Fas

Fas Shigekazu Nagata* Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan * corresponding author tel: 81-6-6879-3310, fax: 81-6-6879-3319, e-mail: [email protected] DOI: 10.1006/rwcy.2000.16004.

SUMMARY Fas is a type I membrane protein belonging to the TNF/NGF receptor family, and is expressed in various tissues and cell lines. Binding of FasL or agonistic anti-Fas antibody to Fas causes apoptosis in Fasbearing cells. That is, the Fas engagement recruits procaspase 8 through an adapter molecule called FADD. Oligomerization of pro-caspase 8 induces processing of pro-caspase 8 to active forms. The caspase 8 then activates other caspases in the downstream of the caspase cascade. These caspases then cleave various cellular substrates to cause morphological changes of the cells, and to degrade chromosomal DNA. The Fas-mediated apoptosis is involved to kill the activated T cells and B cells, thus downregulating the immune reaction. The loss-of-function mutations in Fas in human and mouse cause lymphoproliferation, and accelerate autoimmune diseases.

BACKGROUND

cross-hybridization with human cDNA (WatanabeFukunaga et al., 1992b).

Alternative names CD95, Apo1.

Structure Human Fas is a type I membrane protein with apparent molecular mass of 48 kDa (Itoh et al., 1991; Oehm et al., 1992).

Main activities and pathophysiological roles Fas transduces an apoptotic signal into cells upon engagement by Fas ligand (FasL) or agonistic antiFas antibody (Itoh et al., 1991; Oehm et al., 1992; Nagata and Golstein, 1995; Nagata, 1997).

Discovery

GENE

Fas was discovered as a cell surface antigen that can be recognized by a cytotoxic monoclonal antibody (anti-Fas or anti-Apo1 antibody) against human cellsurface protein (Trauth et al., 1989; Yonehara et al., 1989). The human Fas cDNA was identified from human leukemia cell line by expression cloning using the anti-Fas antibody (Itoh et al., 1991), or by using the amino acid sequence information of the purified protein (Oehm et al., 1992). The mouse Fas cDNA was subsequently isolated from mouse cDNA library by

Accession numbers Human Fas: M67454 (Itoh et al., 1991). Mouse Fas: M83649 (Watanabe-Fukunaga et al., 1992b).

Sequence See Figure 1.

1650 Shigekazu Nagata Figure 1 Nucleotide sequence for human Fas. 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2261 2341 2401 2461 2521

CACGCTTCTG GGCACTGGCA CTCCCGCGGG ATTGCTCAAC CTAGATTATC AATTGAGGAA GCCAATTCTG ATGGGGATGA ATTTTTCTTC AAATAAACTG ACTCTACTGT AATGCACACT GGCTTTGTCT AGAAAACATG ATCCTGAAAC TTCCTGGAGT AAGCCAAAAT AACTGCTTCG AAGATCTCAA AGGACATTAC AGAGTGAAAA AATAGCTGGC CTGAAGAGCC TAAAATCTAG CAAATAGGAG ATTCTGTAGT CCACTCTATG GGCAGGCCAC AACTTTGTTT AATTTAAATA ATATTTCAAT GTGTATGCAT GAGCAGGAGA AATGGCCTAA AAAGGCAAGA AAAGTAGCTT CTTATTTTTC TGGCAGCTTA TACCCTCAAG TGAAAGTTTG CTGTGAAGAT TGGAGGATTT AAATAATGTT

GGGAGTGAGG CGGAACACAC TTGGTGGACC AACCATGCTG GTCCAAAAGT GACTGTTACT CCATAAGCCC ACCAGACTGC CAAATGCAGA CACCCGGACC ATGTGAACAC CACCAGCAAC TCTTCTTTTG CAGAAAGCAC AGTGGCAATA CATGACACTA AGATGAGATC TAATTGGCAT AAAAGCCAAT TAGTGACTCA ACAACAAATT TGTAAATACT AACATATTTG TTGGGAAAAC TGTATGCAGA ATGAATGTAA AATCAATAGA TTTGCCTCTA ATAACTCTGA AGGCTCTACC TGTGAATTCA TTTACTGGCT GTATTACTAG TGCACCCCCA CTGCCCTTAG TGTGACATGT CCCCACCCCC TACATAGCAA TTTCTAAGAT TATTAAATGT AGTTATAAAC TTTTGCCCCT TTTG

