Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes

UV-B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV-B of human granulocytes and keratinocytes, the cells involved in UV-triggered skin inflammation. The intracellular ABA concentration increased in UV-B-exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV-B-induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE(2)), and tumor necrosis factor-α (TNF-α) by UV-B irradiated keratinocytes. Lanthionine synthetase C-like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV-B-triggered release of PGE(2), TNF-α, and NO and ROS production. These results indicate that UV-B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation.     

Molecular modeling of lanthionine synthetase component C-like protein 2: a potential target for the discovery of novel type 2 diabetes prophylactics and therapeutics

The rates of type 2 diabetes (T2D) are rising to epidemic proportions in the US and worldwide. While current T2D medications are efficacious, significant side effects have limited their use and availability. Our laboratory has discovered that abscisic acid (ABA) exerts anti-diabetic effects, in part, by activating peroxisome proliferator-activated receptor γ (PPAR γ). However, since ABA does not bind to the ligand-binding domain (LBD) of PPAR γ, the mechanism of activation of PPAR γ by ABA remains unknown. Lanthionine synthetase component C-like protein 2 (LANCL2) was predicted to be a novel target for the binding and signaling of ABA in human granulocytes and rat insulinoma cells. The goal of this study was to determine whether LANCL2 is a molecular target of ABA and other PPAR γ agonists. To this end we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of LANCL1 as a template. Our molecular docking studies predicted that ABA and other PPAR γ agonists (e.g., rosiglitazone and pioglitazone) share a binding site on the surface of LANCL2. The identification of a binding site for PPAR γ agonists will facilitate the high-throughput virtual screening of large compound libraries and may shed new light on alternative mechanisms of PPAR γ activation.     

LANCL2 Is Necessary for Abscisic Acid Binding and Signaling in Human Granulocytes and in Rat Insulinoma Cells

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic β cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38⁺/LANCL2⁺ HeLa transfected with a chimeric G-protein (Gα_q/i) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G_i. Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.The plant hormone abscisic acid (ABA)⁴ plays a fundamental role in the regulation of plant response to environmental conditions, as well as in plant tissue development (1). Although the ABA biosynthetic pathway in plants and in fungi has been largely detailed, identification of the components of the ABA signaling pathway, particularly of the ABA receptor(s), has remained elusive. Two ABA-binding proteins have been identified in different plant tissues: the chloroplast Mg-chelatase subunit H (2) and, most recently, the G-protein-coupled receptor GCR2, which appears to mediate ABA-controlled stomatal closure and seed dormancy in Arabidopsis (3), although the role of GCR2 in the control of seed germination is still controversial (4–6) and its coupling to a G-protein has been refuted on the basis of sequence analyses (7–8). The Mg-chelatase subunit H was proposed as an intracellular ABA receptor, whereas GCR2 is a plasmamembrane protein, which interacts with the only Gα subunit (GPA 1) present in Arabidopsis (3). Although the Mg-chelatase subunit H does not show any significant homology with mammalian proteins, GCR2 shares a high amino acid identity with the mammalian peptide-modifying lanthionine synthetase C-like protein (LANCL) family (7). The animal LANCL protein family in turn shows structural similarities with the prokaryotic lanthionine synthetase component C proteins (9) involved in the synthesis of lanthionine-containing antimicrobial peptides known as lantibiotics (10).The fact that lantibiotics are not produced in animals suggests that LANCL proteins have a different function than prokaryotic lanthionine synthetase component C proteins. The human genome contains three LANCL genes, LANCL1, LANCL2, and LANCL3, located on chromosomes 2 and 7 and the X chromosome, respectively (11, 12). LANCL1 was the first member of the family to be isolated from human erythrocyte membranes (13). The LANCL2 mRNA was identified in a screening procedure for genes whose down-regulation resulted in anticancer drug resistance; thus, LANCL2 was also called testis-specific Adriamicin sensitivity protein (14). The structural assignment for the human LANCL proteins remains controversial. Based on the presence of seven putative transmembrane domains, LANCL1 and -2 were originally described as new G-protein-coupled receptors (GPCR69A and GPR69B, respectively); however, subsequent studies performed on human epithelial cells overexpressing LANCL1 or LANCL2 fused to the green fluorescent protein (LANCL1-GFP and LANCL2-GFP) showed that LANCL1-GFP is mainly found in the cytosol and in the nucleus, whereas LANCL2-GFP is associated with the plasmamembrane through N-terminal myristoylation (15). Similarly, the debate over the structurally related GCR2 is still open (3–6, 8).ABA has recently been demonstrated to be an endogenous pro-inflammatory hormone in human granulocytes, stimulating several cell functions (phagocytosis, reactive oxygen species and nitric oxide production, chemotaxis, and chemokinesis) through a pathway involving a pertussis toxin (PTX)-sensitive G-protein/receptor complex located on the plasmamembrane, cAMP overproduction, protein kinase A-dependent phosphorylation of the human ADP-ribosyl cyclase CD38, and consequent cADP-ribose (cADPR) generation, leading to an increase of the intracellular Ca²⁺ concentration (16; see also Ref. 17). This signaling pathway is similar to that triggered by ABA in plants (18). Fluorescence microscopy confirmed binding of biotinylated ABA to the granulocyte plasmamembrane. Scatchard plot analysis of [³H]ABA binding demonstrated presence of both high and low affinity ABA binding sites (K_d 11 nm and 500 μm, respectively) on human granulocytes (16). Most recently, nanomolar ABA has been shown to stimulate insulin secretion by human and murine pancreatic β cells and by rat insulinoma cell lines through a signaling pathway similar to the one described in human granulocytes (19). The autocrine release of ABA from glucose-stimulated human and rodent insulin-releasing cells, together with the fact that ABA is also produced by activated inflammatory cells, granulocytes (16), and monocytes (20), suggests that this hormone may contribute to the network of cytokine signals exchanged between inflammatory cells and pancreatic β cells, which is increasingly recognized as a fundamental mechanism in the development of the metabolic syndrome and type II diabetes (21–24).Based on (i) the sequence homology between the putative Arabidopsis ABA-receptor protein GCR2 and the human LANCL protein family, and (ii) the reported association of LANCL2 with the plasmamembrane, we investigated whether LANCL2 might be involved in ABA sensing in mammalian ABA-responsive cells. The results obtained indicate that LANCL2 is indeed, (i) required for ABA binding to the plasmamembrane of human granulocytes and (ii) necessary for the activation of the ABA signaling pathway, leading to the stimulation of the functional responses induced by ABA in human granulocytes and in rat insulinoma cells.     

Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes

The phytohormone abscisic acid (ABA), in addition to regulating several important physiological functions in plants, is also produced and released by human granulocytes and monocytes where it stimulates cell activities involved in the innate immune response.Here we describe the properties of an ABA synthetic analog that competes with the hormone for binding to human granulocyte membranes and to purified recombinant LANCL2 (the human ABA receptor) and inhibits several ABA-triggered inflammatory functions of granulocytes and monocytes in vitro: chemotaxis, phagocytosis, reactive oxygen species production and release of prostaglandin E₂ (PGE₂) by human granulocytes, release of PGE₂ and of monocyte chemoattractant protein-1 by human monocytes. This observation provides a proof of principle that ABA antagonists may represent a new class of anti-inflammatory agents.     

Abscisic Acid Transport in Human Erythrocytes

Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress. Recently, ABA has been shown to be present and active also in mammals, where it stimulates the functional activity of innate immune cells, of mesenchymal and hemopoietic stem cells, and insulin-releasing pancreatic β-cells. LANCL2, the ABA receptor in mammalian cells, is a peripheral membrane protein that localizes at the intracellular side of the plasma membrane. Here we investigated the mechanism enabling ABA transport across the plasmamembrane of human red blood cells (RBC). Both influx and efflux of [³H]ABA occur across intact RBC, as detected by radiometric and chromatographic methods. ABA binds specifically to Band 3 (the RBC anion transporter), as determined by labeling of RBC membranes with biotinylated ABA. Proteoliposomes reconstituted with human purified Band 3 transport [³H]ABA and [³⁵S]sulfate, and ABA transport is sensitive to the specific Band 3 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Once inside RBC, ABA stimulates ATP release through the LANCL2-mediated activation of adenylate cyclase. As ATP released from RBC is known to exert a vasodilator response, these results suggest a role for plasma ABA in the regulation of vascular tone.     

