and modulate antigen-specific immune responses. Herein, we reported for the first time that human metallothionein-3 (MT3), a low molecular weight cysteine-rich metal-binding protein, was a novel promising adjuvant candidate that could help protein antigens to induce rapid, effective, and durable antigen-specific immune responses. In the present study, MT3 was fused to outer membrane protein 19 (Omp19) of Brucella abortus (MT3-Omp19, MO) and C fragment heavy chain (Hc) of tetanus neurotoxin (MT3-Hc, MH), respectively. The results showed that MT3 as a built-in adjuvant increased the Omp19- or Hc-specific antibody responses by 100-1000 folds in seven days after primary immunization. Compared to other commercially available adjuvants, MT3 could stimulate earlier (4 days after primary injection) and stronger (10-100 folds) antibody response with lower antigen dose, and its adjuvanticity relied on fusion to antigen. Although the mechanism was not clear yet, the fusion protein MO was observed to directly activate DCs, promote germinal center formation and improve the speed of Ig class switching. Interestingly, our subsequent study found that other members of the mammalian MT family (human MT1 or murine MT3 for examples) also had potential adjuvant effects, but their effects were lower than human MT3. Overall, this study explored a new function of human MT3 as a novel built-in adjuvant, which may have important clinical application potential in vaccine development against global pandemics.

Luminogenic HiBiT Peptide-Based NanoBRET Ligand Binding Assays for Melatonin Receptors.

The two human melatonin receptors MT1 and MT2, which belong to the G protein-coupled receptor (GPCR) family, are important drug targets with approved indications for circadian rhythm- and sleep-related disorders and major depression. Currently, most of the pharmacological studies were performed using [3H]melatonin and 2-[125I]iodomelatonin (2-[125I]-MLT) radioligands. Recently, NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a sensitive, nonradioactive alternative for quantifying GPCR ligand engagement on the surface of living cells in equilibrium and real time. However, developing such assays for the two melatonin receptors depends on the availability of fluorescent tracers, which has been challenging predominantly owing to their narrow ligand entry channel and small ligand binding pocket. Here, we generated a set of melatonergic fluorescent tracers and used NanoBRET to evaluate their engagement with MT1 and MT2 receptors that are genetically fused to an N-terminal luminogenic HiBiT-peptide. We identified several nonselective and subtype-selective tracers. Among the selective tracers, PBI-8238 exhibited high nanomolar affinity to MT1, and PBI-8192 exhibited low nanomolar affinity to MT2. The pharmacological profiles of both tracers were in good agreement with those obtained with the current standard 2-[125I]-MLT radioligand. Molecular docking and mutagenesis studies suggested the binding mode of PBI-8192 in MT2 and its selectivity over MT1. In conclusion, we describe the development of the first nonradioactive, real-time binding assays for melatonin receptors expressed at the cell surface of living cells that are likely to accelerate drug discovery for melatonin receptors.

Mapping the Melatonin Receptor. 8. Selective MT2 Agonists Derived from 5,6-Dihydroindolo[2,1-a]isoquinolines and Related Systems.

A series of substituted indolo[2,1-a]isoquinolines and indolo[1,2-a]benzoxazines have been prepared, as melatonin analogues, to investigate the nature of the binding site of the melatonin receptor. Agonist and antagonist potency of all the analogues was measured using the [35S]GTPγS binding assay protocol. The binding affinity of the analogues were measured by competition binding studies against the human MT1 (hMT1) and MT2 (hMT2) receptors stably transfected in Chinese Hamster Ovarian (CHO) cells, using 2-[125 I]-iodomelatonin, as a ligand. N-Acetyl 2-(10-methoxy-5,6-dihydroindolo[2,1-a]isoquinolin-12-yl)propyl-1-amine (12 a) binds strongly to both the hMT1 and hMT2 receptors, and shows a preference for the hMT2, as does its propanamido counterpart 12 b. The introduction of two methyl groups into their side chain, analogues 15 a and 15 b, leads to antagonism, in the case of the former, and drastically diminishes its hMT1 binding; an analogous profile is seen for 15 b, which, however, is a partial agonist. Introduction of chlorine or methoxy groups into ring 4 gives compounds, that are weakly binding, with a preference for MT2. Substitution of oxygen for carbon at position 5 gives the indolo[1,2-c]benzoxazines 33, 36 a and b, that bind strongly to the human receptors, 33, 36 b being potent agonists at the melatonin receptors, but do not discriminate between hMT1 and hMT2.

