G protein-coupled receptor 21 in macrophages: An in vitro study.

GPR21 is an orphan and constitutively active receptor belonging to the superfamily of G-Protein Coupled Receptors (GPCRs). GPR21 couples to the Gq family of G proteins and is expressed in macrophages. Studies of GPR21 knock-out mice indicated that GPR21 may be involved in promoting macrophage migration. The aim of this study was to evaluate the role of GPR21 in human macrophages, analyzing (i) its involvement in cell migration and cytokine release and (ii) the consequence of its pharmacological inhibition by using the inverse agonist GRA2. THP-1 cells were activated and differentiated into either M1 or M2 macrophages. GPR21 expression was evaluated at gene and protein level, the signalling pathway was investigated by an IP1 assay, and cytokine release by ELISA. Cell migration was detected by the Boyden chamber migration assay, performed on macrophages derived from both the THP-1 cell line and human peripheral blood monocytes. In addition, we compared the effect of the pharmacological inhibition of GPR21 with the effect of the treatment with a specific GPR21 siRNA to downregulate the receptor expression, thus confirming that GRA2 acts as an inverse agonist of GPR21. GRA2 does not affect cell viability at the tested concentrations, but significantly reduces the release of TNF-α and IL-1ß from M1 macrophages. The analysis of the migratory ability highlighted opposite effects of GRA2 on M1 and M2 macrophages since it decreased M1, while it promoted M2 cell migration. Therefore, the pharmacological inhibition of GPR21 could be of interest for pathological conditions characterized by low grade chronic inflammation.

GPR21 Inhibition Increases Glucose-Uptake in HepG2 Cells.

GPR21 is a constitutively active, orphan, G-protein-coupled receptor, with in vivo studies suggesting its involvement in the modulation of insulin sensitivity. However, its precise contribution is not fully understood. As the liver is both a major target of insulin signalling and critically involved in glucose metabolism, the aim of this study was to examine the role of GPR21 in the regulation of glucose uptake and production in human hepatocytes. In particular, HepG2 cells, which express GPR21, were adopted as cellular models. Compared with untreated cells, a significant increase in glucose uptake was measured in cells treated with siRNA to downregulate GPR21 expression or with the GPR21-inverse agonist, GRA2. Consistently, a significantly higher membrane translocation of GLUT-2 was measured under these conditions. These effects were accompanied by an increased ratio of phAKT(Ser473)/tot-AKT and phGSK-3ß(Ser9)/tot-GSK-3ß, thus indicating a marked activation of the insulin signalling pathway. Moreover, a significant reduction in ERK activation was observed with GPR21 inhibition. Collectively, these results indicate that GPR21 mediates the negative effects on glucose uptake by the liver cells. In addition, they suggest that the pharmacological inhibition of GPR21 could be a novel strategy to improve glucose homeostasis and counteract hepatic insulin resistance.

The signalling mechanisms of a novel mitochondrial complex I inhibitor prevent lipid accumulation and attenuate TNF-α-induced insulin resistance in vitro.

RTC-1 has recently been identified as a member of a new class of anti-diabetic compounds acting through the inhibition of complex I of the mitochondrial respiratory chain (NADH:ubiquinone oxidoreductase) to improve glucose handling and inhibit weight gain in mice fed a high-fat diet (HFD). The exact mechanism by which the reduced activity of NADH:ubiquinone oxidoreductase, in response to RTC-1, promotes these improved metabolic parameters remains to be established. Through extensive in vitro analysis, new molecular insights into these downstream signalling pathways have been obtained. RTC-1-induced inhibition of NADH:ubiquinone oxidoreductase was found to promote glucose uptake in C2C12 myotubes in vitro, through the activation of the Akt substrate of 160kDa (AS160), in response to the increased activity of Akt and AMP-activated protein kinase (AMPK). RTC-1-induced phosphorylation of the AMPK substrate, acetyl-CoA carboxylase (ACC) in vitro, was associated with a decrease in lipid accumulation in 3T3-L1 adipocytes and murine mesenchymal stromal cells (MSC). The novel compound also prevented tumour necrosis factor-alpha (TNF-α)-induced insulin resistance and demonstrated insulin sensitising effects in C2C12 myotubes. Taken together, these results present a systematic analysis of the signalling mechanisms responsible for the potent anti-diabetic and anti-obesogenic effects of this modulator of mitochondrial function, strengthening the potential use of such compounds for the treatment of type 2 diabetes mellitus (T2DM).

Novel mitochondrial complex I inhibitors restore glucose-handling abilities of high-fat fed mice.

