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• W2045449670 abstract "The prompt clearance of cells undergoing apoptosis is critical during embryonic development, normal tissue turnover, as well as inflammation and autoimmunity. The molecular details of the engulfment of apoptotic cells are not fully understood. ced-6 and its human homologue gulp, encode an adapter protein, whose function in engulfment is highly evolutionarily conserved; however, the upstream and downstream components of CED-6 mediated signaling are not known. Recently, ced-1 has been shown to encode a transmembrane protein on phagocytic cells, with two functional sequence motifs in its cytoplasmic tail that are important for engulfment. In this study, using a combination of biochemical approaches and yeast two-hybrid analysis, we present evidence for a physical interaction between GULP/CED-6 and one of the two motifs (NP XY motif) in the cytoplasmic tail of CED-1. The phosphotyrosine binding domain of GULP was necessary and sufficient for this interaction. Since the precise mammalian homologue of CED-1 is not known, we undertook a database search for human proteins that contain the motifs shown to be important for CED-1 function and identified CD91/LRP (low density lipoprotein receptor-related protein) as one candidate. Interestingly, recent studies have also identified CD91/LRP as a receptor involved in the phagocytosis of apoptotic cells in mammals. The GULP phosphotyrosine binding domain was able to specifically interact with one specific NP XY motif in the CD91 cytoplasmic tail. During these studies we have also identified the mouse GULP sequence. These studies suggest a physical link between CED-1 or CD91/LRP and the adapter protein CED-6/GULP during engulfment of apoptotic cells and further elucidate the pathway suggested by the genetic studies. The prompt clearance of cells undergoing apoptosis is critical during embryonic development, normal tissue turnover, as well as inflammation and autoimmunity. The molecular details of the engulfment of apoptotic cells are not fully understood. ced-6 and its human homologue gulp, encode an adapter protein, whose function in engulfment is highly evolutionarily conserved; however, the upstream and downstream components of CED-6 mediated signaling are not known. Recently, ced-1 has been shown to encode a transmembrane protein on phagocytic cells, with two functional sequence motifs in its cytoplasmic tail that are important for engulfment. In this study, using a combination of biochemical approaches and yeast two-hybrid analysis, we present evidence for a physical interaction between GULP/CED-6 and one of the two motifs (NP XY motif) in the cytoplasmic tail of CED-1. The phosphotyrosine binding domain of GULP was necessary and sufficient for this interaction. Since the precise mammalian homologue of CED-1 is not known, we undertook a database search for human proteins that contain the motifs shown to be important for CED-1 function and identified CD91/LRP (low density lipoprotein receptor-related protein) as one candidate. Interestingly, recent studies have also identified CD91/LRP as a receptor involved in the phagocytosis of apoptotic cells in mammals. The GULP phosphotyrosine binding domain was able to specifically interact with one specific NP XY motif in the CD91 cytoplasmic tail. During these studies we have also identified the mouse GULP sequence. These studies suggest a physical link between CED-1 or CD91/LRP and the adapter protein CED-6/GULP during engulfment of apoptotic cells and further elucidate the pathway suggested by the genetic studies. In multicellular organisms, cells die by apoptosis throughout life (1.Gumienny T.L. Lambie E. Hartwieg E. Horvitz H.R. Hengartner M.O. Development. 1999; 126: 1011-1022Crossref PubMed Google Scholar). This is required for processes ranging from embryonic development to routine tissue maintenance. When cells undergo programmed cell death within an organism, the final act is the efficient removal of the apoptotic cells (2.Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1266) Google Scholar). Without such removal, the release of toxic contents of these dying cells could lead to inflammation and subsequent pathologies within the organism (3.Fadok V.A. J. Mammary Gland Biol. Neoplasia. 