2B4 (CD244) is expressed and functional on human eosinophils

Eosinophils are present in parasitic, allergic, various immunological, and malignant disorders as well as in a variety of idiopathic hypereosinophilic syndromes. However, their exact role in some of these conditions remains elusive. They can be activated both in vivo and in vitro by various agonists, such as Igs, lipid mediators, and cytokines. By phenotyping the surface of the eosinophils, it may be possible to better define their function(s) in different pathophysiological settings. In the present work we screened eosinophils with a panel of Abs recognizing CD2 subfamily receptors usually present on a number of hemopoietic cells. We have demonstrated that human peripheral blood eosinophils, but not basophils or neutrophils, express NTB-A. In addition eosinophils express 2B4, CD84, CD58, and CD48, but not signaling lymphocytic activation molecule or CD2, on their surface (FACS). Cross-linking of 2B4 on eosinophils elicited a significant release of eosinophil peroxidase (30 min), IFN-gamma, and IL-4 (18 h). Moreover, activation of eosinophils via 2B4 induced eosinophil-mediated cytotoxicity toward two malignant cell lines, i.e., mouse mastocytoma P815 and EBV-infected 721.221 B cell lines. Cross-linking of 2B4 on the surface of eosinophils or pervenadate treatment elicited ERK and tyrosine phosphorylation, respectively. Furthermore, we showed that eosinophils express slam-associated protein. The demonstration that human eosinophils express a functional 2B4 receptor indicates a broader role for these cells in health and disease.     

Stromal cell derived factor-2 (Sdf2): A novel protein expressed in mouse

The stromal derived factor (SDFs) family comprises a group of molecules generated by stromal cells. SDF1 and SDF4 are chemokines; SDF2 and SDF5 are not yet functionally and structurally defined. In human and mouse, Sdf2 has a paralogous gene, Sdf2l1, whose protein sequences are 78% similar and 68% identical. Human SDF2L1 is an endoplasmic reticulum-stress inducible-gene. In Arabidopsis thaliana, SDF2-like (39% and 37% amino acid sequence identity with Mus musculus Sdf2 and Sdf2l1) has also been implicated in activating the UPR in ER-stress. Here we have cloned, expressed and purified recombinant Sdf2 and raised an anti-Sdf2 antibody. We demonstrated that the protein is expressed in several tissues and is localized in the endoplasmic reticulum. We suggest that Sdf2, initially predicted as a secretory protein because it lacks the canonical ER retention signals in its C-terminal, could be ER-resident through accessory binding proteins or other amino acid sequence motifs, as suggested for the homolog protein SDF2-like. Furthermore, the crystal structure of SDF2-like from Arabidopsis thaliana is a typical β-trefoil containing three MIR motifs; all hydrophobic residues considered important for maintaining the bottom layer of the β-trefoil barrel seem to be conserved in the Sdf2 family. Multiple alignment using 43 sequences for SDF2 and 38 for SDF2L1 paralogous families also revealed a very similar residue conservation profile. Comparing the amino acid sequence and predicted 3D structure with other Sdf2-like proteins we suggest a role of mouse Sdf2 in the Unfolded Protein Response and ER-stress, similar to that of Sdf2l1 from human and mouse and SDF2-like from Arabidopsis thaliana. Chronic ER stress has been associated with many human diseases including cancer and diabetes. Identification of new factors associated with the ER stress pathway can help to identify and define key targets of this response.     

The second PGD(2) receptor CRTH2: structure,properties, and functions in leukocytes

Prostaglandin (PG) D(2) plays a broad range of physiological and pathophysiological functions. Until just a few years ago, it was thought that most of the biological actions of PGD(2) are mediated via the classical PGD(2) receptor DP. Recently, we identified a second PGD(2) receptor, chemoattractant receptor-homologous molecule expressed on T helper (Th)2 cells (CRTH2), with different functions relative to DP. Here, we review the recent findings on the structure, tissue distribution, ligand selectivity, signalling pathways, and functions in leukocytes of this receptor. The data suggest that the PGD(2)/CRTH2 system play important roles in allergic inflammation through its stimulatory effects on Th2 cells, eosinophils, and basophils.     

