The zebrafish activating immune receptor Nitr9 signals via Dap12

Both inhibitory and activating forms of natural killer (NK) cell receptors are found in mammals. The activating receptors play a direct role in the recognition of virally infected or transformed cells and transduce activating signals into the cell by partnering with an adaptor protein, which contains a cytoplasmic activation motif. Activating NK receptors encoded by the mammalian leukocyte receptor complex (e.g., killer cell immunoglobulin-like receptors) and the natural killer complex (e.g., Ly49s) partner with the adaptor protein DAP12, whereas NK receptors encoded in the CD94/NKG2 complex partner with the adaptor protein DAP10. Novel immune-type receptors (NITRs) found in bony fish share several common features with immunoglobulin-type NK receptors. Nitr9 is a putative activating receptor in zebrafish that induces cytotoxicity within the context of human NK cells. One isoform of Nitr9, Nitr9L, is shown here to preferentially partner with a zebrafish ortholog of Dap12. Cross-linking the Nitr9L–Dap12 complex results in activation of the phosphytidylinositol 3-kinase→AKT→extracellular signal-regulated kinase pathway suggesting that the DAP12-based activating pathway is conserved between bony fish and mammals. Sheng Wei and Jun-min Zhou contributed equally to this work.     

Adaptor proteins of blood platelets

Adaptor proteins play a pivotal role in the regulation of signal transduction events elicited after the engagement of cell surface receptors. Platelets exhibit a number of integral membrane receptors capable of initiating a cellular response. These include collagen receptors, von Willebrand factor receptors, the fibrinogen receptor, and a number of G-protein coupled receptors, such as those for thrombin and ADP. The primary function of platelet receptors is the translation of externally applied signals into appropriate responses leading to platelet activation being a prerequisite for normal hemostasis. Multitude of signalling pathways described in platelets is based on the interaction of compounds of many different categories, such as transmembrane receptors, protein kinases, protein phoshatases, G-proteins, transmembrane and cytosolic adaptor proteins, phosphoinositides, cyclic AMP or GMP. Adaptor proteins lack intrinsic effector function, but contain distinct molecular domains, which mediate protein-protein and protein-lipid interactions. These molecules thus serve as a scaffolding, around which effectors and their substrates are assembled into three-dimensional signaling complexes. Adaptor proteins integrate receptor-mediated signals at intracellular levels and couple signaling receptors to cytosolic signaling pathways. While the function of adaptor proteins is well established in immune cells, the knowledge about their role in platelet activation is still at the onset Over the last decade numerous adaptor proteins have been identified in platelets and shown to be involved in accurate assembly of intracellular signaling complexes. Collagen-induced platelet intracellular signaling through GPVI resembles the functional response of B- and T-cell antigen receptors and is the best described in the literature. This review focuses on the structure and functional role of the most extensively studied adaptor proteins during platelet activation induced by physiological agonists.     

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.     

Membrane lipid rafts: new targets for immunoregulation

Engagement of immune receptors by antigen may lead to activation, cell proliferation, differentiation and effector functions. It has recently been proposed that the initiation and propagation of the signaling events taking place in immune cells occur in specialized membrane regions called lipid rafts. These detergent-insoluble glycolipid domains are specialized membrane compartments enriched in cholesterol and glycolipids. They also contain many lipid-modified signaling proteins such as tyrosine kinases of the Src family, GPI (glycosylphosphatidylinositol)-linked proteins as well as adaptor proteins. The confinement of signaling molecules in membrane subdomains suggests that lipid rafts function as platforms for the formation of multicomponent transduction complexes. Indeed, upon receptor binding, immune receptors become raft-associated and additional components of the signaling pathways are recruited to rafts in order to form signaling complexes. It has been speculated that the entry of immune receptors into rafts can regulate cell activation. Accordingly, numerous experiments have provided substantial evidence that raft integrity is crucial for the initiation and maintenance of intracellular signals. Recent studies have also shown that the access and translocation of immune receptors to lipid rafts are developmentally regulated (immature versus mature cells, Th1 versus Th2 lymphocytes) and sensitive to pharmacological agents. The aim of the present review is to summarize the current knowledge of immune receptor signal transduction with particular emphasis on the role of membrane compartments in immune activation. Finally, experimental evidences indicating that these membrane structures may represent clinically relevant potential targets for immune regulation, will be discussed.     