1999; 4: 203-211Crossref PubMed Scopus (68) Google Scholar). In fact, humans with deficiencies in genes involved in the phagocytosis of apoptotic cells and mice with targeted disruptions of specific genes develop symptoms characteristic of autoimmune disease (4.Botto M. Exp. Clin. Immunogenet. 1998; 15: 231-234Crossref PubMed Scopus (85) Google Scholar, 5.Mevorach D. Mascarenhas J.O. Gershov D. Elkon K.B. J. Exp. Med. 1998; 188: 2313-2320Crossref PubMed Scopus (564) Google Scholar, 6.Scott R.S. McMahon E.J. Pop S.M. Reap E.A. Caricchio R. Cohen P.L. Earp H.S. Matsushima G.K. Nature. 2001; 411: 207-211Crossref PubMed Scopus (907) Google Scholar). Although cells that die by either necrosis or apoptosis are eventually engulfed by the same phagocytic effector cells, uptake of apoptotic cells elicits anti-inflammatory cytokine production, in contrast to cells that die by necrosis (7.Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2516) Google Scholar, 8.Reiter I. Krammer B. Schwamberger G. J. Immunol. 1999; 163: 1730-1732PubMed Google Scholar, 9.Ren Y. Stuart L. Lindberg F.P. Rosenkranz A.R. Chen Y. Mayadas T.N. Savill J. J. Immunol. 2001; 166: 4743-4750Crossref PubMed Scopus (85) Google Scholar, 10.Sauter B. Albert M.L. Francisco L. Larsson M. Somersan S. Bhardwaj N. J. Exp. Med. 2000; 191: 423-434Crossref PubMed Scopus (1204) Google Scholar, 11.Green D.R. Beere H.M. Nature. 2000; 405: 28-29Crossref PubMed Scopus (87) Google Scholar). This is most likely due to differences in the signaling mechanisms activated during the recognition and/or processing of dying cells by the phagocyte. Thus, our understanding the molecular mechanism involved in clearance of apoptotic cells is of fundamental importance and could likely have a direct clinical relevance. A number of cell surface receptors have been implicated in the phagocytosis of apoptotic cells in mammals, and these include scavenger receptors, integrins, the phosphatidylserine receptor, and complement receptors (2.Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1266) Google Scholar). There is an apparent redundancy in the receptors required for engulfment, since blocking of individual receptors with antibodies appears to diminish but not completely abrogate engulfment of apoptotic targets. Differences in utilization of these receptors depending on the phagocytic cell as well as their state of activation has also been documented (2.Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1266) Google Scholar, 12.Fadok V.A. Warner M.L. Bratton D.L. Henson P.M. J. Immunol. 1998; 161: 6250-6257PubMed Google Scholar, 13.Pradhan D. Krahling S. Williamson P. Schlegel R.A. Mol. Biol. Cell. 1997; 8: 767-778Crossref PubMed Scopus (126) Google Scholar). Thus, the relative significance of the individual receptors has remained difficult to assess. Moreover, relatively little is known about the cytoplasmic proteins recruited downstream of these receptors that regulate engulfment of apoptotic cells in mammals. However, recent discoveries through elegant genetic studies in the nematode Caenorhabditis elegans system have provided insight into some of the critical players involved in engulfment (14.Ellis R.E. Jacobson D.M. Horvitz H.R. Genetics. 1991; 129: 79-94Crossref PubMed Google Scholar, 15.Hengartner M.O. Cell. 2001; 104: 325-328Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 16.Horvitz H.R. Cancer Res. 1999; 59: 1701s-1706sPubMed Google Scholar). To date, seven genes required for the engulfment of apoptotic cells have been identified in the worm. The recent cloning of all of the seven genes in the worm and the existence of mammalian homologues for six of the seven genes, have provided an exciting opportunity to better delineate the signaling pathways during engulfment of apoptotic cells. The seven genes identified in the worm have been classified into two partially redundant functional pathways, based on the severity of the phenotypes in double-mutants within these two groups. One group of genes consists of the genes ced-2, ced-5, ced-10, and ced-12, and their respective mammalian homologues have been identified as crkII, dock180, rac, and elmo (17.Reddien P.W. Horvitz H.R. Nat. Cell Biol. 2000; 2: 131-136Crossref PubMed Scopus (329) Google Scholar, 18.Albert M. Kim J. Birge R. Nat. Cell Biol. 2000; 2: 899-905Crossref PubMed Scopus (322) Google Scholar, 19.Leverrier Y. Ridley A.J. Curr. Biol. 2001; 11: 195-199Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 20.Tosello-Trampont A. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 21.Wu Y.C. Horvitz H.R. Nature. 1998; 392: 501-504Crossref PubMed Scopus (136) Google Scholar, 22.Gumienny T. Brugnera E. Tosello-Trampont A. Kinchen J. Schedl T. Francis R. Nishiwaki K. Nemergut M. Macara I. Van Aelst L. Qin Y. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Analysis of this group of genes in C. elegans, as well as their homologues in Drosophila and mammals indicate that all of these four cytoplasmic signaling proteins play a role in organizing and controlling cytoskeletal rearrangement during engulfment and cell migration (17.Reddien P.W. Horvitz H.R. Nat. Cell Biol. 2000; 2: 131-136Crossref PubMed Scopus (329) Google Scholar, 18.Albert M. Kim J. Birge R. Nat. Cell Biol. 2000; 2: 899-905Crossref PubMed Scopus (322) Google Scholar, 19.Leverrier Y. Ridley A.J. Curr. Biol. 2001; 11: 195-199Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 20.Tosello-Trampont A. Brugnera E. Ravichandran K.S. J. Biol. Chem. 2001; 276: 13797-13802Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22.Gumienny T. Brugnera E. Tosello-Trampont A. Kinchen J. Schedl T. Francis R. Nishiwaki K. Nemergut M. Macara I. Van Aelst L. Qin Y. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). The receptor(s) that function upstream of this group of genes remains to be identified. The other group includes the genes ced-6/gulp, ced-1, and ced-7/abc1 (23.Wu Y.C. Horvitz H.R. Cell. 1998; 93: 951-960Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 24.Zhou Z. Hartwieg E. Horvitz H.R. Cell. 2001; 104: 43-56Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 25.Liu Q.A. Hengartner M.O. Cell. 1998; 93: 961-972Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 26.Liu Q.A. Hengartner M.O. Curr. Biol. 1999; 9: 1347-1350Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 27.Smits E. Van Criekinge W. Plaetinck G. Bogaert T. Curr. Biol. 1999; 9: 1351-1354Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 28.Luciani M.F. Chimini G. EMBO J. 1996; 15: 226-235Crossref PubMed Scopus (248) Google Scholar). ced-6, as well as its Drosophila and human homologues, encode an evolutionarily conserved adapter protein that is required specifically in the engulfing cells and not in the dying cells. CED-6 and its homologues contain an N-terminal phosphotyrosine binding (PTB) 1The abbreviations used are: PTBphosphotyrosine bindingLRPlow density lipoprotein receptor-related proteinEGFepidermal growth factorSH2Src homology 2GULPenGULfment adaPter proteinGSTglutathione S-transferaseHAhemagglutininGFPgreen fluorescent proteinLZleucine zipper domainCEDcell death abnormal 1The abbreviations used are: PTBphosphotyrosine bindingLRPlow density lipoprotein receptor-related proteinEGFepidermal growth factorSH2Src homology 2GULPenGULfment adaPter proteinGSTglutathione S-transferaseHAhemagglutininGFPgreen fluorescent proteinLZleucine zipper domainCEDcell death abnormal domain, a central leucine zipper, and a proline-rich C-terminal region (25.Liu Q.A. Hengartner M.O. Cell. 1998; 93: 961-972Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 26.Liu Q.A. Hengartner M.O. Curr. Biol. 1999; 9: 1347-1350Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 27.Smits E. Van Criekinge W. Plaetinck G. Bogaert T. Curr. Biol. 1999; 9: 1351-1354Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The conservation of CED-6 function through evolution was best exemplified by the rescue of the engulfment defect in ced-6-deficient worms through expression of human ced-6/gulp as a transgene (26.Liu Q.A. Hengartner M.O. Curr. Biol. 1999; 9: 1347-1350Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This suggested that the interacting partners as well as some of the signaling pathways downstream of CED-6 are likely conserved from