Stimulatory function of paired immunoglobulin-like receptor-A in mast cell line by associating with subunits common to Fc receptors.

Paired Ig-like receptors (PIR) are polymorphic type I transmembrane proteins belonging to an Ig superfamily encoded by multiple isotypic genes. They are expressed on immune cells such as mast cells, macrophages, and B lymphocytes. Two subtypes of PIR have been classified according to the difference in the primary structure of the PIR transmembrane and cytoplasmic regions. These subtypes are designated as PIR-A and PIR-B. In this study, the transmembrane and cytoplasmic regions of the PIR-A subtype were shown to mediate activation signal events such as cytoplasmic calcium mobilization, protein tyrosine phosphorylations, and degranulation in rat mast cell line RBL-2H3. The association of the Fc receptor gamma and beta subunits with PIR-A was shown to be responsible for PIR-A function but not required for membrane expression of PIR-A on COS-7 cells. We further revealed the role of two charged amino acid residues in the transmembrane region, namely arginine and glutamic acid, in PIR-A function and its association with the above subunits. In contrast to the inhibitory nature of the PIR-B subtype, present findings reveal that PIR-A potentially acts as a stimulatory receptor in mast cells, suggesting a mechanism for regulation of mast cell functions by the PIR family.     

The chicken leukocyte receptor complex: a highly diverse multigene family encoding at least six structurally distinct receptor types

The chicken Ig-like receptors (CHIR) have been described as two Ig domain molecules with long cytoplasmic tails containing inhibitory motifs. In this study, we demonstrate that CHIR form a large family, with multiple members showing great sequence variability among members as well as a great diversity in domain organization and properties of the transmembrane and cytoplasmic segments. We characterize various novel receptor types with motifs indicative of inhibitory, activating, or both functions. In addition to the inhibitory receptors with two ITIM, receptors with a single immunoreceptor tyrosine-based switch motif or receptors lacking a cytoplasmic domain were isolated. Activating receptors with a short cytoplasmic domain and a transmembrane arginine assembled with the newly identified chicken FcepsilonRIgamma chain. Three bifunctional receptor types were characterized composed of one or two C2-type Ig-like domains, a transmembrane region with a positively charged residue and combinations of cytoplasmic motifs such as ITIM, immunoreceptor tyrosine-based switch motif, and YXXM. RT-PCR revealed distinct expression patterns of individual CHIR. All receptor types shared a conserved genomic architecture, and in single Ig domain receptors a pseudoexon replaced the second Ig exon. Southern blot analyses with probes specific for the Ig1 domain were indicative of a large multigene family. Of 103 sequences from the Ig1 domain of a single animal, 41 unique sequences were obtained that displayed extensive variability within restricted Ig regions. Fluorescence in situ hybridization localized the CHIR gene cluster to microchromosome 31 and identified this region as orthologous to the human leukocyte receptor complex.     

Identification and characterization of the rat homologue of LAIR-1

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a cell-surface molecule that functions as an inhibitory receptor on various immune cells in both humans and mice. We have cloned a LAIR-1 homologue from the rat that we have named rat LAIR-1. The LAIR-1 gene maps to rat chromosome 1q12 in a region showing conserved synteny with human chromosome 19q13.4 and mouse chromosome 7, where the leukocyte receptor cluster is located. Rat LAIR-1 shows 40 and 71% protein sequence identity with human LAIR-1 and mouse LAIR-1, respectively, has a single Ig-like domain and contains two immunoreceptor tyrosine-based inhibitory motif-like sequences in its cytoplasmic tail. Soluble rat LAIR-1 fusion proteins bind to the same adherent cell lines as human LAIR-1 and mouse LAIR-1, indicating that a putative ligand for all the LAIR-1 molecules is expressed on these cells. Furthermore, we show that rat and mouse LAIR-1 bind the same molecule expressed on human HT29 cells. Since many autoimmune diseases are studied in rat models, identification of rat LAIR-1 allows for in vivo studies on the function of LAIR molecules in these systems.     

Identification of the mouse killer immunoglobulin-like receptor-like (<Emphasis Type="Italic">Kirl</Emphasis>) gene family mapping to Chromosome X

