CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system

The glycosylphosphatidylinositol-anchored CD24 protein is a B cell differentiation Ag that is expressed on mature resting B cells but disappears upon Ag stimulation. We used Burkitt's lymphoma (BL) cells, which are thought to be related to germinal center B cells, to examine the biological effect of Ab-mediated CD24 cross-linking on human B cells and observed 1) induction of apoptosis in BL cells mediated by cross-linking of CD24; and 2) synergism between the cross-linking of CD24 and that of the B cell receptor for Ag in the effect on apoptosis induction. We also observed activation of mitogen-activated protein kinases following CD24 cross-linking, suggesting that CD24 mediates the intracellular signaling that leads to apoptosis in BL cells. Although CD24 has no cytoplasmic portion to transduce signals intracellularly, analysis of biochemically separated glycolipid-enriched membrane (GEM) fractions indicated enhanced association of CD24 and Lyn protein tyrosine kinase in GEM as well as increased Lyn kinase activity after CD24 cross-linking, suggesting that CD24 mediates intracellular signaling via a GEM-dependent mechanism. Specific microscopic cocapping of CD24 and Lyn, but not of other kinases, following CD24 cross-linking supported this idea. We further observed that apoptosis induction by cross-linking is a common feature shared by GEM-associated molecules expressed on BL cells, including GPI-anchored proteins and glycosphingolipids. CD24-mediated apoptosis in BL cells may provide a model for the cell death mechanism initiated by GEM-associated molecules, which is closely related to B cell receptor for Ag-mediated apoptosis.     

Prostaglandin D2 suppresses human NK cell function via signaling through D prostanoid receptor

NK cells play critical roles in immune responses against tumors or virus infections by generating type 1 cytokine and cytotoxicity responses. In contrast, during type 2 dominant immune responses, such as allergic diseases, activities of NK cells are often impaired. These type 2 immune-mediated diseases have been reported to be closely associated with local production of PGD(2). PGD(2) is an eicosanoid primarily synthesized by mast cells and alveolar macrophages, and it functions through two major receptors, D prostanoid receptor (DP) and chemoattractant receptor-like molecule on the Th2 cell. Within the immune system, PGD(2) binding to DP generally leads to suppression of cellular functions. In the current study, we show that: 1) DP is expressed in human NK cells as detected by mRNA analysis and Western blot; 2) PGD(2) inhibits cytotoxicity, chemotaxis, and type 1 cytokine production of human NK cells via signaling through DP; 3) PGD(2) signaling via DP elevates intracellular cAMP levels and the inhibitory effects on NK cells are cAMP dependent; 4) PGD(2) binding to DP suppresses Ca(2+) mobilization triggered by the cross-linking of the activating receptor, CD16. Together, these data uncover a novel mechanism by which PGD(2) functions through DP to suppress type 1 and cytolytic functions of human NK cells, thus contributing to the promotion of a type 2 immune response.     

CD31 (PECAM-1) exists as a dimer and is heavily N-glycosylated.

CD31 (PECAM-1) is a highly abundant cell surface glycoprotein expressed on hemopoietic and endothelial cells where it functions as a homophilic adhesion and signaling receptor. Since dimerization and appropriate glycosylation are important features in the regulation of cell surface interactions and signal transduction, we studied the pattern of glycosylation as well as the ability of CD31 to undergo dimerization, both in solution and when expressed on cell membranes. CD31 is heavily glycosylated, with an approximate carbohydrate content of 21%. Nineteen neutral and thirteen sialylated glycans were identified. Ultracentrifugation analysis showed that soluble recombinant CD31 exists in equilibrium between a monomer and a dimer with an approximate dissociation constant of 12.5 microM. Chemical cross-linking studies of both soluble and membrane-expressed CD31 confirmed that CD31 exists as a dimer. These studies suggest that, like E-cadherin, PECAM-dimerization is likely to play a role in CD31 adhesion and signaling.     