Metformin is the main drug of choice for treating type 2 diabetes, yet the therapeutic regimens and side effects of the compound are all undesirable and can lead to reduced compliance. The aim of this study was to elucidate the mechanism of action of two novel compounds which improved glucose handling and weight gain in mice on a high-fat diet. Wildtype C57Bl/6 male mice were fed on a high-fat diet and treated with novel, anti-diabetic compounds. Both compounds restored the glucose handling ability of these mice. At a cellular level, these compounds achieve this by inhibiting complex I activity in mitochondria, leading to AMP-activated protein kinase activation and subsequent increased glucose uptake by the cells, as measured in the mouse C2C12 muscle cell line. Based on the inhibition of NADH dehydrogenase (IC50 27µmolL(-1)), one of these compounds is close to a thousand fold more potent than metformin. There are no indications of off target effects. The compounds have the potential to have a greater anti-diabetic effect at a lower dose than metformin and may represent a new anti-diabetic compound class. The mechanism of action appears not to be as an insulin sensitizer but rather as an insulin substitute.

The melanocortin 4 receptor: oligomer formation, interaction sites and functional significance.

This study involves the structural and functional properties of the recombinant melanocortin 4 receptor (MC(4)R) expressed in the HEK-293 cell line. Using co-immuno-purification approaches, the receptor appears to be an oligomer, which can be crosslinked through disulphide bonds involving a native cysteine residue (84) to give a dimeric species. This position is located near the cytosolic region of transmembrane segment 2 and it is suggested that this is an interacting interface between MC(4)R monomers. Using co-expression of the native protein and a C84A mutant, it appears that the receptor also forms higher order oligomers via alternative interfaces. Interestingly, disulphide crosslink formation does not occur if the receptor is uncoupled from its G-protein, even though the oligomeric state is preserved. This suggests that the conformational changes, which occur on activation, affect the TM2 interface. The pharmacology of the agonist, NDP-MSH, indicates that the MC(4)R retains high affinity for the ligand in the absence of the G-protein but occupancy for the ligand is increased. The data can be interpreted to suggest that the G-protein exerts a negative allosteric effect on the receptor. Co-expression of one receptor lacking the ability to signal with another, which cannot bind the agonist, restored ligand-dependent activation of the G-protein to situations in which neither receptor on its own could activate the G-protein. Such transactivation suggests meaningful cross talk between the receptor subunits in the oligomeric complex. These studies demonstrate further unique features of the MC(4)R.

An analysis of the phosphoproteome of immune cell lines exposed to the immunomodulatory mycotoxin deoxynivalenol.

The mycotoxin deoxynivalenol (DON) commonly contaminates cereal grains. It is ubiquitous in the Western European diet, although chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, two-dimensional gel electrophoresis was used to identify phosphoproteomic changes in human B (RPMI1788) and T (Jurkat E6.1) lymphocyte cell lines after exposure to modest concentrations of DON (up to 500ng/mL) for 24h. Proteins identified as having altered phosphorylation state post-treatment (C-1-tetrahydrofolate synthase, eukaryotic elongation factor 2, nucleoside diphosphate kinase A, heat shock cognate 71kDa protein, eukaryotic translation initiation factor 3 subunit I and growth factor receptor-bound protein 2) are involved in regulation of metabolic pathways, protein biosynthesis and signaling transduction. All exhibited a greater than 1.4-fold change, reproducible in three separate experiments consisting of 36 gels in total. Flow cytometry validated the observations for eukaryotic elongation factor 2 and growth factor receptor-bound protein 2. These findings provide further insights as to how low dose exposure to DON may affect human immune function and may have potential as mechanism-based phosphoprotein biomarkers for DON exposure.

Interactions of the melanocortin-4 receptor with the peptide agonist NDP-MSH.

