Functional Coupling of the Gαolf Variant XLGαolf with the Human Adenosine A2A Receptor

A recently identified novel Gα_olf variant, XLGα_olf, is shown to functionally couple to the human adenosine A_2A receptor (A_2AR). In Sf9 cells expressing A_2AR, β1, and γ2, co-expression of XLGα_olf increased NECA-induced [³⁵S]GTPγS binding from approximately 130% to 300% of basal levels. Pharmacological characteristics of A_2AR ligands on these cells were evaluated by using [³H]ZM241385- and [³⁵S]GTPγS- binding assays. The rank order of the equilibrium binding constants (K_d or K_i) of adenosine receptor ligands were [³H]ZM241385 ≈ CGS15943 < MRS1220 < < CV1808 ≈ NECA < CGS21680 ≈ adenosine < IBMECA < HEMADO ≈ CPA ≈ CCPA. The rank order of EC₅₀ values for agonists were CV1808 ≈ NECA < adenosine ≈ CGS26180 < IBMECA < HEMADO ≈ CPA ≈ CCPA. This pharmacology is consistent with the literature for A_2AR and suggests that Sf9 cells co-expressing A_2AR, β1, γ2, and XLGα_olf could serve as a heterologous expression system for A_2AR drug screening.     

The Molecular Basis for Recognition of CD1d/α-Galactosylceramide by a Human Non-Vα24 T Cell Receptor

CD1d-mediated presentation of glycolipid antigens to T cells is capable of initiating powerful immune responses that can have a beneficial impact on many diseases. Molecular analyses have recently detailed the lipid antigen recognition strategies utilized by the invariant Vα24-Jα18 TCR rearrangements of iNKT cells, which comprise a subset of the human CD1d-restricted T cell population. In contrast, little is known about how lipid antigens are recognized by functionally distinct CD1d-restricted T cells bearing different TCRα chain rearrangements. Here we present crystallographic and biophysical analyses of α-galactosylceramide (α-GalCer) recognition by a human CD1d-restricted TCR that utilizes a Vα3.1-Jα18 rearrangement and displays a more restricted specificity for α-linked glycolipids than that of iNKT TCRs. Despite having sequence divergence in the CDR1α and CDR2α loops, this TCR employs a convergent recognition strategy to engage CD1d/αGalCer, with a binding affinity (∼2 µM) almost identical to that of an iNKT TCR used in this study. The CDR3α loop, similar in sequence to iNKT-TCRs, engages CD1d/αGalCer in a similar position as that seen with iNKT-TCRs, however fewer actual contacts are made. Instead, the CDR1α loop contributes important contacts to CD1d/αGalCer, with an emphasis on the 4′OH of the galactose headgroup. This is consistent with the inability of Vα24− T cells to respond to α-glucosylceramide, which differs from αGalCer in the position of the 4′OH. These data illustrate how fine specificity for a lipid containing α-linked galactose is achieved by a TCR structurally distinct from that of iNKT cells.     

α2-Adrenergic Agonist-Induced Inhibition of Cyclic Amp Formation in Transfected Cell Lines Using a Microtiter-Based Scintillation Proximity Assay

Abstract

Human embryonic kidney cells (HEK-7) were transfected with the gene for the human α2C receptor (α2C4). Cells were grown in 96-well microtiter plates and cyclic AMP levels were measured by scintillation proximity assay, a modified radioimmunoassay technique. Radioactivity was quantified using a TopCount? Scintillation detector. Cyclic AMP was increased in a dose-dependent manner by the addition of exogenous forskolin. The forskolin-induced enhancement of cyclic AMP was inhibited dose-dependently by the addition of α₂-adrenergic agonists, and this inhibition was blocked by the addition of adrenergic antagonists. The extent of the inhibitory response caused by α₂-adrenoceptor agonists was related to the receptor density in clonal cell lines derived from the transfected parental HEK-7 cells. By using cells grown in microtiter format, and employing the technological advantages of scintillation proximity assay and TopCount? detection, it was possible to simultaneously evaluate the effects of multiple experimental permutations on cellular production of cyclic AMP with minimal disturbance of the cells and minimal and/or automated manipulation of the cyclic AMP formed. This combination of techniques should allow rapid testing of the actions of adrenergic agonists and antagonists on cells transfected with receptors linked to cyclic AMP formation.     