Role of phospholipase C-L2, a novel phospholipase C-like protein that lacks lipase activity, in B-cell receptor signaling

Phospholipase C (PLC) plays important roles in phosphoinositide turnover by regulating the calcium-protein kinase C signaling pathway. PLC-L2 is a novel PLC-like protein which lacks PLC activity, although it is very homologous with PLC delta. PLC-L2 is expressed in hematopoietic cells, but its physiological roles and intracellular functions in the immune system have not yet been clarified. To elucidate the physiological function of PLC-L2, we generated mice which had a genetic PLC-L2 deficiency. PLC-L2-deficient mice grew with no apparent abnormalities. However, mature B cells from PLC-L2-deficient mice were hyperproliferative in response to B-cell receptor (BCR) cross-linking, although B2 cell development appeared to be normal. Molecular biological analysis revealed that calcium influx and NFATc accumulation in nuclei were increased in PLC-L2-deficient B cells. Extracellular signal-regulated kinase activity was also enhanced in PLC-L2-deficient B cells. These mice had a stronger T-cell-independent antigen response. These results indicate that PLC-L2 is a novel negative regulator of BCR signaling and immune responses.     

Identification and pharmacological characterization of a novel human melanin-concentrating hormone receptor, mch-r2.

Melanin-concentrating hormone (MCH) is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G protein-coupled receptor family. Recently an orphan receptor, SLC-1, has been identified as an MCH receptor (MCH-R1). Herein we identify and characterize a novel receptor for human MCH (MCH-R2). The receptor is composed of 340 amino acids encoded by a 1023-base pair cDNA and is 35% homologous to SLC-1. (125)I-MCH specifically bound to Chinese hamster ovary cells stably expressing MCH-R2. MCH stimulated dose-dependent increases in intracellular free Ca(2+) and inositol phosphate production in these cells but did not affect cAMP production. The pharmacological profile for mammalian MCH, [Phe(13),Tyr(19)]MCH, and salmon MCH at MCH-R2 differed compared with MCH-R1 as assessed by intracellular signaling and radioligand binding assays. The EC(50) in signaling assays and the IC(50) in radioligand binding assays of salmon MCH was an order of magnitude higher than mammalian MCH at MCH-R2. By comparison, the EC(50) and IC(50) values of salmon MCH and mammalian MCH at MCH-R1 were relatively similar. Blot hybridization revealed exclusive expression of MCH-R2 mRNA in several distinct brain regions, particularly in the cortical area, suggesting the involvement of MCH-R2 in the central regulation of MCH-mediated functions.     

Siglec-15: an immune system Siglec conserved throughout vertebrate evolution

Siglecs are vertebrate cell-surface receptors that recognize sialylated glycans. Here we have identified and characterized a novel Siglec, named Siglec-15. Siglec-15 is a type-I transmembrane protein consisting of: (i) two immunoglobulin (Ig)-like domains, (ii) a transmembrane domain containing a lysine residue, and (iii) a short cytoplasmic tail. Siglec-15 is expressed on macrophages and/or dendritic cells of human spleen and lymph nodes. We show that the extracellular domain of Siglec-15 preferentially recognizes the Neu5Acalpha2-6GalNAcalpha- structure. Siglec-15 associates with the activating adaptor proteins DNAX activation protein (DAP)12 and DAP10 via its lysine residue in the transmembrane domain, implying that it functions as an activating signaling molecule. Siglec-15 is the second human Siglec identified to have an activating signaling potential; unlike Siglec-14, however, it does not have an inhibitory counterpart. Orthologs of Siglec-15 are present not only in mammals but also in other branches of vertebrates; in contrast, no other known Siglec expressed in the immune system has been conserved throughout vertebrate evolution. Thus, Siglec-15 probably plays a conserved, regulatory role in the immune system of vertebrates.     