Melanocortin-4 receptor (MC4R) has an important regulatory role in energy homeostasis and food intake. Peptide agonists of the MC4R are characterized by the conserved sequence His(6)-Phe(7)-Arg(8)-Trp(9), which is crucial for their interaction with the receptor. This investigation utilized the covalent attachment approach to identify receptor residues in close proximity to the bound ligand [Nle(4),D-Phe(7)]melanocyte-stimulating hormone (NDP-MSH), thereby differentiating between residues directly involved in ligand binding and those mutations that compromise ligand binding by inducing conformational changes in the receptor. Also, recent X-ray structures of G-protein-coupled receptors were utilized to refine a model of human MC4R in the active state (R(*)), which was used to generate a better understanding of the binding mode of the ligand NDP-MSH at the atomic level. The mutation of residues in the human MC4R--such as Leu106 of extracellular loop 1, and Asp122, Ile125, and Asp126 of transmembrane (TM) helix 3, His264 (TM6), and Met292 (TM7)--to Cys residues produced definitive indications of proximity to the side chains of residues in the core region of the peptide ligand. Of particular interest was the contact between D-Phe(7) on the ligand and Ile125 of TM3 on the MC4R. Additionally, Met292 (TM7) equivalent to Lys(7.45) (Ballesteros numbering scheme) involved in covalently attaching retinal in rhodopsin is shown to be in close proximity to Trp(9). For the first time, the interactions between the terminal regions of NDP-MSH and the receptor are described. The amino-terminus appears to be adjacent to a series of hydrophilic residues with novel interactions at Cys196 (TM5) and Asp189 (extracellular loop 2). These interactions are reminiscent of sequential ligand binding exhibited by the beta(2)-adrenergic receptor, with the former interaction being equivalent to the known interaction involving Ser204 of the beta(2)-adrenergic receptor.

Investigation of the structure and function of a Shewanella oneidensis arsenical-resistance family transporter.

The toxic metalloid arsenic is an abundant element and most organisms possess transport systems involved in its detoxification. One such family of arsenite transporters, the ACR3 family, is widespread in fungi and bacteria. To gain a better understanding of the molecular mechanism of arsenic transport, we report here the expression and characterization of a family member, So_ACR3, from the bacterium Shewanella oneidensis MR-1. Surprisingly, expression of this transporter in the arsenic-hypersensitive Escherichia coli strain AW3110 conferred resistance to arsenate, but not to arsenite. Purification of a C-terminally His-tagged form of the protein allowed the binding of putative permeants to be directly tested: arsenate but not arsenite quenched its intrinsic fluorescence in a concentration-dependent fashion. Fourier transform infrared spectroscopy showed that the purified protein was predominantly alpha-helical. A mutant bearing a single cysteine residue at position 3 retained the ability to confer arsenate resistance, and was accessible to membrane impermeant thiol reagents in intact cells. In conjunction with successful C-terminal tagging with oligohistidine, this finding is consistent with the experimentally-determined topology of the homologous human apical sodium-dependent bile acid transporter, namely 7 transmembrane helices and a periplasmic N-terminus, although the presence of additional transmembrane segments cannot be excluded. Mutation to alanine of the conserved residue proline 190, in the fourth putative transmembrane region, abrogated the ability of the transporter to confer arsenic resistance, but did not prevent arsenate binding. An apparently increased thermal stability is consistent with the mutant being unable to undergo the conformational transitions required for permeant translocation.

Proteomic screening of a cell line model of esophageal carcinogenesis identifies cathepsin D and aldo-keto reductase 1C2 and 1B10 dysregulation in Barrett's esophagus and esophageal adenocarcinoma.

Esophageal adenocarcinoma (EA) incidence is increasing rapidly and is associated with a poor prognosis. Identifying biomarkers of disease development and progression would be invaluable tools to inform clinical practice. Two-dimensional polyacrylamide gel electrophoresis was used to screen 10 esophageal cell lines representing distinct stages in the development of esophageal cancer. Thirty-three proteins were identified by MALDI-TOF-MS which demonstrated differences in expression across the cell lines. Western blotting and qRT-PCR confirmed increased cathepsin D and aldo-keto reductases 1C2 and 1B10 expression in metaplastic and dysplastic cell lines. Expression of these proteins was further assessed in esophageal epithelium from patients with nonerosive (NERD) and erosive gastro-esophageal reflux disease, Barrett's esophagus (BE) and EA. When compared with normal epithelium of NERD patients, (i) cathepsin D mRNA levels demonstrated a stepwise increase in expression (p<0.05) in erosive, metaplastic and EA tissue; (ii) AKR1B10 expression increased (p<0.05) 3- and 9-fold in erosive and Barrett's epithelium, respectively; and (iii) AKR1C2 levels increased (p<0.05) in erosive and Barrett's epithelium, but were reduced (p<0.05) in EA. These proteins may contribute to disease development via effects on apoptosis, transport of bile acids and retinoid metabolism and should be considered as candidates for further mechanistic and clinical investigations.

Defined sites of interaction between subunits E (Vma4p), C (Vma5p), and G (Vma10p) within the stator structure of the vacuolar H+-ATPase.