worm to humans. Overexpression of human CED-6 in the macrophage line J774 also promotes engulfment (27.Smits E. Van Criekinge W. Plaetinck G. Bogaert T. Curr. Biol. 1999; 9: 1351-1354Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Presently, there are no known binding partners for CED-6 or its homologues. Interestingly, overexpression of ced-6 in the ced-1- and ced-7-deficient backgrounds partially rescues the engulfment defect (25.Liu Q.A. Hengartner M.O. Cell. 1998; 93: 961-972Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), suggesting that CED-6 might function as a signaling adapter downstream of CED-1 and CED-7, both of which are membrane proteins. However, whether CED-1 or CED-7 interacts with CED-6 and how such an interaction might be mediated has not yet been determined. phosphotyrosine binding low density lipoprotein receptor-related protein epidermal growth factor Src homology 2 enGULfment adaPter protein glutathione S-transferase hemagglutinin green fluorescent protein leucine zipper domain cell death abnormal phosphotyrosine binding low density lipoprotein receptor-related protein epidermal growth factor Src homology 2 enGULfment adaPter protein glutathione S-transferase hemagglutinin green fluorescent protein leucine zipper domain cell death abnormal The molecular nature of ced-1 was revealed very recently. ced-1 encodes a putative receptor required only on the engulfing cells and likely participates in the recognition of apoptotic cells (24.Zhou Z. Hartwieg E. Horvitz H.R. Cell. 2001; 104: 43-56Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). CED-1 contains a large extracellular portion encoding 16 atypical EGF-like repeats, a transmembrane region, and a short cytoplasmic region. Two amino acid sequence motifs in the CED-1 cytoplasmic tail, NP XY and Y XXL motifs, have been shown to be important for function based on the rescue of ced-1-deficient worms using wild type or mutated forms of CED-1. This also suggested that CED-1 could transduce signals during engulfment, since NP XY motifs can serve as binding targets for PTB domains, while the Y XXL motifs can serve as targets for Src-homology 2 (SH2) domains. Currently, the precise mammalian homologue of CED-1 is unknown. The mammalian proteins, MEGF-6 and SREC, identified as most similar to CED-1 (24.Zhou Z. Hartwieg E. Horvitz H.R. Cell. 2001; 104: 43-56Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar), fail to show any homology in the cytoplasmic region with CED-1 and do not contain the NP XY or Y XXL motifs. The homologue of CED-7 (the third member of this group that contains CED-1 and CED-6) is the 12-transmembrane ATP-binding cassette transporter protein ABC1, which appears to be required on the engulfing as well as the dying cells (23.Wu Y.C. Horvitz H.R. Cell. 1998; 93: 951-960Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 28.Luciani M.F. Chimini G. EMBO J. 1996; 15: 226-235Crossref PubMed Scopus (248) Google Scholar, 29.Hamon Y. Broccardo C. Chambenoit O. Luciani M.F. Toti F. Chaslin S. Freyssinet J.M. Devaux P.F. McNeish J. Marguet D. Chimini G. Nat. Cell Biol. 2000; 2: 399-406Crossref PubMed Scopus (457) Google Scholar). However, how CED-7/ABC1 proteins signal and the manner in which they functionally interact with other members of this functional genetic pathway are not known. In this study, using several approaches, we observe the binding of the PTB domain of worm and mammalian CED-6 proteins to the NP XY motif in the cytoplasmic tail of CED-1. The PTB domain of CED-6 appears to be necessary and sufficient to mediate the interaction with CED-1. We also observe the binding of GULP to CD91/LRP, which has recently been found to play a role in the phagocytosis of apoptotic cells (30.Ogden C.A. deCathelineau A. Hoffmann P. Bratton D. Ghebrehiwet B. Fadok V. Henson P.M. J. Exp. Med. 2001; 194: 781-795Crossref PubMed Scopus (931) Google Scholar). These studies further elucidate the pathway suggested by the genetic studies in C. elegans and also confirm and extend them in mammalian cells. The DNA coding sequence for the peptide constructs were cloned by making pairs of oligonucleotides, which contain a 5′-NdeI and a 3′-NotI site and cloned