Apelin Ameliorates TNF-α-Induced Reduction of Glycogen Synthesis in the Hepatocytes through G Protein-Coupled Receptor APJ

Apelin, a novel adipokine, is the specific endogenous ligand of G protein-coupled receptor APJ. Consistent with its putative role as an adipokine, apelin has been linked to states of insulin resistance. However, the function of apelin in hepatic insulin resistance, a vital part of insulin resistance, and its underlying mechanisms still remains unclear. Here we define the impacts of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes. Our studies indicate that apelin reversed TNF-α-induced reduction of glycogen synthesis in HepG2 cells, mouse primary hepatocytes and liver tissues of C57BL/6J mice by improving JNK-IRS1-AKT-GSK pathway. Moreover, Western blot revealed that APJ, but not apelin, expressed in the hepatocytes and liver tissues of mice. We found that F13A, a competitive antagonist for G protein-coupled receptor APJ, suppressed the effects of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes, suggesting APJ is involved in the function of apelin. In conclusion, we show novel evidence suggesting that apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ. Apelin appears as a beneficial adipokine with anti-insulin resistance properties, and thus as a promising therapeutic target in metabolic disorders.     

Inhibition of the ERK Pathway Promotes Apoptosis Induced by 2-Chloro-2′-Deoxyadenosine in the B-Cell Leukemia Cell Line Eheb

2-Chloro-2′-deoxyadenosine (CdA) is a nucleoside analogue active in B-cell chronic lymphocytic leukemia (B-CLL). Although the mechanism of action of CdA has been extensively investigated in leukemic cells, the possibility that this nucleoside analogue interacts with the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway has never been explored. In this study, we show that CdA, at concentrations close to the IC₅₀, activated the ERK pathway in the B-cell line EHEB. Because activation of this pathway is assumed to exert anti-apoptotic effect, we combined CdA with inhibitors of the ERK pathway. The latter were found to enhance CdA-induced apoptosis. These results suggest that the efficacy of CdA could be strengthened by combination with inhibitors of the ERK pathway.     

The RING Domain of TRAF2 Plays an Essential Role in the Inhibition of TNFα-Induced Cell Death but Not in the Activation of NF-κB

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of κB kinase (IKK), as well as in inhibiting apoptosis induced by TNFα. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFα-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-ΔR) has been widely used as a dominant negative in transient overexpression systems to block TNFα-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-ΔR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFα-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells efficiently inhibited the TNFα-induced later phase of prolonged JNK activation, yet failed to inhibit TNFα-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-ΔR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFα-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFα and is essential for suppressing the noncanonical nuclear factor κB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFα-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFα-induced cell death.     

Inhibition of the Ethanol-induced Potentiation of α1 Glycine Receptor by a Small Peptide That Interferes with Gβγ Binding

Previous studies indicate that ethanol can modulate glycine receptors (GlyR), in part, through Gβγ interaction with basic residues in the intracellular loop. In this study, we show that a seven-amino acid peptide (RQH_C7), which has the primary structure of a motif in the large intracellular loop of GlyR (GlyR-IL), was able to inhibit the ethanol-elicited potentiation of this channel from 47 ± 2 to 16 ± 4%, without interfering with the effect of Gβγ on GIRK (G protein activated inwardly rectifying potassium channel) activation. RQH_C7 displayed a concentration-dependent effect on ethanol action in evoked and synaptic currents. A fragment of GlyR-IL without the basic amino acids did not interact with Gβγ or inhibit ethanol potentiation of GlyR. In silico analysis using docking and molecular dynamics allowed to identify a region of ∼350Ų involving aspartic acids 186, 228, and 246 in Gβγ where we propose that RQH_C7 binds and exerts its blocking action on the effect of ethanol in GlyR.     

Regulation of the AGS3·Gαi Signaling Complex by a Seven-transmembrane Span Receptor

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind Gα_i free of Gβγ providing an unusual scaffold for the “G-switch” and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3·Gα_i signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Gα_i1 tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of α₂-adrenergic receptors or μ-opioid receptors reduced AGS3-RLuc·Gα_i1-YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by Gβγ sequestration with the carboxyl terminus of GRK2. Gα_i-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to Gα_i and/or Gα_o indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both Gαβγ and GPR-Gα_i offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.     

