Fractalkine

Fractalkine David R. Greaves1,* and Thomas J. Schall2 1

Sir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, UK ChemoCentryx Inc., 1539 Industrial Road, San Carlos, CA 94070, USA

2

* corresponding author tel: +44-1865-275531, fax: +44-1865-275515, e-mail: [email protected] DOI: 10.1006/rcwy.2000.11021.

SUMMARY Fractalkine is the sole member of the CX3C chemokine subfamily with three amino acids between the C1 and C2 cysteines of the chemokine domain. It is unique among members of the chemokine family in that it exists in a membrane-bound form with the chemokine domain presented on top of a long mucin stalk. The chemokine domain of fractalkine can mediate the chemotaxis of monocytes, T cells, microglia and NK cells. The chemokine domain of fractalkine is a ligand for the chemokine receptor CX3CR1 and the cytomegalovirus receptor US28. Immobilized forms of fractalkine that include the mucin stalk can mediate the tight adhesion of CX3CR1-expressing cells under flow conditions.

BACKGROUND

Discovery Fractalkine was initially identified as an EST sequence from a human brain cDNA library (Bazan et al., 1997). Full-length fractalkine cDNA clones were shown to encode a 373 amino acid type I transmembrane protein. Murine fractalkine shows a high degree of homology with human fractalkine in the chemokine domain and the intracellular domain but much less homology within the mucin stalk (Rossi et al., 1998).

Alternative names Pan et al. (1997) reported a human cDNA sequence identical to that of human fractalkine, which they termed neurotactin.

Structure The 397 amino acid open reading frame of the fractalkine mRNA encodes a 24 amino acid signal peptide,

and the first 76 amino acids of the mature fractalkine protein encode the CX3C chemokine domain. A 241 amino acid mucin stalk is followed by a 19 amino acid transmembrane spanning region and a C-terminal 37 amino acid intracellular domain (Bazan et al., 1997).

Main activities and pathophysiological roles The cellular receptor for fractalkine, CX3CR1, is expressed monocytes, T cells, NK cells, and microglia (Imai et al., 1997; Harrison et al., 1998). Some of the reported activities of fractalkine are outlined below. T Cell and Monocyte Chemotaxis The initial description of fractalkine demonstrated that the chemokine domain of human fractalkine was able to act as a potent chemoattractant for T cells and monocytes but not neutrophils (Bazan et al., 1997). Fractalkine (residues 1±76) and the 95 kDa soluble secreted fractalkine produced by 293 cells transfected with the full-length fractalkine cDNA in an expression vector were equally potent in monocyte and T cell chemotaxis assays (Bazan et al., 1997). NK Cell Chemotaxis Fractalkine induces chemotaxis and the mobilization of intracellular calcium in IL-2-activated NK cells (Al-Aoukaty et al., 1998). Effects on Microglia Recombinant rat fractalkine stimulates chemotaxis and intracellular calcium mobilization in rat microglia (Harrison et al., 1998). The role of fractalkine and CX3CR1 expression within the adult central nervous system or the developing brain has yet to be elucidated. Effects on Neurons Fractalkine has been reported to mediate calcium signaling in rat hippocampal neurons, which were shown

1294 David R. Greaves and Thomas J. Schall to express CX3CR1 RNA (Meucci et al., 1998). The fractalkine treatment of hippocampal neurons was reported to increase the expression of the transcription factor CREB and activate the extracellular response kinases ERK1 and ERK2 (Meucci et al., 1998). HIV Infection The CX3CR1 receptor, previously cloned as the orphan receptor V28 (rat) and CMKBRL1 (human), can function as a coreceptor with CD4 for HIV-1 gp120mediated cell±cell fusion (Combadiere et al., 1998).

Figure 1 Fractalkine-mediated cell adhesion. Immobilized forms of fractalkine that include the CX3C chemokine domain (represented by a green circle) and the mucin stalk (represented by the blue stalks on an orange background) can mediate the tight adhesion of cells expressing the CX3CR1 chemokine receptor. This form of cell adhesion does not require calcium, G protein signaling or the presence of an opposing cell membrane (Imai et al., 1997). (Full colour figure may be viewed online.)