into a modified pEBG eukaryotic expression vector (31.Su H.P. Brugnera E. Van Criekinge W. Smits E. Hengartner M. Bogaert T. Ravichandran K.S. J. Biol. Chem. 2000; 275: 9542-9549Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). GULP and CED-1 cytoplasmic tail expression constructs were obtained by PCR amplification of the relevant cDNAs and cloned into the modified pEBG eukaryotic expression vectors (for expression as a glutathione S-transferase (GST) fusion protein) and pEBB-HA (for expression of hemagglutinin (HA)-tagged proteins) using 5′-NdeI and 3′-NotI restriction sites. To generate the CD16-CED-1·GFP fusion construct, first the cytoplasmic region of CED-1 was amplified and cloned into the pEBB-CD16 vector as described previously (32.Walk S.F. March M.E. Ravichandran K.S. Eur. J. Immunol. 1998; 28: 2265-2275Crossref PubMed Scopus (53) Google Scholar). The enhanced green fluorescent protein (EGFP) coding sequence was subcloned from pEGFP-N1 (CLONTECH, Palo Alto, CA) as a KpnI-NotI fragment in-frame at the 3′ end of the CED-1 coding sequence. The cDNA constructs of CD91/LRP minireceptors have been described previously (37.Li Y. Marzolo M.P. van Kerkhof P. Strous G.J. Bu G. J. Biol. Chem. 2000; 275: 17187-17194Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% bovine calf serum, 5% fetal calf serum, and penicillin/streptomycin/glutamine (Invitrogen). COS-7 cells were transfected using Superfect or Polyfect (Qiagen, Valencia, CA) according to the manufacturer's recommendation. All transient transfections were performed using 2–10 μg of the appropriate expression plasmid and analyzed 24–48 h posttransfection. Cells were lysed on the tissue culture plates with lysis buffer (50 mm Tris, pH 7.6, 150 mm NaCl, 10 μm each aprotinin, leupeptin, pepstatin, AEBSF, 10 mm sodium fluoride, 10 mm sodium pyrophosphate, 1 mm sodium orthovanadate, and 1% Nonidet P-40). GST-tagged proteins were precipitated with glutathione-Sepharose (Amersham Biosciences, Inc.). Precipitates were washed four times with buffer containing 20 mm HEPES, pH 7.6, 150 mm NaCl, 1 μm each aprotinin, leupeptin, pepstatin, AEBSF, 5 mm sodium fluoride, 1 mm sodium orthovanadate, and 0.1% Nonidet P-40. Antibodies for Western blotting GFP, HA, and GST-tagged proteins were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies conjugated to horseradish peroxidase were purchased from Amersham Biosciences, Inc. All immunoblots were developed using enhanced chemiluminescence (Pierce, Rockford, IL). All experiments were performed at least three times. The CytoTrap two-hybrid system was purchased from Stratagene (La Jolla, CA). The ced-6 constructs were cloned in-frame of the hSOS fusion protein (pSos), and the ced-1 constructs were cloned in-frame with the myristoylation membrane localization signal (pMyr). The plasmids were verified by DNA sequencing. Transformation of the Saccharomyces cerevisiae strain, cdc25H, was performed according to the manufacturer's directions. The interaction between the proteins encoded by the introduced plasmids was scored by growth of the yeast transformants at the non-permissive temperature on galactose plates. The specificity of the interactions was verified by growth on glucose plates as well as cotransformation with plasmids encoding mutant proteins or control proteins. The file containing predicted human proteins was downloaded from www.ensembl.org (33.Hubbard T. Birney E. Nature. 2000; 403: 825Crossref PubMed Scopus (23) Google Scholar). The searches were performed on the April freeze dataset using a script written by us, which scans the database for a given motif and removes all sequences that do not contain the motif. The sequences that were not removed were scanned for all the other motifs. The remaining sequences were subjected to BLAST searches to determine the identity of the genes. The mouse GULP sequence was derived from clones represented by GenBankTM accession numbers, AK014093 and AK017798, and the publicly available draft mouse genomic sequence. Comparison with human GULP suggests that AK014093 encodes all but the first two residues of mouse GULP; however, these two were present in AK017798. Additionally, two residues that did