Coordination of Receptor Signaling in Multiple Hematopoietic Cell Lineages by the Adaptor Protein SLP-76

The adaptor protein SLP-76 is expressed in multiple hematopoietic lineages including T cells, platelets, and neutrophils. SLP-76 mediated signaling is dependent on its multiple protein interaction domains, as it creates a scaffold on which key signaling complexes are built. SLP-76 is critical for supporting signaling downstream of both immunoreceptors and integrins. The signaling molecules used both upstream and downstream of SLP-76 are similar among these receptors and across cell types; however, important differences exist. Appreciating how SLP-76 coordinates signal transduction across different cell and receptor types provides insights into the complex interplay of pathways critical for activation of cells of the immune system that are essential for host defense.Adaptor proteins are an important component of many signaling systems both within and beyond the immune system. Unlike enzymes that catalyze the chemical reactions required for signal propagation, adaptor proteins are molecular platforms on which other proteins assemble. Although lacking enzymatic function, adaptor proteins regulate signaling by stabilizing or restricting molecular interactions required for proper enzyme activation and for localizing these key effector molecules appropriately within the cell.Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is an adaptor present in a number of hematopoietic cell lineages including T cells, platelets, neutrophils, mast cells, macrophages, and NK cells. There are two SLP-76 homologs: SLP-65/B cell linker protein (BLNK), and cytokine-dependent hematopoietic cell linker (CLNK), also known as mast cell immunoreceptor signal transducer (MIST). BLNK is expressed in B cells and monocytes, and CLNK is expressed in mast cells, activated T cells, and NK cells. SLP-76 was first identified in T cells as a substrate of T-cell receptor (TCR) stimulated protein tyrosine kinases (PTKs) through “pull down” experiments using immobilized growth factor receptor-bound protein 2 (Grb2) (Jackman et al. 1995). Since then, SLP-76 has been shown to be critically important in the development of T cells and for propagating signals downstream of not only the TCR but also additional receptors present on hematopoietic cells.     

Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction

The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface.     

Activation receptor-induced tolerance of mature NK cells in vivo requires signaling through the receptor and is reversible

NK cell responses are determined by signals received through activating and inhibitory cell surface receptors. Ly49H is an NK cell-specific activating receptor that accounts for the genetic resistance to murine CMV (MCMV). The Ly49H receptor has been shown to interact with two adaptor proteins (DAP12 and DAP10). In the context of MCMV infection, interaction of m157 (the MCMV-encoded ligand for Ly49H) with Ly49H results in activation of Ly49H-expressing NK cells. Chronic exposure of Ly49H with m157, however, induces tolerance in these same cells. The mechanism of this tolerance remains poorly understood. Using a transgenic mouse model, we demonstrate that induction of tolerance in Ly49H(+) NK cells by chronic exposure to m157, in vivo, requires signaling through the Ly49H adaptor protein DAP12, but not the DAP10 adaptor protein. Furthermore, mature Ly49H-expressing NK cells from wild-type mice can acquire a tolerant phenotype by 24 h posttransfer into a transgenic C57BL/6 mouse that expresses m157. The tolerant phenotype can be reversed, in vivo, if tolerant NK cells are transferred to mice that do not express the m157 protein. Thus, continuous activating receptor engagement can induce a transient tolerance in mature NK cells in vivo. These observations provide new insight into how activating receptor engagement shapes NK cell function and has important implications in how NK cells respond to tumors and during chronic viral infection.     