GAAGCGGTTT CCTGAGGCCA CGCTCACTAC GGCATCTGGA GTTAATGCCC ACAGTTGAGA TGTCCTCCAG GTGCCCTGCC AGATGTAGAT CAGAATACCA TGTGACCCTT ACCAAGTGCA CCAATTCCAC AGAAAGGAAA AATTTATCTG AGTCAAGTTA AAGAATGACA CAACTTCATG CTTTGTACTC GAAAATTCAA CAGTTCTGAG GCTTGGTTTT TAGATTTTTA AAACTTCATC GGATGAAAGA TCAGTGTATG AGAAGCTATG AATTACCTCT GAAGATCATA TCAAAGACCT CATAGAAAAC CAAAACTACC AGCTTTGCCA AACATGGAAA AAATTCTAGC CATGAACCCA GAAAATGTTC TGGTAAAATC TTAAGATTCT GAATTTTAAG TGAAGCAGAT TGTGTTTGGA

Chromosome location and linkages Human chromosome 10q24.1 (Inazawa et al., 1992) Mouse chromosome 11 (Watanabe-Fukunaga et al., 1992b) linked to lpr mutation.

PROTEIN

Accession numbers Human Fas: PID g105364, g182410 (Itoh et al., 1991) Mouse Fas: PID g346698, g193226 (WatanabeFukunaga et al., 1992b).

Description of protein Human Fas is comprised of 319 amino acids with a signal sequence of 15 amino acids at the N-terminus

ACGAGTGACT CCCCTTGCTG GGAGTTCGGG CCCTCCTACC AAGTGACTGA CTCAGAACTT GTGAAAGGAA AAGAAGGGAA TGTGTGATGA AGTGCAGATG GCACCAAATG AAGAGGAAGG TAATTGTTTG ACCAAGGTTC ATGTTGACTT AAGGCTTTGT ATGTCCAAGA GAAAGAAAGA TTGCAGAGAA ACTTCAGAAA TATATGCAAT TTACTGGGTA ATATCTCATG AAGAGTAAAT TTAAGATTAT TTAGTACAAA ACCTTTTGCT GATAATTCTA TTTATGTAAA TTGCACAGTT ATTAAATTAT TACTTCTTTC CCTCTCCATT TATCACCAAA CTGGTTTGGA TGTTTGCAAT AATAATGTCC ATCATCTGGA CCTTACTACT AAATAATATT ACCTGGAACC ATTATAAAAT

TGGCTGGAGC CCCAGGCGGA AAGCTCTTTC TCTGGTTCTT CATCAACTCC GGAAGGCCTG ACCTAGGGAC GGAGTACACA AGGACATGGC TAAACCAAAC TGAACATGGA ATCCAGATCT GGTGAAGAGA TCATGAATCT GAGTAAATAT TCGAAAGAAT CACAGCAGAA AGCGTATGAC AATTCAGACT TGAAATCCAA TAGTGTTTGA CATTTTATCA ATTCTGCCTC GCAGTGGCAT GCTCTGGCAT TGTCTATCCA GAAATATCAG GAGATTTTAC GTATATGTAT TATTGGTGTC AATGTTTGAC TCAGGCATCA TTTGCCTTGG AAATACTTAA GATACTAACT CAAAGATGAT CATGTAAAAC TTTAGGAATT ATCCTACGTT TATATTTCTG ACCTAAAGAA ATAGGTAAAA

CTCAGGGGCG GCTGCCTCTT ACTTCCCACC ACGTCTGTTG AAGGGATTGG CATCATGATG TGCACAGTCA GACAAAGCCC TTAGAAGTGG TTTTTTTGTA ATCATCAAGG AACTTGGGGT AAGGAAGTAC CCAACCTTAA ATCACCACTA GGTGTCAATG CAGAAAGTTC ACATTGATTA ATCATCCTCA AGCTTGGTCT AAAGATTCTT TTTATTAGCG CAAGGATGTT GCTAAGTACC CTAACATATG CAGGCTAACC TTACTGAACA CATATTTCTA TTGAGTGCAG ATATTATACA TATTATATAT AAAGCATTTT TGCTCATCTT TAGTCCACCA GCTCTCAGAG AAAATAGATT CTGCTACAAA GCTCTTGTCA TAAATATCTT TAAATGTAAA CTTCCATTTA GTACGTAATT

(Itoh et al., 1991; Oehm et al., 1992). A single transmembrane domain of 17 amino acids divides the molecule into the extracellular region of 157 amino acids, and the cytoplasmic region of 145 amino acids. The extracellular region can be further divided into three cysteine-rich subregions.

Relevant homologies and species differences The extracellular region of Fas has a homology (25± 30% homology) with that of other TNF/NGF receptor family members (Itoh et al., 1991; Oehm et al., 1992; Smith et al., 1994). The cytoplasmic region of Fas contains a domain of about 80 amino acids that shows a homology with the corresponding region of the TNF type I receptor (Itoh and Nagata, 1993; Tartaglia et al., 1993) and the death receptors DR3 and DR4 (Ashkenazi and Dixit, 1998). The domain is called the `death domain'. The amino acid

Fas 1651 sequence of mouse Fas has an identity of 49.3% with human Fas (Watanabe-Fukunaga et al., 1992b). There is no species-specificity among human, mouse, and rat (Takahashi et al., 1994).

