Mayumi Kakizaki

Identification and Molecular Characterization of Fractalkine Receptor CX3CR1, which Mediates Both Leukocyte Migration and Adhesion

Leukocyte trafficking at the endothelium requires both cellular adhesion molecules and chemotactic factors. Fractalkine, a novel transmembrane molecule with a CX3C-motif chemokine domain atop a mucin stalk, induces both adhesion and migration of leukocytes. Here we identify a seven-transmembrane high-affinity receptor for fractalkine and show that it mediates both the adhesive and migratory functions of fractalkine. The receptor, now termed CX3CR1, requires pertussis toxin-sensitive G protein signaling to induce migration but not to support adhesion, which also occurs without other adhesion molecules but requires the architecture of a chemokine domain atop the mucin stalk. Natural killer cells predominantly express CX3CR1 and respond to fractalkine in both migration and adhesion. Thus, fractalkine and CX3CR1 represent new types of leukocyte trafficking regulators, performing both adhesive and chemotactic functions.

Molecular Cloning of a Novel Human CC Chemokine Secondary Lymphoid-Tissue Chemokine That Is a Potent Chemoattractant for Lymphocytes and Mapped to Chromosome 9p13

By searching the Expressed Sequence Tag (EST) data base, we identified partial cDNA sequences potentially encoding a novel human CC chemokine. We determined the entire cDNA sequence which encodes a highly basic polypeptide of 134 amino acids total with a putative signal peptide of 23 amino acids. The predicted mature protein of 111 amino acids has the four canonical cysteine residues and shows 21–33% identity to other human CC chemokines, but has a unique carboxyl-terminal extension of about 30 amino acids which contains two extra cysteine residues. The mRNA was expressed strongly in tissues such as the lymph nodes, Appendix, and spleen. The recombinant protein, which was produced by the baculovirus system and purified to homogeneity, was a highly efficient chemoattractant for certain human T cell lines and a highly potent one for freshly isolated peripheral blood lymphocytes and cultured normal T cells expanded by phytohemagglutinin and interleukin 2. Unlike most other CC chemokines, however, this novel chemokine was not chemotactic for monocytes or neutrophils, suggesting that it is specific for lymphocytes. From these results, we designated this novel CC chemokine as SLC fromsecondary lymphoid-tissuechemokine. SLC fused with the secreted form of alkaline phosphatase (SLC-SEAP) was used to characterize the SLC receptor. Binding of SLC-SEAP to freshly isolated lymphocytes was blocked by SLC (IC50, 0.12 nm) but not by any other CC chemokine so far tested, suggesting that resting lymphocytes express a class of receptors highly specific for SLC. By using somatic cell hybrids, radiation hybrids, and selected yeast and bacterial artificial chromosome clones, we mapped the SLC gene (SCYA21) at chromosome 9p13 and between chromosomal markers, D9S1978(WI-8765) and AFM326vd1, where the gene for another novel CC chemokine termed ELC from EBI1-ligandchemokine (SCYA19) also exists. Collectively, SLC is a novel CC chemokine specific for lymphocytes and, together with ELC, constitutes a new group of chemokines localized at chromosome 9p13.

Dual Functions of Fractalkine/CX3C Ligand 1 in Trafficking of Perforin + /Granzyme B + Cytotoxic Effector Lymphocytes That Are Defined by CX3CR1 Expression

Fractalkine/CX3C ligand 1 and its receptor CX3CR1 are known to mediate both cell adhesion and cell migration. Here we show that CX3CR1 defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B, which include NK cells, γδ T cells, and terminally differentiated CD8+ T cells. In addition, soluble fractalkine preferentially induced migration of cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic effector lymphocytes with membrane-bound fractalkine promoted subsequent migration to the secondary chemokines, such as macrophage inflammatory protein-1β/CC ligand 4 or IL-8/CXC ligand 8. Thus, fractalkine expressed on inflamed endothelium may function as a vascular regulator for cytotoxic effector lymphocytes, regardless of their lineage and mode of target cell recognition, through its ability to capture them from blood flow and to promote their emigration in response to other chemokines.

Identification of Single C Motif-1/Lymphotactin Receptor XCR1

Single C motif-1 (SCM-1)/lymphotactin is a member of the chemokine superfamily, but retains only the 2nd and 4th of the four cysteine residues conserved in other chemokines. In humans, there are two highly homologous SCM-1 genes encoding SCM-1α and SCM-1β with two amino acid substitutions. To identify a specific receptor for SCM-1 proteins, we produced recombinant SCM-1α and SCM-1β by the baculovirus expression system and tested them on murine L1.2 cells stably expressing eight known chemokine receptors and three orphan receptors. Both proteins specifically induced migration in cells expressing an orphan receptor, GPR5. The migration was chemotactic and suppressed by pertussis toxin, indicating coupling to a Gα type of G protein. Both proteins also induced intracellular calcium mobilization in GPR5-expressing L1.2 cells with efficient mutual cross desensitization. SCM-1α bound specifically to GPR5-expressing L1.2 cells with a K d of 10 nm. By Northern blot analysis, GPR5 mRNA of about 5 kilobases was detected strongly in placenta and weakly in spleen and thymus among various human tissues. Identification of a specific receptor for SCM-1 would facilitate our investigation on its biological function. Following the set rule for the chemokine receptor nomenclature, we propose to designate GPR5 as XCR1 from XC chemokine receptor-1.

