{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T08:19:34Z","timestamp":1792657174753,"version":"4.5.0"},"reference-count":35,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5475","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2000,6,30]]},"abstract":"<jats:p>Heterozygous mutations encoding abnormal forms of the death receptor Fas dominantly interfere with Fas-induced lymphocyte apoptosis in human autoimmune lymphoproliferative syndrome. This effect, rather than depending on ligand-induced receptor oligomerization, was found to stem from ligand- independent interaction of wild-type and mutant Fas receptors through a specific region in the extracellular domain. Preassociated Fas complexes were found in living cells by means of fluorescence resonance energy transfer between variants of green fluorescent protein. These results show that formation of preassociated receptor complexes is necessary for Fas signaling and dominant interference in human disease.<\/jats:p>","DOI":"10.1126\/science.288.5475.2354","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:50:55Z","timestamp":1027763455000},"page":"2354-2357","source":"Crossref","is-referenced-by-count":558,"title":["Fas Preassociation Required for Apoptosis Signaling and Dominant Inhibition by Pathogenic Mutations"],"prefix":"10.1126","volume":"288","author":[{"given":"Richard M.","family":"Siegel","sequence":"first","affiliation":[{"name":"Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John K.","family":"Frederiksen","sequence":"additional","affiliation":[{"name":"Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David A.","family":"Zacharias","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute and Department of Pharmacology, University of California, San Diego School of Medicine, La Jolla, CA 92093, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francis Ka-Ming","family":"Chan","sequence":"additional","affiliation":[{"name":"Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michele","family":"Johnson","sequence":"additional","affiliation":[{"name":"Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Lynch","sequence":"additional","affiliation":[{"name":"Immunex Corporation, 51 University Street, Seattle, WA 98101, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roger Y.","family":"Tsien","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute and Department of Pharmacology, University of California, San Diego School of Medicine, La Jolla, CA 92093, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael J.","family":"Lenardo","sequence":"additional","affiliation":[{"name":"Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.immunol.17.1.331"},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.281.5381.1305"},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.immunol.17.1.221"},{"key":"e_1_3_1_5_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.272.46.28889"},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1084\/jem.185.8.1487"},{"key":"e_1_3_1_7_2","unstructured":"A. M. Chinnaiyan K. O'Rourke"},{"key":"e_1_3_1_7_3","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(95)90541-3"},{"key":"e_1_3_1_8_2","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1995.tb00245.x"},{"key":"e_1_3_1_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/384638a0"},{"key":"e_1_3_1_10_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.8.4345"},{"key":"e_1_3_1_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0968-0004(97)01085-2"},{"key":"e_1_3_1_12_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.7539157"},{"key":"e_1_3_1_13_2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJM199611283352204"},{"key":"e_1_3_1_14_2","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(95)90013-6"},{"key":"e_1_3_1_15_2","doi-asserted-by":"publisher","DOI":"10.7326\/0003-4819-130-7-199904060-00020"},{"key":"e_1_3_1_16_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.8.4552"},{"key":"e_1_3_1_17_2","doi-asserted-by":"publisher","DOI":"10.1172\/JCI5121"},{"key":"e_1_3_1_18_2","doi-asserted-by":"publisher","DOI":"10.1086\/302333"},{"key":"e_1_3_1_19_2","unstructured":"R. M. Siegel and M. J. Lenardo unpublished data."},{"key":"e_1_3_1_20_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.288.5475.2351"},{"key":"e_1_3_1_21_2","unstructured":"Fas truncation mutants were created by polymerase chain reaction (PCR) mutagenesis with appropriate primers and Pwo high-fidelity polymerase (Roche Molecular Biochemicals). For the AU-1\u2013tagged receptors a template with DNA coding for the DTYRYI AU-1 epitope tag sequence previously inserted into the region upstream of Fas CRD1 was used. For HA tagging mutations were cloned into the Eco RI\u2013Xho I sites of a modified pCDNA3 vector containing the leader sequence of p60 followed by sequences coding for the YPYDVPDYA HA epitope tag. Point mutations were created with the Quickchange technique (Stratagene) substituting Pwo for Pfu polymerase. Mutations were verified by restriction enzyme mapping and automated sequencing."},{"key":"e_1_3_1_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0968-0004(97)01164-X"},{"key":"e_1_3_1_23_2","unstructured":"Murine BW thymoma cell were transfected by electroporation with 10 \u03bcg of the indicated plasmids and 5 \u03bcg of the GFP reporter plasmid. After 18 hours apoptosis was induced with Fas mAb APO-1 (500 ng\/ml) or 5% v\/v supernatant containing soluble FasL\/leucine zipper fusion protein. Viable cell loss was quantitated as described (13). Cell loss induced by APO-1 for singly transfected receptors was 29% for full-length Fas (1\u2013317) 2.9% for Fas 43\u2013317 \u20131.7% for Fas 67\u2013317 and \u20138.1% for vector-transfected controls."