Efficiency of Escherichia coli Jm109 and genetical engineering strains (E. coli MT2, E. coli MT3) in cadmium removal from aqueous solutions

Metallothioneins (MTs) are proteins with high metal binding affinity that exist in many organisms. Recently, this feature of MTs has been used in many areas, including environmental studies. In this study, the human MT2, and MT3 genes were cloned into Escherichia coli Jm109 to remove cadmium (Cd) from aqueous solutions. Minimum inhibition concentrations (MIC) of Cd for E. coli Jm109, E. coli MT2, and E. coli MT3 were determined as 154, 212, and 250 mg L −1 , respectively. 0.5, 5, and 25 mg L −1 Cd were applied to the strains. The maximum Cd removal was at the concentration of 0.5 mg L −1 . The optimal conditions for temperature, pH, and contact time were 37 °C, 7, and 24 h, respectively. E. coli MT3 was the most effective strain in Cd bioremoval. Scanning electron microscope (SEM) images showed that the morphological structures of the Cd treated cells were disrupted. The Energy dispersive spectroscopy (EDS) peaks of the Cd treated cells clearly showed Cd absorption into the cells. Scanning transmission electron microscope (STEM) images showed that there were clusters reflecting the accumulation of Cd in the MT3 cells. The appearance of attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) spectrum peaks of hydroxyl, carboxyl, amine, and sulfoxide groups in MT3 cells exposed to Cd confirmed that these groups bind Cd. As a result, it can be said that the MT3 gene can be used as an effective tool in Cd removal from waters. • The Cd removal capacities of genetically modified Escherichia coli were high. • E. coli MT3 was more effective in Cd removal than E. coli Jm109 and E. coli MT2. • SEM and STEM images showed that E. coli MT3 absorbs more Cd than other strains. • ATR-FTIR spectrum peaks confirmed that MT3 cells bind Cd. • MT3 can be used as an effective tool in Cd removal from water.

Modeling the Heterodimer Interfaces of Melatonin Receptors.

Melatonin receptors are Class A G protein-coupled receptors (GPCRs) that regulate a plethora of physiological activities in response to the rhythmic secretion of melatonin from the pineal gland. Melatonin is a key regulator in the control of circadian rhythm and has multiple functional roles in retinal physiology, memory, immunomodulation and tumorigenesis. The two subtypes of human melatonin receptors, termed MT1 and MT2, utilize overlapping signaling pathways although biased signaling properties have been reported in some cellular systems. With the emerging concept of GPCR dimerization, melatonin receptor heterodimers have been proposed to participate in system-biased signaling. Here, we used computational approaches to map the dimerization interfaces of known heterodimers of melatonin receptors, including MT1/MT2, MT1/GPR50, MT2/GPR50, and MT2/5-HT2C. By homology modeling and membrane protein docking analyses, we have identified putative preferred interface interactions within the different pairs of melatonin receptor dimers and provided plausible structural explanations for some of the unique pharmacological features of specific heterodimers previously reported. A thorough understanding of the molecular basis of melatonin receptor heterodimers may enable the development of new therapeutic approaches against aliments involving these heterodimeric receptors.

Melatonin MT1 and MT2 receptor ERK signaling is differentially dependent on Gi/o and Gq/11 proteins.