T-cadherin as a receptor regulating angiogenesis and blood vessel remodeling

The search for a membrane receptor responsible for hormone-like effects of low density lipoproteins (LDL) has revealed two proteins (Mol. wt. 105 and 130 kDa) in the membrane fraction of human aortic smooth muscle cells. These proteins were identified as mature T-cadherin and its unprocessed precursor. T-cadherin was originally cloned from chick embryo brain, where it was implicated in axon guidance in the developing nervous system. Our study on the T-cadherin distribution in human organs and tissues has indicated that T-cadherin is specifically expressed in nervous and cardiovascular system. However, physiological significance of T-cadherin expression in the vasculature, as well as intracellular signaling pathways mediating its effects remain obscure. This review summarizes our current knowledge about intracellular signaling utilized by T-cadherin and discusses possible functions of T-cadherin in the vasculature.     

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) peceptor-1 down-modulates VPF/VEGF receptor-2-mediated endothelial cell proliferation, but not migration, through phosphatidylinositol 3-kinase-dependent pathways

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) achieves its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGF receptor-1) and KDR (VEGF receptor-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with these two receptors intact, we developed a chimeric receptor system in which the N terminus of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR (EGDR) and Flt-1 (EGLT). We observed that KDR, but not Flt-1, was responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration. Moreover, Flt-1 showed an inhibitory effect on KDR-mediated proliferation, but not migration. We also demonstrated that the inhibitory function of Flt-1 was mediated through the phosphatidylinositol 3-kinase (PI-3K)-dependent pathway because inhibitors of PI-3K as well as a dominant negative mutant of p85 (PI-3K subunit) reversed the inhibition, whereas a constitutively activated mutant of p110 introduced the inhibition to HUVEC-EGDR. We also observed that, in VPF/VEGF-stimulated HUVECs, the Flt-1/EGLT-mediated down-modulation of KDR/EGDR signaling was at or before intracellular Ca(2+) mobilization, but after KDR/EGDR phosphorylation. By mutational analysis, we further identified that the tyrosine 794 residue of Flt-1 was essential for its antiproliferative effect. Taken together, these studies contribute significantly to our understanding of the signaling pathways and biological functions triggered by KDR and Flt-1 and describe a unique mechanism in which PI-3K acts as a mediator of antiproliferation in primary vascular endothelium.     

CD33-related sialic-acid-binding immunoglobulin-like lectins in health and disease

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are members of the Ig superfamily that bind sialic acids in different linkages in a wide variety of glycoconjugates. These membrane receptors are expressed in a highly specific manner, predominantly within the haematopoietic system. The CD33-related Siglecs represent a distinct subgroup that is undergoing rapid evolution. The structural features of CD33-related Siglecs and the frequent presence of conserved cytoplasmic signalling motifs point to roles in regulating leukocyte functions that are important during inflammatory and immune responses. In this review, we summarise ligand binding preferences and describe recent progress in elucidating the functional roles of CD33-related Siglecs in the immune system. We also discuss the potential for targeting novel therapeutics against these surface receptors.     

Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors

Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane. Certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization. Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary signaling in the regulation of body weight.     

Expression of OB receptor splice variants during prenatal development of the mouse

In adult animals, signaling through the leptin receptor (OB-R) has been shown to play a critical role in fat metabolism. However, it is not known when these receptors are first expressed and what their role may be during embryonic development. To date, at least 6 splice variants of the OB-R have been identified. Although the function of each of these individual splice variants are unknown, only one of them, ob-rL ,encodes a receptor with a long intracellular domain that is implicated in OB-R signaling. In this study we have used in situ hybridization to examine the localization of OB-R splice variants during embryonic development of C57B1/6J mice. Using a probe, ob-r, that recognizes all of the splice variants, ob-r mRNA was found to be distributed in developing bone, mesenchyme, notochord and liver. In addition, epithelial structures including leptomeninges, choroid plexi and hair follicles also expressed ob-r. No ob-r mRNA was detected in the CNS. ob-rL, expression was only detected in notochord, bone and mesenchyme. The differential expression of these two mRNA isoforms suggests that the extracellular and intracellular domains of the OB receptor perform different biological functions.     