High dose M-CSF partially rescues the Dap12-/- osteoclast phenotype

Osteoclasts are macrophage derived cells and as such are subject to regulation by molecules impacting other members of the immune system. Dap12 is an adaptor protein expressed by NK cells and B and T lymphocytes. Dap12 also mediates maturation of myeloid cells and is expressed by osteoclasts which are dysfunctional in its absence. We find Dap12-/- osteoclast precursors fail to differentiate, in vitro, and the abnormality is partially rescued by high dose M-CSF. The relative paucity of osteoclast number, even in presence of high dose cytokine, is attended by dampened proliferation of precursor cells and their failure to normally migrate towards the osteoclast-recognized matrix protein, osteopontin. Furthermore, Dap12-/- osteoclasts generated in high dose M-CSF fail to normally organize their cytoskeleton. The incapacity of Dap12 null cells to undergo normal osteoclast differentiation is not due to blunted stimulation of major RANK ligand (RANKL) or M-CSF induced signaling pathways. On the other hand, when plated on osteopontin, Dap12-/- pre-osteoclasts do not activate the tyrosine kinase, Syk, which normally binds to the adaptor protein and transmits downstream signals. Attesting to the importance of the Dap12/Syk complex, Syk deficient macrophages do not undergo normal osteoclastogenesis. Furthermore, the same cells plated onto osteopontin, adhere poorly and fail to phosphorylate c-Src or Pyk2, two kinases central to organization of the osteoclast cytoskeleton.     

A TIR Domain Protein from E. faecalis Attenuates MyD88-Mediated Signaling and NF-κB Activation

Toll-like receptor signaling, mediated by functional Toll/interleukin-1 receptor (TIR) domains, plays a critical role in activating the innate immune response responsible for controlling and clearing infection. Bacterial protein mimics of components of this signaling pathway have been identified and function through inhibition of interactions between Toll-like receptors (TLRs) and their adaptor proteins, mediated by TIR domains. A previously uncharacterized gene, which we have named tcpF (for TIR domain-containing protein in E. faecalis) was identified in the genome of Enterococcus faecalis V583, and predicted to encode a protein resembling mammalian and bacterial TIR proteins. We overexpressed and purified TcpF from E. coli and found that the recombinant protein could bind to phosphatidylinositol phosphates in vitro, suggesting a mechanism by which TcpF may be anchored to the plasma membrane in close proximity to TIR domains of TLRs and adaptor proteins. Purified TcpF was also found to interact specifically with the TIR adaptor protein MyD88, and this interaction was dependent on the BB loop domain in the Box 2 region of TcpF. Despite no evidence of TcpF being a secreted protein, recombinant TcpF was effectively able to enter RAW264.7 cells in vitro although the mechanism by which this occurs remains to be determined. Overexpression of TcpF in mammalian cells suppressed the NF-κB activation induced by bacterial lipoteichoic acid. A mutant lacking the tcpF gene was attenuated for survival in macrophages, with increased ability to activate NF-κB compared to the wild type strain. Complementation in trans restored growth, and inhibition of NF-κB, to that of wild type levels. No appreciable difference in bacterial persistence, dissemination or pathogenesis was observed between the wild type and mutant in a mouse peritonitis model however, which suggested either a subtle role for TcpF or functional overlap with other redundant factor(s) in this virulence model.     

Single-spanning transmembrane domains in cell growth and cell-cell interactions

As a whole, integral membrane proteins represent about one third of sequenced genomes, and more than 50% of currently available drugs target membrane proteins, often cell surface receptors. Some membrane protein classes, with a defined number of transmembrane (TM) helices, are receiving much attention because of their great functional and pharmacological importance, such as G protein-coupled receptors possessing 7 TM segments. Although they represent roughly half of all membrane proteins, bitopic proteins (with only 1 TM helix) have so far been less well characterized. Though they include many essential families of receptors, such as adhesion molecules and receptor tyrosine kinases, many of which are excellent targets for biopharmaceuticals (peptides, antibodies, et al.). A growing body of evidence suggests a major role for interactions between TM domains of these receptors in signaling, through homo and heteromeric associations, conformational changes, assembly of signaling platforms, etc. Significantly, mutations within single domains are frequent in human disease, such as cancer or developmental disorders. This review attempts to give an overview of current knowledge about these interactions, from structural data to therapeutic perspectives, focusing on bitopic proteins involved in cell signaling.     