not align between human GULP and AK014093 are found to align between human GULP and AK017798, but were confirmed by the mouse genomic draft sequences. The C-terminal 20-amino acids of AK017798 appear to be derived from genomic intron sequences and did not align with the human GULP or the AK014093 clone. While the genetic studies clearly show a role for CED-6 in engulfment of apoptotic cells, the binding partners of CED-6 are not known. Specifically, we were interested in interacting partners for the CED-6 PTB domain. These previous studies had identified the C. elegans, Drosophila melanogaster, and Homo sapiens ced-6 genes, but the identity of the murine ced-6 gene was not known (26.Liu Q.A. Hengartner M.O. Curr. Biol. 1999; 9: 1347-1350Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 27.Smits E. Van Criekinge W. Plaetinck G. Bogaert T. Curr. Biol. 1999; 9: 1351-1354Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). During the course of the studies described below, we identified the murine ced-6 homologue from database searches. By assembling two murine ced-6 expressed sequence tags and comparison with the draft mouse genomic sequence we have compiled the cDNA sequence encoding murine CED-6 (Fig. 1). The mouse ced-6 gene encodes a 304-amino acid protein with the same overall architecture as the human and worm CED-6 proteins: an N-terminal PTB domain, immediately followed by a leucine zipper domain and a C-terminal proline-rich region. The mouse and human CED-6 proteins are highly homologous (93% amino acid identity) with only a two-residue difference within the PTB domain. Comparison of C. elegans CED-6 with the mammalian CED-6 proteins reveals 34 extra residues at the N-terminal end and an additional 150 amino acids at the C-terminal region (Fig. 1). Previously, the human CED-6 protein was simply referred to as hCED-6 (26.Liu Q.A. Hengartner M.O. Curr. Biol. 1999; 9: 1347-1350Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 27.Smits E. Van Criekinge W. Plaetinck G. Bogaert T. Curr. Biol. 1999; 9: 1351-1354Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). To avoid confusion between worm and mammalian proteins, we have renamed the mammalian CED-6 proteins as GULP (enGULfment adaPter protein). PTB domains have previously been shown to be capable of interacting with sequences that contain a core NP XY motif, although the specificity of particular PTB domains depends on neighboring sequences (38.Borg J.P. Margolis B. Curr. Top. Microbiol. Immunol. 1998; 228: 23-38PubMed Google Scholar, 39.Siegal G. Nat. Struct. Biol. 1999; 6: 7-10Crossref PubMed Scopus (13) Google Scholar). The recent cloning of the c ed-1 gene reveals that it encodes a transmembrane receptor containing cytoplasmic NP XY and Y XXL motifs (potential binding sites for both PTB and SH2 domains, respectively) that were shown to be critical for CED-1-dependent phagocytosis (24.Zhou Z. Hartwieg E. Horvitz H.R. Cell. 2001; 104: 43-56Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Since CED-1 and CED-6 belong to the functional genetic pathway (14.Ellis R.E. Jacobson D.M. Horvitz H.R. Genetics. 1991; 129: 79-94Crossref PubMed Google Scholar, 24.Zhou Z. Hartwieg E. Horvitz H.R. Cell. 2001; 104: 43-56Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar) and CED-6 contains a PTB domain (25.Liu Q.A. Hengartner M.O. Cell. 1998; 93: 961-972Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), we tested whether the CED-1 NPLY sequence (surrounding tyrosine 965) may serve as a ligand for the CED-6/GULP PTB domains. We generated eukaryotic expression vectors encoding GST fused to a 13-amino acid CED-1 sequence containing the NP XY motif. The ability of HA-tagged GULP to bind to this CED-1-derived peptide was tested. After transient transfection into COS-7 cells, the GST·CED-1 peptide was precipitated using glutathione beads, and the coprecipitation of HA-GULP was assessed by immunoblotting. As shown in Fig. 2B, GULP was coprecipitated with the GST-tagged CED-1 peptide. However, CED-1 peptides carrying the mutation of the asparagine to alanine in the −3 position (relative to the tyrosine) disrupted the interaction. Since SH2 domain-mediated interactions require