Molecular cloning of a novel C or γ type chemokine, SCM-1

From human PBMC stimulated with PHA, we have isolated cDNA clones encoding a novel cytokine named SCM‐1, which is significantly related to the CC and the CXC chemokines but has only the 2nd and the 4th of the four cysteines conserved in these proteins. Its gene is also distinctly mapped to human chromosome 1. SCM‐1 is strongly induced in human PBMC and Jurkat T cells by PHA stimulation. Among various human tissues, SCM‐1 is expressed most strongly in spleen. SCM‐1 is found to be 60.5% identical to lymphotactin, a recently described murine lymphocyte‐specific chemokine, which also retains only two cysteines. SCM‐1 and lymphotactin may thus represent the human and murine prototypes of a novel C or γ type chemokine family.

JEAP, a Novel Component of Tight Junctions in Exocrine Cells

Tight junctions (TJs) consist of transmembrane proteins and many peripheral membrane proteins. To further characterize the molecular organization of TJs, we attempted here to screen for novel TJ proteins by the fluorescence localization-based expression cloning method. We identified a novel peripheral membrane protein at TJs and named it junction-enriched and -associated protein (JEAP). JEAP consists of 882 amino acids with a calculated molecular weight of 98,444. JEAP contained a polyglutamic acid repeat at the N-terminal region, a coiled-coil domain at the middle region, and a consensus motif for binding to PDZ domains at the C-terminal region. Exogenously expressed JEAP co-localized with ZO-1 and occludin at TJs in polarized Madin-Darby canine kidney cells, but not with claudin-1, JAM, or ZO-1 in L cells. Endogenous JEAP localized at TJs of exocrine cells including pancreas, submandibular gland, lacrimal gland, parotid gland, and sublingual gland, but not at TJs of epithelial cells of small intestine or endothelial cells of blood vessels. The present results indicate that JEAP is a novel component of TJs, which is specifically expressed in exocrine cells.

Two actions of frabin: direct activation of Cdc42 and indirect activation of Rac

Frabin is an actin filament-binding protein which shows GDP/GTP exchange activity specific for Cdc42 small G protein and induces filopodium-like microspike formation and c-Jun N-terminal kinase (JNK) activation presumably through the activation of Cdc42. Frabin has one actin filament-binding (FAB) domain, one Dbl homology (DH) domain, first pleckstrin homology (PH) domain adjacent to the DH domain, one cysteine-rich FYVE domain, and second PH domain from the N-terminus to the C-terminus in this order. Different domains of frabin are involved in the microspike formation and the JNK activation, and the association of frabin with the actin cytoskeleton through the FAB domain is necessary for the microspike formation, but not for the JNK activation. We have found here that frabin induces the formation of not only filopodium-like microspikes but also lamellipodium-like structures in NIH3T3 and L fibroblasts. We have analysed the mechanism of frabin in these two actions and found that frabin induces filopodium-like microspike formation through the direct activation of Cdc42 and lamellipodium-like structure formation through the Cdc42-independent indirect activation of Rac small G protein. The FAB domain of frabin in addition to the DH domain and the first PH domain is necessary for the filopodium-like microspike formation, but not for the lamellipodium-like structure formation. The FYVE domain and the second PH domain in addition to the DH domain and the first PH domain are necessary for the lamellipodium-like structure formation. We show here these two actions of frabin in the regulation of cell morphology.

Molecular cloning of mXCR1, the murine SCM-1/lymphotactin receptor

Single C motif‐1 (SCM‐1)/lymphotactin is a C‐type member of the chemokine superfamily. Previously, we identified its specific receptor XCR1. Here we isolated the murine homologue of XCR1 (mXCR1). To demonstrate its biological activity, we produced recombinant mouse SCM‐1 by the baculovirus expression system. B300‐19 murine pre‐B cells expressing mXCR1 responded to mSCM‐1 in chemotactic and calcium‐mobilization assays. mXCR1 mRNA was weakly expressed in spleen and lung of normal C57BL/6 mice. In spleen, CD8+ cells and NK1.1+ cells were found to express mXCR1. Identification of mXCR1 will now allow us to study the role of this unique cytokine system in the mouse models of inflammation and immunity.