Vacuolar H(+)-ATPases (V-ATPases) are multi-subunit membrane proteins that couple ATP hydrolysis to the extrusion of protons from the cytoplasm. Although they share a common macromolecular architecture and rotational mechanism with the F(1)F(0)-ATPases, the organization of many of the specialized V-ATPase subunits within this rotary molecular motor remains uncertain. In this study, we have identified sequence segments involved in linking putative stator subunits in the Saccharomyces V-ATPase. Precipitation assays revealed that subunits Vma5p (subunit C) and Vma10p (subunit G), expressed as glutathione-S-transferase fusion proteins in E. coli, are both able to interact strongly with Vma4p (subunit E) expressed in a cell-free system. GST-Vma10p also associated with Vma2p and Vma1p, the core subunits of the ATP-hydrolyzing domain, and was able to self-associate to form a dimer. Mutations within the first 19-residue region of Vma4p, which disrupted interaction with Vma5p in vitro, also prevented the Vma4p polypeptide from restoring V-ATPase function in a complementation assay in vivo. These mutations did not prevent assembly of Vma5p (subunit C) and Vma2p (subunit B) into an inactive complex at the vacuolar membrane, indicating that Vma5p must make multiple interactions involving other V-ATPase subunits. A second, highly conserved region of Vma4p between residues 19 and 38 is involved in binding Vma10p. This region is highly enriched in charged residues, suggesting a role for electrostatic effects in Vma4p-Vma10p interaction. These protein interaction studies show that the N-terminal region of Vma4p is a key factor not only in the stator structure of the V-ATPase rotary molecular motor, but also in mediating interactions with putative regulatory subunits.

MTSEA prevents ligand binding to the human melanocortin-4 receptor by modification of cysteine 130 in transmembrane helix 3.

We have investigated the effect of the sulfhydryl-reactive reagent, methyl thiosulfonate ethylammonium (MTSEA), on ligand binding to the human melanocortin-4 (MC4) receptor stably expressed in HEK-293 cells. MTSEA inhibited binding of the agonist, 125I-NDPalpha-MSH, and the antagonist, 125I-SHU9119, in a concentration-dependent manner. Pre-incubation of cells with either the agonist or antagonist protected from subsequent MTSEA inhibition of radioligand binding. Mutation of Cys130 in transmembrane helix 3 to alanine, whilst not affecting ligand binding, led to a complete loss of the inhibitory effect of MTSEA. Since other types of sulfhydryl-reactive reagents had no effect on ligand binding, we conclude that covalent modification of Cys130 by MTSEA disrupts ligand binding by neutralising a close-by negative charge, most likely on Asp126.

Interaction of inhibitors of the vacuolar H(+)-ATPase with the transmembrane Vo-sector.

The macrolide antibiotic concanamycin A and a designed derivative of 5-(2-indolyl)-2,4-pentadienamide (INDOL0) are potent inhibitors of vacuolar H(+)-ATPases, with IC(50) values in the low and medium nanomolar range, respectively. Interaction of these V-ATPase inhibitors with spin-labeled subunit c in the transmembrane V(o)-sector of the ATPase was studied by using the transport-active 16-kDa proteolipid analogue of subunit c from the hepatopancreas of Nephrops norvegicus. Analogous experiments were also performed with vacuolar membranes from Saccharomyces cerevisiae. Membranous preparations of the Nephrops 16-kDa proteolipid were spin-labeled either on the unique cysteine C54, with a nitroxyl maleimide, or on the functionally essential glutamate E140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). These residues were previously demonstrated to be accessible to lipid. Interaction of the inhibitors with these lipid-exposed residues was studied by using both conventional and saturation transfer EPR spectroscopy. Immobilization of the spin-labeled residues by the inhibitors was observed on both the nanosecond and microsecond time scales. The perturbation by INDOL0 was mostly greater than that by concanamycin A. Qualitatively similar but quantitatively greater effects were obtained with the same spin-label reagents and vacuolar membranes in which the Nephrops 16-kDa proteolipid was expressed in place of the native vma3p proteolipid of yeast. The spin-label immobilization corresponds to a direct interaction of the inhibitors with these intramembranous sites on the protein. A mutational analysis on transmembrane segment 4 known to give resistance to concanamycin A also gave partial resistance to INDOL0. The results are consistent with transmembrane segments 2 and 4 of the 16-kDa putative four-helix bundle, and particularly the functionally essential protonation locus, being involved in the inhibitor binding sites. Inhibition of proton transport may also involve immobilization of the overall rotation of the proteolipid subunit assembly.

Evidence for the proximity of the extreme N-terminus of the neurokinin-2 (NK2) tachykinin receptor to Cys167 in the putative fourth transmembrane helix.