Selective Inhibition of Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) Identifies a Central Role for VEGFR‐2 in Human Aortic Endothelial Cell Responses to VEGF

Abstract

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR‐family proteins consist of VEGFR‐1/Flt‐1, VEGFR‐2/KDR/Flk‐1, and VEGFR‐3/Flt‐4. Among these, VEGFR‐2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1–3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR‐specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR‐2 tyrosine kinase, ZM323881 (5‐[[7‐(benzyloxy) quinazolin‐4‐yl]amino]‐4‐fluoro‐2‐methylphenol), to explore the role of VEGFR‐2 in endothelial cell function. Consistent with its reported effects on VEGFR‐2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR‐2, but not of VEGFR‐1, epidermal growth factor receptor (EGFR), platelet‐derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR‐1 and VEGFR‐2, but not VEGFR‐3, in the absence or presence of ZM323881. Inhibition of VEGFR‐2 blocked activation of extracellular regulated‐kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR‐1‐specific ligand, placental growth factor (PlGF). Inhibition of VEGFR‐2 also perturbed VEGF‐induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor‐2 inhibition also reversed VEGF‐stimulated phosphorylation of CrkII and its Src homology 2 (SH2)‐binding protein p130^Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR‐2 thus blocked all VEGF‐induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR‐2 is critical for VEGF signaling and/or that VEGFR‐2 may function in a heterodimer with VEGFR‐1 in human vascular endothelial cells.     

Akt/TSC/mTOR Activation by the KSHV G Protein-Coupled Receptor: Emerging Insights into the Molecular Oncogenesis and Treatment of Kaposi’s Sarcoma

Kaposi’s sarcoma (KS) is an enigmatic vascular neoplasm that has reached epidemic proportions in parts of the developing world and is a leading cause of morbidity and mortality among the AIDS population. Unfortunately, KS is still difficult to manage therapeutically, especially in its most advanced clinical manifestations. The recent identification of the KS-associated human herpesvirus (KSHV or HHV8) as its viral etiologic agent has prompted renewed interest in the molecular pathogenesis of this disease. Emerging evidence now points to a single KSHV gene, vGPCR, as essential for KS development, providing a unique opportunity to expose new targets for the treatment of this tumor. In this regard, recent work has identified the Akt/TSC/mTOR signaling cascade as a critical pathway in vGPCR sarcomagenesis. Indeed, pharmacological inhibition of mTOR with rapamycin has shown promising results in preventing vGPCR tumorigenesis in an animal model for KS. These observations are further validated by coincident reports demonstrating the efficacy of rapamycin (sirolimus) as an immunossuppresive and anti-tumoral solution for posttransplant KS patients. Collectively, these data suggest that inhibition of the Akt/TSC/mTOR signaling pathway may provide a novel molecular-based approach for the treatment of patients who currently have a paucity of therapeutic options.     

α-Hemoglobin Stabilizing Protein (AHSP), a Kinetic Scheme of the Action of a Human Mutant,AHSPV56G

A kinetic analysis has been made of the interaction of α-Hb chains with a mutant α-hemoglobin stabilizing protein, AHSP^V56G, which is the first case of an AHSP mutation associated with clinical symptoms of mild thalassemia syndrome. The chaperone AHSP is thought to protect nascent α chains until final binding to the partner β-Hb. Rather than protecting α chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with α-Hb. For both AHSP^WT and AHSP^V56G, the binding to α-Hb is quite rapid relative to the α-β reaction, as expected because the chaperone binding must be quite competitive to complete the α chain folding process before α-Hb binds irreversibly to β-Hb. The main kinetic difference is a dissociation rate of AHSP^V56G·α-Hb some four times faster relative to AHSP·α-Hb. Considering a role of protein folding, the AHSP^V56G apparently does not bind long enough (0.5 s versus 2 s for the WT) to complete the structural modifications. The overall replacement reaction (AHSP·α-Hb + β-Hb → AHSP + αβ) can be quite long, especially if there is an excess of AHSP relative to β-Hb monomers.     

A novel Gαs-binding protein,Gas-2 like 2, facilitates the signaling of the A2A adenosine receptor

The A_2A adenosine receptor (A_2AR) is a G-protein-coupled receptor that contains a long cytoplasmic carboxyl terminus (A_2AR-C). We report here that Gas-2 like 2 (G2L2) is a new interacting partner of A_2AR-C. The interaction between A_2AR and G2L2 was verified by GST pull-down, co-immunoprecipitation, immunocytochemical staining, and fluorescence resonance energy transfer. Expression of G2L2 increased the intracellular cAMP content evoked by A_2AR in an A_2AR-C-dependent manner. Immunoprecipitation and pull-down assays demonstrated that G2L2 selectively bound to A_2AR-C and the inactive form of Gαs to facilitate the recruitment of the trimeric G protein complex to the proximal position of A_2AR for efficient activation. Collectively, G2L2 is a new effector that controls the action of A_2AR by modulating its ability to regulate the Gαs-mediated cAMP contents.     