Fractalkine and Cell Adhesion In the original description of fractalkine it was noted that HEK 293 cells transfected with an expression vector containing the full-length fractalkine cDNA exhibited tight binding of human monocytes and T cells (Bazan et al., 1997). A recombinant form of the extracellular portion of fractalkine containing the CX3C chemokine domain and the mucin stalk immobilized on a glass slide was shown to mediate the tight binding of cells expressing the CX3CR1 chemokine receptor (Imai et al., 1997). It was shown that the CX3C chemokine domain must be attached to the mucin stalk to mediate the tight binding of CX3CR1expressing cells. Further work by Fong et al. (1998) and Haskell et al. (1999) has shown that immobilized fractalkine can mediate the rapid flow arrest of CX3CR1-expressing cells independent of G protein activation (Figure 1).

GENE AND GENE REGULATION

Accession numbers GenBank: Human fractalkine: U91835 Mouse fractalkine: AF074912 Rat fractalkine: AF030358

G

Ca2+

α β γ

CX3CR1 Ca2–

ADHESION

(–) G protein coupling (–) Extracellular calcium (–) Opposing cell membrane

Cells and tissues that express the gene Fractalkine is expressed by endothelial cells, some macrophages and dendritic cells, and microglia.

PROTEIN

Chromosome location

Accession numbers

The human fractalkine gene (SCYD1) has been mapped to chromosome 16q13 by fluorescence in situ hybridization (Nomiyama et al., 1998). The murine fractalkine gene has been mapped to mouse chromosome 8 (Rossi et al., 1998).

GenPept: Human fractalkine: NP 002987 Mouse fractalkine: AAB71763 Rat fractalkine: AAC33834

Relevant linkages

See Figure 2.

The human fractalkine gene is part of a chemokine gene mini locus on chromosome 16q13. The fractalkine gene lies 5.5 kb 30 of the MDC gene and 28 kb 50 of the TARC gene (Nomiyama et al., 1998).

Sequence Discussion of crystal structure Mizoue et al. (1999) reported the solution structure of the chemokine domain of fractalkine (residues 1±76)

Fractalkine 1295 Figure 2 Amino acid sequence for human, mouse, and rat fractalkine. Signal sequence underlined.

determined by NMR. In contrast to other CC and CXC chemokines that exist as homodimers in solution, the fractalkine chemokine module is monomeric. Other structural differences between fractalkine and other chemokine structures were noted, which are likely to be important in the interaction between fractalkine and its cellular receptor CX3CR1.

Important homologies Fractalkine is the sole member of the CX3C chemokine family. While fractalkine was identified by virtue of amino acid homology with members of the CC chemokine family, the three-dimensional structure of the chemokine domain of fractalkine is different from that of CC chemokines (Mizoue et al., 1999).

Posttranslational modifications The mucin stalk is extensively glycosylated.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Human endothelial cells express fractalkine in response to inflammatory signals. Neurons have

been shown to express fractalkine mRNA (Nishiyori et al., 1998). A subset of mononuclear phagocytes and dendritic cells express fractalkine.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Stimuli that elicit fractalkine secretion by HUVEC cells include IL-1 and TNF, and lipopolysaccharide.

RECEPTOR UTILIZATION Fractalkine binds with high affinity to the chemokine receptors CX3CR1 (Imai et al., 1997) and the cytomegalovirus receptor US28 (Kledal et al., 1998).

IN VITRO ACTIVITIES

In vitro findings Soluble fractalkine mediates the chemotaxis of monocytes, T cells, NK cells, and microglia in vitro. Immobilized extracellular forms of fractalkine mediate the tight adhesion of CX3CR1-expressing cells through a calcium-independent mechanism that does not involve integrins or G protein signaling (Imai et al., 1997).

1296 David R. Greaves and Thomas J. Schall

Bioassays used Monocyte chemotaxis.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Knockout mouse phenotypes To date, no fractalkine gene knockout mouse has been reported.

Interactions with cytokine network Fractalkine expression by HUVEC cells is upregulated by the proinflammatory cytokines IL-1 , TNF, and lipopolysaccharide (Bazan et al., 1997).

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects Fractalkine can exist as a membrane-bound and a cleaved soluble form. Anti-N-terminal fractalkine antibodies stain a wide range of cell types in normal and diseased human tissues. Normal levels of soluble fractalkine have not been reported.