Functional characterization of a novel serotonin receptor (5-HTap2) expressed in the CNS of Aplysia californica

Serotonin has been shown to be a neuromodulator in the Aplysia californica CNS. The diversity of serotonin actions is due to the existence of several different receptor subtypes. In this study we report the cloning of a full-length cDNA, coding for a novel serotonin receptor (5-HTap2). The receptor protein bears the characteristics of G protein-coupled receptors. It shares 68% and 34% of its amino acid sequence identity with the 5-HTlym receptor from Lymnaea stagnalis and the mammalian 5-HT1A receptor, respectively. When transfected in HEK 293 cells, 5-HTap2 was negatively coupled to adenylate cyclase. Ligand binding analysis indicated that the order of potencies of various drugs for the inhibition of [3H]LSD binding was: methiothepin > metergoline > 5-CT > PAPP > 5-HT > ketanserin > NAN-190 > 8-OH-DPAT > clozapine. RT-PCR amplification of RNA isolated from different tissues indicated that this receptor is expressed in the CNS and in bag cells. The expression of 5-HTap2 restricted to the CNS suggests an important role for this receptor in the modulation of neuronal functions in Aplysia. Moreover, the high expression of 5-HTap2 in the bag cells, associated with its pharmacological profile, suggests that this receptor may be implicated in modulating the afterdischarge during the egg-laying behavior.     

A novel cell surface antigen, 4C8, is expressed on human eosinophils

BACKGROUND: A novel monoclonal antibody, anti-4C8, reacted with human peripheral lymphocytes and monocytes but not with neutrophils. In this study, we investigated whether the 4C8 antigen is expressed on human peripheral eosinophils. METHODS: Expression of the 4C8 antigen on eosinophils was analyzed by flow cytometry and molecular analysis of the antigen was performed with eosinophils by Western blotting. RESULTS: Among human peripheral granulocytes, the 4C8 antigen was expressed on CD16-negative cells but not on CD16-positive cells. The 4C8 antigen also appeared to be expressed on eosinophils. To confirm the latter finding, eosinophils were purified from peripheral blood. On flow cytometric analysis, anti-4C8 antibody reacted with purified eosinophils. On Western blotting analysis, anti-4C8 reacted with a single band of 80 kDa in lysates from purified eosinophils. The correlation between the percentage of eosinophils determined by May-Giemsa staining and the percentage of 4C8-positive/CD16-negative cells among granulocytes was good (r = 0.91, P < 0.0001). CONCLUSIONS: Only a few cell surface antigens are available to distinguish human peripheral eosinophils from neutrophils. The novel cell surface antigen, 4C8, is a useful new marker of human eosinophils.     

Rat basophilic leukemia cells (RBL-2H3) generate prostaglandin D2 (PGD2) after regulated upon activation, normal T-cell expressed and secreted (RANTES) activation

Increasing evidence indicates that local neurogenic inflammation, possibly in response to different stimuli, may be involved in sensory nerve sensitization, migraine generation and some other precipitating events leading to neuronal dysfunction in the brain. In addition, mast cells generate eicosanoids that are linked to asthma and other inflammatory diseases. Regulated upon activation, normal T-cell expressed and secreted (RANTES) is a small protein and a prototype member of the CC chemokine-beta subfamily with chemoattractant and inflammatory properties. In this study we used the RBL-2H3 cell line to determine whether or not these cells generate prostaglandin D2 (PGD2) after treatment with RANTES. After 4 hours of incubation, RBL-2H3 cells cultured with RANTES at 20 ng/mL released large amounts of PGD2 in a dose-response manner compared to control. Moreover, RBL-treated RANTES generated a large quantity of histamine. Our study confirms once again the proinflammatory action of RANTES, in this case acting on the stimulation of the arachidonic acid cascade product PGD2.     

G Protein-coupled receptor kinase 2 (GRK2): A novel modulator of insulin resistance

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key, integrative node in many signalling pathways. Besides its canonical role in the modulation of the signalling mediated by many G protein-coupled receptors (GPCR), this protein can display a very complex network of functional interactions with a variety of signal transduction partners, in a stimulus, cell type, or context-specific way. We review herein recent data showing that GRK2 can regulate insulin-triggered transduction cascades at different levels and that this protein plays a relevant role in insulin resistance and obesity in vivo, what uncovers GRK2 as a potential therapeutic target in the treatment of these disorders.     

Purification and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli.

The adenosine A(2a) receptor belongs to the seven transmembrane helix G-protein-coupled receptor family, is abundant in striatum, vasculature and platelets and is involved in several physiological processes such as blood pressure regulation and protection of cells during anoxia. For structural and biophysical studies we have expressed the human adenosine A(2a) receptor (hA2aR) at high levels inserted into the Escherichia coli inner membrane, and established a purification scheme. Expression was in fusion with the periplasmic maltose-binding protein to levels of 10-20 nmol of receptor per L of culture, as detected with the specific antagonist ligand [(3)H]ZM241385. As the receptor C-terminus was proteolyzed upon solubilization, a protease-resistant but still functional receptor was created by truncation to Ala316. Addition of the sterol, cholesteryl hemisuccinate, allowed a stable preparation of functional hA2aR solubilized in dodecylmaltoside to be obtained, and, increased the stability of the receptor solubilized in other alkylmaltosides. Purification to homogeneity was achieved in three steps, including ligand affinity chromatography based on the antagonist xanthine amine congener. The purified hA2aR fusion protein bound [(3)H]ZM241385 with a K(d) of 0.19 nm and an average B(max) of 13.7 nmol x mg(-1) that suggests 100% functionality. Agonist affinities for the purified solubilized receptor were higher than those for the membrane-bound form. Sufficient pure, functional hA2aR can now be prepared regularly for structural studies.     