G protein-coupled receptors (GPCRs) transmit extracellular signals into cells by activating G protein- and β-arrestin-dependent pathways. Extracellular signal-regulated kinases (ERKs) play a central role in integrating these different linear inputs coming from a variety of GPCRs to regulate cellular functions. Here, we investigated human melatonin MT1 and MT2 receptors signaling through the ERK1/2 cascade by employing different biochemical techniques together with pharmacological inhibitors and siRNA molecules. We show that ERK1/2 activation by both receptors is exclusively G protein-dependent, without any participation of β-arrestin1/2 in HEK293 cells. ERK1/2 activation by MT1 is only mediated though Gi/o proteins, while MT2 is dependent on the cooperative activation of Gi/o and Gq/11 proteins. In the absence of Gq/11 proteins, however, MT2 -induced ERK1/2 activation switches to a β-arrestin1/2-dependent mode. The signaling cascade downstream of G proteins is the same for both receptors and involves activation of the PI3K/PKCζ/c-Raf/MEK/ERK cascade. The differential G protein dependency of MT1 - and MT2 -mediated ERK activation was confirmed at the level of EGR1 and FOS gene expression, two ERK1/2 target genes. Gi/o /Gq/11 cooperativity was also observed in Neuroscreen-1 cells expressing endogenous MT2 , whereas in the mouse retina, where MT2 is engaged into MT1 /MT2 heterodimers, ERK1/2 signaling is exclusively Gi/o -dependent. Collectively, our data reveal differential signaling modes of MT1 and MT2 in terms of ERK1/2 activation, with an unexpected Gi/o /Gq/11 cooperativity exclusively for MT2 . The plasticity of ERK activation by MT2 is highlighted by the switch to a β-arrestin1/2-dependent mode in the absence of Gq/11 proteins and by the switch to a Gi/o mode when engaged into MT1 /MT2 heterodimers, revealing a new mechanism underlying tissue-specific responses to melatonin.

Metallothionein 2A gene polymorphisms in relation to diseases and trace element levels in humans.

Human metallothioneins are a superfamily of low molecular weight intracellular proteins, whose synthesis can be induced by essential elements (primarily Zn and Cu), toxic elements and chemical agents, and stress-producing conditions. Of the four known isoforms in the human body MT2 is the most common. The expression of metallothioneins is encoded by a multigene family of linked genes and can be influenced by single nucleotide polymorphisms (SNPs) in these genes. To date, 24 SNPs in the MT2A gene have been identified with the incidence of about 1 % in various population groups, and three of them were shown to affect physiological and pathophysiological processes. This review summarises current knowledge about these three SNPs in the MT2A gene and their associations with element concentrations in the body of healthy and diseased persons. The most investigated SNP is rs28366003 (MT2A −5 A/G). Reports associate it with longevity, cancer (breast, prostate, laryngeal, and in paranasal sinuses), and chronic renal disease. The second most investigated SNP, rs10636 (MT2A +838G/C), is associated with breast cancer, cardiovascular disease, and type 2 diabetes. Both are also associated with several metal/metalloid concentrations in the organism. The third SNP, rs1610216 (MT2A −209A/G), has been studied for association with type 2 diabetes, cardiomyopathy, hyperglycaemia, and Zn concentrations. Metallothionein concentrations and MT2A polymorphisms have a potential to be used as biomarkers of metal exposure and clinical markers of a number of chronic diseases. This potential needs to be studied and verified in a large number of well-defined groups of participants (several hundreds and thousands) with a focus on particular physiological or pathological condition and taking into consideration other contributing factors, such as environmental exposure and individual genetic and epigenetic makeup.

Two indoleamines are secreted from rat pineal gland at night and act on melatonin receptors but are not night hormones.

At night, the pineal gland produces the indoleamines, melatonin, N-acetylserotonin (NAS), and N-acetyltryptamine (NAT). Melatonin is accepted as a hormone of night. Could NAS and NAT serve that role too? Concentration-response measurements with overexpressed human melatonin receptors MT1 and MT2 ; mass spectrometry analysis of norepinephrine-stimulated secretions from isolated rat pineal glands; analysis of 24-hour periodic samples of rat blood. We show that NAT and NAS do activate melatonin receptors MT1 and MT2 , although with lower potency than melatonin, and that in vitro, melatonin and NAS are secreted from stimulated, isolated pineal glands in roughly equimolar amounts, but secretion of NAT was much less. All three were found at roughly equal concentrations in blood during the night. However, during the day, serum melatonin fell to very low values creating a high-amplitude circadian rhythm that was absent after pinealectomy, whereas NAS and NAT showed only small or no circadian variation. Blood levels of NAS and NAT were insufficient to activate peripheral melatonin receptors, and they were invariant, so they could not serve as circulating hormones of night. However, they could instead act in paracrine circadian fashion near the pineal gland or via other higher-affinity receptors.