IN THERAPY

Preclinical ± How does it affect disease models in animals? There have been no reports on fractalkine expression in animal disease models. Recently, it was shown that treatment with an anti-fractalkine receptor monoclonal antibody blocked leukocyte infiltration in the Wistar-Kyoto rat model of crescentic glomerulonephritis (Feng et al., 1999).

References Al-Aoukaty, A., Rolstad, B., Giaid, A., and Maghazachi, A. A. (1998). MIP-3alpha, MIP-3beta and fractalkine induce the locomotion and the mobilization of intracellular calcium, and activate the heterotrimeric G proteins in human natural killer cells. Immunology 95, 618±624.

Bazan, J. F., Bacon, K. B., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves, D. R., Zlotnik, A., and Schall, T. J. (1997). A new class of membrane-bound chemokine with a CX3C motif. Nature 385, 640±644. Combadiere, C., Salzwedel, K., Smith, E. D., Tiffany, H. L., Berger, E. A., and Murphy, P. M. (1998). Identification of CX3CR1. A chemotactic receptor for the human CX3C chemokine fractalkine and a fusion coreceptor for HIV-1. J. Biol. Chem. 273, 23799±23804. Feng, L., Chen, S., Garcia, G. E., Xia, Y., Siani, M. A., Botti, P., Wilson, C. B., Harrison, J. K., and Bacon, K. B. (1999). Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication. Kidney Int. 56, 612±620. Fong, A. M., Robinson, L. A., Steeber, D. A., Tedder, T. F., Yoshie, O., and Patel, D. D. (1998). Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow. J. Exp. Med. 188, 1413± 1419. Harrison, J. K., Jiang, Y., Chen, S., Xia, Y., Maciejewski, D., McNamara, R. K., Streit, W. J., Salafranca, M. N., Adhikari, S., Thompson, D. A., Botti, P., Bacon, K. B., and Feng, L. (1998). Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia. Proc. Natl Acad. Sci. USA 95, 10896±10901. Haskell, C. A., Cleary, M. D., and Charo, I. F. (1999). Molecular uncoupling of fractalkine-mediated cell adhesion and signal transduction. J. Biol. Chem. 274, 10053±10058. Imai, T., Hieshima, K., Haskell, C., Baba, M., Nagira, M., Nishimura, M., Kakizaki, M., Takagi, S., Nomiyama, H., Schall, T. J., and Yoshie, O. (1997). Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 91, 521±530. Kledal, T. N., Rosenkilde, M. M., and Schwartz, T. W. (1998). Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirus-encoded broad-spectrum receptor US28. FEBS Lett. 441, 209±214. Meucci, O., Fatatis, A., Simen, A. A., Bushell, T. J., Gray, P. W., and Miller, R. J. (1998). Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity. Proc. Natl Acad. Sci. USA 95, 14500±14505. Mizoue, L. S., Bazan, J. F., Johnson, E. C., and Handel, T. M. (1999). Solution structure and dynamics of the CX3C chemokine domain of fractalkine and its interaction with an N-terminal fragment of CX3CR1. Biochemistry 38, 1402±1414. Nishiyori, A., Minami, M., Ohtani, Y., Takami, S., Yamatomo, J., Kawaguchi, N., Kume, T., Akaike, A., and Satoh, M. (1998). Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from neuron to microglia? FEBS Lett. 429, 167±172. Nomiyama, H., Imai, T., Kusuda, J., Miura, R., Callen, D. F., and Yoshie, O. (1998). Human chemokines fractalkine (SCYD1), MDC (SCYA22) and TARC (SCYA17) are clustered on chromosome 16q13. Cytogenet. Cell Genet. 81, 10±11. Pan. Y., Lloyd, C., Zhou, H., Dolich, S., Deeds, J., Gonzalo, J. A., Vath, J., Gosselin, M., Ma, J., Dussault, B., Woolf, E., Alperin, G., Culpepper, J., Gutierrez-Ramos, J. C., and Gearing, D. (1997). Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation [Published erratum appears in Nature 389, 100]. Nature 387, 611±617. Rossi, D. L., Hardiman, G., Copeland, N. G., Gilbert, D. J., Jenkins, N., Zlotnik, A., and Bazan, J. F. (1998). Cloning and characterization of a new type of mouse chemokine. Genomics 47, 163±170.