CRTH2, an orphan receptor of T-helper-2-cells, is expressed on basophils and eosinophils and responds to mast cell-derived factor(s).

We have recently cloned a putative chemoattractant receptor, named CRTH2, which is preferentially expressed on human T-helper- (Th) 2 but not Th1 cells. In this study, we demonstrated that CRTH2 is also highly expressed on peripheral blood basophils and eosinophils. Our search for a CRTH2 ligand identified mast cells as the possible producers of a ligand. When stimulated with an anti-FcepsilonR1 antibody, cord blood-derived mast cells secreted factor(s) that induced Ca(2+) mobilization in CRTH2-expressing K562 cells but not in mock transfected cells. These findings implied the involvement of CRTH2 in mast cell-mediated immune responses such as allergic reactions.     

A novel IgA receptor expressed on a murine B cell lymphoma.

The specificity and properties of a novel IgA receptor expressed on the surface of a tissue culture-adapted B cell lymphoma, T560, that originated in murine gut-associated lymphoid tissue, have been explored. Like the IgA receptors of murine T and splenic B cells studied by others, the T560 IgA receptor is trypsin sensitive and neuraminidase resistant and is up-regulated on T560 cells by exposing them overnight to high concentrations of polymeric IgA. Unlike them, the T560 IgA receptor is inhibited by low concentrations of IgM and high concentrations of IgG2a and IgG2b, binds at pH 4.0 but not at pH 8.0, is down-regulated by activation of protein kinase C and is sensitive to phosphatidylinositol-specific phospholipase C, indicating that it is glycosyl phosphatidylinositol-linked to the cell membrane. It is not a cell-bound form of galactosyl transferase, does not appear to bind to Ig through carbohydrate residues and does not react specifically with antibody to secretory component. It may be a completely new, cross-reactive receptor, perhaps related in some way to the polymeric Ig receptor or to the receptor for IgA expressed on the apical surface of Peyer's patch M cells, which is known to cross-react with IgG. Alternatively, it may be homologous to the highly IgA-specific Fc alpha R of T cells but, perhaps because of its glycosyl phosphatidylinositol linker, may have an ability to move and interact with other Ig receptors on the cell surface such that Ig bound to them are cross-inhibitory.     

Synthesis of alkyne derivatives of a novel triazolopyrazine as A(2A) adenosine receptor antagonists

A novel [1,2,4]triazolo[1,5-a]pyrazine core was synthesized and coupled with terminal acetylenes. The structure-activity relationship of the alkynes from this novel template was studied for their in vitro and in vivo adenosine A(2A) receptor antagonism. Selected compounds from this series were shown to have potent in vitro and in vivo activities against adenosine A(2A) receptor. Compound 12, in particular, was found to be orally active at 3mg/kg in both a mouse catalepsy model and a 6-hydroxydopamine-lesioned rat model.     

The zymogen granule protein 2 (GP2) binds to scavenger receptor expressed on endothelial cells I (SREC-I)

The pancreatic zymogen granule membrane protein (GP2) is expressed by pancreatic acinar cells and M cells of the ileum. GP2 is the closest related homologue of the urine resident Tamm–Horsfall protein (THP). Recently, it was shown that THP is a ligand of various scavenger receptors (SRs). Therefore, we were interested, if GP2 has similar properties.cDNA of different SRs was stably transfected into a murine thymoma cell line. GP2 was recombinantly expressed, purified and biotinylated. Binding or uptake of GP2 by transfected cells or monocyte-derived dendritic cells (moDCs) was analyzed by flow-cytometry.GP2 is a binding partner of the scavenger receptor expressed on endothelial cells I (SREC-I) but not of SR-AI and SR-BI. The dissociation constant (K_d) of GP2 binding to SREC-I is 41.3 nM. SREC transfected cells are able to internalize GP2. moDCs express SREC-I and also bind and internalize GP2. Inhibition of SREC-I on moDCs with anti-SREC-I antibodies does not result in a decreased GP2 binding.Interaction of GP2 with SREC-I and uptake might have profound effects in antigen clearance and mediation of the immune response. In addition to SREC-I other presently unknown receptors for GP2 on DCs might be involved in this process.     