Natural killer (NK) inhibitory receptors, which recognize major histocompatability complex (MHC) proteins in humans, are known as killer Ig-like receptors (KIRs) and are encoded by a multi-gene immunoglobulin (Ig) superfamily. In a screen for genes differentially expressed in the mouse thymus, we discovered the first close rodent homologue of the NK receptor KIR family, which we named KIR- Like (Kirl). KIRL1 shares 40% amino acid identity with primate KIR family members, with the majority of the homology contained within the Ig-like ectodomains. KIRL1 is more similar to the KIRs than to any other known member of the Ig domain-containing leukocyte receptor superfamily. This highly significant homology suggests that the KIR family did not arise independently in primates, as has been previously suggested, but rather evolved from a primordial gene already present in the common rodent/primate ancestor. KIRL1 lacks the cytoplasmic protein motifs that mediate inhibition in KIRs (immunoregulatory tyrosine inhibiting motif, ITIM); KIRL1 also lacks the transmembrane activation signature (a conserved K residue involved in association with the immunoregulatory tyrosine activating motif-containing DAP12 molecule) found in some KIRs. Nevertheless, we hypothesize that Kirl1 is functional, for the following reasons: (1) Kirl1 mRNA is expressed at high levels in immature thymocytes; (2) Kirl1 is regulated during thymocyte development; (3) KIRL1 protein is detected in thymus. We also show that the mouse genome contains a closely related, transcribed gene, which we name Kirl2. Kirl2 encodes a KIR-like molecule with three Ig-like domains and also lacks tyrosine-based immunoregulatory motifs in its cytoplasmic region.     

Identification and characterization of S2V, a novel putative siglec that contains two V set Ig-like domains and recruits protein-tyrosine phosphatases SHPs

We describe the molecular cloning and characterization of S2V, a novel sialic acid binding immunoglobulin-like lectin. The cDNA of S2V encodes a type 1 transmembrane protein with four extracellular immunoglobulin-like (Ig-like) domains and a cytoplasmic tail bearing a typical immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif. A unique feature of S2V is the presence of two V-set Ig-like domains responsible for the binding to sialic acid, whereas all other known siglecs possess only one. S2V is predominantly expressed in macrophage. In vivo S2V was tyrosine-phosphorylated when co-expressed with exogenous c-Src kinase. Upon tyrosine phosphorylation, S2V recruits both Src homology 2 (SH2) domain-containing protein-tyrosine phosphatases SHP-1 and SHP-2, two important inhibitory regulators of immunoreceptor signal transduction. These findings suggest that S2V is involved in the negative regulation of the signaling in macrophage by functioning as an inhibitory receptor. When expressed in COS-7 cells, S2V was able to mediate sialic acid-dependent binding to human red blood cells, suggesting that S2V may function through cell-cell interaction.     

Ermap, a gene coding for a novel erythroid specific adhesion/receptor membrane protein

Ermap (erythroid membrane-associated protein), a gene coding for a novel transmembrane protein produced exclusively in erythroid cells, is described. It is mapped to murine Chromosome 4, 57 cM distal to the centromere. The initial cDNA clone was isolated from a day 9 murine embryonic erythroid cell cDNA library. The predicted peptide sequence suggests that ERMAP is a transmembrane protein with two extracellular immunoglobulin folds, as well as a highly conserved B30.2 domain and several phosphorylation consensus sequences in the cytoplasmic region. ERMAP shares a high homology throughout the entire peptide with butyrophilin, a glycoprotein essential for milk lipid droplet formation and release. A GFP-ERMAP fusion protein was localized to the plasma membrane and cytoplasmic vesicles in transiently transfected 293T cells. Northern blot analysis and in-situ hybridization demonstrated that Ermap expression was restricted to fetal and adult erythroid tissues. ERMAP is likely a novel adhesion/receptor molecule specific for erythroid cells.     

Internalization of a major group human rhinovirus does not require cytoplasmic or transmembrane domains of ICAM-1.

Intercellular adhesion molecule-1 (CD54), a cell adhesion molecule and the receptor for the major group of rhinoviruses, is a class 1 membrane protein with five Ig-like domains in its extracellular region, a transmembrane domain, and a short cytoplasmic domain. The amino-terminal domains (D1 and D2) are sufficient for virus binding and the first is most important (1). We have investigated whether other extracellular domains, transmembrane or cytoplasmic domains are required for virus entry as determined by postinfection virion protein biosynthesis. We demonstrate that cytoplasmic, transmembrane, and Ig-like domains 3, 4, and 5 are not essential for rhinovirus entry into transfected COS cells. The efficiency of rhinovirus infection directly correlates with the efficiency of rhinovirus binding and a form of intercellular adhesion molecule-1 that is glycophosphatidyl-inositol anchored, and thus does not extend into the inner leaflet of the membrane bilayer or the cytoplasm efficiently supports virus entry.     

Cloning and expression of a rat neuromedin K receptor cDNA

Functional cDNA clones for rat neuromedin K receptor were isolated from a rat brain cDNA library by cross-hybridization with the bovine substance K receptor cDNA. Injection of the mRNA synthesized in vitro from the cloned cDNA into Xenopus oocytes elicited electrophysiological responses to tachykinins, with the most potent sensitivity being to neuromedin K. Ligand-binding displacement in membranes of mammalian COS cells transfected with the cDNA indicated the rank order of affinity of the receptor to tachykinins: neuromedin K greater than substance K greater than substance P. The hybridization analysis showed that the neuromedin K receptor mRNA is expressed in both the brain and the peripheral tissues at different levels. The rat neuromedin K receptor consists of 452 amino acid residues and belongs to the family of G protein-coupled receptors, which are though to have seven transmembrane domains. The sequence comparison of the rat neuromedin K, substance P, and substance K receptors revealed that these receptors are highly conserved in the seven transmembrane domains and the cytoplasmic sides of the receptors. They also show some structural characteristics, including the common presence of histidine residues in transmembrane segments V and VI and the difference in the numbers and distributions of serine and threonine residues as possible phosphorylation sites in the cytoplasmic regions. This paper thus presents the first comprehensive analysis of the molecular nature of the multiple peptide receptors that exhibit similar but pharmacologically distinguishable activities.     