Autocrine abscisic acid plays a key role in quartz-induced macrophage activation

Inhalation of quartz induces silicosis, a lung disease where alveolar macrophages release inflammatory mediators, including prostaglandin-E(2) (PGE(2)) and tumor necrosis factor α (TNF-α). Here we report the pivotal role of abscisic acid (ABA), a recently discovered human inflammatory hormone, in silica-induced activation of murine RAW264.7 macrophages and of rat alveolar macrophages (AMs). Stimulation of both RAW264.7 cells and AMs with quartz induced a significant increase of ABA release (5- and 10-fold, respectively), compared to untreated cells. In RAW264.7 cells, autocrine ABA released after quartz stimulation sequentially activates the plasma membrane receptor LANCL2 and NADPH oxidase, generating a Ca(2+) influx resulting in NFκ B nuclear translocation and PGE(2) and TNF-α release (3-, 2-, and 3.5-fold increase, respectively, compared to control, unstimulated cells). Quartz-stimulated RAW264.7 cells silenced for LANCL2 or preincubated with a monoclonal antibody against ABA show an almost complete inhibition of NFκ B nuclear translocation and PGE(2) and TNF-α release compared to controls electroporated with a scramble oligonucleotide or preincubated with an unrelated antibody. AMs showed similar early and late ABA-induced responses as RAW264.7 cells. These findings identify ABA and LANCL2 as key mediators in quartz-induced inflammation, providing possible new targets for antisilicotic therapy.     

Abscisic acid is an endogenous stimulator of insulin release from human pancreatic islets with cyclic ADP ribose as second messenger

Abscisic acid (ABA) is a plant stress hormone recently identified as an endogenous pro-inflammatory cytokine in human granulocytes. Because paracrine signaling between pancreatic beta cells and inflammatory cells is increasingly recognized as a pathogenetic mechanism in the metabolic syndrome and type II diabetes, we investigated the effect of ABA on insulin secretion. Nanomolar ABA increases glucose-stimulated insulin secretion from RIN-m and INS-1 cells and from murine and human pancreatic islets. The signaling cascade triggered by ABA in insulin-releasing cells sequentially involves a pertussis toxin-sensitive G protein, cAMP overproduction, protein kinase A-mediated activation of the ADP-ribosyl cyclase CD38, and cyclic ADP-ribose overproduction. ABA is rapidly produced and released from human islets, RIN-m, and INS-1 cells stimulated with high glucose concentrations. In conclusion, ABA is an endogenous stimulator of insulin secretion in human and murine pancreatic beta cells. Autocrine release of ABA by glucose-stimulated pancreatic beta cells, and the paracrine production of the hormone by activated granulocytes and monocytes suggest that ABA may be involved in the physiology of insulin release as well as in its dysregulation under conditions of inflammation.     

Computational modeling-based discovery of novel classes of anti-inflammatory drugs that target lanthionine synthetase C-like protein 2

Background

Lanthionine synthetase component C-like protein 2 (LANCL2) is a member of the eukaryotic lanthionine synthetase component C-Like protein family involved in signal transduction and insulin sensitization. Recently, LANCL2 is a target for the binding and signaling of abscisic acid (ABA), a plant hormone with anti-diabetic and anti-inflammatory effects.

Methodology/Principal Findings

The goal of this study was to determine the role of LANCL2 as a potential therapeutic target for developing novel drugs and nutraceuticals against inflammatory diseases. Previously, we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of lanthionine synthetase component C-like protein 1 (LANCL1) as a template. Using this model, structure-based virtual screening was performed using compounds from NCI (National Cancer Institute) Diversity Set II, ChemBridge, ZINC natural products, and FDA-approved drugs databases. Several potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked compound (NSC61610) in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Our findings showed that the lead compound, NSC61610, activated peroxisome proliferator-activated receptor gamma in a LANCL2- and adenylate cyclase/cAMP dependent manner in vitro and ameliorated experimental colitis by down-modulating colonic inflammatory gene expression and favoring regulatory T cell responses.

Conclusions/Significance

LANCL2 is a novel therapeutic target for inflammatory diseases. High-throughput, structure-based virtual screening is an effective computational-based drug design method for discovering anti-inflammatory LANCL2-based drug candidates.     