Siglec-7: a sialic acid-binding lectin of the immunoglobulin superfamily

The Siglecs are a recently discovered family of sialic acid-binding lectins of the immunoglobulin (Ig) superfamily. We report a molecule showing homology to the six first reported Siglecs, with the closest relationship to Siglec-3(CD33), Siglec-5, and Siglec-6(OBBP-1). The extracellular portion has two Ig-like domains, with the amino-terminal V-set Ig domain including amino acid residues known to be involved in sialic acid recognition by other Siglecs. The cytoplasmic domain has putative sites of tyrosine phosphorylation shared with some Siglecs, including an Immuno-receptor Tyrosine-based Inhibitory Motif (ITIM). Expression of the full-length cDNA induces sialic acid-dependent binding to human erythrocytes. A recombinant chimeric form containing the extracellular Ig domains selectively recognizes the sequence Neu5Acalpha2-6Galbeta1-4Glc, and binding requires the side chain of sialic acid. Mutation of an arginine residue predicted to be critical for sialic acid binding abolishes both interactions. Taken together, our findings justify designation of the molecule as Siglec-7. Analysis of bacterial artificial chromosome (BAC) clones spanning the known human genomic location of Siglec-3 indicates that the Siglec-7 gene is also located on chromosome 19q13.3-13.4. Human tissues show strong expression of Siglec-7 mRNA in spleen, peripheral blood leukocytes, and liver. The combination of an extracellular sialic acid binding site and an intracellular ITIM motif suggests that this molecule is involved in trans-membrane regulatory signaling reactions.     

Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec‐5 and Siglec‐14

The intracellular chaperone heat‐shock protein 70 (Hsp70) can be secreted from cells, but its extracellular role is unclear, as the protein has been reported to both activate and suppress the innate immune response. Potential immunomodulatory receptors on myelomonocytic lineage cells that bind extracellular Hsp70 are not well defined. Siglecs are Ig‐superfamily lectins on mammalian leukocytes that recognize sialic acid‐bearing glycans and thereby modulate immune responses. Siglec‐5 and Siglec‐14, expressed on monocytes and neutrophils, share identical ligand‐binding domains but have opposing signaling functions. Based on phylogenetic analyses of these receptors, we predicted that endogenous sialic acid‐independent ligands should exist. An unbiased screen revealed Hsp70 as a ligand for Siglec‐5 and Siglec‐14. Hsp70 stimulation through Siglec‐5 delivers an anti‐inflammatory signal, while stimulation through Siglec‐14 is pro‐inflammatory. The functional consequences of this interaction are also addressed in relation to a SIGLEC14 polymorphism found in humans. Our results demonstrate that an endogenous non‐sialic acid‐bearing molecule can be either a danger‐associated or self‐associated signal through paired Siglecs, and may explain seemingly contradictory prior reports on extracellular Hsp70 action.     

Paired Siglec receptors generate opposite inflammatory responses to a human-specific pathogen

Paired immune receptors display near-identical extracellular ligand-binding regions but have intracellular sequences with opposing signaling functions. While inhibitory receptors dampen cellular activation by recognizing self-associated molecules, the functions of activating counterparts are less clear. Here, we studied the inhibitory receptor Siglec-11 that shows uniquely human expression in brain microglia and engages endogenous polysialic acid to suppress inflammation. We demonstrated that the human-specific pathogen Escherichia coli K1 uses its polysialic acid capsule as a molecular mimic to engage Siglec-11 and escape killing. In contrast, engagement of the activating counterpart Siglec-16 increases elimination of bacteria. Since mice do not have paired Siglec receptors, we generated a model by replacing the inhibitory domain of mouse Siglec-E with the activating module of Siglec-16. Siglec-E16 enhanced proinflammatory cytokine expression and bacterial killing in macrophages and boosted protection against intravenous bacterial challenge. These data elucidate uniquely human interactions of a pathogen with Siglecs and support the long-standing hypothesis that activating counterparts of paired immune receptors evolved as a response to pathogen molecular mimicry of host ligands for inhibitory receptors.     

