Biological Consequences of Vanadium Effects on Formation of Reactive Oxygen Species and Lipid Peroxidation.

Lipid peroxidation (LPO), a process that affects human health, can be induced by exposure to vanadium salts and compounds. LPO is often exacerbated by oxidation stress, with some forms of vanadium providing protective effects. The LPO reaction involves the oxidation of the alkene bonds, primarily in polyunsaturated fatty acids, in a chain reaction to form radical and reactive oxygen species (ROS).

Targeting Epigenetic Changes Mediated by Members of the SMYD Family of Lysine Methyltransferases.

A comprehensive understanding of the mechanisms involved in epigenetic changes in gene expression is essential to the clinical management of diseases linked to the SMYD family of lysine methyltransferases. The five known SMYD enzymes catalyze the transfer of donor methyl groups from S-adenosylmethionine (SAM) to specific lysines on histones and non-histone substrates. SMYDs family members have distinct tissue distributions and tissue-specific functions, including regulation of development, cell differentiation, and embryogenesis.

Glycoprotein G-protein Coupled Receptors in Disease: Luteinizing Hormone Receptors and Follicle Stimulating Hormone Receptors.

Signal transduction by luteinizing hormone receptors (LHRs) and follicle-stimulating hormone receptors (FSHRs) is essential for the successful reproduction of human beings. Both receptors and the thyroid-stimulating hormone receptor are members of a subset of G-protein coupled receptors (GPCRs) described as the glycoprotein hormone receptors. Their ligands, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and a structurally related hormone produced in pregnancy, human chorionic gonadotropin (hCG), are large protein hormones that are extensively glycosylated.

Polyoxometalates function as indirect activators of a G protein-coupled receptor.

The luteinizing hormone receptor (LHR), a G protein-coupled receptor (GPCRs), can initiate signaling in the presence of some vanadium-containing compounds as a result of vanadium compound interactions with the membrane lipids and/or the cell membrane lipid interface. The ability of LHR expressed in CHO cells to initiate signaling in the presence of highly charged and water-soluble polyoxovanadates (POV) including Na3[H3V10O28] (V10) and two mixed-valence heteropolyoxovanadates, K(NH4)4[H6V14O38(PO4)]·11H2O (V14) and [(CH3)4N]6[V15O36(Cl)] (V15), was investigated here. Interactions of the vanadium compounds with CHO cells decreased the packing of membrane lipids, drove aggregation of LHR and increased signal transduction by LHR.

Effects of vanadium(IV) compounds on plasma membrane lipids lead to G protein-coupled receptor signal transduction.

Luteinizing hormone receptors (LHR), expressed at physiological numbers <30,000 receptors per cell, translocate to and signal within membrane rafts following binding of human chorionic gonadotropin (hCG). Similarly LHR signal in cells when treated with bis(maltolato)oxovanadium(IV) (BMOV), bis(ethylmaltolato)oxovanadium(IV) (BEOV) or VOSO, which decrease membrane lipid packing. Overexpressed LHR (>85,000 receptors per cell) are found in larger clusters in polarized homo-transfer fluorescence resonance energy transfer (homo-FRET) studies that were not affected by either hCG or vanadium compounds.

Fluorescence Observation of Single-Cell cAMP Signaling by G Protein-Coupled Receptors.

We present complementary flow cytometric and microscopic imaging methods, both utilizing a membrane-targeted cAMP sensor protein ICUE3, to examine hormone-dependent signaling by the luteinizing hormone (LH) receptor in individual cells. This receptor, a seven transmembrane domain protein belonging to the GPCR family, signals by activating adenylate cyclase to increase cAMP levels. The ICUE3 sensor protein exhibits fluorescence energy transfer between its CFP and YFP moieties and the ratio of CFP emission to YFP sensitized emission (YFPSE) increases with cAMP concentration.

Continuous Fluorescence Depletion Anisotropy Measurement of Protein Rotation.

Protein rotation in viscous environments can be measured by fluorescence depletion anisotropy (FDA) which combines long lifetimes of chromophore triplet states with the sensitivity of fluorescence excitation and detection. FDA achieves sensitivity well beyond that attainable by the more common technique of time-resolved phosphorescence anisotropy (TPA). We have now combined benefits of both time-domain and frequency-domain FDA into a single continuous technique (CFDA).

Luteinizing hormone receptors are confined in mesoscale plasma membrane microdomains throughout recovery from receptor desensitization.

We examined the involvement of membrane microdomains during human luteinizing hormone (LH) receptor recovery from receptor desensitization after removal of bound hormone. Lateral motions of individual desensitized LH receptors expressed on the surface of Chinese hamster ovary cells and transient association of these receptors with detergent-resistant membrane (DRM) microdomains isolated using isopycnic sucrose gradient ultracentrifugation were assessed. Single particle tracking experiments showed untreated individual LH receptors to be confined within cell-surface membrane compartments with an average diameter of 199 ± 17 nm and associated with membrane fractions characteristic of bulk plasma membrane.

Raft localization of type I Fcε receptor and degranulation of RBL-2H3 cells exposed to decavanadate, a structural model for V2O5.