residues C-terminal to the tyrosine, we mutated the glutamine to an alanine, in the +3 position relative to the tyrosine, to test if this motif was binding to GULP in a manner similar to SH2 domain ligands. Mutation of glutamine at the +3 position did not appear to affect binding to GULP (Fig. 2B). To determine whether GULP can interact with the NP XY motif in the intact CED-1 cytoplasmic tail and to determine the relative contribution of NP XY versus Y XXL motifs, a construct encoding the entire cytoplasmic region of CED-1 fused to a GST tag was generated. In addition, mutations of the asparagine of the NP XY motif (N962A mutant) and the tyrosine in the Y XXL motif (Y1019A mutant) were also performed (Fig. 2C). When coexpressed with GULP in COS-7 cells, both the wild type and the Y1019A mutant bound to GULP, but the N962A mutant of CED-1 showed a severe reduction in binding (Fig. 2D), suggesting that the interaction most likely occurs through GULP binding to the NP XY motif on CED-1. We have previously identified a leucine zipper domain immediately following the PTB domain of GULP, which mediates homodimerization (31.Su H.P. Brugnera E. Van Criekinge W. Smits E. Hengartner M. Bogaert T. Ravichandran K.S. J. Biol. Chem. 2000; 275: 9542-9549Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). We tested whether dimerization of GULP is required for interaction with CED-1 using a leucine zipper mutant of GULP (mLZ), shown in Fig. 2E, that we have shown fails to dimerize (31.Su H.P. Brugnera E. Van Criekinge W. Smits E. Hengartner M. Bogaert T. Ravichandran K.S. J. Biol. Chem. 2000; 275: 9542-9549Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). This mutant LZ construct bound CED-1 comparable with wild type GULP (Fig. 2F), suggesting that under these conditions, the leucine zipper-dependent dimerization is not critical for the CED-1/GULP interaction. Next, we wished to test whether GULP can interact with membrane-bound CED-1. Our multiple efforts to express full-length CED-1 on the cell surface were unsuccessful, and the reason for this unclear. To assess the interaction of GULP with a membrane-bound CED-1, we fused the cytoplasmic tail of CED-1 to the extracellular domain of CD16 (34.Kolanus W. Romeo C. Seed B. Cell. 1993; 74: 171-183Abstract Full Text PDF PubMed Scopus (301) Google Scholar). We added a GFP tag to the C terminus of the CD16-CED-1 sequence to verify that the fusion protein was expressed on the cell surface (data not shown) as well as providing a tag for immunoblotting. When coexpressed, GULP was able to coprecipitate the CD16-CED-1·GFP fusion protein (Fig. 3A). We also tested whether the isolated PTB domain of GULP can interact with CED-1. Two different GULP PTB constructs (one that terminates just prior to the leucine zipper and another that contains the core PTB sequence based on alignment with other PTB domains) were both capable of precipitating the CD16-CED-1·GFP fusion protein. Taken together, these data suggest that GULP, via its PTB domain, can interact with a membrane-bound CED-1. We then tested whether the C. elegans CED-6 PTB domain can also interact with the NP XY sequence motif in the CED-1 cytoplasmic tail. Although the PTB domains of GULP and CED-6 are quite similar, the worm protein has an additional 35 residues N-terminal to the homology with GULP PTB. Thus, we generated two versions of CED-6 PTB coding for amino acids 1–200 and 36–200 (Fig. 3C). Both of these constructs were able to interact with the wild type CED-1 cytoplasmic tail as shown in Fig. 3D. The interaction of CED-6 PTB was severely diminished with the N962A mutant of CED-1. These data suggested that the PTB domain of GULP and CED-6 have similar ligand binding characteristics, which is consistent with the observation that the human GULP was able to rescue the cell corpse engulfment defect in the ced-6 mutant worm (26.Liu Q.A. Hengart" @default.
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• W2045449670 title "Interaction of CED-6/GULP, an Adapter Protein Involved in Engulfment of Apoptotic Cells with CED-1 and CD91/Low Density Lipoprotein Receptor-related Protein (LRP)" @default.
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