Melatonin and Oxidative Stress in the Diabetic State: Clinical Implications and Potential Therapeutic Applications.

All living organisms exhibit circadian rhythms, which govern the majority of biological functions, including metabolic processes. Misalignment of these circadian rhythms increases the risk of developing metabolic diseases. Thus, disruption of the circadian system has been proven to affect the onset of type 2 diabetes mellitus (T2DM). In this context, the pineal indoleamine melatonin is a signaling molecule able to entrain circadian rhythms. There is mounting evidence that suggests a link between disturbances in melatonin production and impaired insulin, glucose, lipid metabolism, and antioxidant capacity. Besides, several genetic association studies have causally associated various single nucleotide polymorphysms (SNPs) of the human MT2 receptor with increased risk of developing T2DM. Taken together, these data suggest that endogenous as well as exogenous melatonin may influence diabetes and associated metabolic disturbances not only by regulating insulin secretion but also by providing protection against reactive oxygen species (ROS) since pancreatic β-cells are very susceptible to oxidative stress due to their low antioxidant capacity.

Melatonin receptor structures shed new light on melatonin research.

The tryptophan derivative melatonin is an evolutionary old molecule that is involved in a pleiotropy of physiological functions. In humans, age-related decline of circulating melatonin levels and/or dysregulation of its circadian synthesis pattern have been associated with several disorders and disease states. Several molecular targets have been proposed for melatonin since its discovery, in 1959. Among them, melatonin MT1 and MT2 receptors are the best characterized melatonin targets, mediating melatonin effects in a variety of tissues. They belong to the superfamily of G protein-coupled receptors. Two back-to-back articles published in the "Nature" Journal earlier this year present the first crystal structures of the human MT1 and MT2 in its inactive states. Here, we will briefly outline the discovery path of melatonin receptors until their structural elucidation and discuss how these new findings will guide future research toward a better understanding of their function and rational drug design.

XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity.

The human MT11 and MT22 melatonin receptors are G protein-coupled receptors (GPCRs) involved in the regulation of circadian rhythm and sleep patterns3. Drug development efforts target both receptors for treatment of insomnia, circadian rhythm and mood disorders, and cancer3, while MT2 has also been implicated in type 2 diabetes (T2D)4,5. Here we report the X-ray Free Electron Laser (XFEL) structures of the human MT2 receptor in complex with agonists 2-phenylmelatonin (2-pmt) and ramelteon6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants, H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-pmt. Comparison of the MT2 structures with MT18 reveals that, despite the fact that the orthosteric ligand-binding site residues are conserved, there are notable conformational variations as well as differences in [3H]-melatonin dissociation kinetics that provide new insights into the selectivity between melatonin receptor subtypes. In addition to the membrane-buried lateral ligand entry channel that is also observed in MT1, the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data supporting a prominent role for the intramembrane ligand entry in both receptors, while simultaneously suggesting the possibility of an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.

Structural basis of ligand recognition at the human MT1 melatonin receptor.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest family of G-protein coupled receptor subtypes3. Drugs targeting serotonin receptors, including 5-HT1A, 5-HT1D, and 5-HT1e, are used for numerous disorders4. In addition, serotonin receptors have high basal activation and are subject to lipid regulation, but the structural basis for lipid regulation, basal activation, and serotonin pan agonism remains unclear. Here we report five structures of serotonin receptor-G protein complexes: three 5-HT1A complexes either in the apo state, bound to serotonin, or bound to the antipsychotic drug, aripiprazole; one 5-HT1D complex with serotonin and one 5-HT1e complex with a 5-HT1e and 5-HT1F selective agonist, BRL-54443. Unexpectedly, phospholipid PI4P was found at the interface of 5-HT1A with G-protein and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterols, particularly at 5-HT1A, where cholesterols are directly involved in shaping the ligand pocket that determines the aripiprazole specificity. Within the apo 5-HT1A ligand-binding pocket are structured water molecules that mimic 5-HT to activate the receptor. Together, these results address a long-standing question of how lipids and water molecules regulate GPCRs and reveal the basis for pan agonism of serotonin and determinants of drug recognition of serotonin receptors.