Vanadium oxides (VOs) have been identified as low molecular weight sensitizing agents associated with occupational asthma and compromised pulmonary immunocompetence. Symptoms of adult onset asthma result, in part, from increased signal transduction by Type I Fcε receptors (FcεRI) leading to release of vasoactive compounds including histamine from mast cells. Exposure to (VOs) typically occurs in the form of particles which are insoluble.

The anti-diabetic bis(maltolato)oxovanadium(IV) decreases lipid order while increasing insulin receptor localization in membrane microdomains.

The effects of treatment with bis(maltolato)oxovanadium(IV) (BMOV) on protein localization in membrane microdomains were investigated by comparing the effects of insulin and treatment with BMOV on the lateral motions and compartmentalization of individual insulin receptors (IR). In addition, effects of insulin and BMOV on the association of IR, phosphorylated IR (pIR) and phosphorylated insulin receptor substrate-1 (pIRS-1) with chemically-isolated plasma membrane microdomains on rat basophilic leukemia (RBL-2H3) cells were evaluated. Single particle tracking experiments indicate that individual quantum dot-labeled IR on RBL-2H3 cells exhibit relatively unrestricted lateral diffusion of approximately 1 × 10(-10) cm(2) s(-1) and are confined in approximately 475 nm diameter cell-surface membrane compartments.

Insulin receptors and downstream substrates associate with membrane microdomains after treatment with insulin or chromium(III) picolinate.

We have examined the association of insulin receptors (IR) and downstream signaling molecules with membrane microdomains in rat basophilic leukemia (RBL-2H3) cells following treatment with insulin or tris(2-pyridinecarbxylato)chromium(III) (Cr(pic)(3)). Single-particle tracking demonstrated that individual IR on these cells exhibited reduced lateral diffusion and increased confinement within 100 nm-scale membrane compartments after treatment with either 200 nM insulin or 10 μM Cr(pic)(3). These treatments also increased the association of native IR, phosphorylated insulin receptor substrate 1 and phosphorylated AKT with detergent-resistant membrane microdomains of characteristically high buoyancy.

Actin-dependent clustering of insulin receptors in membrane microdomains.

Recent evidence suggests that, after binding insulin, insulin receptors (IR) interact with specialized, cholesterol-containing, membrane microdomains and components of the actin cytoskeleton. Using single particle tracking techniques, we examined how binding of insulin, depletion of membrane cholesterol and disruption of actin filaments affect the lateral diffusion of individual quantum dot-labeled native IR on live rat basophilic leukemia 2H3 cells. We also examined the effects of similar treatments on IR clustering and multivalent insulin binding on these cells using both photon counting histogram analysis and polarization-based fluorescence resonance energy homo-transfer imaging.

Fluorescence correlation spectroscopic examination of insulin and insulin-like growth factor 1 binding to live cells.

We used fluorescence correlation spectroscopy to examine the binding of insulin, insulin-like growth factor 1 (IGF1) and anti-receptor antibodies to insulin receptors (IR) and IGF1 receptors (IGF1R) on individual 2H3 rat basophilic leukemia cells. Experiments revealed two distinct classes of insulin binding sites with K(D) of 0.11 nM and 75 nM, respectively.

Restricted lateral diffusion of luteinizing hormone receptors in membrane microdomains.

Single particle tracking was used to evaluate lateral motions of individual FLAG-tagged human luteinizing hormone (LH) receptors expressed on CHO cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared with LH receptors on untreated cells, LH receptors on cells treated with 100 nm hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Similar to LH receptors labeled with Au-hCG, LH receptors labeled with gold-deglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nm hCG.

Antidiabetic vanadium compound and membrane interfaces: interface-facilitated metal complex hydrolysis.

The interactions of metabolites of the antidiabetic vanadium-containing drug bis(maltolato)oxovanadium(IV) (BMOV) with lipid interface model systems were investigated and the results were used to describe a potentially novel mechanism by which these compounds initiate membrane-receptor-mediated signal transduction. Specifically, spectroscopic studies probed the BMOV oxidation and hydrolysis product interaction with interfaces created from cetyltrimethylammonium bromide (CTAB) which mimics the positively charged head group on phosphatidylcholine. (1)H and (51)V NMR spectroscopies were used to determine the location of the dioxobis(maltolato)oxovanadate(V) and the maltol ligand in micelles and reverse micelles by measuring changes in the chemical shift, signal linewidth, and species distribution.

Effects of vanadium-containing compounds on membrane lipids and on microdomains used in receptor-mediated signaling.

There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium (V) compounds which have been shown in animal models to enhance insulin responsiveness.

Constitutively-active human LH receptors are self-associated and located in rafts.

Several naturally occurring mutations in human luteinizing hormone receptors (LHR) at position 578 are associated with constitutive activation of the receptor. To determine whether human LHRs that signal in the absence of ligand are self-associated, fluorescence resonance energy transfer (FRET) between receptors was evaluated. Values for FRET between wild type LHR in the absence of ligand were less than 1% and increased significantly to over 11% after exposure to hCG.