Protein phosphorylation and binding of a 14-3-3 protein in Vicia guard cells in response to ABA

Under drought stress, ABA promotes stomatal closure to prevent water loss. Although protein phosphorylation plays an important role in ABA signaling, little is known about these processes at the biochemical level. In this study, we searched for substrates of protein kinases in ABA signaling through the binding of a 14-3-3 protein to phosphorylated proteins using Vicia guard cell protoplasts. ABA induced binding of a 14-3-3 protein to proteins with molecular masses of 61, 43 and 39 kDa, with the most remarkable signal for the 61 kDa protein. The ABA-induced binding to the 61 kDa protein occurred only in guard cells, and reached a maximum within 3 min at 1 microM ABA. The 61 kDa protein localized in the cytosol. ABA induced the binding of endogenous vf14-3-3a to the 61 kDa protein in guard cells. Autophosphorylation of ABA-activated protein kinase (AAPK), which mediates anion channel activation, and ABA-induced phosphorylation of the 61 kDa protein showed similar time courses and similar sensitivities to the protein kinase inhibitor K-252a. AAPK elicits the binding of the 14-3-3 protein to the 61 kDa protein in vitro when AAPK in guard cells was activated by ABA. The phosphorylation of the 61 kDa protein by ABA was not affected by the NADPH oxidase inhibitor, H(2)O(2), W-7 or EGTA. From these results, we conclude that the 61 kDa protein may be a substrate for AAPK and that the 61 kDa protein is located upstream of H(2)O(2) and Ca(2+), or on Ca(2+)-independent signaling pathways in guard cells.     

Myristoylation of human LanC-like Protein 2 (LANCL2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin

Human LANCL2, also known as Testis-specific Adriamycin Sensitivity Protein (TASP), is a member of the highly conserved and widely distributed lanthionine synthetase component C-like (LANCL) protein family. Expression studies of tagged LANCL2 revealed the major localization to the plasma membrane, juxta-nuclear vesicles, and the nucleus, in contrast to the homologue LANCL1 that was mainly found in the cytosol and nucleus. We identified the unique N-terminus of LANCL2 to function as the membrane anchor and characterized the relevant N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site. Interestingly, the non-myristoylated protein was confined to the nucleus indicating that the myristoylation targets LANCL2 to the plasma membrane. Cholesterol depletion by methyl-β-cyclodextrin caused the partial dissociation of overexpressed LANCL2 from the plasma membrane in vitro, whereas in vivo we observed an enhanced cell detachment from the matrix. We found that overexpressed LANCL2 interacts with the cortical actin cytoskeleton and therefore may play a role in cytoskeleton reorganization and in consequence to cell detachment. Moreover, we confirmed previous data that LANCL2 overexpression enhances the cellular sensitivity to the anticancer drug adriamycin and found that this sensitivity is dependent on the myristoylation and membrane association of LANCL2.     

Protein kinase cascade involved in rapid ABA-signaling in guard cells of Vicia faba

Protein kinases are involved in signal transduction for environmental stress responses. In response to drought and salinity, a 48-kDa protein kinase (AAPK; abscisic acid-activated protein kinase (AAPK) in guard cells is activated by abscisic acid (ABA) and phosphorylates several targets such as the carboxy-terminus of inward-rectifying K+ channel and heterogeneous mRNA binding protein to adopt to the changing environment. The AAPK expressed specifically in guard cells, and recombinant AAPK was phosphorylated only with the extract from ABA-treated guard cells but not from untreated cells. This indicates the presence of an AAPK kinase (AAPKK), which is activated by ABA and phosphorylates AAPK preceding the activation of AAPK. Both AAPK and AAPKK are involved in the protein kinase cascade for the rapid ABA-signaling.     