Carbamate Insecticides Modulate G Protein‐Dependent Signaling in Cells Expressing Melatonin Receptors

Melatonin targets MT1 and MT2 melatonin receptors that are expressed in brain as well as peripheral tissues in order to signal “time‐of‐day” messages for the synchronization of circadian rhythms. In mammals, the homeostatic balance of glucose metabolism and insulin release is modulated by Gαi protein‐dependent melatonin receptor (MTR) signaling in pancreatic β‐cells (Kemp et al. 2002). In‐silico screening paired with competition binding has demonstrated that carbamate insecticides with similar pharmacophores to melatonin have affinity for the mammalian MT1 and MT2 melatonin receptors (Jones et al. 2015 EB Abs.; Popovska‐Gorevski et al. 2017; Glatfelter et al. 2017 EB Abs.). Therefore we hypothesize that exposure to these environmental chemicals can disrupt melatonin receptor signaling. The objective of this study was to examine the effect of carbamate insecticides on G protein dependent‐signaling pathways on human MT1 and MT2 melatonin receptors stably expressed in CHO cells and in rat INS‐1 β‐cells expressing MT1. The apparent efficacy of carbamates (ie., carbaryl, fenobucarb, bendiocarb, and carbofuran) assessed by affinity ratios derived from competition with 2‐[125I]‐iodomelatonin binding, with or without G protein inactivation by GTP, suggested antagonist efficacy on hMT1 receptors (no affinity shift with GTP; KiGTP/KiControl:0.59 – 2.3) and agonist efficacy on hMT2 receptors (rightward affinity shift with GTP; KiGTP/KiControl: 4.8 – 11). The same insecticides tested at 10 μM did not modify forskolin‐stimulated cAMP levels in CHO‐hMT1 cells but inhibited stimulated cAMP formation in CHO‐hMT2 cells (47%–72% of melatonin effect on stimulation). Carbaryl (10 pM – 100 μM) acted as a partial agonist at both hMT1 (log IC50= −5.8 ± 0.3 M; Emax= 55% melatonin effect) and hMT2 (log IC50 = −10.0 ± 0.4 M; Emax= 67% melatonin effect) receptors with over 10,000 times higher potency for hMT2 compared to hMT1. Next, carbaryl at 10 μM, but not at 1 μM, inhibited forskolin‐stimulated insulin release from INS‐1 pancreatic β‐cells by 25% (96% melatonin effect) as determined by radioimmunoassay. These data suggest that carbamate insecticides with similar structures to melatonin can alter G protein‐dependent signaling in tissues that express melatonin receptors. Exposure to these chemicals could potentially contribute to the development of diabetes or other metabolic disorders as disruption of β‐cell melatonin receptor signaling and insulin release are linked to increased risk of disease (Tuomi et al. 2016).Support or Funding InformationSupported by ES 023684 to MLD and RVR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non-24-hour sleep-wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT1 and MT2 , belonging to the G protein-coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT1 and MT2 . Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT1 and MT2 by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT1 or MT2 . None of the mABs cross-reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR-KO mice as control. MT2 expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta-cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.

Structural basis of ligand recognition at the human MT1 melatonin receptor

Structural basis of ligand recognition at the human MT1 melatonin receptor

XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity

XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity

Hamster Melatonin Receptors: Cloning and Binding Characterization of MT₁ and Attempt to Clone MT₂.