Neurokinin-2 receptor (NK(2)R) binding of [(3)H]-SR48968, a piperidinyl antagonist, is inhibited by methanethiosulfonate ethylammonium (MTSEA) in a time- and concentration-dependent manner. By the systematic alanine replacement of putative loop and transmembrane region cysteine residues (Cys(4), Cys(81), Cys(167), Cys(262), Cys(281), Cys(308), and Cys(309)), we have determined that MTSEA perturbs [(3)H]-SR48968 binding by modifying Cys(167) in transmembrane helix 4. Data were substantiated using glycine, serine, and threonine substitutions of Cys(167). MTSEA preferentially modifies cysteine residues that are in proximity to a negatively charged environment. Hence, aspartate and glutamate residues were systematically substituted with leucine or valine, respectively, and the inhibitory effects of MTSEA on [(3)H]-SR48968 binding were reevaluated to determine those acidic residues close to the MTSEA binding crevice. Most significantly, substitution of Asp(5) in the receptor's extreme N-terminus abolished the effects of MTSEA on [(3)H]-SR48968 binding. Therefore, our data would suggest close association of the extreme N-terminus with the extracellular surfaces of helices 4 and 3 in the NK(2)R in forming a binding crevice for MTSEA. The inhibition of SR48968 binding appears to result from loss of the SR48968 binding conformation of Gln(166) induced by MTSEA when it is coupled to Cys(167). Hence, it is proposed that there is mutually exclusive hydrogen bonding of SR48968 and MTSEA to Gln(166).

Met-204 and Tyr-205 are together important for binding GLP-1 receptor agonists but not their N-terminally truncated analogues.

A mutagenesis study to systematically analyse residues spanning the first extracellular loop of the GLP-1 receptor identified a double mutant, Met-204/Tyr-205-Ala/Ala, which displayed: markedly reduced affinity for the natural agonist GLP-1; slightly reduced affinity for its analogue exendin-4; and unaltered affinity for several N-terminally truncated analogues of GLP-1 and exendin-4. This suggests that the locus is important for the formation of the binding site for the N-terminal residues of peptide agonists.

Expression, purification and spectroscopic studies of full-length Kir3.1 channel C-terminus.

A polypeptide corresponding to the full-length C-terminal cytoplasmic domain of a G-protein-regulated inwardly rectifying potassium channel (Kir3.1) bearing a hexahistidine (His6) tag was produced by DNA recombinant overexpression techniques in Escherichia coli. This permitted the isolation of approximately 5 mg of pure protein per liter of bacterial culture. Further purification by size exclusion chromatography (SEC) of the C-terminal domain revealed that it exists predominantly as a dimer. The secondary structure was estimated using circular dichroism measurements that indicated the presence of approximately 35% beta-sheet and approximately 15% alpha-helix. G-protein betagamma subunits incubated with His-tagged Kir3.1 C-terminal domain, bound to immobilized metal affinity chromatography (IMAC) resin, copurified with the peak of specifically eluted recombinant protein. These observations demonstrate that full-length Kir3.1 C-terminus can be purified in a stable conformation capable of binding proteins known to activate Kir3 channels and may contain elements involved in channel assembly.

A protein chemical approach to channel structure and function: the proton channel of the vacuolar H(+)-ATPase.

The vacuolar H(+)-ATPase provides a channel through which protons can be pumped across the bilayer. It is a complex assembly of about 20 subunits made up from 13 different polypeptide chains. The proton channel is located in the bilayer and therefore must be formed from one or both of the two intramembraneous subunits, called in yeast Vphlp (100 kDa) and Vma3p (16 kDa). Electron microscopy and the use of water soluble and hydrophobic chemical probes in conjunction with mutagenesis to cysteine or glutamic acid residues, suggest that the membrane sector consists of a single Vphlp subunit in association with a hexameric complex of the four-helical bundle Vma3p subunit. This hexamer encloses a large central pore lined by the first transmembrane helix. This pore appears to be impermeable, however; instead, a glutamic acid residue critical to transport function is located on the outside of the hexamer, deeply buried in the membrane and accessible to probes and inhibitors resident in the hydrophobic phase of the bilayer. The stoichiometry and chemistry of inhibitor binding supports the postulate that the mechanism of action involves rotation of the hexamer in the plane of the bilayer. Mutagenesis data on the Vphlp subunit indicate that it is vital to proton transport. It is likely, therefore, that the proton channel is formed at the interface of the Vphlp and Vma3p subunits, the protons moving via a network of interacting charged amino acid side-chains.