Cytoplasmic tail deletion converts membrane immunoglobulin to a phosphatidylinositol-linked form lacking signaling and efficient antigen internalization functions

Membrane-bound immunoglobulin (mIg) is the antigen receptor on B lymphocytes mediating early events in antigen presentation and signal transduction. Wild-type human mIgM constructs transfected into the murine B-cell lymphoma A20 are expressed as transmembrane proteins with antigen presentation and signaling functions comparable to the endogenous mIgG2A; the transfected wild-type mIgM is internalized rapidly after anti-Ig cross-linking. Transfected constructs lacking the normal three-amino acid cytoplasmic tail are expressed exclusively as phosphatidylinositol-linked proteins, lack both antigen presentation and signal transduction functions, and are internalized slowly following anti-Ig binding. The molecular mass of the cytoplasmic tail-deleted phosphatidylinositol-linked Ig molecule is consistent with cleavage of the transmembrane residues during processing. Cytoplasmic domains may therefore regulate the mode of expression of membrane proteins and thereby influence their functional capabilities.     

A novel interleukin-17 receptor-like protein identified in human umbilical vein endothelial cells antagonizes basic fibroblast growth factor-induced signaling

We have previously utilized a combination of high throughput sequencing and genome-wide microarray profiling analyses to identify novel cell-surface proteins expressed in human umbilical vein endothelial cells. One gene identified by this approach encodes a type I transmembrane receptor that shares sequence homology with the intracellular domain of members of the interleukin-17 (IL-17) receptor family. Real-time quantitative PCR and Northern analyses revealed that this gene is highly expressed in human umbilical vein endothelial cells and in several highly vascularized tissues such as kidney, colon, skeletal muscle, heart, and small intestine. In addition, we also found that it is also highly expressed in the ductal epithelial cells of human salivary glands, seminal vesicles, and the collecting tubules of the kidney by in situ hybridization. This putative receptor, which we have termed human SEF (hSEF), is also expressed in a variety of breast cancer tissues. In co-immunoprecipitation assays, this receptor is capable of forming homomeric complexes and can interact with fibroblast growth factor (FGF) receptor 1. Overexpression of this receptor inhibits FGF induction of an FGF-responsive reporter gene in human 293T cells. This appears to occur as a result of specific inhibition of p42/p44 ERK in the absence of upstream MEK inhibition. This inhibitory effect is dependent upon a functional intracellular domain since deletion mutants missing the IL-17 receptor-like domain lack this inhibitory effect. These findings are consistent with the recent discovery of the zebrafish homologue, Sef (similar expression to fgf genes), which specifically antagonizes FGF signaling when ectopically expressed in zebrafish or Xenopus laevis embryos. Based on sequence and functional similarities, this novel IL-17 receptor homologue represents a potential human SEF and is likely to play critical roles in endothelial or epithelial functions such as proliferation, migration, and angiogenesis.     

Plexin-A1 and plexin-B1 specifically interact at their cytoplasmic domains

Semaphorin 3A (Sema3A) is a member of semaphorins and functions as an axonal repulsive guidance molecule. Neuropilin-1 and plexin-As form receptor complexes for Sema3A and plexin-As are thought to initiate the intracellular signaling cascade. However, the molecule by which plexin-As transduce their signal is not well understood. We searched molecules that interact with intracellular domains of plexin-A1 by yeast two-hybrid screening and identified a 349 amino acid fragment of plexin-B1 as a plexin-A1 interacting protein. We, then, cloned mouse plexin-B1 and confirmed their interaction in a mammalian expression system. Plexin-B1 physically associated with plexin-A1, but not with plexin-A2 or A3. Northern blot analysis showed the expression of both plexin-A1 and B1 in adult brain. We propose that plexin-A1 and B1 interact in the adult brain and transduce Sema3A signaling in cooperation.