A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins

The Toll-like receptor 4 (TLR4) is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR) signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3) and nuclear factor kappaB (NFkappaB) respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.     

Diversity of the human LILRB3/A6 locus encoding a myeloid inhibitory and activating receptor pair

Leukocyte immunoglobulin-like receptor (LILR)B3 and LILRA6 represent a pair of inhibitory/activating receptors with identical extracellular domains and unknown ligands. LILRB3 can mediate inhibitory signaling via immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail whereas LILRA6 can signal through association with an activating adaptor molecule, FcRγ, which bears a cytoplasmic tail with an immunoreceptor tyrosine-based activation motif. The receptors are encoded by two highly polymorphic neighboring genes within the leukocyte receptor complex on human chromosome 19. Here, we report that the two genes display similar levels of single nucleotide polymorphisms with the majority of polymorphic sites being identical. In addition, the LILRA6 gene exhibits copy number variation (CNV) whereas LILRB3 does not. A screen of healthy Caucasians indicated that 32 % of the subjects possessed more than two copies of LILRA6, whereas 4 % have only one copy of the gene per diploid genome. Analysis of mRNA expression in the major fractions of PBMCs showed that LILRA6 is primarily expressed in monocytes, similarly to LILRB3, and its expression level correlates with copy number of the gene. We suggest that the LILRA6 CNV may influence the level of the activating receptor on the cell surface, potentially affecting signaling upon LILRB3/A6 ligation.     

Protein-tyrosine kinases and adaptor proteins in FcepsilonRI-mediated signaling in mast cells

Mast cells function as the initiator of the allergic reaction and play a role in the innate immune system. Aggregation of the high affinity IgE receptor (FcepsilonRI) on mast cells triggers degranulation with the release of chemical mediators such as histamine, production of cytokines and leukotrienes. FcepsilonRI signals by activating proximal non-receptor type of protein-tyrosine kinases, Lyn, Syk, Btk and Fyn. Activated tyrosine kinases then phosphorylate their specific substrates which include other enzymes and adaptor proteins and assemble these cytoplasmic signaling molecules for cellular activation. The adaptor proteins have multiple domains that allow binding to effector molecules and therefore act as positive or negative regulators controlling FcepsilonRI signaling. Deletion of the adaptor proteins such as LAT, SLP-76 or Gab2 resulted in decreased FcepsilonRI-mediated anaphylactic reaction in vivo. Functional analysis of adaptor proteins has raised the possibility that they may be new targets for the discovery of anti-allergic drugs.     

Single-spanning transmembrane domains in cell growth and cell-cell interactions: More than meets the eye?

As a whole, integral membrane proteins represent about one third of sequenced genomes, and more than 50% of currently available drugs target membrane proteins, often cell surface receptors. Some membrane protein classes, with a defined number of transmembrane (TM) helices, are receiving much attention because of their great functional and pharmacological importance, such as G protein-coupled receptors possessing 7 TM segments. Although they represent roughly half of all membrane proteins, bitopic proteins (with only 1 TM helix) have so far been less well characterized. Though they include many essential families of receptors, such as adhesion molecules and receptor tyrosine kinases, many of which are excellent targets for biopharmaceuticals (peptides, antibodies, et al.). A growing body of evidence suggests a major role for interactions between TM domains of these receptors in signaling, through homo and heteromeric associations, conformational changes, assembly of signaling platforms, etc. Significantly, mutations within single domains are frequent in human disease, such as cancer or developmental disorders. This review attempts to give an overview of current knowledge about these interactions, from structural data to therapeutic perspectives, focusing on bitopic proteins involved in cell signaling.Key words: bitopic membrane proteins, transmembrane domains, transmembrane signaling, helix-helix interactions, receptors     

CD300 heterocomplexes, a new and family-restricted mechanism for myeloid cell signaling regulation