A novel ryanodine receptor expressed in pancreatic islets by alternative splicing from type 2 ryanodine receptor gene

Cyclic ADP-ribose (cADPR), a potent Ca²⁺ mobilizing intracellular messenger synthesized by CD38, regulates the opening of ryanodine receptors (RyRs). Increases in intracellular Ca²⁺ concentrations in pancreatic islets, resulting from Ca²⁺ mobilization from RyRs as well as Ca²⁺ influx from extracellular sources, are important in insulin secretion by glucose. In the present study, by screening a rat islet cDNA library, we isolated a novel RyR cDNA (the islet-type RyR), which is generated from the RyR2 gene by alternative splicing of exons 4 and 75. When the expression vectors for the islet-type and the authentic RyRs were transfected into HEK293 cells, the islet-type RyR2 as well as the authentic one showed high affinity [³H]ryanodine binding. Intracellular Ca²⁺ release in the islet-type RyR2-transfected cells was enhanced in the presence of cADPR but not in the authentic RyR2-transfected cells. The islet-type RyR2 mRNA was expressed in a variety of tissues such as in pancreatic islets, cerebrum, and cerebellum, whereas the authentic RyR2 mRNA was predominantly expressed in heart and aorta. These results suggest that the islet-type RyR2 may be an intracellular target for cADPR signaling.     

A novel fibroblast growth factor receptor-5 preferentially expressed in the pancreas(1)

Using the polymerase chain reaction on human embryonic cDNAs, we isolated a cDNA encoding a novel 504 amino acid protein, termed fibroblast growth factor receptor (FGFR)-5, which is highly homologous to known FGFRs. The NH(2)-terminal portion of FGFR5 contains a putative secretory signal sequence, three typical immunoglobulin-like domains, six cysteines, and an acidic box, but no HAV motif. The COOH-terminal portion of FGFR5 contains one transmembrane domain but no intracellular kinase domain. Recombinant FGFR5 expressed in COS-7 cells is not secreted, but recombinant truncated FGFR5 lacking the predicted transmembrane domain is secreted. Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) do not bind to FGFR5. Among 23 adult human tissues, FGFR5 mRNA is preferentially expressed in the pancreas. These results suggest that FGFR5 may provide a binding site for some other fibroblast growth factors and may regulate some pancreatic function.     

Characterization of FGFRL1, a novel fibroblast growth factor (FGF) receptor preferentially expressed in skeletal tissues

Clones for a novel transmembrane receptor termed FGFRL1 were isolated from a subtracted, cartilage-specific cDNA library prepared from chicken sterna. Homologous sequences were identified in other vertebrates, including man, mouse, rat and fish, but not in invertebrates such as Caenorhabditis elegans and Drosophila. FGFRL1 was expressed preferentially in skeletal tissues as demonstrated by Northern blotting and in situ hybridization. Small amounts of the FGFRL1 mRNA were also detected in other tissues such as skeletal muscle and heart. The novel protein contained three extracellular Ig-like domains that were related to the members of the fibroblast growth factor (FGF) receptor family. However, it lacked the intracellular protein tyrosine kinase domain required for signal transduction by transphosphorylation. When expressed in cultured cells as a fusion protein with green fluorescent protein, FGFRL1 was specifically localized to the plasma membrane where it might interact with FGF ligands. Recombinant FGFRL1 protein was produced in a baculovirus system with intact disulfide bonds. Similar to FGF receptors, the expressed protein interacted specifically with heparin and with FGF2. When overexpressed in MG-63 osteosarcoma cells, the novel receptor had a negative effect on cell proliferation. Taken together our data are consistent with the view that FGFRL1 acts as a decoy receptor for FGF ligands.     

5-Hydroxy-2-(2-phenylethyl)chromone (5-HPEC): A novel non-nitrogenous ligand for 5-HT2B receptor

Chromones are a class of natural products found in almost every known terrestrial plant with over 4000 naturally occurring derivatives having been isolated and structurally elucidated. Recently, 5-hydroxy-2-(2-phenylethyl)chromone (5-HPEC), isolated from Imperata cylindrical, showed neuroprotective activity against glutamate induced excitotoxicity in primary cultures of rat cortical cells. In comparison to other naturally occurring neuroprotective chromones, 5-HPEC contains fewer hydroxyl groups. Here we report our most recent characterization on this interesting natural product against a number of CNS receptors for the purpose to identify the potential molecular targets that may be related to its biological activity. Based on our studies, including radiobinding assays, calcium flux functional assays and molecular modeling studies, 5-HPEC may represent a type of novel nonnitrogenous ligands to the 5-HT2B receptor.