},{"key":"e_1_3_1_24_2","doi-asserted-by":"crossref","unstructured":"A. Miyawaki and R. Y. Tsien Methods Enzymol. in press; L. Stryer Annu. Rev. Biochem. 47 819 (1978); R. M. Siegel et al. \u201cMeasurement of molecular interactions in living cells by fluorescence resonance energy transfer between variants of the green fluorescent protein \u201d Science 's STKE (2000) (www.stke.org\/cgi\/content\/full\/OC_sigtrans;2000\/38\/pl1).","DOI":"10.1126\/stke.2000.38.pl1"},{"key":"e_1_3_1_25_2","unstructured":"Fas receptors with COOH-terminal in-frame fusions to CFP and YFP (at position 210 in place of the death domain) were cotransfected into 293T HEK cells and they were appropriately expressed on the cell surface (18). In-frame CFP and YFP fusions with Fas and other TNF family receptors were generated by standard PCR cloning techniques; correct protein expression was confirmed by Western blotting and fluorescence microscopy. 293T cells were transfected with 1 \u03bcg of the indicated YFP fusion protein constructs and 2 \u03bcg of the indicated CFP constructs. After 24 to 36 hours cells were harvested in phosphate-buffered saline and analyzed on a FACSvantage cytometer (Becton Dickinson) with a krypton laser tuned to 413 nm for CFP and an air-cooled laser tuned to 514 nm for YFP. CFP was detected with a 470 nm\/20 nm bandpass filter. YFP and FRET were detected with 546 nm\/10 nm bandpass filters with signals from the 514- and 413-nm lasers respectively. Cells were sequentially illuminated with the 514- and 413-nm lasers so that all three signals could be detected from each cell. Compensation was applied so that there was no FRET signal visible from cells transfected with CFP or YFP alone. We collected 50 000 events from each sample and analyzed the data with the FlowJo software package (Treestar Inc.). Another characteristic of FRET is the dequenching of donor fluorescence after photobleaching of the acceptor. This dequenching can be converted to a measurement of FRET efficiency (E%) which is related to the distance between two molecules by the F\u00f6rster equation [see (23)]. For FRET efficiency measurements CFP emission intensities from cotransfected cells were measured on a fluorescence microscope before and after bleaching the YFP with 5 min of illumination through a 505- to 545-nm bandpass filter. We corrected for direct bleaching of cells transfected with the CFP fusion partner alone. FRET efficiencies were calculated using the formula E% = [1 \u2013 (CFP emission before YFP bleach\/CFP emission after YFP bleach)] \u00d7 100%."},{"key":"e_1_3_1_26_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.270.1.387"},{"key":"e_1_3_1_27_2","doi-asserted-by":"publisher","DOI":"10.1038\/329219a0"},{"key":"e_1_3_1_28_2","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.18.6.3321"},{"key":"e_1_3_1_28_3","doi-asserted-by":"publisher","DOI":"10.1038\/7701"},{"key":"e_1_3_1_28_4","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1994.tb06326.x"},{"key":"e_1_3_1_29_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.274.53.38241"},{"key":"e_1_3_1_29_3","doi-asserted-by":"crossref","first-page":"4622","DOI":"10.4049\/jimmunol.156.12.4622","volume":"156","author":"Papoff G.","year":"1996","unstructured":"Papoff G., et al., J. Immunol. 156, 4622 (1996).","journal-title":"J. Immunol."},{"key":"e_1_3_1_30_2","unstructured":"293T cells were transfected with Fugene 6 (Boehringer Mannheim) according to the manufacturer's instructions. Cells were lysed in 150 mM NaCl 20 mM tris-Cl (pH 7.5) 1 mM EDTA 5 mM iodoacetamide 2 mM dithiothreitol 10% glycerol 1% Triton X-100 and protease inhibitors (Boehringer Mannheim). After preclearing with protein G\u2013agarose beads (Boehringer Mannheim) and normal mouse immunoglobulin G proteins were immunoprecipitated with 1 mg anti-GFP (Roche Molecular Biochemicals) and protein G\u2013agarose beads. Immune complexes were washed three times with lysis buffer. AU-1 was immunoprecipitated with 2 \u03bcl of anti\u2013AU-1 (Covance) and protein A beads. Proteins were electrophoresed on Tris\/Glycine gels (Novex) transferred to nitrocellulose membranes and blotted with the indicated antibodies. Bands were visualized with SuperSignal WestDura (Pierce). Densitometry was performed with one-dimensional image analysis software (Kodak)."},{"key":"e_1_3_1_31_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1074-5521(99)80051-9"},{"key":"e_1_3_1_32_2","unstructured":"We thank D. Martin L. Zheng and D. Smith for plasmid construction; K. Holmes R. Swofford and D. Stephany for excellent technical assistance; M. Peter and B. Herman for valuable reagents; T. Waldmann R. Germain H. Metzger J. O'Shea C. Jackson and F. Hornung for critical reading of the manuscript and helpful comments; and S. Starnes for editorial assistance. S. Straus J. Puck C. Jackson and J. Dale of the NIH ALPS working group provided clinical material and encouragement that were essential for this work. Supported by a Cancer Research Institute\/Miriam and Benedict Wolf fellowship (F.K.-M.C.) NIH grant NS27177 (R.Y.T.) and a fellowship in the HHMI\/NIH scholar program (M.J.)."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.288.5475.2354","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T10:15:37Z","timestamp":1705140937000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.288.5475.2354"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2000,6,30]]},"references-count":35,"journal-issue":{"issue":"5475","published-print":{"date-parts":[[2000,6,30]]}},"alternative-id":["10.1126\/science.288.5475.2354"],"URL":"https:\/\/doi.org\/10.1126\/science.288.5475.2354","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2000,6,30]]}}}