For many years, it was of interest to identify the sequences encoding the two melatonin receptors (MT1 and MT2) from various species. After publishing the basic molecular characterization of the human, rat, mouse, sheep, and platypus MT1, MT2, or Mel1c receptors, we began cloning the genes from other animals, such as birds, bats, and vipers. The goal was to advance the receptor crystallization, which could greatly contribute the understanding of the sequence/stability relationship. European hamster MT1 receptor was cloned for the first time from this gender, was expressed in stable form in cells, and its binding characterized with a sample of 19 melatonin ligands. Siberian hamster (Phodopus sungorus) expresses a non-functional MT2. We observed that unlike this hamster, the European hamster (Cricetus cricetus) does not have a stop codon in the MT2 sequence. Thus, we undertook the tedious task of cloning the MT2 receptor. We partially succeeded, sequencing the complete exon 2 and a fragment of exon 1 (from putative amino acids 12 to 38 and 77 to 323), after several years of efforts. In order to show that the protein parts we cloned were capable to sustain some binding capacities, we designed a chimeric MT2 receptor using a consensus sequence to replace the unknown amino acids, based on other small rodent MT2 sequences. This chimeric construct could bind melatonin in the nanomolar range. This work is meant to be the basis for attempts from other laboratories of the community to determine the complete natural sequence of the European hamster MT2 receptor. The present work is the first to show that, among the hamsters, if the Siberian is a natural knockout for MT2, the European one is not.

Carbamate Insecticides Bind to a Novel Allosteric Site on hMT1 Melatonin Receptors

In silico screening paired with 2‐[125I]‐iodomelatonin competition binding has implicated carbamate insecticides as potential endocrine disruptors due to their affinity for human (h) MT1 and MT2 melatonin receptors (Popovska‐Gorevski et al. 2017). hMT1 and hMT2 receptor modelling predicts that these compounds may be binding to the orthosteric site and/or to previously uncharacterized hydrophobic pockets proximal to the melatonin‐binding pocket. The location of the specific binding site for carbamate insecticides is currently unknown. The goal of this study was to assess the mechanism(s) by which they inhibit orthosteric ligand binding. Melatonin (1 μM), luzindole (a prototypical non‐selective MT1/MT2 competitive melatonin receptor antagonist, 100 μM), and four carbamate insecticides (carbaryl, fenobucarb, bendiocarb, and carbofuran, 1 mM) were screened for binding to allosteric sites by estimating the Maximal Fractional Inhibition (MFI) of a saturating 2‐[125I]‐iodomelatonin concentration (1200 pM) binding to CHO‐hMT1 and CHO‐hMT2 cell membranes. All six compounds at hMT2 and melatonin at hMT1 completely inhibited the saturating concentration of radioligand (MFI≥60%) suggesting an orthosteric binding mechanism, while luzindole and the carbamate insecticides only partially inhibited binding at hMT1 (MFI<60%), alluding to binding through an allosteric mechanism. To confirm these results, full competition binding curves in CHO‐hMT1 and CHO‐hMT2 cells were performed using five different concentrations of 2‐[125I]‐iodomelatonin (50–1200 pM) to assess binding to the receptor orthosteric site (α=0) and to determine the degree of cooperativity (α>0.01) between orthosteric and allosteric ligand binding. For both hMT1 and hMT2 receptors, melatonin (MT1 KB=0.131 nM, MT2 KB=0.154 nM) completely inhibited binding of saturating concentrations of radioligand confirming that both melatonin and 2‐[125I]‐iodomelatonin bind to the same site. Luzindole (MT1 KB=354 nM, MT2 KB=11.3 nM), and the carbamate insecticide with the highest MT1 and MT2 melatonin receptor affinities, carbaryl (MT1 KB=3790 nM, MT2 KB=389 nM), also completely inhibited 1200 pM 2‐[125I]‐iodomelatonin binding at hMT2 confirming that they bind to the orthosteric site. Interestingly, for luzindole and carbaryl at hMT1, the levels of maximal fractional inhibition of binding decreased with increasing concentrations of radioligand. These patterns show that luzindole and carbaryl bind to an allosteric site on hMT1 with strong negative cooperativity (αluzindole=0.037±0.013, n=3; αcarbaryl=0.017±0.001, n=3). Interactions of carbamate insecticides with a melatonin receptor allosteric site may provide novel mechanisms by which environmental melatonin receptor modulators disrupt signaling of the endogenous ligand.Support or Funding InformationSupported by ES 023684 to MLD and RVR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.