Cloning and characterization of Siglec-10, a novel sialic acid binding member of the Ig superfamily, from human dendritic cells

The Siglecs (sialic acid-binding Ig-like lectins) are a subfamily of I-type lectins, which specifically recognize sialic acids. Nine members of the family have been identified thus far. We have obtained a novel cDNA clone from a human dendritic cell cDNA library encoding a protein with sequence and structural features of the Siglec family, hence designated as Siglec-10. The full-length Siglec-10 cDNA encodes a type 1 transmembrane protein containing four extracellular immunoglobulin-like domains, a transmembrane region, and a cytoplasmic tail with two classical immunoreceptor tyrosine-based inhibitory motifs. The N-terminal V-set Ig domain has most of the amino acid residues typical of the Siglecs. Siglec-10 shows the closest homology to Siglec-5 and Siglec-3/CD33. Various cells and cell lines including monocytes and dendritic cells express Siglec-10. High levels of mRNA expression were seen in peripheral blood leukocytes, spleen, and liver. When expressed on COS-7 cells, Siglec-10 was able to bind human red blood cells and soluble sialoglycoconjugates in a sialic acid-dependent manner. The identification of Siglec-10 as a new Siglec family member and its expression profile, together with its sialic acid-dependent binding capacity, suggest that it may be involved in cell-cell recognition by interacting with sialylated ligands expressed on specific cell populations.     

Cloning,expression, and characterization of fugu CD4, the first ectothermic animal CD4

We have cloned and sequenced the first ectothermic animal CD4 gene from fugu, Takifugu rubripes, using a public database of the third draft sequence of the fugu genome. The fugu CD4 gene encodes a predicted protein of 463 amino acids containing four extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tail. Fugu CD4 shares low identity of about 15–20% with avian and mammalian CD4 proteins. Unlike avian and mammalian CD4, fugu CD4 lacks the Cys pair of the first Ig-like domain, but has a unique possible disulfide bond in the third domain. These differences suggest that fugu CD4 may have a different structure that could affect binding of major histocompatibility complex class II molecules and subsequent T-cell activation. In the putative fugu cytoplasmic region, the protein tyrosine kinase p56^lck binding motif is conserved. The predicted fugu CD4 gene is composed of 12 exons, differing from other CD4 genes, but showing conserved synteny and many conserved sequence motifs in the promoter region. RT-PCR analysis demonstrated that the fugu CD4 gene is expressed predominantly in lymphoid tissues. We also show that fugu CD4 can be expressed on the surface of cells via transfection. Molecular characterization of CD4 in fish provides insights into the evolution of both the CD4 molecule and the immune system.     

Chicken Ig-like receptor B2, a member of a multigene family, is mainly expressed on B lymphocytes, recruits both Src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 and SHP-2, and inhibits proliferation

Ig-like inhibitory receptors have been the focus of intensive research particularly in mouse and human. We report the cloning and characterization of three novel inhibitory chicken Ig-like receptors (CHIR) that display a two Ig-domain extracellular structure, a transmembrane region lacking charged residues and a cytoplasmic domain containing two ITIM. The localization of all receptors to a small genomic region and the hybridization pattern indicated that they belong to a multigene family. The genomic structure of the extracellular domain with two exons encoding the signal peptide and single exons for each Ig domain resembled that of all human leukocyte Ig-like receptors and killer cell Ig-like receptors, whereas the exons encoding the C terminus displayed a structure closely resembling killer cell Ig-like receptor genes. A mAb generated against one receptor designated CHIR-B2 reacted with all B cells and a small T cell subset, but not with monocytes, thrombocytes, or various leukocyte-derived cell lines. The mAb immunoprecipitated a 46-kDa protein from bursal cells and transfected cells. The Src homology 2 domain containing protein tyrosine phosphatase (SHP)-2 bound to CHIR-B2 even in unstimulated cells, whereas pervanadate treatment induced the tyrosine phosphorylation and recruitment of several CHIR-B2-associated proteins including SHP-1 and increased levels of SHP-2. Moreover, mAb cross-linking of CHIR-B2 reduced the proliferation of a stable transfected cell line. Together, we have identified a multigene family containing multiple CHIR including one receptor designated CHIR-B2 that is mainly expressed on B lymphocytes and inhibits cellular proliferation by recruitment of SHP-1 and SHP-2.