Affinity for ligand(s) Fas ligand binds to Fas with a Kd of about 1.0 nM.

Cell types and tissues expressing the receptor Fas is ubiquitously expressed in various tissues and cells (Watanabe-Fukunaga et al., 1992b; LeithaÈuser et al., 1993). In particular, Fas is expressed in the thymus, activated T cells, hepatocytes, and heart. Lymphomas of T cell and B cell origin express a high level of Fas, constitutively (Falk et al., 1992; Debatin et al., 1994).

Regulation of receptor expression Expression of Fas in mature T cells is upregulated by activation with phorbol myristate acetate and ionomycin. A cis-regulatory element of NFB at positions ÿ295 to ÿ286 in the 50 flanking region of human Fas chromosomal gene is responsible for the induced expression of Fas in T cells (Chan et al., 1999). Oncogene p53 upregulates the expression of Fas (Owen-Schaub et al., 1995). p53-responsive elements in the intron 1 and 50 promoter flanking region of human Fas gene seem to be responsible for the p53-dependent expression of Fas (Muller et al., 1999).

Release of soluble receptors The soluble Fas can be produced by alternative splicing (Cascino et al., 1995; Hughes and Crispe, 1995; Papoff et al., 1996). Patients with nonhematopoietic malignancies or ATL (adult T cell leukemia) have a high level of soluble Fas in the serum (Sugawara et al., 1997). There is a soluble decoy receptor (DcR3) to which FasL binds with an affinity similar to that with which it binds the authentic Fas receptor (Pitti et al., 1999). The DcR3 gene is amplified in about half of human lung cancer and colon cancer cells.

SIGNAL TRANSDUCTION

Associated or intrinsic kinases The cytoplasmic region of Fas and its signaling molecule, FADD, is constitutively phosphorylated (Kennedy and Budd, 1998). Several kinases including RIP (receptor-interacting protein) and p59fyn were reported to interact with the cytoplasmic region of the Fas receptor (Stanger et al., 1995; Atkinson et al., 1996). However, the cells deficient in RIP kinase are potent to transduce the apoptotic signal, suggesting that RIP is not essential for the Fas-mediated apoptotic signal transduction (Ting et al., 1996).

Cytoplasmic signaling cascades A cascade of caspases (cysteine proteases) is activated by Fas engagement (Enari et al., 1996; Nagata, 1997; Tewari and Dixit, 1995) (Figure 2). Binding of Fas ligand or agonistic anti-Fas antibody to Fas activates Fas. Fas ligand is a trimer, and the agonistic anti-Fas antibodies are IgM (immunoglobulin pentamer), or IgG3 that tends to aggregate. The F(ab0 )2 fragment of anti-Apo1 antibody or other isotypes does not activate Fas (Dhein et al., 1992), suggesting that Fas must be trimerized or aggregated to the higher order to transduce the signal (Takahashi et al., 1996). Trimerization of Fas by FasL or agonistic anti-Fas or Apo1 antibody causes recruitment of caspase 8 to Fas through an adapter called FADD (Boldin et al., 1995, 1996; Chinnaiyan et al., 1995; Muzio et al., 1996). This activates caspase 8, which then sequentially activates other caspases such as caspases 3, 6, and 7 in the downstream of the caspase cascade (Hirata et al., 1998; Kawahara et al., 1998). Caspases thus activated would cleave various cellular substrates to progress the apoptotic program (Martin and Green, 1995; Nagata, 1997). One of the caspase substrates is ICAD (inhibitor of caspase-activated DNase)/DFF45 (DNA fragmentation factor 45) (Liu et al., 1997; Sakahira et al., 1998). The cleavage of ICAD by caspase 3 leads to the activation of a DNase (CAD), which causes the DNA fragmentation seen in most apoptotic cells (Enari et al., 1998). This signal transduction cascade was confirmed by knockingout the respective signaling molecules. That is, the cells lacking FADD or caspase 8 do not undergo apoptosis induced by Fas activation (Varfolomeev et al., 1998; Yeh et al., 1998; Zhang et al., 1998a). The cells lacking caspase 3 or ICAD do not show DNA fragmentation induced by Fas activation (Woo et al., 1998; Zhang et al., 1998b).