Application of a Novel Method for the Comparison of DNA Binding Parameters of the Two Human Thyroid Hormone Receptor Subtypes hTRα1 and hTRβ1

Abstract

DNA-binding characteristics of the two human thyroid hormone receptors α₁ and β₁ (hTRα₁ and hTRβ₁) were studied by applying the recently developed solid-phase scintillation technique. Biotinylated double stranded oligonucleotides containing thyroid hormone response elements (TRE) were immobilized to streptavidin coated scintillating microtiter plates. The TRE:s consisted of variants of the consensus core sequence AGGTCA as monomers or as dimers in direct repeats. Equilibrium binding of radioactive labelled hTRα₁ and hTRβ₁ were studied. Metabolically ³⁵S-labelled hTR (in vitro translated cDNA) as well as hTR expressed in the baculovirus-system and labelled with ¹²⁵I-triiodothyronine (¹²⁵I-T₃) were used. In binding saturation experiments, the affinity for the TRE:s investigated did not differ greatly between hTRα₁ and hTRβ₁. No significant effects of T₃ on the amplitude of DNA binding of either hTRα₁ or hTRβ₁ to the single site response elements could be demonstrated. Receptor binding to direct repeats was stimulated by the hormone in the case of the hTRβ₁. The hTRα₁ binding to direct repeats was not significantly altered by T₃. The single site octameric variant of a TRE. TAAGGTCA, was observed to bind tighter to the hTR:s as compared to the hexameric variant AGGTCA. In the binding competition format, with one response element immobilized and other (un-biotinylated) added to the reaction mixture, there was a larger dymanic range for the affinity constants (IC50) as compared to the affinity constants (K_d) obtained in the binding saturation experiments. The present quantitative results confirm previous reports obtained with qualitative methods like gel shift assays. The method described here is applicable in basic research concerning characterisation of DNA binding of nuclear receptors. It also lends itself to automatization in high capacity formats.     

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A_2A receptor (A_2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A_2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A_2AR C-terminus from 412 to 316 amino acids (A_2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A_2AR levels. As cAMP production is downstream of the first activation event—coupling of G protein to its receptor—investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gα_s proteins, we characterize the association of A_2AR and A_2AΔ316R to Gα_s with and without GDP or GTPγs using surface plasmon resonance (SPR). Gα_s affinity for A_2AR was greatest for apo-Gα_s, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A_2AR C-terminus (A_2AΔ316R) decreased the affinity of the unliganded receptor for Gα_s by ～20%, suggesting small changes to binding can greatly impact downstream signaling.     

Physical and Epitope Analysis of a Recombinant Human T-Cell Receptor Vα/Vβ Construct Support the Similarity to Immunoglobulin

The genetic organization and protein structure of T-cell receptors (TCR) and immunoglobulins (Ig) are remarkably similar. Through recombinant, physical, and peptide-based immunological studies we demonstrated that rabbit antisera generated against a recombinant single-chain TCR (scTCR) react with defined peptide epitopes of their constituent TCR and chains. These antisera cross-react with the light-chain Mcg as well as with peptides duplicating its covalent structure. Conversely, rabbit antisera generated to human light chains cross-reacted with the recombinant scTCR. Rabbit anti- antibodies purified on an scTCR affinity column bound to T-cell lines and to T and B lymphocytes from peripheral blood. Circular dichroism analysis demonstrated plots characteristic of -sheets for both Mcg and recombinant scTCR. Antisera directed against TCR -chain synthetic peptides reacted with scTCR, Mcg light-chain protein, synthetic peptides from regions of sequence homology in -chains, and Mcg. Based upon this homology and the serological cross-reactions which reflect conformational determinants, we suggest that the V/V antigen-binding domain of this particular monoclonal scTCR construct is substantially similar to the conformational structure of light chains.     

Cinnamic Acid Protects the Nigrostriatum in a Mouse Model of Parkinson’s Disease via Peroxisome Proliferator-Activated Receptorα

Neurochemical Research - Parkinson’s disease (PD) is the second most common devastating human neurodegenerative disorder and despite intense investigation, no effective therapy is available...