The CD300 family of myeloid immunoglobulin receptors includes activating (CD300b, CD300e) and inhibitory members (CD300a, CD300f), as well as molecules of uncertain function presenting a negative charge within their transmembrane domain (CD300c, CD300d). In this paper, we establish that CD300c is a functional immune receptor able to deliver activating signals upon ligation in RBL-2H3 mast cells. CD300c signaling is partially mediated by a direct association with the immune receptor tyrosine-based activation motif-bearing adaptor FcεRγ. The existence of complementary transmembrane-charged residues in certain CD300 receptors suggested the formation of heterodimers within this family. Indeed, we proved the interaction between CD300b and CD300c in transfected COS-7 cells and demonstrated that it has important functional consequences. Unexpectedly, dimmer formation was dependent on the immunoglobulin domains rather than the charged transmembrane residues. Concordantly, all CD300 members were found to interact with each other, even with themselves, forming both homo- and heterodimers. We found that the combination of CD300 receptors in a complex differentially modulates the signaling outcome, strongly suggesting a new mechanism by which CD300 complexes could regulate the activation of myeloid cells upon interaction with their natural ligands.     

Exposure to silver nanoparticles affects viability and function of natural killer cells,mostly via the release of ions

Natural killer (NK) cells play a crucial role in linking innate and adaptive immune responses, especially during viral infections and tumor surveillance. They have two major effector functions: the killing of stressed/abnormal cells and the release of cytokines. Their activity is regulated via inhibitory and activating surface receptors. At the same time that the production and use of engineered nanoparticles is steadily increasing, the risk for exposure to silver nanoparticles (AgNPs) from consumer products or biomedical applications is growing. Given this, we assessed the effects of 20-nm big AgNPs on NK cells, which represent an important part of the immune system. Our study involved overnight exposure of human blood NK cells to different concentrations of AgNPs, and silver (Ag) ion controls, and analyzing them for viability, surface receptor expression, intracellular markers, cytokine release, and killing potential. Exposure to AgNPs, but not to Ag ion controls, reduced the viability and the cytotoxic potential after polyriboinosinic-polyribocytidylic acid stimulation of NK cells and increased the expression of the inhibitory receptor CD159a. Exposure to AgNPs and Ag ion controls reduced the expression of the activating receptors CD335 and of CD16 and increased the expression of the activating receptor CD314. Overall, exposure to AgNPs changes NK cells’ function and phenotype and may present a risk for modulating human immune responses, which should be further investigated.     

The activating Ly49W and inhibitory Ly49G NK cell receptors display similar affinities for identical MHC class I ligands

The Ly49 receptor family plays an important role in the regulation of murine natural killer (NK) cell effector function. They recognize cell surface-expressed class I MHC (MHC-I) and are functionally equivalent to the killer Ig-related receptors (KIRs) in human NK cells. Ly49s exist in activating and inhibitory forms with highly homologous extracellular domains, displaying greater variability in the stalk regions. Inhibitory Ly49s can recognize self-MHC-I and therefore mediate tolerance to self. The role of activating Ly49 receptors is less clear. Some activating Ly49 receptors have been shown to recognize MHC-I molecules. The binding affinity of activating Ly49 receptors with MHC-I is currently unknown, and we sought to examine the affinities of two highly related receptors, an activating and an inhibitory Ly49 receptor, for their shared MHC-I ligands. The ectodomain of inhibitory Ly49G of the BALB/c mouse strain is highly similar to the Ly49W activating receptor in the nonobese diabetic (NOD) mouse. Recombinant soluble Ly49G and W were expressed, refolded, and analyzed for binding affinity with MHC-I by surface plasmon resonance. We found that Ly49G and Ly49W bound with similar affinity to the same MHC-I molecules. These results are a first determination of an activating Ly49 receptor affinity for MHC-I and show that, unlike prior results obtained with activating and inhibitory KIR receptors, functional homologues to Ly49 receptors, activating and inhibitory Ly49, can recognize common MHC-I ligands, with similar affinities.