Fluorescence photobleaching recovery using total internal reflection interference fringes.

Lateral diffusion measurements on cell membrane molecules, most commonly accomplished through fluorescence photobleaching recovery (FPR or FRAP), provide information on such molecules' size, environment, and participation in intermolecular interactions. However, difficulties arise in FPR measurements of lateral dynamics of materials, such as visible fluorescent protein (VFP) fusion proteins, where fluorescent intracellular species contribute to the fluorescence recovery signal and thus distort measurements intended to reflect surface molecules only. A new method helps eliminate these difficulties.

Compartmentalization of the Type I Fc epsilon receptor and MAFA on mast cell membranes.

The Mast cell Function-associated Antigen (MAFA) is a membrane glycoprotein on rat mast cells (RBL-2H3) expressed at a ratio of approximately 1:30 with respect to the Type I Fc epsilon receptor (Fc epsilon RI). Despite this stoichiometry, clustering MAFA by its specific mAb G63 substantially inhibits secretion of both granular and de novo synthesized mediators induced upon Fc epsilon RI aggregation. Since the Fc epsilon RIs apparently signal from within raft micro-environments, we investigated possible co-localization of MAFA within these membrane compartments containing aggregated Fc epsilon RI.

Luteinizing hormone receptors translocate to plasma membrane microdomains after binding of human chorionic gonadotropin.

Receptor-mediated signal transduction by G protein-coupled receptors can involve redistribution of plasma membrane receptors into membrane structures that are characterized by insolubility in Triton X-100 and low buoyant density in sucrose gradients. Here we describe the translocation of wild-type (wt) rat LH receptors (LHR-wt) from the bulk membrane into membrane microdomains (rafts) after the binding of human chorionic gonadotropin (hCG). In sucrose gradient ultracentrifugation of plasma membranes from cells stably expressing FLAG-tagged LHR-wt, receptors were located in high-density membrane fractions before binding of hormone and in low-density fractions after hCG treatment.

High probe intensity photobleaching measurement of lateral diffusion in cell membranes.

Lateral diffusion measurements, most commonly accomplished through Fluorescence Photobleaching Recovery (FPR or FRAP), provide important information on cell membrane molecules' size, environment and participation in intermolecular interactions. However, serious difficulties arise when these techniques are applied to weakly expressed proteins of either of two types: fusions of membrane receptors with visible fluorescent proteins or membrane molecules on autofluorescent cells. To achieve adequate sensitivity in these cases, techniques such as interference fringe FPR are needed.

Chimeric GnRH-LH receptors and LH receptors lacking C-terminus palmitoylation sites do not localize to plasma membrane rafts.

Luteinizing hormone and gonadotropin releasing hormone receptors (LHR and GnRHR, respectively) are G protein-coupled receptors with important functions in reproduction. We have developed chimeric GnRHR-LHR that contain the full GnRHR coupled to various forms of the LH receptor C-terminus to explore the role of the LH receptor C-terminus in raft localization of the receptor and signaling. Addition of the full-length LHR C-terminus to GnRHR resulted in localization of the resting chimeric receptor in the bulk membrane rather than plasma membrane rafts as has been reported for the wild-type GnRHR [A.

Membrane organization of luteinizing hormone receptors differs between actively signaling and desensitized receptors.

Signaling by the luteinizing hormone/choriogonadotropin receptor (LHR) is of considerable interest because of its requirement for successful reproduction. Time-resolved phosphorescence anisotropy and fluorescence resonance energy transfer were used to investigate the organization of endogenous LHRs in porcine follicular membranes in two distinct signaling states, active and desensitized. Desensitized LHRs exhibited approximately 3-fold slower rotational correlation times compared with active LHRs (59 +/- 4 and 21 +/- 9 mus, respectively), suggesting that with agonist-dependent desensitization the receptors are organized into larger protein complexes.

Self-association and raft localization of functional luteinizing hormone receptors.

Membrane motions of LH receptors following binding of hormone agonists are consistent with hormone-driven aggregation. It is increasingly apparent that G protein-coupled receptors, including the LH receptor, are engaged in dynamic interactions with one another and other membrane components. These interactions are governed, in part, by a number of factors including whether the receptor has bound ligand, whether the receptor is capable of transducing a hormone-mediated signal, and the nature of the membrane environment within which the receptor is found.

Interactions of the mast cell function-associated antigen with the type I Fcepsilon receptor.

Clustering the mast cell function-associated antigen (MAFA), a membrane glycoprotein expressed on 2H3 cells, by its specific monoclonal antibody G63 substantially inhibits secretion normally triggered by aggregating these cells' Type I Fcepsilon receptor (FcepsilonRI). To explore possible MAFA-FcepsilonRI interactions giving rise to this inhibition, we have studied by time-resolved phosphorescence anisotropy the rotational behavior of both MAFA and FcepsilonRI as ligated by various reagents involved in FcepsilonRI-induced degranulation and MAFA-mediated inhibition thereof. From 4 to 37 degrees C the rotational correlation times (mean+/-S.