1652 Shigekazu Nagata Figure 2 Fas-induced apoptosis. Binding of Fas ligand to Fas induces trimerization of Fas, which recruits procaspases 8 via an adapter called FADD/MORT1, and induces its processing to the mature form. In one signaling pathway, caspase 8 directly activates caspase 3 downstream of the caspase cascade. In another pathway, caspase 8 cleaves Bid, a proapoptotic member of the Bcl2 family. The cleaved Bid then enters mitochondria to cause release of cytochrome C, which activates caspase 9 together with Apaf-1. The caspase 9 then activates caspase 3. Caspase 3, thus activated, cleaves various cellular substrates to induce morphological changes of cells and nuclei. One of the caspase 3 substrates is ICAD/DFF-45, which is complexed with CAD, a specific DNase. Cleavage of ICAD by caspase 3 releases CAD from ICAD, and CAD then enters nuclei to cause DNA fragmentation.

DOWNSTREAM GENE ACTIVATION

Transcription factors activated Transcription factors are not activated by Fas engagement, although NFB was reported to be activated in some cell lines (Rensing et al., 1995).

Genes induced The Fas activation quickly leads to cleavage of chromosomal DNA (Itoh et al., 1991), and does not induce gene expression.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors Activation of Fas causes apoptotic cell death in most cases (Nagata, 1997). It may also cause necrotic cell death in certain conditions (Vercammen et al., 1998).

Phenotypes of receptor knockouts and receptor overexpression mice

Many other signaling cascades initiated by the activated Fas leading to apoptosis have been proposed. For example, ceramide was postulated to mediate the Fas signal by activating the Ras/MAP kinase pathway (Cifone et al., 1994; Gulbin et al., 1995; Tepper et al., 1995). However, recent analysis suggests that ceramide is not activated during Fasmediated apoptosis, and ceramide is probably not involved in this signaling pathway (Hofmann and Dixit, 1998; Hsu et al., 1998; Watts et al., 1997). Other pathways proposed involving DAXX, JNK, or Bid (Brenner et al., 1997; Yang et al., 1997; Li et al., 1998; Luo et al., 1998) also need to be confirmed.

Mouse mutation lpr (lymphoproliferation) is a lossof-function mutation of Fas (Watanabe-Fukunaga et al., 1992a) (Figure 3). In one allele (lpr), an early transposable element (ETn) is inserted in intron 2 of the Fas chromosomal gene (Adachi et al., 1993). The Fas transcript prematurely terminates in intron 2. However, the inhibition of expression is not complete, as demonstrated by the presence of full-length Fas mRNA, albeit at a low level, suggesting that lpr is a leaky mutation. In the other allele (lprcg), a point mutation that causes replacement of an amino acid from isoleucine to asparagine is introduced in the death domain of the Fas cytoplasmic region, which abolishes the ability of Fas to transduce the apoptotic signal (Watanabe-Fukunaga et al., 1992a). Mice carrying the lpr mutation develop lymphadenopathy and splenomegaly, produce autoantibodies, and suffer

Fas 1653 Figure 3 Mutations of Fas in lpr-mice and in human patients with Canale±Smith syndrome. (a) A mutation in the Fas gene in lpr mice. The structure of mouse Fas chromosomal gene is schematically shown. In lpr mice an early transposable element (ETn) carrying poly(A) addition signal on a long terminal repeat (LTR) is inserted in intron 2 of the gene. This causes premature termination of Fas transcript. (b) Point mutations in the Fas death domain in human Canale±Smith syndrome and lprcg mice. The amino acid sequences of the death domain of human and mouse Fas are aligned. The amino acid replacements in patients of Canale±Smith syndrome are indicated on the upper line, while the mutation in lprcg mice is shown in the lower line.

wild

lpr

from autoimmune diseases such as nephritis and arthritis (Cohen and Eisenberg, 1991). Fas knockout mice were also established, and the mice shows more accelerated and pronounced lymphadenopathy and splenomegaly than lpr mice (Adachi et al., 1995).

Human abnormalities Human patients with Canale±Smith syndrome or autoimmune lymphoproliferative syndrome show phenotypes (lymphadenopathy, splenomegaly, and autoimmune diseases) similar to those in lpr mice, and carry loss-of-function mutations in the Fas gene (Fisher et al., 1995; Rieux-Laucat et al., 1995; Nagata, 1998). Mutations in the Fas death domain in this syndrome are shown in Figure 3b.

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain Administration of soluble Fas (the extracellular region of Fas fused to the Fc region of human immunoglobulin) blocks development of CTL-induced hepatitis in a mouse model system (Kondo et al., 1997).

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LICENSED PRODUCTS Monoclonal antibodies (clones CH-11, UB-2, and ZB-4) against human Fas are available from Medical & Biological Laboratories (MBL), Nagoya, Japan. The CH-11 antibody works as an agonist (Yonehara et al., 1989), while the ZB-4 antibody works as an antagonist. Monoclonal antibody against mouse Fas (clone Jo2) (Ogasawara et al., 1993) is available form PharMingen (San Diego, CA, USA). The Jo2 antibody works as an agonist.