Identification of features regulating OST1 kinase activity and OST1 function in guard cells

The phytohormone abscisic acid (ABA) mediates drought responses in plants and, in particular, triggers stomatal closure. Snf1-related kinase 2 (SnRK2) proteins from several plant species have been implicated in ABA-signaling pathways. In Arabidopsis (Arabidopsis thaliana) guard cells, OPEN STOMATA 1 (OST1)/SRK2E/SnRK2-6 is a critical positive regulator of ABA signal transduction. A better understanding of the mechanisms responsible for SnRK2 protein kinase activation is thus a major goal toward understanding ABA signal transduction. Here, we report successful purification of OST1 produced in Escherichia coli: The protein is active and autophosphorylates. Using mass spectrometry, we identified five target residues of autophosphorylation in recombinant OST1. Sequence analysis delineates two conserved boxes located in the carboxy-terminal moiety of OST1 after the catalytic domain: the SnRK2-specific box (glutamine-303 to proline-318) and the ABA-specific box (leucine-333 to methionine-362). Site-directed mutagenesis and serial deletions reveal that serine (Ser)-175 in the activation loop and the SnRK2-specific box are critical for the activity of recombinant OST1 kinase. Targeted expression of variants of OST1 kinase in guard cells uncovered additional features that are critical for OST1 function in ABA signaling, although not required for OST1 kinase activity: Ser-7, Ser-18, and Ser-29 and the ABA-specific box. Ser-7, Ser-18, Ser-29, and Ser-43 represent putative targets for regulatory phosphorylation and the ABA-specific box may be a target for the binding of signaling partners in guard cells.     

Myristoylation of human LanC-like protein 2 (LANCL2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin

Human LANCL2, also known as Testis-specific Adriamycin Sensitivity Protein (TASP), is a member of the highly conserved and widely distributed lanthionine synthetase component C-like (LANCL) protein family. Expression studies of tagged LANCL2 revealed the major localization to the plasma membrane, juxta-nuclear vesicles, and the nucleus, in contrast to the homologue LANCL1 that was mainly found in the cytosol and nucleus. We identified the unique N-terminus of LANCL2 to function as the membrane anchor and characterized the relevant N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site. Interestingly, the non-myristoylated protein was confined to the nucleus indicating that the myristoylation targets LANCL2 to the plasma membrane. Cholesterol depletion by methyl-beta-cyclodextrin caused the partial dissociation of overexpressed LANCL2 from the plasma membrane in vitro, whereas in vivo we observed an enhanced cell detachment from the matrix. We found that overexpressed LANCL2 interacts with the cortical actin cytoskeleton and therefore may play a role in cytoskeleton reorganization and in consequence to cell detachment. Moreover, we confirmed previous data that LANCL2 overexpression enhances the cellular sensitivity to the anticancer drug adriamycin and found that this sensitivity is dependent on the myristoylation and membrane association of LANCL2.     

ABA signal transduction at the crossroad of biotic and abiotic stress responses

Abscisic acid (ABA) regulates key processes relevant to seed germination, plant development, and biotic and abiotic stress responses. Abiotic stress conditions such as drought induce ABA biosynthesis initiating the signalling pathways that lead to a number of molecular and cellular responses, among which the best known are the expression of stress-related genes and stomatal closure. Stomatal closure also serves as a mechanism for pathogen defence, thereby acting as a platform for crosstalk between biotic and abiotic stress responses involving ABA action. Significant advances in our understanding of ABA signal transduction have been made with combination of approaches including genetics, biochemistry, electrophysiology and chemical genetics. Molecular components associated with the ABA signalling have been identified, and their relationship in the complex network of interactions is being dissected. We focused on the recent progress in ABA signal transduction, especially those studies related to identification of ABA receptors and downstream components that lead ABA signal to cellular response. In particular, we will describe a pathway model that starts with ABA binding to the PYR/PYL/RCAR family of receptors, followed by inactivation of 2C-type protein phosphatases and activation of SnRK2-type kinases, and eventually lead to activation of ion channels in guard cells and stomatal closure.     

Overexpression of Arabidopsis acyl‐CoA‐binding protein ACBP2 enhances drought tolerance

