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.