Arabidopsis thaliana acyl‐CoA‐binding protein 2 (ACBP2) is a stress‐responsive protein that is also important in embryogenesis. Here, we assign a role for ACBP2 in abscisic acid (ABA) signalling during seed germination, seedling development and the drought response. ACBP2 was induced by ABA and drought, and transgenic Arabidopsis overexpressing ACBP2 (ACBP2‐OXs) showed increased sensitivity to ABA treatment during germination and seedling development. ACBP2‐OXs also displayed improved drought tolerance and ABA‐mediated reactive oxygen species (ROS) production in guard cells, thereby promoting stomatal closure, reducing water loss and enhancing drought tolerance. In contrast, acbp2 mutant plants showed decreased sensitivity to ABA in root development and were more sensitive to drought stress. RNA analyses revealed that ACBP2 overexpression up‐regulated the expression of Respiratory Burst Oxidase Homolog D (AtrbohD) and AtrbohF, two NAD(P)H oxidases essential for ABA‐mediated ROS production, whereas the expression of Hypersensitive to ABA1 (HAB1), an important negative regulator in ABA signalling, was down‐regulated. In addition, transgenic plants expressing ACBP2pro:GUS showed beta‐glucuronidase (GUS) staining in guard cells, confirming a role for ACBP2 at the stomata. These observations support a positive role for ACBP2 in promoting ABA signalling in germination, seedling development and the drought response.     

Purification and characterization of a barley aleurone abscisic acid-binding protein

A protein designated ABAP1 and encoded by a novel gene (GenBank accession number AF127388) was purified and shown to specifically bind abscisic acid (ABA). ABAP1 protein is a 472-amino acid polypeptide containing a WW protein interaction domain and is induced by ABA in barley aleurone layers. Polyclonal antiidiotypic antibodies (AB2) cross-reacted with purified ABAP1 and with a corresponding 52-kDa protein associated with membrane fractions of ABA-treated barley aleurones. ABAP1 genes were detected in diverse monocot and dicot species, including wheat, tobacco, alfalfa, garden pea, and oilseed rape. The recombinant ABAP1 protein optimally bound (3)H-(+)-ABA at neutral pH. Denatured ABAP1 protein did not bind (3)H-(+)-ABA, nor did bovine serum albumin. The maximum specific binding as shown by Scatchard plot analysis was 0.8 mol of ABA mol(-1) protein with a linear function of r(2) = 0.94, an indication of one ABA-binding site with a dissociation constant (K(d)) of 28 x 10(-9) m. ABA binding in aleurone plasma membranes showed a maximum binding capacity of 330 nmol of ABA g(-1) protein with a K(d) of 26.5 x 10(-9) m. The similarities in the dissociation constants for ABA binding of the recombinant protein and that of the plasma membranes suggest that the protein within the plasma membrane fraction is the native form of ABAP1. The stereospecificity of ABAP1 was established by the incapability of ABA analogs and metabolites, including (-)-ABA, trans-ABA, phaseic acid, dihydrophaseic acid, and (+)-abscisic acid-glucose ester, to displace (3)H-(+)-ABA bound to ABAP1. However, two ABA precursors, (+)-ABA aldehyde and (+)-ABA alcohol, were able to displace (3)H-(+)-ABA, an indication that the structural requirement of ABAP1 at the C-1 position is not strict. Our data show that ABAP1 exerts high binding affinity for ABA. The interaction is reversible, follows saturation kinetics, and has stereospecificity, thus meeting the criteria for an ABA-binding protein.     

Granulocyte-macrophage colony-stimulating factor binding sites and oxidative metabolism in human granulocytes

We investigated the interaction between GM-CSF and its receptor on human granulocytes and on several human tumor cell lines. Specific high-affinity binding for GM-CSF was characterized by Scatchard plot analysis. The specific radioactivity of the 125I-labeled derivative of rH. GM-CSF was determined by self-displacement analysis and calculated to be 30 microCi/micrograms. The maximum concentration of binding sites (B max) in granulocytes was 40 fmol/mg protein (2,200 molecules GM-CSF bound/cell) and the dissociation constant (KD) was 0.42 nM. No binding sites for GM-CSF were found in two lung cancer cell lines, SCLC-16HV and NCI-N417 or in the urinary bladder carcinoma cell line 5637, whereas the promyelocytic leukemia cell line HL60 was positive for GM-CSF binding. Time course experiments showed maximum binding of GM-CSF in granulocytes after an incubation period of 60 min and a decrease in binding after an incubation period of 2 h. In parallel, we found a maximum biological signal when granulocytes were preincubated for 90 min with GM-CSF, and a decrease after an incubation time of 120 min. Preincubation of the cells with rH. GM-CSF induced an enhancement of the production of activated oxygen species by the cells in response to PMA.